BILAN de l’activité de recherche et des résultats obtenus par l’unité (Partie I : Bilan scientifique/Scientific Report)

Chimie et Biologie des Membranes et des Nanoobjets

                                       

UMR 5248 CNRS, Université Bordeaux 1, ENITAB

2006-2009  

 

                                        Directeur de rédaction : Erick Dufourc Rédacteurs : Christine Lopes-Monteiro, Patricia Dulor, Axelle Grélard, Claude Manigand, AnneMarie Elié, Emmanuel Geneste, Jean-Pierre Aimé, Alain Brisson, Patrick Cottin, Bernard Desbat, Juan Elezgaray, Bernard Gallois, Léon Ghosez, Gilles Guichard, Ivan Huc, Olivier Lambert, Jochen Lang, Fernando Léal-Calderon, Reiko Oda, Jean-Marie Schmitter, Maria Urdaci. Pessac le 9 October 2009

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Summary

GENERAL CONSIDERATIONS

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CBMN at Glance Aims of the Laboratory ADMINISTRATIVE & FINANCIAL REPORT Organisation Human resources Finances Hygiene & Safety measures Management & quality considerations

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DIFFUSION OF SCIENTIFIC & TECHNICAL KNOWLEDGE Teaching & research training Management of platforms Formation of people Development of applied science Diffusion of science

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DETAILED SCIENTIFIC ACTIVITY REPORT 2006-2009 Highlights, Prizes Detailed scientific report of CBMN teams Detailed scientific report of groups willing to join CBMN

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SCIENTIFIC PRODUCTION LISTS

113

Annexe 1: Hygiène & Sécurité Annexe 2: Plan de Formation

161 167

Excel Charts for Scientific Report (only on paper support)

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GENERAL CONSIDERATIONS The laboratory « Chemistry & Biology of Membranes & Nanoobjects” (CBMN) has been created in January 2007 as UMR 5248. This is a mixed unit in the sense that it is attached to CNRS (Institutes of Chemistry & Biological Sciences), the University of Bordeaux (Chemistry & Biology departments) and the National School of Engineers in Technical Agronomy, ENITAB. In order to present a view of the activity on a quadrennial period, the scientific production has been taken from 2006; in fact the teams that constitute the laboratory were already working together before the official creation. In its present form, the laboratory is organized in 4 departments that are further organized in 12 teams. The team is the effective “building block” of the UMR in the sense that it has complete responsibility for science and finances, within the laboratory project. After the general presentation of the Unit, the team activities will be presented, within their respective department. The total number of scientists and faculty members is 37. There are 20 engineers, technicians and administrative people and an average yearly number of Master, PhD and post doc of 60. About 40 undergraduate students are also present in the lab for short training periods. The Laboratory has three (3) geographical locations: IECB building (B13), B8 building and ENITAB. The three places are within 100 m to 1 km distance. 2007 has been the year were all the laboratory organization has been set up: team management, laboratory council, hygiene and safety committee, continuous training, web site, internal regulation, automated holidays management, etc. Funding of the lab is growing up from year to year due to the extreme dynamism of teams. The consolidated 2009 budget is of more than 9 million Euros of which 40% is obtained from competitive granting agencies. As it will be seen later, the scientific activity has been growing along the years and the quality of research has increased. As an indicator (to be taken with caution as always), in the previous activity report the global impact factor (number of papers times the journal impact factor) was 3.4 for the entire Laboratory, it is now of 4.3. Patenting activity, diffusion of science, platform labelling and other activities also contribute indicating that the choice of creating a laboratory at the interface of chemistry, biology and physics was a wise move. The laboratory is going to further expand by increasing its size by 50 % and addressing more questions in the field of nutrition and health with the aim of scientific excellence. As it will be seen in the Project part, CBMN is going to grow up and make marked structural changes. Two of existing groups will merge together. 4 other groups will join the Laboratory among which one is going to also merge into existing groups. The 14 teams then obtained will be structured into 3 departments (see project part). A new building of about 3000 m2 will host two of the departments, at the beginning of 2010. Scientific reports from these new groups willing to join will be presented after the actual CBMN team reports.

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CBMN at glance (2007-2009) Laboratory surfaces Personnel*** Permanent Non-permanent PhD Students Research Teams PhD & HDR Publications Patents Grants public & private**** Technical Platforms

2007 3000 m2 95 49 46 33 11 19* 148* 4* 1700 k€ 5

2009 3500 m2 111 57 54 38 12 18** 145** 3** 4100k€ 6

*2006-07. **2008-june09; ***does not include undergraduate students ****Does not include grants obtained for multisite technical platforms (NMR, X-rays, etc.)

Aims of the Laboratory Its mission is to bring a fundamental knowledge of complex biological phenomena by analyzing them at several time and spatial scales, from the molecule to the cell and to the organism. Beside the fundamental aspects of research, the laboratory develops also aspects very applied towards the cellular adhesion, the nanochips, the delivery of active molecules, the valorisation of probiotic bacteria, diabetes and colloids in food and pharmacy. We develop fundamental aspects on molecular modelling of biomolecules, structural biology (NMR, X-rays, Electron microscopy, vibrational spectroscopies, mass spectrometry, etc.), chemical and physicochemical conception of biomimetic scaffolds, production and test of biomolecules that may be used in understanding or curing various biological malfunctions. Transfer of knowledge is also very important and it is in the objectives of the laboratory to patent discoveries and develop them either in collaboration with well established industrial partners or participate in the construction and running of start-ups.

ADMINISTRATIVE & FINANCIAL REPORT Organisation The effective organization chart is presented in the following pages with development of items.

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Organisation de l’UMR 5248 « Chimie et Biologie des Membranes et des Nanoobjets » Chimie Physique des Systèmes membranaires Formation

Secrétariat Gestion

Internet Communication

Desbat, Elezgaray, Oda

Biophysique et Biochimie structurale

Relations Université

Directeur Erick Dufourc

Directoire Scientifique

Dufourc, Schmitter, Gallois

Directeur Adjoint Alain Brisson

Conseil de Laboratoire

Responsable Administrative Patricia Dulor

Animation Scientifique

Hygiène et Sécurité

June 2009

Plateau Technique

Services Techniques

Chimie biomimétique et médicinale Huc, Ghosez

Biologie cellulaire, Microbiologie et Biochimie des membranes Brisson, Lambert, Lang, Urdaci

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Organisation de l’UMR 5248, Services Formation

B. Desbat

Secrétariat Gestion

P. Dulor (resp. adm.) , E. Dupouyet

Internet Communication

C. Buré, JM. Chevalier, E. Geneste**, R. Oda, V. Zhendre

Relations Université

A. Brisson, F. Godde, J. Lang, JM Schmitter, M. Urdaci

Directoire Scientifique

A. Brisson, B. Desbat, E. Dufourc, B. Gallois, J. Elezgaray, I. Huc, O lambert, J. Lang, R. Oda, JM Schmitter, M. Urdaci

Conseil de Laboratoire

K. Bathany, MN Bennassy, P. Bonnafous, A. Brisson, S. Castano, F. Caubet, G. Charpentier, X. De Hatten, M. Denayrolles, J. Dessolin, E. Dufourc, E. Dupouyet, F. Godde, F. Jean-François, M. Laguerre, S. Trepout

Animation Scientifique

I. Huc, E. Dufourc

Hygiène et Sécurité

A.M. Elie, A. Grélard, C. Manigand

Plateau Technique

RMN

A. Grélard, Y. Chollet**

SM

K. Bathany, C. Buré

ME

J. Lai Kee Him, S. Tan

VIB

B. Desbat

MM

M. Laguerre

Electrop

G. Charpentier

Services Techniques

AM Elie, J. Merias, F. Pedaros-Caubet **UMS 3033 7

Organisation de l’UMR 5248, Départements Scientifiques Départements

Direction

Chimie Physique des Systèmes Membranaires

Biophysique et Biochimie Structurale

Chimie Biomimétique et Médicinale

Biologie Cellulaire, Microbiologie et Biochimie des Membranes

Equipes

Membres permanents

Desbat

B. Desbat, S. Lecomte, S. Castano, J. Géan

Oda

R. Oda

Elezgaray/Laguerre

J. Dessolin , J. Elezgaray, M. Laguerre,

Schmitter

JM Schmitter, K. Bathany, C. Buré, E. Geneste, S. Chaignepain

Gallois

B. Gallois, T. Garnier, B. d’Estaintot, C. Manigand, JM Chevalier, J. Chaudière

Dufourc

B. Odaert, A. Grelard, I Pianet, E. Dufourc

Huc

Y. Ferrand, F. Godde, I. Huc

Ghosez

L. Ghosez

Brisson

A. Brisson, A. Bouter, J. Lai Kee Him, C. Gounou

Lambert

O. Lambert, P. Bonnafous, JC. Taveau

Lang

J. Lang, G. Charpentier, V. Lagrée, R. Benoit, P. Scotti, MN Benassy, A. Milochau,

Urdaci

M. Urdaci, P. Bressolier, R. Caubet, M. Denayrolles B. Grossiord, F. Caubet, AM Elie 8

Three instances are used by the directors to help running the laboratory

1. Scientific Advisory Council It is constituted by group leaders and advises during monthly meetings the directors on the scientific policy of the Laboratory. Members Alain BRISSON, PR

Maria URDACI, PR

Bernard GALLOIS, DR

Ivan HUC, DR

Bernard DESBAT, DR

Juan ELEZGARAY, DR

Reiko ODA, CR

Jean-Marie SCHMITTER, PR

Erick DUFOURC, DR

Jochen LANG, DR

Léon GHOSEZ, PR

Olivier LAMBERT, DR

Minutes of meetings are available in the CBMN Intranet

2. Laboratory Advisory Council The Laboratory Advisory Council is compulsory by CNRS and Ministry of Research status. It advises the directors on the scientific policy, the management of resources and the general organization and running of the Laboratory. It is composed of representatives of all personnel categories who are both elected or nominated by the direction. NAME Surname BATHANY Katell BENASSY Marie-Noëlle BONNAFOUS Pierre BRISSON Alain CASTANO Sabine CHARPENTIER Gilles DE HATTEN Xavier DENAYROLLES Muriel DESSOLIN Jean DUFOURC Erick DUPOUYET Evelyne GODDE Frédéric LAGUERRE Michel CALA Olivier PEDARROS-CAUBET Françoise TREPOUT Sylvain

Quality IE UBx 1 TCN UBx 1 MdC UBx 1 PR UBx 1 MdC UBx 1 PR UBx 1 Post-doctorant MdC ENITAB CR CNRS DR CNRS TCN CNRS MdC UBx 1 DR CNRS PhD TCN UBx 1 PhD

Statut Elected Nominated Nominated Adjunct Director Nominated Elected Elected Elected Elected Director Elected Nominated Elected Elected Nominated Elected

Minutes of meetings are available in the CBMN Intranet

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3. Hygiene & Safety Council The Hygiene & Safety Council is compulsory by CNRS and Ministry of Research status. It helps the directors on all matters dealing with hygiene and safety measures. Hygiene & Safety Council NAME Surname 1 2 3 4 5 6 7 8 9 10 11 12 13 14

ELIE GRELARD MANIGAND BONNAFOUS DESSOLIN De HATTEN TREPOUT DUFOURC

Anne -Marie Axelle Claude Pierre Jean Xavier Sylvain Erick

Quality IE, ACMO site ENITAB IE, ACMO site IECB IE, ACMO site B8 MdC CR Post-Doctorant Doctorant Directeur Médecin du travail CNRS Resp H &S DR15 CNRS Médecin du travail UBx1 Resp H & S UBx1 Médecin du travail ENITAB Resp H & S ENITAB

Human resources The personnel are composed of 37 CNRS Scientists and Faculty members. During the quadrennial, two scientists and one faculty member retired and we could hire or welcome in mutation from other laboratories, two scientists and four faculty members. Also three engineers or administrative people left for the UMS 3033 and fortunately, we could recruit or welcome in mutation from other laboratories 4 engineers, technicians or administrative staff amounting the number of permanent people to 57 as for June 2009. The laboratory is very dynamic in recruiting non permanent people because the other half of the personnel is composed of PhD students and post-doctoral fellows. In addition we welcome about 40 undergraduate students per year for short research training periods. In total the number of Laboratory members’ ranges from 100 to 140 on a yearly basis. A point that is important to emphasize is the fact that we have only 2 administrative staff for running all finances and human resources. This is clearly too small and, as it will be presented in the project, the priority number one of the laboratory is to strengthen the administrative staff.

Finances (details in Excel charts (3.3) and the end of document) As it can be seen in the chart below, funding originates from three main sources: i) recurrent “State” grants, ii) grants from others sources and iii) salaries for permanent people. State grants (mean of 200 k€) that are attributed to the Laboratory by CNRS and the Ministry of research depending on the number of Scientists and Faculty members represents between 2 and 5 % of the total budget. Salaries represent about 5 million € per year. Both items did not evolve much from 2007 to 2009. The other grants (ANR, Europe, industry, charity agencies, etc.) are obtained on a competitive basis and are evolving rapidly between 2007 and 2009. Thanks to the dynamism of teams that are successful in applications they represent 40% of the total budget in 2009. Mode of repartition Recurrent “State” funding They are attributed to the teams in January on the following basis: 60% go directly to the groups depending of the number of members (number of points): each scientist, faculty member, PhD, Post doc and engineers or technicians participating in effective research is worth of one point. 10

CBMN Budget 6 000 5 000

Keuros

4 000 3 000

State Grants (SE)

2 000

Other Grants (RP)

1 000

Salaries

0 2007

2008

2009

Year

Master students and undergraduate trainees staying for less than six months are not counted. The date for counting is the 1st of January. Teams that do not have engineers or technicians are given a virtual point for compensation. In July an adjustment is made depending whether or not teams have negotiated other grants. Other grants Grants obtained by members are entirely given back to the team to which they belong. The team defines its proper policy for spending grants. The UMR does not take overheads on these grants. This is mainly due to the fact that during the past quadrennial most of the teams where indebted for 15% to the UMS 3033 (IECB) for participation in running coasts. A different policy will be defined for the next quadrennial. Infrastructure grants The UMR is hosted by the University of Bordeaux and ENITAB. Costs for general running (electricity, water, heating/air conditioning, etc) are directly managed by trusties.

Management & quality considerations Automated orders The UMR has installed a system that allows an automated management of orders (machines, chemicals, supplies, etc.). Due to the different geographical locations, a network of computers with the appropriate secured software has been installed where teams are located. Supervisors (permanent team members) have been trained and habilitated to enter orders on the account of the research group. There is hence only one computer manipulation which is of course validated by the Laboratory accounting person. This procedure simplifies the operation and gives management visibility to the research group and efficiency for global managing. Since 2008 the following supervisors have been mandated. Céline Gounou-Tessier Myriam Delamarre Michel Laguerre Béatrice Langlois d’Estaintot

Sophie Lecomte Marie-Noëlle Benassy Benoit Odaert

Katell Bathany Alexandra Milochau Yann Ferrand

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Holiday management. Holiday that personnel can regularly take is also managed automatically through the laboratory intranet. Asking for holiday is made through the web site and is validated by email warning to the group leader or hierarchy responsible. This is a very flexible procedure that allows knowing whether personnel are in holidays, mission, training, etc. This is of particular importance for insurance coverage reasons. Internet, Communication (http://www.cbmn.u-bordeaux.fr) A dynamic (in the sense of data bases) and interactive internet site has been built-up by Emmanuel Geneste. It is modifiable by laboratory members depending on their authorization level. Among the much functionality, Laboratory, group or members’ publications are automatically managed and interfaced with the web of science. The “chargé de communication” in the UMR are Reiko Oda et Vanessa Zhendre. In particular they organize regularly laboratory open days such as « fête de la science ».

Hygiene & Safety measures (see Annexe 1 for details) The UMR has 3 engineers responsible for Hygiene & Safety, called ACMO, who are located in the three building where there are CBMN laboratories: Anne-Marie Elié (ENITAB), Claude Manigand (B8) et Axelle Grélard (IECB). In addition, other people are members of the Hygiene & Safety council (see above) and help the direction in all these matters. This committee meets at least once a year. There are more frequent meetings between the ACMO and the direction. The ACMO are doing a fantastic job in ensuring that working in a laboratory in made in safe conditions. Together with the direction they organized several aspects of the laboratory life. Welcome to new members The welcome and training of new members takes different forms: A synthetic form “New member form” allows checking personal social insurance, where the activity is to be conducted, and authorizations to access laboratories. In can be found in the Intranet, and has to be filled by the newcomer and handed at the administration after having seen all key people (group leader, webmaster, ACMO, etc.). The new member has then been made aware of all Laboratory regulations and can be registered in the Labintel (CNRS registry) and Web site. The UMR internal regulation. It is available in intranet and is given to all newcomers. It contains in detail all instructions about laboratory rules. Welcome document: this is a practical manual for Hygiene and Safety rules in the laboratory that is handed to all newcomers. Welcome day: because new members often arrive in October, in the fall, half a day is dedicated to the welcome and explanation of the Laboratory regulations and safety measures to new members. This comes in addition of the welcome document. The director, group leaders and ACMO are giving short talks to inform the new members of general and specific rules for Hygiene and Safety in the laboratory or in specific experiments. A visit of the building where the new member will work is organized with the corresponding ACMO, to become familiar will all security apparatus and exits. New members arriving at other dates, all along the year receive a specific formation by the ACMO of the site. Internet: a web page dedicated to H & S is present on the CBMN web site. Report on Hygiene and security problems and actions to solve them. All accidents or incidents are reported on a H & S book available on all geographical sites. The ACMO together with the direction take actions to resolve potential malfunctions. Some actions need repairs or installation of security apparatus (extraction systems, life blankets, etc.). A detailed report is made in Annexe 1. There have been only minor incidents except for a fire that occurred in November 19th 2008 in the IECB site (2nd floor). A fume hood has been destroyed in the chemistry laboratory and intervention of fireman (Talence brigade) was necessary. Nobody was injured due to the selfcontrol of two post-docs that were working when the fire occurred and who warned the fire brigade. The entire lab was unusable due to accumulation of dust. The accident has evidenced the insufficient number of anti-fire blankets, powder extinguishers and sand-buckets. These defects 12

have been corrected. The laboratory has been refurbished due to the conjugated efforts of the UMS 3033 (IECB) and our UMR.

DIFFUSION OF SCIENTIFIC & TECHNICAL KNOWLEDGE Teaching & research training The laboratory has 20 Faculty members teaching in both the Chemistry and the Biology departments at the University of Bordeaux. There are all responsible for defining the program of lectures (UE, teaching units) at all levels in University (Licence to Master). Some professors or research directors have the responsibility of an entire Master (those of Biochemistry and Chemical Biology or Cell Biology and Pathophysiology for instance) or training of professors for technical institutes (CAPET). Some CBMN Professors are directors of graduate schools (Biology and Health for instance). Practical training of undergraduate students on high-tech machines (NMR spectrometers, Mass spectrometry, X-rays, electron microscopy, vibrational spectroscopy, etc.) is currently conducted by CBMN faculty members. The Laboratory welcomes on a yearly basis about 40 undergraduate students and 15 PhD theses.

Management of platforms 1)

2)

3)

4)

5)

Various teams ensure the maintenance and management of technological platforms. NMR for UMS 3033 (IECB): Erick Dufourc’s team. 6 machines (100 MHz WB, 300 MHz NB with sample changer, 300 MHz WB, 400 MHz NB, 500 MHz WB, 700 MHz NB). Approximately 60 people (UMR and UMS) have access to the platform after being properly trained by the team. The team also ensures studies for both academic and industrial external users (TOTAL, SANOFI, etc.). In 2006 the team was at the initiative of setting up the Aquitaine NMR network, whose role is to foster regional scientific exchanges. This networking was very useful in building up the funding (Aquitaine region, Ministry of Research, Europe, CNRS, INRA) and the negotiation of two spectrometers (600 & 700 MHz). Both machines have been successfully installed in July 2007 at IECB and in March 2008 at INRA. The team succeeded also in bringing up a scientific program and a budget to acquire, as part of the national TGIR NMR, a 800 MHz machine. Bordeaux is now part of this national NMR large scale facility with Paris, Lille, Grenoble, Lyon and Orleans. The team ensures the local contact and running for external users. Electron Microscopy for UMS 3033 (IECB): Alain Brissons and Olivier Lambert’s teams. 2 machines CM-120 and Tecnai-20 FEI). In 2008, Olivier Lambert was at the initiative of setting up an Electron Microscopy network for Aquitaine region. The team also ensures studies for both academic and industrial requests (SANOFI, etc.). Their expertise in electron tomography is present in the “Pôle de competence Imagerie 3D en Aquitaine” supported by MIB (Institute Carnot). A plan is made for installing during the quadrennial an advanced 300kV cryoTEM instrument designed for high resolution tomography. Mass Spectrometry for UMS 3033 (IECB) and Plateforme de Génomique Fonctionnelle (PGF). Jean-Marie Schmitter‘s team. 5 instruments (MALDI and ESI ionisation). In the fall 2009, the team will be building another site at PGF dedicated to mass spectrometry applied to “omics” (proteomics, lipidomics) with access to a complementary set of six tandem mass spectrometers. X-Rays for UMS 3033 (IECB). Reiko Oda, Ivan Huc and Bernard Gallois’s teams. 3 machines SAXS/WAXS small molecules and protein crystallography. Ivan Huc and Bernard Gallois were at the initiative of setting up a X-rays crystallography network for Aquitaine region. Optical and Vibrational spectroscopy. Bernard Desbat’s team. 3 machines. The team is setting up a platform for both Raman and IR imaging (funding for 3 additional machines). 13

6)

Molecular Modeling. Juan Elezgaray and Michel Laguerre’s team. Access to large scale facility computing on national networks. The team serves as an interface for UMR users for large scale computing.

Formation of people The laboratory is taking care, through the responsible for continuous formation, Bernard Desbat, of permanent formation of members (See details in Annexe 2). Also, specific formations are proposed to internal members and external people. They take the form of advanced schools (NMR, Vibrational spectroscopy, Mass spectrometry) or workshops that are recognized by both CNRS or Ministry or Research.

Development of applied science The UMR has strong connections with the industry (food and wine industry, pharmaceutical industries) for transferring basic knowledge into applied technology. Several CIFRE (grants through the Ministry of Industry) conventions have been passed with companies. Two industrial “startup” companies in the field of pharmaceutical tools were at the initiative or involving UMR members: FluoFarma (Michel Laguerre, Léon Ghosez) and AnnexinTools (Alain Brisson).

Diffusion of science The Laboratory members have a strong implication in congress or advanced schools. During the last quadrennial they organized entirely or in part 26 international events. Very often the group leaders were presidents of the organizing or scientific committees. In December 2008 a group of young scientists and faculty members has been organizing in coordination with the direction the first CBMN scientific day. All groups presented a selection of their activities with an appropriate language for exchange between chemists biologists and physicists. This initiative was a success as it really fostered discussions and collaboration between teams of very different discipline. The second edition will take place in the 3rd of December 2009.

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DETAILLED SCIENTIFIC REPORT OF TEAMS 2006-2009 SUMMARY SOME HIGHLIGHTS (COVERS, PRIZES) ************************ CBMN SCIENTIFIC DEPARTMENTS CELL BIOLOGY, MICROBIOLOGY & MEMBRANE BIOCHEMISTRY • Team Alain BRISSON: Molecular Imaging & Nanobiotechnology • Team Olivier LAMBERT: Architecture of membrane Complexes & Cell Processes • Team Jochen LANG: Molecular Dynamics & Functional Proteomics of Secretory Cells • Team Maria URDACI: Interactions Probiotic Bacteria-Hosts BIOPHYSICS & STRUCTURAL BIOCHEMISTRY • Team Erick DUFOURC: Structure & Dynamics of Membrane Assemblies • Team Bernard GALLOIS: Crystallography of Biological Macromolecules • Team Jean-Marie SCHMITTER: Mass Spectrometry of Biological Macromolecules BIOMIMETIC & MEDICINAL CHEMISTRY • Team Ivan HUC: Biomimetic & Bioorganic Supramolecular Chemistry • Team Léon GHOSEZ: Medicinal Chemistry PHYSICAL-CHEMISTRY OF MEMBRANOUS SYSTEMS • Team Bernard DESBAT: Biochemical systems studied by Vibrational Spectroscopies & Optical Methods • Team Juan ELEZGARAY: Molecular Modeling & Numerical Imaging • Team Reiko ODA: Self-Assembly of Amphiphilic Molecules ************************ TEAMS OR SCIENTISTS WILLING TO JOIN CBMN FOR THE NEXT QUADRENNIAL • Team Gilles GUICHARD: Biomimetic Chemistry o (with Karine GIONNET)

• Team Fernando LEAL-CALDERON: Transfer & Flow of Energetic Fluids o (with Pascale SUBRA-PATERNAULT, Patrick SAUVANT, Chrystel FAURE, Claude ATGIE)

• Team Patrick COTTIN: Proteolysis Growth & Muscle Development (Joining Olivier Lambert’s Team) • Team Jean-Pierre AIME: Molecular Physics & Hertzian Optics o Cyril PETIBOIS joining Bernard DESBAT’s Team o Victor MAURIZOT joining Ivan HUC’s Team

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SOME HIGHLIGHTS COVERS Several hot papers have been published by UMR members. Here are some journal or books covers illustrating some of these works.

Huc’s Book

Richter, R. P., Berat, R., and Brisson, A. R. (2006) Formation of solid‐ supported lipid bilayers: An integrated view, Langmuir 22, 3497‐ 3505.

Diller, A., Loudet, C., Aussenac, F., Raffard, G., Fournier, S., Laguerre, M., Grelard, A., Opella, S. J., Marassi, F. M., and Dufourc, E. J. (2009) Bicelles: A natural 'molecular goniometer' for structural, dynamical and topological studies of molecules in membranes, Biochimie 91, 744‐751.

Desbat & Castano’s Book Gillies, E., Dolain, C., Léger, J.‐M. , Huc, I. (2006), Amphipathic helices from aromatic amino acid oligomers, J. Org. Chem. 71 , 7931

Berni, E., Garric, J., Lamit, C., Kauffmann, B., Leger, J. M., and Huc, I. (2008) Interpenetrating single helical capsules, Chemical Communications, 1968‐1970.

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PRIZES AND AWARDS • • •

Huc, I. (2008) Jecker Prize of the French Academy of Sciences. Huc, I. (2009) Invited Professor, Nagoya University. Jean-François F. (2009) (Dufourc’s team). Best PhD Thesis prize, GERLI (National). ********************************************

CBMN DEPARTMENTS CBMN was organized in 4 departments as seen above during the past quadrennial. Teams belong to one of the four and are attached to Biology or Chemistry institutes at University of Bordeaux 1 or CNRS. Exception is made for one team that is attached to the Ministry of Agriculture because it is located in a School of Engineers where all members are heavily involved into Engineer’s formation. Because the unitary building block of the Laboratory is the team, reports are presented team by team, as they appear in departments. In order to have a team overview at glance, two tables summarize the team composition during the last quadrennial and the scientific production, respectively. To prevent artificial expansion of production lists (several teams may have contributed to the same production), publications are grouped and numbered for the entire Laboratory in the Scientific Production lists section. Nonetheless, the 5 most significant publications are explicitly given in each team report. These publications are also present in the Report Excell charts. In the same chart the 10 and 15 best publications are also given by reference number. This chart is attached at the end of the paper support or joined as a separate file (CBMN_3_3) on electronic support. Other indicator such as PhD theses defended, Grant amounts per year, Patents and Congress organizations in which the team has been involved are also given in the Production section.

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DEPARTMENT: CELL BIOLOGY, MICROBIOLOGY & MEMBRANE BIOCHEMISTRY This department was initially constituted of 3 teams (January 2007). The direction of a new young team was given to Olivier Lambert in January 2008. Team Name Molecular Imaging & Nano-biotechnology Architecture of membrane Complexes & Cell Processes Molecular Dynamics & Functional Proteomics of Secretory Cells Interactions Probiotic Bacteria-Hosts

Laboratory Head Alain Brisson, PR UBx1 Olivier Lambert, DR CNRS Jochen Lang, PR UBx1 Maria Urdaci, PR ENITAB

This department deals with cell adhesion, vesicular transport and membrane fusion processes. More applied aspects are also developed in relation with human health (diabetes, cancer, etc.) nutrition and nano-biotechnologies. Themes: • • • • • • • • •

Cell adhesion, multimembranous complexes, solid proteoliposomes Drug nanovectors, functional nanovectors Protein anchoring platforms on membrane surfaces Probiotic bacteria mode of action. Antimicrobial substances. Human Health & Nutrition. Bacterial adhesion on the intestinal tract. Biofilm reconstruction. Receptors responsible for immune-modulation. Transport in beta-cells, exocytose, diabetes. Glycotoxicity & RNA/protein expression. Molecular mechanisms for vesicular/fusion transport of beta-cells. Membrane Fusion mediated by proteins.

Technical Approaches: • • • • •

Cryo-Electron Microscopy, Atomic Force Microscopy, Quartz Microbalance (QCMD). Fluorescence Microscopy. Molecular Biology. Cell cloning. Mutagenesis. Microbiology. Bacterial cloning. Antimicrobial activity tests & toxicity tests. Electrophysiology (extracellular & patch)

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TEAM « MOLECULAR IMAGING AND NANOBIOTECHNOLOGY » Team Leader: Alain BRISSON, Professor CE2, University Bordeaux 1. Team Composition (2006-2009): Anthony Bouter, MCF UB1 Céline Gounou, AI UB1 Joséphine Lai-Kee-Him, IE CNRS Sisareuth Tan, AI CNRS Stéphane Mornet Post-doc EU (*2006) Rémi Bérat, Post-doc UB1 (2008-09) Anne Simon, Post-doc CNRS 2007

Anne Simon, Post-doc UB1 2009 Nick Buzhynskyy, PhD EU (*2006) Rémi Bérat, PhD UB1 (*2007) Boris Garnier, PhD UB2 (2007-2009) Nicolas Arraud, PhD UB1 Fabrice Casenave BTS UB1

**Pierre Bonnafous, MCF UB1 **Olivier Lambert, DR2 CNRS **Jean-Christophe Taveau, PR2 UB1 **Sylvain Trépout PhD UB1 **Olivier Le Bihan (2007-2009) **Mathilde Dubois (2007-2008)

* up to, ** on leave from LIMNT on 01/01/08 for the team AMCCP *** B.Sc. and M.Sc. trainees (stay between 2 and 6 months): 10

Team Production (2006-2009): Publications, peer reviewed, (ACL) Publications, peer reviewed, not yet in bases (ACLN) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI) Publications, peer reviewed in published national colloquia (ACTN) Oral communications in congress (COM)

21 1 30

Books or Book chapters (OS) Congress/Workshop organization Patents

1 1 1

8

Defended PhD Theses or HDR

3

2

Grants : public & private k€/year

200

1

Scientific activity Résumé Les recherches développées dans l’équipe MINBT ont deux orientations principales, fortement interconnectées, l’une portant sur des études fondamentales de complexes protéines-membranes et l’autre sur des applications dans le domaine des nanobiotechnologies. Certains processus cellulaires, tels que l’apoptose, l’activation plaquettaire ou la fusion cellulaire, sont caractérisés par la réorganisation de la membrane plasmique et l’exposition de molécules de phosphatidylsérine (PS) du feuillet interne au feuillet externe des membranes. L’Annexine-A5 (Anx5), protéine soluble de la famille des annexines, se lie aux membranes exposant de la PS de manière Ca2+–dépendante, ce qui explique son utilisation comme marqueur des processus associés à l’exposition de PS. Toutefois, le (ou les) rôle de l’Anx5 au niveau des processus de réorganisation membranaire est encore mal défini. Le MINBT développe un ensemble de projets ayant pour objectifs de préciser le rôle de l’Anx5 dans les processus de réorganisation membranaire et de caractériser les membranes exposant de la PS, principalement les microparticules plasmatiques pro-coagulantes. Pour ces études, le MINBT développe des outils moléculaires appropriés –protéines recombinantes fonctionnalisées, nanoparticules d’or- et des approches physico-chimiques nouvelles. Les projets de NanoBioTechnologie concernent le développement d’applications dans les domaines du diagnostic ou de la délivrance de produits d’imagerie ou de médicaments. Ces projets exploitent les propriétés uniques de liaison de l’Anx5 à des surfaces membranaires. Abstract The research activities developed in the MINBT team have two main orientations, dealing with basic studies of protein-membrane complexes and on biotechnological applications of these complexes. A number of cellular processes, e.g. apoptosis, blood platelet activation or cell fusion, are characterized by a reorganization of plasma membrane and the exposure of phosphatidylserine (PS) molecules from the inner to the outer leaflet of the plasma membrane. Annexine-A5 (Anx5), a soluble protein of the annexin family, binds to PS-exposing membranes in a Ca2+–dependent manner, which explains its use as a marker of processes associated with PS exposure. The role(s) of Anx5 in membrane reorganization processes is still ill defined. The MINBT develops several research projects aiming at elucidating the role of Anx5 in membrane reorganization processes, and at characterizing PS-exposing membranes, principally plasmatic microparticles. For these studies, the MINBT develops original molecular tools –recombinant proteins, functionalized gold nanoparticles- and novel physico-chemical approaches. The second research orientation of MINBT, in NanoBioTechnology, concerns the development of applications for diagnosis or drug delivery. These projects exploit the unique binding properties of Anx5 to membrane surfaces. 19

I-

Sttudies of processes of membrane reo organizatioon with P PS exposu ure and ch haracterizaation of PS--exposing membranes m s

phatidylserin ne exposure and a role of An nx5 in skeleta al muscle form mation I-1 Phosp P Plasma membbranes are coomposed of two t asymmetrric phospholiipids leaflets. In normal conditions, c phosphatiidylserine (PS S) is located exclusively e inn the inner leaaflet of the plaasma membraane. Exposure of PS on the outer membrrane leaflet plays a central role during myogenesis, m b being required d in i myoblastts differentiattion. Differenntiated myobblasts fuse into * * * * Serum myocytess, leading too functional muscular ceells. We set up depletion * * conditionns (serum deepletion and high cell deensity) induccing A. in vitro v reconstitutioon of myogenesiis. Differentiation n * High cell of monnonucleated myoblasts (left) was induced in vitroo myoblastt differentiatioon from rat skkeletal musclee C2C12 cell line l density * ated and ledd to multinuclea myotubes (riight). Nuclei aree * in vitro and a we study the dynamicss and kineticss of PS expossure markedd by *. B. PS exxposure for diffferentiating and d by meanss of fluoresceent-derivativess of recombinnant AnxA5. We apoptootic myoblasts. PS was stained by Cy5 labeledd found thaat PS exposurre starts 24h after a differenttiation inductiion, AnxA55 (white). Mitotrracker (green), a fluorescent dyee specificc to mitochondrria, indicates ap poptotic state off is maxim mal at 48h and a decreasess after 72h for f this in vitro cells. C. endogenouss AnxA5 detecttion in C2C122 reconstituuted cell moddel of myogenesis. Apoptootic cells exp pose skeletaal muscle cell bby western blot.. D. subcellularr PS also, inducing i recoggnition by maacrophages and leading to th heir distrib bution of AnxA A5-GFP into C2C12 duringg eliminatioon. We show wed that apooptotic myobllasts expose PS differeentiation steps. AnxA5-GFP is distributed inn cytoplaasm and nucleuss 24, 48 and 72 2h after medium m continuouusly along thhe plasma meembrane surfa face whereas the switch from growth to ddifferentiation con nditions. exposure of PS in differentiating d g myoblasts is i localized and transient. This differrence could explain whyy differentiatting minated by macrophages. m S Since the rolee of myoblastts are not elim PS expossure in differenntiation is cruucial and sincee AnxA5 exhib bits specific interaction i with PS, we stuudied the putattive involvem ment of endoggenous AnxA A5 in myobblasts’ differentiation and d/or myotube formation. The T presencee of endogennous AnxA5 in c was chaaracterized byy western bloot analysis. We C2C12 cell observed that the leveel of AnxA5 expression e is nearly the saame wth or in differentiation con nditions, indiccating that diffferentiation in nduction is during abbout ten days either in grow not relateed to a variaation of AnxxA5 expressioon. To study the subcelluular distribution of AnxA5 5, C2C12 myoblastts were transffected by AnxxA5-GFP exppressing plasm mid. We obserrved that AnxxA5-GFP exp pression is stable froom 24h afterr transfection up to aboutt ten days. AnxA5-GFP A e exhibits a cyytoplasmic an nd nuclear distributioon. As no variations off expression level or su ubcellular distribution of AnxA5 occu ur during differentiiation, we proopose that AnnxA5 does noot play a role in this proceess. In contraast, endogenou us AnxA5 relocalizees at the plasm ma membranee of myotubess as observed by immunosttaining, suggeesting a role of o Anx5 in contractioon or excitatioon function off mature musccle cells. I-2 Function of Anx5 in membrane repair On-goingg studies concern the particiipation of Anxx5 in membrane repair proccesses. This prroject is not developed further heere for confideentiality reasoons. I-3 Charracterization of plasmatic microparticles P Plasmatic miccroparticles arre membrane fragments prresent in circuulating bloodd, which origiinate from apoptoticc or activated cells from vaarious organs or tissues. Th he interest in plasmatic miccroparticles sttems from the fact thhat most of thhem expose PS molecules and a constitutee therefore a natural n surfacee for blood co oagulation. A correlaation has beenn established between b a highh level of plassmatic microparticles and riisks of thromb bosis. This explains the need for diagnostic methods m of plaasmatic micro oparticles. How wever, the deevelopment of accurate m to about 1 µ µm- and comp position of diagnostic methods is hampered byy the heterogeeneity in size –from 50 nm plasmaticc microparticlees. O objective is to developp a quantitativve assay of thee PS-exposingg surface of pllasmatic micro Our oparticles, using fluoorescently-labbeled Anx5 molecules m prodduced at MINBT. The apprroach has beenn validated with w Anx5functionaalized gold nannoparticles (seee II-2-B) on both b apoptoticc membranes and plasma m membranes. Th his project has receivved support from f the ANR R-Emergence program (Pro oject MP-NpA AuA5 Dec. 20007-Feb. 2010 0) and the Doctoral School “Bioloogie-Santé” with w a PhD felllowship in Sep pt. 2007. T first phasse of this projject has consisted in establishing a methhod for measuuring the surfaace of PSThe containinng membraness sensitive enough for quanntifying the amount a of microparticles inn plasma sam mples. The physiologgical amount of PS in plassmatic micropparticles is of the order of few ng/ml. A novel flow cytometry approachh has been devveloped and opptimized whicch is based on n the measurem ment of the biinding of fluo orescentlylabeled Anx5 A to glasss microspherees functionalizzed with supp ported lipid bilayers b (SLB Bs). This meth hod has a 2 20

sensitivity better than 10 pg/ml, thus significantly better than previously published procedures. The second phase of this project will concern the measurement of PS-containing membranes first in model systems of liposomes and next in plasma samples from control patients and later from patients with defined pathologies.

II- Nanobiotechnological applications II-1 Development of 2D anchoring platforms for antibodies, proteins and cells The development of 2D platforms for anchoring antibodies, proteins or cells, constitutes a major research area of our team. Several projects aim at exploiting the properties of Anx5 and Anx5-derived proteins to self-assemble in 2D arrays on lipid membranes to immobilize biological entities in a controlled manner. At the origin of this research line is the finding that chimerical Anx5 molecules made of Anx5 coupled chemically to a peptide (e.g. an RGDcontaining peptide) or fused to a protein (e.g. a domain of protein A) conserve the main membrane-binding Figure 2. Cell adhesion on an Anx5-RGD anchoring platform properties of Anx5. This has led to a patent filed in 2005, with PCT extension in May 2006 (Patent WO2005114192). Cell platforms have been realized, with both human endothelial cells and mouse stem cells, from 2D matrices of complexes made of Anx5 coupled to several cell adhesion peptides (Figure 2). The controlled immobilization of proteins on solid supports is a major challenge in the development of antibody and protein chips. In this context, we have produced chimerical proteins made of Anx5 fused to a fragment of protein A from Streptococcus aureus, called ZZ, which is responsible for the binding of protein A to the Fc fragment of immunoglobulins (IgGs). We have verified that the Anx5-ZZ proteins combine the properties of Anx5 and of protein A.

Figure 3. Layer-by-layer construction of a 2D platform of IgG-Ab sandwiches The approach has now been validated with the layer-by-layer construction of antibody / antigen / antibody sandwiches in the case of several test protein systems such as ovalbumin (Figure 3) and the prostate specific antigen. The strategy presents the following advantages over the classical ELISA approach: i) the orientation of the antibodies (IgG1 in Figure3) and the successive molecular layers is optimized, due to the oriented binding of Anx5-ZZ on the supported membrane; ii) the density of IgG1, Ag and IgG2 is maximal and can be tuned by varying the surface density of Anx5-ZZ; iii) the native state of the proteins is preserved, as there is no direct contact between proteins and solid support; iv) the layer-by-layer construction of protein assemblies is highly efficient, as it relies on spontaneous self-assembly and molecular recognition processes. The current phase of this project concerns the development of bead-based detection methods.

II-2 Development of functionalized nanoparticles and nanovectors II-2-A Formation of supported lipid bilayers SLBs present a twofold interest, in basic science as models of biological membranes and in nanobiotechnology as biocompatible surfaces. Our long-lasting efforts to elucidate and control the processes of 21

lipid bilayer formation by deposition of lipid vesicles on solid supports have led to a general scheme, which is valid for silica, mica and TiO2 supports (Langmuir 2006, 22, 3497-3505). Basically, the process is driven by electrostatic interactions between the lipid vesicles and the surface, which is negatively charged in the case of silica, glass, mica or titanium oxide. The process of SLB formation is tunable, by adjusting the lipid vesicle composition and the calcium concentration. II-2-B Synthesis of functionalized gold nanoparticles The development of inorganic particles functionalized with proteins or protein fragments constitutes a major challenge in nanobiotechnology, for their various applications as contrasting agents or as drug delivery systems. The MINBT team has developed an original strategy for synthesizing gold nanoparticles functionalized with Anx5 and Anx5-derived proteins (Figure 4). This work has led to a patent application (Patent WO2007122259), adding up to the patent previously deposited covering applications of Anx5-derived proteins. This constitutes the intellectual propriety supporting the project of start-up “AnnexinTools” (see III).

Figure 4. Left : Scheme of Anx5-functionalized gold particle (Anx5-Au-NP): right : labeling of apoptotic bodies with Anx5-Au-NPs, by EM. II-2-C Development of drug delivery systems The MINBT develops protein-functionalized nanovectors for encapsulation and delivery of various types of compounds, principally contrast agents for MRI and active principles for drug delivery. Two types of nanovectors are developed, based on either polymers or liposomes. Proteins used for functionalization are based on Anx5 or Anx5-derived proteins. The polymer-based nanovectors have been developed in collaboration with expert teams from the LCPO-ENSCPB, initially with R. Borsali’s group and now with S. Lecommandoux’s group. The liposome-based nanovectors are developed within MINBT. These activities are supported by two ANR grants and one EU-FP7 grand, where MINBT acts as partner. The formulation of liposomes and the coupling of proteins at liposome surface have been optimized. The development of magnetic liposomes has been realized by passive encapsulation of magnetic particles. The development of such systems is characterized by cryo-Electron Microscopy and physico-chemical methods. Main targets of interest are instable atheroma plaques and inflammation sites.

II-3 Development of membrane proteins-containing lipid bilayers on nanostructured supports (FP7- ASMENA project) This project is part of a EU-FP7 project which plans on creating new platforms for drug screening based on in vitro measurements of functional and conformational changes in membrane proteins. Our contribution in this project deals with the development of membrane proteins-containing lipid bilayers suspended on, or attached to, nanostructured supports. Our initial approach has consisted in depositing a lipid bilayer stabilized by a 2D crystalline AnxA5 matrix on nanostructured silicon wafers by the Langmuir-Blodgett method, adapting a method previously published by the post-doc in charge of the project (Ref 4 - 2007). The surfaces were characterized by AFM. We demonstrated that a proteo-lipid bilayer covers the substrate spanning the pores. Very interestingly, we showed that 2D crystals of AnxA5 bound to a lipid monolayer can be transferred

Figure 5. Schematic representation of a suspended proteolipid bilayer.AFM images of proteo-lipid bilayer onto a nanostructured support (left, 6x6x1 µm) and of AnxA5 2D crystal suspended over a pore (right, 275x275x3 nm). 22

onto a nanostructured support by the Langmuir-Schaefer method. 2D crystals of AnxA5 bound to a lipid monolayer suspended over pores were imaged by AFM, though at low resolution (Figure 5).

III- Project on spore proteins A project dealing with spore proteins is currently performed.. This project is not developed here as we consider depositing a patent.

IV- Project of creation of a start-up A project of creation of a start-up has been initiated in 2008, headed by R. Bérat, former PhD student at MINBT, and A. Brisson. This project, called AnnexinTools, is based on two patents describing the production of chimeric proteins derived from Anx5 and of gold nanoparticles functionalized with Anx5-derived proteins, for applications in diagnosis. This project has received an award from the National Concours de Création d’Entreprises Innovantes (Emergence program) in 2008 and is incubated at the IRA (Incubateur Régional d’Aquitaine) since May 2008. The AnnexinTools team receives training support from Unitech technopole and IRA.

5 significant publications: 1. 2. 3.

4. 5.

Ralf P. Richter, Rémi Bérat, Alain R. Brisson (2006). The formation of solid-supported lipid bilayers – an integrated view. Langmuir 22, 3497-3505. Boris Garnier, Anthony Bouter, Céline Gounou, Klaus G. Petry and Alain R. Brisson. Annexin A5-functionalized liposomes for targeting phosphatidylserine-exposing membranes. Bioconjugate Chemistry (in press). Nikolay Buzhynskyy, Marcin Golczak, Joséphine Lai Kee Him, Olivier Lambert, Béatrice Tessier, Céline Tessier, Thierry Granier, Béatrice Langlois d’Estaintot, Bernard Gallois, Jean-Marc Chevalier, Joanna Bandorowicz-Pikula, Slawomir Pikula and Alain R. Brisson. Annexin-A6 presents two modes of association with with phospholipid membranes. J. Struct. Biol. (in press). Vanessa Schmidt, Cristiano Giacomelli, François Lecolley, Joséphine Lai-Kee-Him, Alain R. Brisson and Redouane Borsali (2006). Diblock Copolymer Micellar Nanoparticles Decorated with Annexin-A5 Proteins. J. Am. Chem. Soc. 128, 9010-9011. Rémi Bérat, Murielle Rémy-Zolghadry, Céline Gounou, Claude Manigand, Sisareuth Tan, Carmen Saltó, Ernest Arenas, Laurence Bordenave and Alain R. Brisson (2007). Peptide-presenting 2D protein matrix on supported lipid bilayers: an efficient platform for cell adhesion. Biointerphases, 2(4), 165-172.

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) A. Brisson coordonne et participe aux enseignements suivants de Biochimie et Biophysique au sein des UFR des Sciences Biologiques des Universités de Bordeaux-1 et Bordeaux-2 : Licence Biologie Cellulaire et Physiologie. L2-Module « Méthodes d’analyse des macromolécules biologiques » L3-Module « Biochimie structurale et physico-chimie des macromolécules biologiques » Master mention Biologie Santé : Spécialité Biochimie Structurale-Interface Biologie-Physique-Chimie. M1-Module « Imagerie moléculaire et cristallographie des rayons X » M1-Module « Nanotechnologie en biologie » M2-Module « Méthodes d’imagerie pour la biologie » M2-Module « Complexes et interactions moléculaires » Licence Chimie L3-Licence Professionnelle en Chimie Industrielle « Spécialité Matériaux » ENSCPB Option Micro-et Nano-Technologie ; Module « Nanobiotechnologie ». A. Bouter participe à des enseignements de Biologie cellulaire, Biochimie, Immunologie, Imagerie pour la Biologie et Bioinformatique en licence BCP, licence BO et master de Biochimie Structurale-IBPC.

Managment and running of a technological platform. The LIMNT and teams ensure the daily maintenance and manage the Electron Microscopy platform for UMS 3033 (IECB). The team brings scientific and engineering competence for running 2 microscopes (CM-120 and Tecnai-20 FEI).

23

TEAM « ARCHITECTURE OF MEMBRANE COMPLEXES & CELL PROCESS » Team Leader: Olivier Lambert, Research Director (01/10/2009), CNRS. Team Composition (2008-2009) (team created the 01012008): Jean-Christophe Taveau, Pr Bx1 Mathilde Dubois, PostDoctorante ANR Antnoy Desert Doctorant

Pierre Bonnafous MCF Bx1 Sarah Aventin, Technicienne ANRS

Olivier Le Bihan, Doctorant Sylvain Trépout, Doctorant

B.Sc. and M.Sc. trainees (stay between 2 and 6 months): 8

Team Production (2006-2009): Publications, peer reviewed, (ACL) Publications, peer reviewed, not yet in bases (ACLN) Invited conferences congress (INV) Oral communications in congress (COM) Poster communications in congress (AFF)

16 2 2 15 9

Books or Book chapters (OS) Patents Defended PhD Theses or HDR Grants : public & private k€/year

1 1 1 50 k€

Scientific activity Résumé En lien avec la compréhension de processus cellulaires reposant sur une action concertée d’assemblages de macromolécules, nous étudions l'architecture cellulaire 3D à une résolution nanométrique, par une des approches les plus prometteuses qu’est la "cryo-tomographie électronique" (cryoTE). Cette technique est très attractive pour des études structurales d’assemblages supramoléculaires ou bien subcellulaires à partir d’extraits, de coupes cellulaires ou directement sur cellule entière. Au cours de ces dernières années, nous avons implanté au CBMN la cryoTE (technique d’acquisition et analyse d’images spécifiques). Nous avons développé une méthodologie pour l’étude des complexes protéiques membranaires que nous avons appliquée à l’étude de l’organisation structurale d’un système bactérien d’efflux multidrogues comprenant trois protéines membranaires OprM-MexA-MexB. Dans ce cadre, nous avons décrit l’organisation structurale du système bipartite OprM-MexA reconstitué dans deux membranes lipidiques mimant les membranes interne et externe de la paroi bactérienne et avons proposé un mécanisme de formation du système tripartite pour lequel le complexe OprM-MexA serait un intermédiaire bipartite d’assemblage. Le champ d’application de la cryo-ET ne se limite pas à la Biologie. Nous avons également montré son importance pour des études structurales à l’interface de la Biologie et de la Chimie. Plus spécialement, nous avons apporté des informations 3D inédites en décrivant l’évolution de la morphologie de nanoparticules hybrides au cours de leur synthèse. De plus, dans un contexte de bio-fonctionnalisation, nous avons analysé les interactions nanoparticules –membranes et décrit un mécanisme d’internalisation "spontanée" de nanoparticules de silice dans des liposomes.

Abstract In connection with the understanding of cellular processes based on concerted action macromolecule assemblies, we develop approaches to visualize the 3D cellular architecture at a nanometric resolution using one of the most promising approaches, the cryo - electron tomography (cryoTE). This technique is very attractive for structural studies of supramolecular assemblies from subcellular extracts, cell section or whole cell. In recent years, we have implemented at CBMN this cryoTE approach (including tomographic acquisition and specific image analysis sofwares). We have developed a methodology for the study of membrane protein complexes. We studied the structural organization of a bacterial efflux multidrug system which is composed of three membrane proteins OprM-MexB-MexA. We described the structural organization of the bipartite system Mexa-OprM reconstituted in two lipid membranes mimicking the internal and external membranes of the bacterial cell wall and proposed a mechanism of the formation of tripartite system for which an intermediate bipartite would be the OprM-MexA complex. The scope of the cryo-ET is not limited to biology. We also showed its importance for studies at the interface of Biology and Chemistry. More specifically, we have provided first-time 3D information describing the evolution of the morphology of hybrid nanoparticles during their synthesis. Moreover, in the context of bio-functionalisation, we analyzed the nano-membrane interactions and describes a mechanism for a"spontaneous" internalization of silica nanoparticles in liposomes. 24

• Creation of the Team: Architecture of membrane complexes and cell process The team has been created in 2008 (01/01/2008). We aim at elucidating the 3D structure of membrane proteins in controlled or native cellular environments using Cryo Transmission Electron Microscopy (CryoTEM) and Cryo-Electron Tomography (Cryo-ET) approach. This project initiated in the group of Alain Brisson has gathered three researchers, J.-C. Taveau (Prof. Univ. Bordeaux-1), Pierre Bonnafous (MCF Univ. Bordeaux1) and O. Lambert.

• Structural analysis of membrane complexes

• Methodology Transmission Electron microscopy (TEM) and cryo-electron microscopy (cryo-TEM) are used to image ultrastructure of tissues and cells as well as to provide information on the structure of purified molecules including membrane proteins. Cryo-TEM allows the visualization of hydrated and unstained specimens embedded within a thin (< 200 – 300 nm) layer of amorphous ice that represents a state close to the native state. The structure determination of membrane protein in lipid environment can be carried out using cryo electron microscopy combined with the recent development of data collection and image processing. We describe a protocol to study assemblies or stacks of membrane protein reconstituted into a lipid membrane using both cryo electron tomography and image analysis which is an suitable approach for solving 3D structure of membrane complexes. Cryo-ET consists in combining Cryo-TEM with tomographic methods. Tomographic data acquisition consists in the record of a tilt series of images, i.e., a series of micrographs (2D projections) of a given specimen recorded at different tilt angles under comparable imaging conditions. 3D reconstruction involves a 1st step of alignment of 2D projections to a common coordinate system, and a 2nd step of backprojection which gives a 3D volume of densities, called tomogram, from weighted back-projection or algebraic algorithms. A procedure of volume alignment and volume averaging derived from single particle reconstruction methods can be carried out on sub-volumes of individual membrane complex to improve the signal to noise ratio providing a more detailed architecture. The cryoET approach raises high interest in structural biology because it allows in principle to image unfixed and untreated cells at sub-molecular resolution (at least for thin areas). The Cryo-ET approach has been applied to the 3D structure determination of protein-membrane complexes and synthetic nano-objects.

•

Multidrug efflux pump Pseudomonas aeruginosa is a Gram-negative bacterium that causes opportunistic infections in immunocompromised patients and exhibits natural and acquired resistance to diverse antibiotics. OprM-MexAMexB efflux system which belongs to RND system (Resistance Nodulation and Division) is constitutively expressed and is capable of exporting a large variety of drug out of the cell. OprM-MexA-MexB a tripartite pump composed of three membrane proteins located within the outer membrane (OprM), the periplasmic space (MexA) and the inner membrane (MexB) respectively. Although these three proteins are believed to form a complex channel allowing the extrusion of drugs directly into the extracellular medium, the structure of this whole complex inserted into lipid membranes remains unknown.

25

The reconstitution of the whole pump ab initio being a very important challenge, we first focus on individual compound OprM and MexA and the bipartite complex OprMMexA (coll. Pr. A. Ducruix Paris). As both proteins are membrane proteins maintained soluble in the presence of detergent, we have studied the physico-chemical conditions suitable for the formation of the bipartite complex using Quartz crystal microbalance with monitoring Dissipation (QCM-D) and cryo-TEM. These studies were performed on silica support (QCM-D) and on silica nanoparticles to have similar chemical support. The solid support has the advantage to allow the control of the protein orientation

 

and then provide reliable information on OprM-MexA interaction. We showed that the complex can be formed in the presence of detergent and remains stable after detergent removal. Next we have analysed the structure of MexA after its incorporation via its palmitoyl anchor into a lipid membrane. Our Cryo-TEM images indicate that the domain of MexA which was not solved in the X-ray model was likely located close the lipid membrane. Based on these works and on our former electron crystallographic study of OprM (Lambert et al., J Struct. Biol., 2005), we performed the reconstitution of complexes of OprM and MexA into proteoliposomes by detergent removal. Stacks of protein layers with a constant height of 21 nm, separated by lipid bilayers were obtained at stoichiometry of 1:1 (w/w). Using cryo electron tomography, we showed that these protein layers were composed of OprM-MexA complexes selfassembled into regular arrays. Image processing of extracted sub-tomograms depicted the architecture of the bipartite complex sandwiched between two lipid bilayers mimicking outer and inner membranes. The intermembrane distance of 21 nm indicated that the height of the bipartite complex is greater than the modeled height of the whole efflux pump in which OprM and MexB are brought in contact. The peculiar structure of the OprM-MexA bipartite complex yields insights into a possible mechanism of tripartite system assembly (Trépout S., Taveau J-C, Benabdelhak H., Ducruix A., Frangakis A and Lambert O Multidrug efflux pump structure revealed by cryoelectron tomography, submitted to J. Mol. Biol.). 26

• Cell adhesionn: VE-cadheriin junctions Interrcellular juncttions are specialized regionns of cell mem mbranes whicch control the cohesion of tissues. t In adherens junctions, caddherin molecuules form hom mophilic interaactions betweeen cells with identical phenotypes via t cytoplasmic regions are their extrracellular reggions, while their linked too the actin cytoskeleton c T via α- and β-catenins. The molecular process responsible forr the assembbly of adhereens ural junctions is still largeely unknown, as is the deetailed structu molecular edifi fices. descriptioon of these complex supram We have h started a project aiminng at elucidatting the structu ure of the supramolecullar assemblyy formed byy the cadheerin m annd in a nativee environmen nt ( molecules on model membrane G Using recombbinant cadheerin coll. D. Gulino Grenoble). um molecules (with a His-tag extensionn) from vascuular endotheliu w have stud died (VE) assembled onto Ni-NTA lipidd liposome, we yothe 3D sttructure of theese pseudo-adherens juncttions using cry ET. A procedure p of volume aliggnment derivved from sin ngle particle reconstruction r n methods was carried out on sub-volum mes of individdual cadherin hexamers. Thhe resulting averaged a volu ume reveals the rough architecture of the caddherin hexam mer ment sandwichhed between two lipid meembranes. Fuurther alignm refinemennts are in progress to provide a more detailed architectuure of cadherrin hexamer and a to describbe the hexam merhexamer interactions. •

Cryyo electron n tomography at Bioloogy–Chemiistry interfaace • Morphologica M al characterissation of hybrrid nanoparticcle.

We have h studied the t growth of polystyrene nodules n on siliica particles using u cryo-elecctron tomograaphy (coll. Pr S. Ravvaine and Pr. E. Duguet). This T was the most suitablee approach forr imaging polyystyrene nodu ules at the early stage of their polymerizatio p on since theyy are swollen n of styrene monomers annd highly seensitive to orphology dehydratiion. 3-D recoonstructions have allowedd evaluating over the pollymerisation time, the mo regularityy, the polydisppersity of latexx size on a sinngle silica seed d and the conttact angles. Polystyrene synthesis on silica beads (diameter ( 170 nm). Cryo-EM image (A) and a sterreoscopic view of 3D D reconstructioon of a hybrid particle. The small polystyrene p noodules appear randomly r distributed around a the silica particle (Taveeau et al., Soft Matter 2008).

•

Meechanisms of o nano-uptake into ceell

Our current interest is to develoop novel methhodologies to image nanopaarticles withinn cellular com mpartments w is to unnderstand the impact of and also use nanopartiicles to studyy cell process.. The overall goal of this work synthetic nanoparticless on human heealth. • Mechanism M of nanoparticlle transport across lipid meembranes Nanooparticle transsport across cell c membranee plays a cruciall role in thee developmennt of drug delivery systems as a well as in the toxicity response r induuced by nanopartiicles. As hyydrophilic naanoparticles interact i with lipidd membranes and are able to t induce mem mbrane perturbatiions, hypothhetic mechaanisms baseed on membranne curvature or hole forrmation havee been proposedd for activatinng their transm migration. We have studied thhe transport of o hydrophilic silica nanopaarticles into largee unilamellar neutral DOP PC liposomes via an internalizzation process. The strong adhesive a interaactions of lipid membrane m onto the silica naanoparticle triiggered liposome deformation until the forrmation of a curved neck. Theen the rupturee of this membbrane neck ledd to the 2 27

complete engulfment of the nanoparticle. Using cryo-ET we determined 3D structures of intermediate steps of this process unveiling internalized silica nanoparticles surrounded by a supported lipid bilayer. This engulfing process was achieved for a large range of particle size (from 35 nm to 200 nm in diameter). These original data provide interesting highlights for nanoparticle transport across lipid membrane and could be applied to biotechnology development. Characterisation of synthetic vectors and study of gene transfer mechanism The design and the rational development of new synthetic vectors for gene transfer require a characterisation of the physicochemical properties of supramolecular assemblies in regards to their transfection efficiency in vitro and in vivo. Various assemblies made of amphiphilic polymers and lipid derivatives mixed with plasmid DNA and siRNA respectively that are very promising for gene therapy have been characterized by cryo electron microscopy (Bello-Roufai et al., 2007; Desigaux et al., 2007) (coll. Bruno Pitard, Nantes). A better understanding of the identified key-steps, including endocytosis, endosomal escape and nuclear delivery is required for further developments to improve their efficacy. We have developed a labelling protocol using aminated nanoparticles as markers for plasmid DNA to examine the intracellular route of lipoplexes using transmission electron microscopy. Cellular uptake and the mechanism of endosomal escape of DNA molecules were monitored by providing snapshots of morphological changes of lipoplexes, membrane reorganisations and endosomal membrane ruptures (Le Bihan,O., Chèvre,R., Mornet,S., Garnier,B., Pitard,B., Lambert,O. Probing the mechanism of action of cationic lipid/DNA lipoplexes at a nanometric scale. submitted to Nucleic Acid Res).

5 significant publications: 1. Le Bihan O, Bonnafous P, Marak L, Bickel T, Trépout S, Mornet S, De Haas F, Talbot H, Taveau J-C, Lambert O. Nanoparticle transport across phospholipid membrane. J Struct. Biol. Accepted for publication. 2. Taveau J.-C., Nguyen D., Perro A., Ravaine S., Duguet E., Lambert O. (2008). New insights into the nucleation and growth of PS nodules on silica nanoparticles by 3D cryo-electron tomography Soft Matter, 4, 311-315. 3. Trépout S., Mornet S., Benabdelhak H., Ducruix A., Brisson A., Lambert O. (2007). Membrane protein selectively oriented on solid support and reconstituted into a lipid membrane. Langmuir 23, 2647-2654 4. Trépout S., Taveau J-C., Mornet S., Benabdelhak H., Ducruix A., Lambert O. (2007). Organization of reconstituted lipoprotein MexA onto supported lipid membrane Eur. Biophys. J. 36, 1029-1037. 5. Bello Roufai M., Lambert O., Pitard B. (2007). Relationships between the physicochemical properties of a amphiphilic triblock copolymers/DNA complexes and their intramuscular transfection efficiency. Nucleic Acids Res. 35, 728-739.

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Teaching of bioinformatics and bioimaging. Licence, Master 1 & 2 levels. Teaching of biochemistry. Licence. Co-responsible for the Master “Bioinformatique” University Bordeaux Co-responsible for the Mention “Biologie , Santé“ University Bordeaux Setting up a local network of electron microscopy (January 2009): RESAME (réseau Aquitain de microscopie électronique) Partial organization: “Ecole thématique du CNRS Nano-objets aux interfaces (NOIS 2007) ” May 2007 Lacanau, (120 participants).

Implication in running EM platform. The team ensures in part with the team of Alain Brisson the maintenance of the electron microscopes for UMS 3033 (IECB). The team brings scientific competence for running the two cryoEM machines (FEI CM120, tecnai F20 200kV FEG) and performing electron tomography. The team also ensures studies for both academic and industrial requests (SANOFI, etc.). Our expertise in electron tomography is present in the “Pôle de competence Imagerie 3D en Aquitaine” supported by MIB (Institut Carnot). In 2009 the team was at the initiative of setting up the Aquitaine EM network, whose role is to develop regional technical and scientific exchanges. This network will help the building up of funding for renewing EM equipments or/and to work together to acquire the last generation of electron microscope (following the example of Aquitaine NMR Network).

28

TEAM « MOLECULAR DYNAMICS & FUNCTIONAL PROTEOMICS OF SECRETORY CELLS » Team Leader: Jochen LANG, PR1, Université de Bordeaux 1. Team Composition (2006-2009): Bogdan CATARGI, PU-PH, UB2-CHU Gilles CHARPENTIER, PR2 UB1 Stéphanie CHEVALLIER MCU UB1 Valérie LAGREE, MCU UB1 Benoît ROGER, MCU UB1

Pier SCOTTI, MCU UB1 Alexandra MILOCHAU, AI UB1 Marie-N. BENASSY, Tech UB1 Charline BLATCHE, Tech UB1 Mathilde DUBOIS, postdoc

Matthieu RAOUX PostDoc Frédéric BOAL, PhD Florence GRISE, PhD Julien PAPIN, PhD Benoît HASTOY, PhD

Diploma students (6 months): 8

Team Production (2006-2009): Publications, peer reviewed, (ACL) Publications, peer reviewed,not in data bases (ACLN) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI) Oral communications in congress (COM)

17 1

Poster communications in congress (AFF) Books or Book chapters (OS)

2 1

1 1

Congress/Workshop organization PhD Theses defended or HDR

1 2

4

Grants : public&private k€/year

50 k€

SCIENTIFIC ACTIVITY Résumé Les membranes biologiques sont largement impliquées dans des événements cellulaires majeurs tels la signalisation et le transport. La fusion vésicule-membrane plasmique est de fait un processus fondamental qui intervient non seulement lors de la sécrétion mais aussi dans la neurotransmission, cicatrisation, la cytodiérèse ou encore la différenciation cellulaire. De plus ce processus d’exocytose se retrouve au cœur de certaines pathologies chroniques incapacitantes telle que le diabète de type 2. Cette pathologie complexe, qui affecte 5% de la population européenne, découle en partie d’une déficiente dans la sécrétion d’insuline. Les cellules β-pancréatiques, sécrétant l’insuline, constituent ainsi un modèle de choix pour étudier le transport vésiculaire et la fusion avec la membrane plasmique, tant d’un point de vue mécanistique que d’un point de vue de sa régulation. Nos travaux sur les protéines impliquées dans le transport vésiculaire et l’exocytose dans les cellules pancréatiques ont pour objectifs principaux de : a) décortiquer les interactions moléculaires impliquées à tous les niveaux de l’exocytose (interaction entre les différentes protéines, régulation de ces interactions, modélisation des interactions dans les membranes et à l’interface membrane/cytosol via des approches physico-chimiques), b) étudier la régulation de l’expression et la fonction de ces protéines clés dans le cadre de la sécrétion de l’insuline et des syndromes diabétiques et. En ce qui concerne l’objectif (a) nous avons pu déterminer plusieurs éléments essentiels : i) mode d’action de la latrophiline, récepteur membranaire qui interfère avec l’exocytose ii) identification du senseur calcique impliqué dans le déclenchement de l’exocytose et caractérisation de son fonctionnement iii) définition du rôle de deux domaines de la protéine CSP, un chaperon moléculaire interagissant avec le senseur calcique et les protéines SNARES au cours de l’exocytose iv) description de la dynamique de conformation des domaines transmembranaires des protéines SNARE. Pour ce qui est de notre objectif de comprendre les dysfonctionnements dans la sécrétion qui surviennent lors d’une exposition prolongée à des taux élevés de glucose, appelée « glucotoxicité », nous avons pu démontrer : i) des changements dans les niveaux d’exocytose ii) le rôle prépondérant de la signalisation via l’AMPc sur l’exocytose d’insuline. Sur la période concernée par l’évaluation nous avons été capables de développer au sein de l’équipe de nouvelles approches expérimentales telles que des systèmes de mesure électrochimiques et électrophysiologiques sur cellules uniques.

Abstract Membrane delimited activities participate in major cellular events such as signaling and transport. The insulin-secreting β-cells have provided a powerful model to dissect mechanisms and regulation of vesicular transport and fusion with the plasma membrane, a process fundamental not only to secretion, but also to 29

neurotrannsmission, wound healing, migration, m cyttokinesis and cellular c differrentiation. Mooreover, these events are linked to a major lifeloong incapacitaating disease, Type 2 diabeetes, which afffects 5% of thhe population in Europe i innsulin secretio on as an impoortant facet. O Our studies on n proteins and its coomplex etioloogy includes insufficient involved in vesicular transport t and exocytosis in pancreatic β--cells have tw wo major goalls: a) gaining insight in t relevant prroteins at diffferent stages of o exocytosis, the regulatoryy events and understand u the interpplay between the the interaactions in physicochemical terms in mem mbranes and att the membranne/cytosol inteerface; b) to investigate the expreession and function of these proteins to diiabetic syndro omes. Ass far as goal (a) ( is concernned, we have been able to (i) determine the mode off action of latrrophilin, a receptor coupled to exocytosis, e (iii) to determinne the relevaant calcium sensor s in insuulin exocytossis and to i characterrize its functioonal behaviorr, (iii) to estabblish the role of two domaains of CSP, a chaperone interacting with calccium sensors and SNARE proteins duriing exocytosiis and (iv) to establish thee dynamics of SNARE protein trransmembranee domains. In our attempt to understandd the secretorry dysfunction n after prolonnged exposurre to elevated d levels of ( termed gllucotoxicity, we w have beenn able to show w (i) changes at the level oof exocytosis and a (ii) to glucose (b), reveal thee prominent roole of cAMP-signaling in general and at the level of exxocytosis in particular. Duuring the evalluation periodd we were alsso able to imp plement electtrochemical annd electrophy ysiological approachhes as well as qPCR q and preeparation of prrimary cells in n the laboratorry.

• GENERAL INTRODUCTION

Inssulin transporrt and secretioon from panccreatic β-cells proceeds by exocytosis thhat is fusion of vesicle (donor) membranes m w with the targeet plasma meembrane. Mem mbrane fusionn during vesiicular transpo ort and in exocytosiis requires a conserved c set of proteins, thhe SNARE proteins and its involvement in LDCV exo ocytosis in β-cells haas been show wn1. Althoughh the general machinery m is known, its precise p functioon, interaction ns and the sequence of events is still largely unclear. u Panccreatic β-cellss have alwayss provided a vvaluable mod del for the p hormonne is tightly regulated. study of exocytosis off large dense core vesicles and the release of this potent c thhe sole trigger, Althoughh calcium constitutes physiologgical release iss profoundly amplified a by other o metabolissm-secretion coupling c factoors such as cA AMP. Interestinngly, a numbeer of evidencees indicate thhat βcells in geeneral and exoocytosis in paarticular are altered in a majoor disease, i.e. diabetes (typee 2). Duuring the evaaluation periood we have been able to ennlarge our paanel of techniques by acquuiring the expeertise to preppare islets and a primary cells (subsequeent to an exxchange of a postdoc withh JC Jonas’ grroup in Louvaain) and by esstablishing several electrophhysiological annd electrocheemical approaaches (details seee below).

• MEC CHANISMS AND A REGULA ATION OF EXOCYTOSIS X S Latrophillin, a GPCR linked l to exoccytosis T black-wiidow spider toxin The t α-latrottoxin constitutees one of the most potent release factoors at the synaapse. We hadd previously demonstratedd its FIG. 1: Effect off LTX on siingle vesicle exocytosis e action allso on β-cellls, underscoriing the numeerous (amperometry). A: traaces of native cellls stimulated with h 1 or 0.1 nM α-LTX and cellss expressing eitheer LPH or LPH--TD1 and resemblannces betweenn neurons andd these endocrine expossed to 0.1 nM α-L LTX. B: LTXN4C ((1 nM) stimulates secretion cells, andd clearly dem monstrated the involvement of a in nattive cells. C: statiistics. specific G-protein G couppled receptor, named latropphilin (LPH) 2. However, thhe prevailing view v claimedd that t but servves only as ancchoring point for the toxin tthat subsequen ntly forms this recepptor is not actiivated by the toxin pores in the membranne and thus allows the innflux of releaase-inducing cations. In collaboration with w Yuri mperial Collegge London, UK) U we addressed this topicc in several cclonal β-cell liines using Ushkaryoov’s group (Im full-lengtth as well as truncated recceptors, the laatter being no o longer capaable to transm mit downstream m signals. N Furtherm more we emplooyed wild-typpe and mutantt toxin (LTXN4C ) that is deevoid of pore formation. A series of 1

Lang, J., Molecular M mechanisms and regulaation of insulin exxocytosis as a parradigm of endocrrine secretion. Euur. J. Biochem., 19 999. 259: p. 3-17. 2 Lang, J., Y. Y Ushkaryov, A. Grasso, and C.B B. Wollheim, Ca2++-independent inssulin exocytosis innduced by α-latrootoxin requires la atrophilin, a G proteinn-coupled receptoor. EMBO J., 19998. 17: p. 648-657. 3 30

pharmacological, cell biology and electrophysiological experiments (in collaboration with P. Vacher, INSERM U916, Institut Bergonié) clearly demonstrated that the toxin initially activates LPH, thereby stimulates phospholipase C with subsequent rise in cytosolic calcium [Ca2+]i and inactivates repolarizing BK-channels thus keeping [Ca2+]i up (ref. 15). A second transmembrane toxin-binding protein had been described, PTPσ, that could concentrate the toxin and thus favors pore formation. In a series of pharmacological and imaging experiments we clearly demonstrated that PTPσ could only enhance toxin-induced secretion and recruitment of intracellular calcium sensors in the presence of LPH (ref. 14). Synaptotagmins, the calcium sensors Ca2+ constitutes the sole trigger of exocytosis and a group of vesicular transmembrane proteins, the synaptotagmins, constitute a calcium-sensor in exocytosis via their two cytosolic C2 domains that bind cooperatively to Ca2+ and phospholipids or SNARE proteins in-vitro. We had previously shown that this applies not only for the release of synaptic vesicles at the synapse but also for the exocytosis of LDCVs occurring at much lower calcium levels and underlining the ubiquitous occurrence of this mechanism 3. However, the precise nature of the synaptotagmin isoform implicated remained to be demonstrated as the neuronal isoforms of synaptotagmin, syt1 and syt2, are only expressed in clonal, but not in primary β-cells and calciumsensitive syt3 and 4 are absent from LDCVs 4. In contrast, syt7, 8 and 9 had all been advocated as LDCV calcium sensors. As antibodies were neither available on a collaborative basis nor from a commercial source, we raised polyclonal antipeptide antibodies against FIG. 2 : Stimulation of living cells by low micromolar Ca2+ translocates the C2A, but not the C2B domain of Syt9. A: Images a syt7, 8, 9 and 11. Using confocal imaging and and c were taken 0 sec and, b and d 3 sec after stimulation. B: subcellular fractionations we could demonstrate that quantification. C and D: Molecular simulation of C2B domains of the different isoforms of syt7 are restricted to syt1 (C) and syt9 (D). endosomal-lysosomal compartments, whereas syt8 is localized outside the secretory pathway and calcium-insensitive in living cells (ref. 6 and 16). In contrast, syt9 is localized on LDCVs and we have examined in detail the calcium-sensitivity of its membrane translocation by biochemical approaches, the use of neurotoxins and fluorescent videomicroscopy in living cells (ref. 8). Our data indicate that only the C2A domain is Ca2+sensitive and translocates to the plasma membrane, but not to vesicular membranes. Moreover, in living cells membrane binding occurs in the absence of SNARE proteins and thus interactions with phospholipids are sufficient. In contrast to C2A, the C2B domain is calcium-insensitive, despite the fact that its structure is largely comparable to the archetypical C2B domain of syt2 as evidenced by molecular dynamics (collaboration with N. Taib/M. Laguerre, CBMN). Interestingly, C2B diminishes the overall calcium affinity of C2A, thus conferring specific properties to the endocrine syt, and plays in addition a role in endocytosis (ref. 8). It is currently a major debate whether the C2A or the C2B domains mediate the Ca2+-effects on exocytosis. We therefore resorted to a cellular knockdown and concomitant expression of siRNA-resistant mutants of the Ca2+-coordinating residues in the C2 domains. Biochemical secretion assays and electrophysiological analysis performed in collaboration with S. Collins in the group of P. Rorsman (U. Oxford, UK) clearly demonstrated that only the C2A domain is required for vesicle fusion. Moreover, knockdown of syt9 resulted in dramatic loss of LDCVs without interfering with endosomal compartments suggesting that syt9 is specifically required for the generation or the stability of LDCVs (Ms submitted). Establishing the subcellular localization of syt11 proved to be more challenging and in stark contrast to all known synaptotagmins, we could finally attribute syt11 to the intermediate compartment (between ER and cis-Golgi). In collaboration with A. Lajoix (CNRS UMR-5232, Montpellier), we could also demonstrate a similar localization in primary β-cells. This localization FIG. 3 : Co-localisation of syt11 (green) with ERGIC (red), the marker of the intermediate compartment

3

Lang, J., M. Fukuda, H. Zhang, K. Mikoshiba, and C.B. Wollheim, The first C2 domain of synaptotagmin is required for exocytosis of insulin from pancreatic β-cells: action of synaptotagmin at low micromolar calcium. EMBO J., 1997. 16: p. 5837-5846. 4 Gut, A., C.E. Kiraly, M. Fukuda, K. Mikoshiba, C.B. Wollheim, and J. Lang, Expression and localisation of synaptotagmin isoforms in endocrine β-cells: their function in insulin exocytosis. J. Cell Sci., 2001. 114: p. 1709-1716. 31

is very unusual as all other known syts are present on post-Golgi vesicles. In addition, transport from the ER to the Golgi is Ca2+-independent, suggesting that syt11 may be involved in another function. We therefore decided to address the question by examining its interactions and resorted to pulldown and either subsequent mass spectrometry (JM Schmitter, CBMN) or quantitative immunoblotting. Surprisingly, syt11 interacted with snd1, a membrane-localized protein known to be part of the RISC complex, with FMRP, part of stress granules, and the endomembrane R-SNARE Vti1. In collaboration with B. Ochoa’s group, which had cloned mammalian snd1, we further substantiated these interactions and showed that both, FMRP and snd1 binding depend on the C2B domain of syt11 and the N-terminal half of snd1. In view of our data we hypothesized that syt11 may be linked to either stress granules or P-bodies and may intervene in RISC function. Studies are currently ongoing concerning the effect of knockdown of syt11 or overexpression of a scavenging cytosolic syt11-C2B on the distribution of stress granule markers during puromycine treatment. We have also started a collaboration with C. Grosset (INSERM U 889, UB2) to test a putative interaction with RISC (via FMRP, snd1) using FUNReg. In summary, these data may indicate a novel link between vesicular traffic and regulation of RNA levels. This project should be ready for submission of a Ms in end 2009. CSP, a chaperone in exocytosis We continued our previous work on the cysteine string protein Csp in order to understand its domain architecture and function in exocytosis. Csp contains an archetypal J-domain, a linker region, the name-giving cysteine string and a variable C-terminus that interfere to a variable degree with exocytosis 5 though the molecular mechanisms are unknown. Using a novel isoform with restricted expression and a distinct cysteine FIG. 4: Interaction between Csp and Syt9. A : Calcium string, Cspβ, we demonstrated that cysteine dependence of Cspα binding to syt9 for in vitro.. B : The glutamate prenylation is not required for membrane residue E93 in the linker domain of Cspα is required for in vitro attachment, as previously thought, but governs binding of Syt9. C: Mutation E93V modifies the local hydrophobicity targeting to distinct subcellular membranes, i.e. of the linker domain. Golgi vs. LDCVs (ref. 10). We had previously shown that the variable C-terminus permits homo-dimerisation, a typical event for chaperones, and binding to the vesicular SNARE synaptobrevin 2 6. However, we had no clue about the molecular function of the linker domain, whose importance had been established by us previously 5. Immuno-precipitations, pull-down experiments and the use of FRET/FLIM established that the linker binds to the calcium sensor syt9 via the C2A domain in a Ca2+dependent fashion and binding requires the residue E93 in the Csp linker. Its mutation to Val abolishes a charged hook on the surface of Csp according to molecular modeling (M. Laguerre, UMR CBMN), interactions with syt9 and its function in exocytosis. This work permits to place the chaperone function in the Ca2+-stimulation period during exocytosis. A manuscript has been submitted. Dynamics of SNARE transmembrane domains Despite the important functions of protein transmembrane domains, their structure and dynamics are often scarcely known. The SNARE proteins VAMP/synaptobrevin and syntaxin 1 are implicated in membrane fusion and the transmembrane domains of SNARE proteins have been shown to be crucial fro membrane fusion beyond a simple anchoring role. Its dynamics were addressed in a collaborative project with R. Oda, B. Desbats and M. Laguerre (CBMN). Using different spectroscopic approaches we observed a marked sensitivity of their transmembrane domain structure in regard to the lipid/peptide ratio. In the dilute condition, peptides corresponding to the complete transmembrane domain fold into an α helix inserted at ~35° to the normal of the membranes, an observation in line with molecular simulations. Upon an increase in the peptide/lipid ratio, the peptides readily exhibited transition to β-sheet structure. Moreover, the insertion angle of these β-sheets increased to 54° and was accompanied by a derangement of lipid acyl chains. For both proteins the transition from α-helix to β-sheet was reversible under certain conditions by increasing the peptide/lipid ratio. This phenomenon was observed in different model systems including multi-bilayers and small unilamellar vesicles. In addition, differences in peptide structure and transitions were observed when using distinct lipids (DMPC, DPPC or DOPC) thus indicating parameters influencing transmembrane domain structure and conversion from helices

5

Zhang, H., W.L. Kelley, L.H. Chamberlain, R.D. Burgoyne, and J. Lang, Mutational analysis of cysteine-string protein function in insulin exocytosis. J. Cell Sci., 1999. 112: p. 1345-1351. 6 Boal, F., H. Zhang, C. Tessier, P. Scotti, and J. Lang, The variable C-terminus of cysteine string proteins modulates exocytosis and protein-protein interactions. Biochemistry, 2004. 43: p. 16212-16223. 32

to sheets (ref. 1). This unprecedented dynamic behavior of a transmembrane domain may have important functional consequences during membrane fusion by disturbing the bilayer and thus favoring lipid mixing. We have also set-up the procedure to obtain high-purity amphiphilic full-length SNARE proteins in a buffer suitable for reconstitution and spectroscopic approaches (see Project).

• GLUCOTOXICITY, SIGNALING AND EXOCYTOSIS Type 2 diabetes is a progressive disease due to a gradual deterioration of β-cell function in the presence of insulin resistance. The ensuing hyperglycemia worsens the insulin resistance and further impairs β-cell function, thus creating a vicious circle. Indeed, islets exposed to chronic high glucose concentrations become insensitive to subsequent glucose stimulation. Chronically elevated glucose may exert its deleterious effects at different points of the sequence of events connecting elevation of extracellular glucose concentration to insulin release7. Indeed, previous FIG. 5: Dynamics of SNARE transmembrane domains. A : Upon reports demonstrated alterations in β-cell increased TMD/lipid ration, peptides adopt β-sheet conformation. differentiation, leading to reduced glucose B: Hypothetical change in TMD conformation during vesicle fusion. detection, modifications in glucose metabolism, in calcium handling and an increased rate of apoptosis. Moreover, decreased responsiveness to stimuli such as arginine or sulfonylureas in islets from type 2 diabetic patients have been reported and raises the question of whether glucotoxicity may also impede late steps in insulin secretion. We had therefore investigated the influence of glucotoxicity on distal steps of insulin secretion using the well differentiated clonal β-cell line INS-1E and human islets in collaboration with P. Vacher (INSERM U916, Institut Bergonié), an islet transplantation group (F. Pattou, INSERM U859, Lille) and an obesity research group (N. Moustaïd-Moussa, U Tenn, US), the latter supported by a Fulbright exchange grant. In our hands, these cells provide a useful model with minimal apoptosis and changes in calcium handling that mimics the behaviour of primary cells. More importantly, functional studies using intact and semi-permeabilized cells clearly demonstrated a defect in the late steps of exocytosis. These alterations were accompanied by altered FIG. 6: Glucose regulates cAMP- and Ca2+-dependent pathways in βexpression of several key proteins essential for cells. u or d, up- or downregulated between 5.5 and 20 mM glucose; S, U exocytosis in clonal β-cells and in human islets or D, stable, up- or downregulated between two glucose concentrations, (ref. 9). Thus, this model permits to investigate the combination indicates thus the profile between 5.5, 11 and 20 mM vesicular transport/exocytosis and the glucose. NA, not affected by glucose. *, validated by qPCR. regulation of the expression of its components in a major disease. Strikingly, altered Ca2+-handling in glucotoxicity depended on cAMP-signalling known to be important for full secretion competence and amplification of exocytosis in β-cells. Glucose and incretin hormones physiologically regulate β-cell function, gene expression and insulin exocytosis via calcium and cAMP. We have therefore investigated long-term effects of glucose on these pathways with special regard to the incretin GLP-1 in clonal β-cells and in human or rat islets. GLP-1 or forskolin mediated increases in cytosolic calcium, cAMPlevels or insulin secretion were largely reduced in clonal β-cells cultured at elevated glucose levels. Statistical analysis of transcription profiles identified cAMP pathways as major targets in glucotoxicity. These findings were confirmed by quantitative PCR and unraveled a marked down-regulation of the calcium-sensitive adenylyl cyclase ADCY8 in INS-1E cells, in rat and in human islets. GLP-1 as well as glucose-induced calcium signaling relied on ADCY8 as shown by knock-down. Moreover, re-expression of ADCY8, but not of GLP-1 receptor, 7

Lang, J., PIPs and pools in insulin secretion. Trends Endocrinol. Metab., 2003. 14: p. 297-299. 33

recoveredd GLP-1 and forskolin signnaling in gluccotoxicity. Sim milarly, activaation of the ddownstream taarget CRE dependedd on ADCY88. Finally, capacitance c m measurements revealed thaat cAMP-indduced amplifiication of exocytosiis proceeds via ADCY8. Thus, T cAMP-m mediated path hways are proofoundly remoodeled in glu ucotoxicity and hereein the calciuum-sensitive ADCY8 A playys a central role r in glucoose and GLP--1 signaling. Our data contributee further to thhe understandding of isoforrm specific sig gnaling in genneral and to eexocytosis in particular and proviide new insighhts in β-cell faailure. A manuuscript has been submitted.

•

ELECTROPHY L YSIOLOGY

We installed electrophhysiological approachhes in pancreaatic β-cells: paatch clamp experimeents (whole cell configurration and perforatedd patch) as well as exxtracellular recordingg using microeelectrode arraays (MEA). Patch clam mp experimennts are designned to study β-cell exxocytosis andd, when neceessary, the populatioons of ionic channels. Constitutive C exocytosiis is evaluateed by the dettermination of global calcium currrent density with w respect to the cell capacitance.. Regulated exxocytosis is c determineed by the inncrease of capacitance evoked either by applying a a series of depolarizzing pulses to activatee voltageFIG nylyl cyclase cyclase8 is8s required is required for amplificati on of FIG7:7: Aden nylyl for am mplification of ex xocytosis dependennt calcium chhannels or by b infusing exocytosis ycyclases. adenylyl cycresentative clases. A: capac repre esentative capaccitance by adenylylby A: repr citance recordings s of INSfree calciium into the cell c via the paatch pipette recordings INS-1E I cells tran sfected (sh-CON) with psh1E cells oftra ansfected with pssh-NC N)-NC or (sh-CON) psh-4 (shorrADCY8). Apsh-4 solution. wasdialysis (shExocytosis ADCY8).was by 1.5 dialy ysis free withcalcium 1.5 µM Mthrough free wExocytosis elicited by d elicitedwith µ µM calcium throug the in patch ette in or thepresenc absencce or presence off/100 1 µM e of 1 µM FSK/ the patch pigh ipette the pipe abssence µM m provide noon-invasive The MEA system FSK/100 µMSummary mary in capacitance during IBMX B: SIBMX B:ofSumm cha anges ofinchanges capaciitance during exxocytosis t study simuultaneously recordinggs, allowing to exocytosis ite elicited ehree different by con thhree different elicited by th nditions: iterativee conditions: depolarizations serative (Depol) up to sixty cells or group of cells. Stimulated depolarizations s (Depol) or intra of 1.5 in µMstandar free ca alcium or intracellu ular dialysis of acellular 1.5 µM dialysis free callcium rd buffer 2+ w scarce isolaated spikes annd bursts of cells show absence in (Ca standard (Ca2+) or in pre theesence ence of 1μμM FS SK/100 ) in bu theeffer absence of 1orμprese MF FSK/100 M IBM MX (Ca2+ spikes. Quantification Q n of the reesponse is I/FSK). μM IBMX (Caa2+ I/FSK). ensured by determinning the freqquency of s the ampllitude of each spike remains constant. spiking, since

5 signifficant publiications: 1. W Wissam, Y., S. S Federman, A. A Milochau, S. S Walid, M. Laguerre, L B. Desbat, R. Oda,, and J. Lang, Reversible transition between α-heliix and β-sheet conformation c of a transmembrrane domain. Biochim. B Biophyys. Acta - Biom membranes, in press. 2. G Grise, F., N. Taib, T C. Monterrrat, V. Lagreee, and J. Lang, Distinct roles of the C2A annd the C2B dom main of the vesicular Ca C 2+ sensor synnaptotagmin 9 in i endocrine β-cells. Biochem. J., 2007. 403: p. 483-492. 3. D Dubois, M., P. Vacher, B. Rogger, D. Huyghe, B. Vandewallle, J. Kerr-Contte, F. Pattou, N.. Moustaid-Mou ussa, and J. Lang, Glucotoxicity inhibbits late steps off insulin exocyttosis. Endocrino ology, 2007. 1448: p. 1605-16144. B Boal, F., S. Le Pevelen, C. Czziepluch, P. Scootti * and J. Lan ng*, Cysteine-sttring protein isoform β(Cspβ) is targeted 4. to the tranns-Golgi netwoork as a non-paalmitoylated CSSP in clonal β-cells. Biochim m. Biophys. Actta - Mol. Cell Res., R 2007. 1773: p. 109-19. * equal contribution L Lajus, S., P. Vacher, V D. Hubber, M. Duboiss, M.N. Benassy, Y. Ushkaryyov, and J. Laang, α-Latrotoxxin induces 5. exocytosiss by inhibition of o voltage-depeendent K+ channnels and by stiimulation of L--type Ca2+ channnels via latrop philin in βcells. J. Biiol. Chem., 20006. 281.: p. 55222-5531.

ng, researcch & educcation train ning, diffu usion of sccience (con ngress, worrkshops, Teachin scientiffic open dayys etc.) Teachingg (ca. 1200h eqq TD per annuum) Baachelor of Scieence: in chargge of 4 Coursees (UE); Masteer of Science: in charge off 3 Courses (U UE) Coo-director Specialty Cell Bioology, Develop opment and Pa athophysiology gy In charge of Loggistics/Techniical Resourcess for Teaching g (Bachelor inn Biology) R; since I/2007) Doctoral School: Direccteur-Adjoint (ca. 330 thésaards, 480 HDR Workshops during “Fêête de la Sciennce » 2007, 20008 and 2009 S Meeting of the Dooctoral Schooll (ca. 350 partticipants) Annual Scientific Congresss organization: Club Exo-E Endocytose 2006

3 34

TEAM « INTERACTIONS BETWEEN PROBIOTIC BACTERIA AND HOSTS» Team Leader: Maria Urdaci, PR1 (ENITA de Bordeaux). Team composition (2006-2009) : Muriel Denayrolles, MC ENITAB Pascale Cavard, AJT ENITAB, 10% Benoit Grossiord, MC ENITAB Aurélie Baliarda, Post-Doc Anne Marie Elie, IE ENITAB Soledad Arias, Post-Doc Philippe Bressollier, PR ULim Borja Sanchez, Post-Doc Roland Caubet, MC UB1 Hedi Harizi, Post-Doc Françoise Pedarros, T UB1 Marie Lefevre Post-Doc Annie Monistrol, AT ENITAB, 10% Bastien Massias, Post-Doc Myriam Delamarre, S ENITAB, 20% Silvia Racedo, Post-Doc Michèle Dayde, AJT ENITAB, 10% Taroub Bouzaine, PhD B.Sc. and M.Sc. trainees (stay between 2 and 6 months): 13

Imen Ben Abdelmalek, PhD Bastien Massias, PhD Imene Jaziri, PhD Thu Nguyet, PhD Naima Saad, PhD Armelle Ballade, PhD Belsene Talbi, PhD Imene Ben Slimene PhD

Team Production (2006-2009): Publications, peer reviewed, (ACL) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI) Oral communications in congress (COM) Poster communications in congress (AFF)

30 2 2

Books or Book chapters (OS) Patents Defended PhD Theses or HDR

4 16

Grants : public & private k€/year

1 1 6 75 k€

Scientific activity Résumé Les probiotiques sont des microorganismes vivants qui, administrés par voie orale, confèrent un effet bénéfique sur la santé de l’hôte. Bien que ces effets bénéfiques soient reconnus depuis longtemps, les bases scientifiques restent, en partie, à élucider. Notre objectif final est de mieux comprendre le mécanisme d’action des probiotiques, y compris au niveau cellulaire. Nous sélectionnons des souches de Bacillus et de Lactobacillus en utilisant divers critères, comme la capacité d’adhésion à des cellules épithéliales ou au mucus, les propriétés d’immunomodulation et la capacité à produire des substances antimicrobiennes. L’étude de l’innocuité de certaines souches candidates a été très poussée. Un intérêt accru a été donné à la caractérisation des constituants bactériens potentiellement impliqués dans l’effet bénéfique. Les éléments de surface ou les composés secrétés par les bactéries sont en première ligne dans l’interaction avec l’hôte. Plus spécifiquement, nous avons purifié et caractérisé des protéines de surface, secrétées, et des exopolysaccharides (EPS) à partir de plusieurs souches probiotiques. L’implication des composants de la surface bactérienne dans le processus d’adhésion a été démontrée. De nouvelles protéines capables d’adhérer à la mucine et/ou à la fibronectine ont été décelées, certaines possédant aussi des motifs conservés qui présagent de l’interaction avec hôte. Nous avons apporté une nouvelle hypothèse quant à la manière dont certaines protéines peuvent se retrouver à la surface des bactéries. Notre recherche sur les substances antimicrobiennes nous a amené à la découverte d’un nouveau lantibiotique qui a fait l’objet d’une prise de brevet. Nous avons également étudié le pouvoir immunomodulateur des souches; certaines ont été sélectionnées pour induire un patron de cytokines anti-inflammatoires. L’étude de l’impact des préprobiotiques et d’autres additifs sur la microflore de l’hôte a été réalisée via la mise au point de méthodes moléculaires d’analyse de la composition de microflores complexes. Un nouveau programme informatique pour l’analyse des profils complexes (ProReg XL Tool) ainsi qu’une nouvelle technologie (CSbyDG) ont été développés. Abstract Probiotics are living microorganisms administered in an adequate amount that confer a health benefit to the host. Although beneficial effects have been recognized for a long time, some fundamental aspects remain to be clarified. Our final objective is a better understanding of the mechanism of action of probiotics, particularly including the cellular level. We select Bacillus and Lactobacillus strains by using various criteria, such as their adhesion capacity to epithelial cells or to the mucus, their immuno-modulatory properties and their capacity to produce antimicrobial substances. The safety evaluation of certain candidate strains has been pushed forward. A special interest is 35

given to the characterrization of thee bacterial coonstituents po otentially implicated in thee beneficial effect. The o compoundss secreted by microbes aree in first line for interactioon with the host. h More elements of surface, or oteins, secreteed proteins annd exopolysaccharides specificallly, we have purified and characterizedd surface pro (EPS) of several probiotic strains. We W have demoonstrated the involvement i o bacterial suurface compou of unds in the w proteins able a to adheree to mucin and/or a fibroneectin were iddentified, som me having adhesion process. New W have clariified, issuing a new hypotthesis, the conservedd motifs that predict an innteraction witth the host. We occurrencce of certain proteins p on thhe bacterial suurface. Our research on anttimicrobial su ubstances led d us to the discoveryy of a new Laantibiotic, thaat has been paatented. We allso studied thhe immunomoodulatory pro operties of some straains; several of o these were chosen becauuse induces anti-inflamma a atory cytokin nes. The impaact of preprobiotics and other additives a on the host miccroflora was investigated by means off molecular microflora m t A new computer c proggram for the analysis a of com mplex profiless (ProReg XL L Tool) as welll as a new analysis tools. technologgy (CSbyDG) has been devveloped.

• Straiin selection n and charaacterization n Straains We have h isolated a large numbber of potentiially probioticc Lactobacilluus and Bacilluus strains. Strrains were identifiedd to species leevel using mollecular methods (16S rDNA A and rpoB seequencing, specific-PCR). Molecular M typing (R RAPD, PCR-R RFLP, Pulsedd field electrrophoresis…) was used too discriminatee strains. Isollates were screened using variouus criteria succh as their addhesion capaccity to the eppithelial cells or to the mu ucus, their modulatory properties and their t capacity to produce an ntimicrobial suubstances. immunom Exoppolysacchariddes (EPS) EPSss may confer interesting prroperties to prrobiotic strain ns, such as im mmunomodulaation, antiviral and anti-tumooral activities, and biofilm formation. Lactic Acid Baacteria (LAB)) and Bacillus were screenedd for EPS prooduction and polymers weere biochemiccally characterrized ws EPSs werre retrieved and a genetic determinants d w were (size andd sugar compposition). New studied. Strains S possesss various EPS S operons andd, for some, their t expressioon studied by RTPCR, wass constitutive.. Safetty study A coomplete safetyy study of som me Bacillus strains was reaalized in collaaboration withh the lab of S. S Cutting, (Univ. off London). Strrains tested were w safe in reegard of resultts obtained inn different anim mal models. Moreover, M recent EU U criteria form mulated by thee EFSA were used u for the ch hoice of strainns.

•

Baccterial adheesion and coompounds implicated d

I has been coommonly sugggested that thee adhesion cappacity of a prrobiotic to the mucus or It too epithelial ceells is an impoortant criteria to help strainn persistence, ppathogen excllusion and m may contributte to the imm munostimulattion and host cross-talkinng. However, bacterial addhesion is a complex c proccess involving g different meechanisms. Inn several Gram m positive B. clausii / Caco2 cells bacteria, intera actions with coomponents off the gastro-inttestinal tract (m mucins, epithelial cells, extracelluular matrix prroteins…) are mediated by a set of surfaace proteins annd non proteinnaceous molecular entities succh as teichoïc and lipoteeichoïc acids, and exopollysaccharides. Some probiotic properties arre thought to occur on thee bacterial su urface. We haave demonstraated the ment of bacteriial surface com mpounds in thhe adhesion prrocess. Surfacce proteins havve been involvem characterrized (coll J.M M. Schmitter) using u tandem mass spectrom metry. New prroteins able too adhere to mucin and/or fibronnectin were iddentified, som me having con nserved motiffs (SH3 domaains, for example)) that predict an interactionn with the hoost. Secreted proteins SDS‐PAGE of surfaace proteins

P I+ g ate

PI+ = 1.03 %

12h

P I+ g ate

PI+ = 2.96 %

16h

P I+ g ate

14,25

3

9h

PI+ = 6.98 %

0,56 1,11

% of red fluorescen nce (PI) cw-GAPDH/Cells 100E-12 (U/Cell)

6

0,87 1,29

12

G row th tim e (h)

9

102,24

2 20h

P I+ g ate

P I+ g ate

PI+ = 19.83 %

48h

P I+ g ate

PI+ = 94.14 %

20

16

PI+ = 13.96 %

24h

1,03 1,87 15,27 2,96 3,27 12,53 6,98 3,79 18,84 13,96 9,38 16,89 19,83

24

« m e m b ra n e  p e rm e a b ility »

P I, F L 3 (re la tiv e  flu o re s c e n c e )     

115,67

F DA, F L 1 (relative fluorescence)

76,3

Evolution off L. plantarum 299v 2 cell membranes permeability evolution wa associated GAPDH G and cell wall activity duri ring growth pha ase. (a) Representatio on of cw-GAPDH activity, membranes permeability p (% % of PI fluorescence) and metabolic activity (mean fluorrescence of FD DA) at different stag ges in the growth h phase. (b) Dot plots FL1 versus FL3 showing bi-parametricc analysis of metabolic m activity and membrane m integrity ty during growth of L. plantarum p 299v.

« Metabolic ac tivity »

3 36

were also characterized for some probiotic strains. Fonctional study of proteins has been initiated. We have clarified, issuing a new hypothesis, the occurrence of certain proteins on the bacterial surface. Glyceraldehyde-3-Phosphate Deshydrogenase (GAPDH), a cell-wall associated housekeeping enzyme, has been selected as a model study. GAPDH concentration on cell wall was growth time dependent. Flow cytometry analysis using double labelling of the probiotic L. plantarum 299v cells with anti-GAPDH antibodies and PI, established unambiguously that cells with impaired membranes manifest more cw-GAPDH than unaltered cells.

Production of antimicrobial substances (AS) It has been proposed that production of AS by probiotics is one of the mechanisms by which they protect host against pathogenic microorganisms and is considered as a selection criteria. Strains are screened by antimicrobial production against bacteria and fungi. New methods for rapid characterization of AS has been established by using mass spectrometry (coll JM. Schmitter). Bacillus subtilis 3 produces amicoumacine that possess anti-inflammatory and anti-bacterial activities. We have developed an ELISA of Amicoumacin-related compound suitable for a rapid and simple screening test for producer strains as well as to study in vivo metabolism and bioavailability of these compounds. In collaboration with Sanofi-Aventis we have studied the AS produced by a commercial Bacillus probiotic strain. Our research led us to the discovery of a new Lantibiotic, that has been patented. Its purification and structural analysis by mass spectroscopy and NMR were completed with S cloning of genetic determinants responsable of its production. NMR CH structural study of this peptide in micellar medium and ligand interactions ║ Asn Ala Dha CH S Arg Dhb Leu Trp were conducted in collaboration with B. Odaert (E. Dufourc team). Studies Phe Tyr Ala │ Gly Phe Lys Ala Ala Abu Ala Ala Ala NH of the lantibiotic target, lipid precursors of the bacterial cell wall, have Pro Gly S S been realized (IBBMC, UMR 8619, Orsay).

•

Immunomodulation and molecular cross-talking

The gastro-intestinal immune system forms the largest immune system. Probiotic supplementation may enhance host defense against infectious diseases, stimulating cytokine/chemokine production by the innate immune system. In relation with industrial partners, our strain collection was screened in order to design new probiotics with immunomodulatory and/or anti-inflammatory capacities. Six strains were selected for further studies. The selection of probiotic strains based on their capacity to decrease proinflammatory cytokine/chemokine responses and to restore Strains downregulate TNF-α induced Th1/Th2 balance disruption is a promising avenue expression on inflammatory mediators by for the development of new therapeutic agents PBMC stimulated with TNF-α (25 ng/mL). against GI chronic inflammatory conditions. We also determined the “anti-inflammatory” potential of selected probiotic strains (coll I.Pinchuk, Univ. Texas). In vivo, inflammation models were used (coll, J. Fioramonti, INRA Toulouse) and interesting results were obtained with two strains. Probiotics modulate some molecular pathways within intestinal and immune cells by interacting with certain receptors, mainly Toll-like receptors (TLRs) and NOD proteins. Untreated 30 + BCU The signal is transmitted to the nucleus via different pathways, 25 + Bli which generally involves the consecutive action of several kinases + BcO/C 20 (MAPKs, PI-3K and GSK-3). The signal finally arrives in the + BcSIN 15 nucleus, where it induces changes in the genetic expression and 10 produces physiological and/or immune changes in the cell. We have 5 studied de TLR activation in the intestinal cells (Caco 2) using 0 different probiotic strains. Activation of the TLRs is in relationship TLR2 TLR3 TLR4 TLR5 TLR9 NOD1 NOD2 with the strain used.

•

Impact of food and feed additives on the host microflora. Molecular analysis of complex microflora. The aim of this research program, started in 2006 under industrial contract, was to screen feed additives as growthpromoters substitutives. Molecular tools (PCR-DGGE mainly) were applied to study in vivo the impact of diverse feed additives (probiotics, prebiotics, fibers, essential oils…) on the host microflora. A large research program is in progress to explore pig and chicken gut microflora. We also use these molecular methods to explore probiotic

ScAmy54 Active and calcium binding sites

Ca2+

37

effects onn animal moddels (Prod’Innnov project). A new compu uter program for the analysis of compleex profiles (ProReg XL X Tool) as well w as a new technology t (C CSbyDG) has been developed. M Moreover, we are a looking for enzymes to be used in prebiotic producction. Two neew amylases (ScAmy54 ( and ScAm my 43) produucing specific oligosacchariides, essentiallly maltotriose, have been been characteerized and their struccture resolvedd.

Esseential oils and a microbiaal inhibitionn. Essenntial oils perrmeabilize bacterial membbranes by form ming pores resulting r in a rapid efflux x of small molecules. Their actioon is dependaant of growthh phase and in n relation witth membrane fluidity mod difications. a and a requires precise p quantittative informaation about Studies att sub-lethal cooncentrations are the most appropriated five grow wth parameters (µExp, µStatt, µAdapt, TAdappt, CFUMax). Classical metthods are nott efficient an nd lead to me mathematiccal approachess that could bee useful in incompplete data. Thuus, we have deeveloped som Thymol achieviing this goal using u techniquues that monittor the conducctivity changee of a microbiial culture. The ressults obtained shows inhibittions in differrent growth paarameters deppending on oils, bacteria and groowth phase annd could be usseful tools in several s areas of o microbiologgy, such as th he study of antimiccrobial agentss, to improvee the controll of food pathogens by tthe appropriaate use of protectors, the estabblishment of models m in preedictive microobiology as w well as in fun ndamental b stresss. studies concerning bacterial

5 signifficant publiications: 1. Sáncheez B, S. Ariaas, S. Chaigneepain, M. Den nayrolles, J.M. Schmitter, P.. Bressollier aand MC. Urd daci. 2009. Identificattion of surface proteins involvved in the adhhesion of a prob biotic Bacillus cereus strain tto mucin and fibronectin. f Microbioloogy 155, 1708-1716. 2. Urdaci,, MC, Sánchezz B. 2009. Som me immunomoduulatory effects of probiotic baccteria might bee due to porcinee neutrophil elastase innhibitor, a serpinn present in MR RS broth. Immuunol. Lett. 121, 99-100. 3. Sánchez, B., Bressolieer P., Urdaci MC. M 2008. Expoorted proteins in n Gram-positive probiotic bactteria as means of o bacteriaF Immunool. Med. Mic. 54 4: 1-17. host adhession and intercoommunication. FEMS 4. Massiass B, MC. Urdaaci. 2009. ProR Reg XL Tool: ann easy-to-use co omputer tool suuite for rapidly regrouping a hiigh number of identicaal electrophorettic profiles. BioTechniques. 46: 441-450. 5. Bouhsss A., B. Al-Daabbagh, M. Vinncent, B. Odaeert, M. Aumon nt-Nicaise, P. Bressollier, B M M. Desmadril, D. D MenginLecreulx, MC. Urdaci and a J. Gallay. 2009. 2 Specific interactions of clausin, a new w lantibiotic, wiith lipid precurrsors of the bacterial cell c wall. Biophyysical J. 97(5). In press.

Teachin ng, researcch & educattion trainin ng, diffusio on of science (congresss, workshop ps, scientiffic open dayys etc.) A the mem All mbers of our team are fully fu implicated in teachinng activities. Teaching courses c of Microbioology, Biotechhnology, Biocchemistry andd Molecular Biology B are performed p in two engineering High Schools (ENITAB ( andd ISTAB) andd also at the University (B Bordeaux andd Limoges). K Knowledge is generally completed by large sesssions of pracctical work annd some tutoriials, like scienntific projects.. Some teaching staff is “ coontinue”, maiinly provided for industriaal staffs and in several prrofessional also invoolved in the “formation Masters. Our implicattion in such a professionall formation allso requires thhe following of numbers of o student performinng training peeriods in instituutions involveed in agricultu ure and food sciences s (Mainnly bio-industtry or food companiees). Our interrest in food sciences s allow wed us to pro opose new formations relaated to food safety s and preservattion, also openned to staff meembers of the industry or otther professioon. Some of uss also do teach hing at the internatioonal level as interventions i i the Master 2 research in in n the universitty of Tunis (114 h for 4 yeaars), in the universityy of Bangkokk (15 h in 2009), in the univversity of Ho Chi Minh in Vietnam (twoo weeks for 6 years) or Universitty of Louvainn-la-Neuve in Belgium (4 h for 6 years).. The unit is also a implicateed in the ASIA AINVEST program “Quality in the food inddustry” financced by the Eu uropean Com mmission for enhancing Vietnamese V n the agro-induustry (this traiining is distrib buted to a capacity for exporting food by boossting the qualiity attitude in public mainly consisteed of executivves, leaders annd people in charge of com mpany): a weeek every threee months ooperation décentralisée regions Aquitaiine-Sousssince 20008. We are alsso in charge a teaching coooperation (“co Massa-Drrâa”) with thee university off Agadir, Moroocco (9 missio ons/year sincee 2008). - Partial organization: o “Fête de la sccience: Les bactéries qui nous font du bieen” CapSciencces, October 2008. 2

pation to th he « Pôle dee Compétittivité » Prod’Innov. Particip The laborratory is impllicated in this regional polee. The team “d developpemennt of news prrobiotics for human h and animal heealth” obtaineed this label and the projeect is granted for 4 years by b the DGE. Different reg gional and national industries i as well w as two laaboratories aree implicated. In I this contexxt, confidential data are not presented in this meemory. Moreoover, some staaff of the team m participate in i workshops//meetings on the strategy of o the pole on a monnthly basis.

3 38

DEPARTMENT: BIOPHYSICS & STRUCTURAL BIOCHEMISTRY Three teams constitute this department Team Name Structure & Dynamics of Membrane Assemblies Crystallography of Biological Macromolecules Mass Spectrometry of Biological Macromolecules

Laboratory Head Erick Dufourc, DR CNRS Bernard Gallois, DR CNRS Jean-Marie Schmitter, PR UBx1

This department deals with structure, dynamics and mode of action of peptides, proteins and lipids. Molecules are isolated or under the form of complex membrane assemblies: model and natural membranes, colloidal and non covalent complexes. Systems under study are under the form of crystals, powders, soft matter, in solution or in the gas state. Themes: • • • • • • • • •

Lipids & nanoobjects magnetically orientable (bicelles & liposomes) Lipid domains (Rafts). Cholesterol & plant sterols. Signaling lipids Peptides and membrane proteins involved in cancer, in apoptosis and with antimicrobial properties. Natural colloids: tannins-proteins, lipoaminoacid carriers of active molecules. Enzymes and transcription factors involved in biosynthesis of tannins. Interactions host/virus of m lettuce mosaic. Protein aggregation: Prion HET-s, lithostatin. Proteomic analysis of membrane proteins. Clinical proteomics & peptidomics. Strains of Bacillus with antimicrobial activities.

Technical Approaches: • • • • • • •

Mass Spectrometry, Maldi-Tof, Esi-Tof, Q-Tof, MS/MS. X-Rays Crystallography High field liquid and solid state NMR. Magic angle sample spinning. Circular Dichroism, IR spectroscopy, Dynamic light scattering. Chemical syntheses and isotopic labeling of lipids and peptides H/D isotopic exchange on membrane proteins. Protein expression and labeling. Crystal growth.

39

TEAM « STRUCTURE & DYNAMICS OF MEMBRANE ASSEMBLIES » Team Leader: Erick Dufourc, Research Director, CNRS. Team Composition (2006-2009): Axelle Grélard, IE CNRS (50%) Isabelle Pianet, HDR, IR CNRS (50%) Cécile Loudet, Doctorante Marc-Antoine Sani, Doctorant Yann André, Doctorant

Isabelle Lebars, CR1 CNRS (up to 2008) Anna Diller, Post-Doctorante CNRS Sébastien Buchoux, Doctorant Olivier Cala, Doctorant Vania Rodrigues de Lima, Doctorante

Benoît Odaert, CR1 CNRS Marie Garnier, Doctorante Vanessa Zhendre, Doctorante Jeannot Toupet, Doctorant Christian Bijani, Doctorant

B.Sc. and M.Sc. trainees (stay between 2 and 6 months): 10

Team Production (2006-2009): Publications, peer reviewed, (ACL) 59 Edition of Books (DO) 1 Invited conferences congress (INV) 15 Congress/Workshop organization 8 Publications, peer reviewed in published international 11 Defended PhD Theses or HDR 7 colloquia (ACTI) Oral communications in congress (COM) 24 Grants : public & private k€/year 50 k€ Poster communications in congress (AFF) 29 Special funding for NMR equipment* 3140 k€ Books or Book chapters (OS) 4 *Funding obtained by the team for IECB and for Aquitaine NMR allowed installing both 700 & 800 MHz (2007, 2009)

Scientific activity Résumé La biophysique membranaire et colloïdale que nous développons se décline en 3 thèmes, les lipides, les peptides ou protéines effectrices de membranes et les colloïdes en nutrition et santé. La RMN des liquides et des solides est aussi portée par le groupe en support à nos études et à celles d’autres équipes (UMR et extérieur). Plus spécifiquement, nous avons produit et caractérisé des nanoobjets membranaires lipidiques, les bicelles, qui ont des propriétés d’orientation macroscopique remarquables dans les champs magnétiques, ce qui leur confère le qualificatif de « goniomètre moléculaire » très intéressant pour la biologie structurale. Nous avons aussi mis en évidence le rôle « universel » des stérols de plantes, de champignons et de mammifères dans la régulation de la dynamique des membranes et proposé une explication « évolutionniste »aux modification subtile des structures en passant d’un règne à l’autre. Nous avons également montré que les phophoinositides jouent un rôle essentiel dans la co-régulation de la dynamique membranaire qui est impliquée dans les processus de croissance et de division cellulaire. Deux nouveaux mécanismes d’action des peptides antibiotiques sur les membranes ont été découverts : celui du « coin électrostatique » des peptides acides, cyliques et acylés et celui du « pore désordonné » pour les peptides basiques se structurant en domaines à feuillet beta sur les membranes. Nous avons démontré que les tannins condensés contenus en partie dans les raisins et le vin forment des colloïdes selon des règles dépendant de leur structure et de leur degré de polymérisation. Nous avons aussi mis en évidence leur forte interaction avec les protéines de la salive riches en proline « les éponges à tannins » pour moduler la sensation d’astringence perçue lors de leur ingestion. Enfin des travaux en collaboration des membres du groupe ont permis de caractériser par RMN des liquides les interactions très spécifiques entre protéines et acides nucléiques. Abstract Development of membrane and colloid biophysics is split into 3 parts: lipids, peptides or proteins that affect membranes and colloids in health and food. Solid and liquid state NMR is also hold by the group for its own research and also for other groups in the UMR or abroad. More specifically, we have produced and characterized membranous lipidic nanoobjects, bicelles, that show remarkable macroscopic orientation properties in magnetic fields. This makes them ideal “molecular goniometers” for structural biology. We also evidenced the “universal” role of sterols from plants, fungi and mammals in the regulation of membrane dynamics and proposed a possible “evolutionist” explanation for their minute structural modifications on going from plants to mammals. We have also shown that phosphoinositides play an essential role in co-regulating the membrane dynamics in mechanisms of cell growth and division. Two new mechanisms of action of antibiotic peptides on membranes have been evidenced: the “electrostatic lipophilic wedge” for acidic, cyclic and acylated peptides and the “distorted pore” for peptides that form betasheeted patches on membranes. We have demonstrated that wine and grape tannins form colloids in water40

alcoholic media, whose properties depend on their 3D structure and their polymerization number. We also characterized their strong complex formation with saliva proteins rich in Proline residues, the latter being now called a “tannin sponge”. This specific interaction modulates the astringency feeling upon ingestion. Collaborative works of some group members, taking advantage of the liquid state NMR facility, afforded a minute description of protein-nucleic acid interactions.

• Lipids Biphenyl Bicelles We have synthesized phophophatidylcholines bearing two phenyl rings in one of their fatty acyl chains. Theses lipids when mixed to very short chain lipids make discoid nanoobjects (100 nm) that can be macroscopically oriented in magnetic fields in a way similar to smectic liquid crystals (discs normal to the field). They have been characterized by multinuclear NMR, electron microscopy and small angle X-rays scattering (coll R. Oda). Their remarkable thermal and orientation stability allows embedding in the bicelle membrane proteins, peptides, sterols and other lipids, which opens an entire field for structural biology of lipophylic molecules.

Biphenyl Bicelles. Molecular structures, schematics and electron micrographs of bicelles (Loudet et al. Biophys J, 2007)

Sterols from plants, fungi and mammals Plant and fungus sterols enter in the composition of lipid domains « rafts » that are implicated in the polarized growth. We have shown using solid state NMR of deuterium labeled lipids that theses sterols possess remarkable regulating properties of membrane dynamics. They maintain the membrane in a “liquid ordered” state throughout large temperature ranges, allowing the membranes of these organisms to cope with large temperature variations. We have proposed that modification of the sterol aliphatic chain in a way to reinforce Regulation of temperature-driven membrane dynamics by plant sterols. Central panel: order parameter as a function of temperature and 2H-NMR spectrum of a liquid-ordered, lo, state. Left panel: schematics of solid-ordered, so (gel), and liquid-disordered, ld (fluid), membrane states. Right panel: schematics of the lo (raft) membrane state together with the structures of plant sterols. (Beck et al. FASEB 2007)

membrane cohesion through increase of van der Walls forces occurred during evolution and is responsible for the greater temperature adaptation of plants and fungi, compared to mammals. Lipid signaling and “in cell” solid state NMR. Phosphatidylinositols are considered as lipid signals in the nuclear membrane and are involved in cell division. We were able to probe the dynamics of many membranes in entire cells coming from sea urchin, used as models to study cell division and its malfunctions in cancer processes (coll. B Larijani, Cancer Research UK). Incubation of cells or cell organelles with appropriate deuterated lipids afforded measuring membrane dynamics by pushing solid state NMR to its limits in sensitivity (100 nanomoles of labeled lipids). A remarkable fluidity was reported, in spite of elevated amounts of sterol, prompting the phosphoinositide lipids as a counterbalance for the otherwise known rigidifying effect of sterols. .

Probing entire cell dynamics . De-membranated sea urchin sperm (right) with Nuclear Enveloppe Remnants (arrows ) and the corresponding solid state 2H-NMR spectra (left bottom ).(Garnier et al . PLOS one , 2009 )

• Peptides involved in apoptosis, cancer and of antimicrobial action Membranous peptides involved in cancer & apoptosis Solid phase synthesis and purification of 15N-labelled peptides involved in cancer (neu/ErbB2) and apoptosis (Bax, Bcl2) have been carried out in rather large quantities, needed for their study in membrane media. In collaboration with JM Schmitter we even developed a very efficient and rapid chromatographic method to quantitatively recover hydrophobic peptides and lipids after having been mixed within membranes. After having shown that the transmembrane part of the tyrosine kinase receptor contained a pi-helix (structural trigger for 41

dimerization) we demonstrated, by reconstitution studies in bicelle membranes, that a flat membrane topology was needed for « coiled-coil » dimerization, a key step for cell signaling and cell growth. No dimer was observed in highly curved surfaces. On the other hand, we have demonstrated that peptides allowing the anchoring of Bax and Bcl2 proteins are disordered in solution but acquire a helical structure at the surface of mitochondrion membrane models. In collaboration with Pr. G. Gröbner (Umea Univ, Sweeden) we have developed a method to show the specific interaction of apoptotic peptides with cardiolopin, a key lipid in the apoptosis cascade. It is based on 31P-NMR under moderate magic angle spinning of entire functional mitochondria affording molecular identification of lipid species. Antimicrobial Peptides Small antimicrobial peptides often produced by « Electrostatic wedge » insects exhibit the potency to kill microorganisms by direct action on the plasma membrane, thus representing a potential alternative in the struggle for finding new antibiotic species. A number of mechanisms have been evidenced for peptides structuring themselves as amphipathic helices at membranes. After synthesis and/or purification from bacterial cultures, we evidenced new mechanisms for acidic, cyclic lipopeptides or basic betasheeted short peptides. Surfactin is a potent antibacterial peptide and has been studied against membranes The electrostatic lipophilic wedge. SF molecules mimicking bacteria. Using solid and liquid state NMR, IR penetrate into membranes due to hydrophobic forces, spectroscopy (coll. B. Desbat), and electron microscopy electrostatic repulsion then occurs between peptide negative charges and acidic lipid heads. Large increase (coll. A. Brisson) we have shown that unexpectedly this in local membrane curvature happens and destabilize acidic peptide interacts preferentially with negatively membranes (Buchoux et al. Biophys J, 2008) charged lipids to restructure membranes into small vesicles. A minute deciphering of interactions led us to propose the “electrostatic lipophilic wedge” as a working molecular mechanism. On the other hand amphiphilic peptides that are secreted during stress are very potent antibacterial/antifungal species and show remarkable action against plasmodium. In collaboration with MH Metz-Boutigue (Inserm, Strasbourg), B. Desbat and J. Elezgaray we could decode using NMR, IR, patch clamp and molecular dynamics the minute mechanism of action of cateslytin, a very powerful short and peptide belonging to this family. Very interestingly cateslytin forms antiparallel beta-sheet patches at membranes surfaces mimicking bacteria and promote the formation of rigid membrane domains. In fungi-like membranes a specific interaction with ergosterol is evidenced. Rigid/fluid line defects are proposed as to be appropriate for membrane crossing though a 10 Å “distorted pore”, as evidenced from molecular dynamics and patch clamp electrophysiological measurements.

The distorted pore. Cateslytin forms antiparallel betasheeted patches at bacterial/fungi like membrane surfaces. Rigid/fluid phase boundary defects occur through which arginin residue penetrate and allow the transient formation of a distorted pore, in a mechanism akin to molecular electroporation. (Jean-François et al. Biochemistry, Biophys J, 2008, FasebJ 2009)

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Colloids for health and food

Colloidal tannins Tannins can be found in grapes and wine and represent a particular interest for food industry and medical research (antioxidant properties). In collaboration with E. Fouquet & I. Pianet (ISM, Bordeaux) we have undertaken the preparation of large quantities of condensed polyphenols (dimers, trimers and tetramers of the monomeric units catechin and/or epicatechin) using new and original synthetic approaches allowing control of both stereochemistry and polymerization degree. The structural analysis of their colloidal complexes by liquid, diffusion, high resolution magic angle spinning NMR and molecular dynamics (coll. M. Laguerre) demonstrated that the greater the polymeric degree, the greater the condensed tannin dynamics (internal rotations between monomeric units). This leads to critical micellar concentrations much higher for dimers and trimers than for monomers. The nanometer Tannin colloids . Micelle formation in a water-alcoholic medium as a micelles are stable for polymerization degrees greater than function of time in a molecular dynamics simulation. A) catechin monomer, B) Catechin-catechin dimer . (Pianet et al. Langmuir 2008) 2 whereas micelles made of monomers further aggregate with time to form precipitates. This peculiar colloidal behavior controls their bioavalability and suggests a poorer effect of monomeric tannins. Colloidal tannins-saliva protein complexes When ingested, tannins form colloidal complexes with human saliva proteins and in particular with proline rich proteins (PRP) that are known to form collagen helices. After having synthesized entire such proteins or protein fragments in the laboratory, their interaction with monomeric and polymeric tannins has been followed with NMR (liquid, diffusion, HRMAS), light scattering and molecular dynamics (coll. M. Laguerre). The numerous binding sites have been identified (mostly on the hydrophilic face of proline residues), the stoechiometry and the affinity constants have been determined. The property of sequestering tannins has been put forward to name theses proteins as “tannins sponges” and is responsible for the feeling of astringency (removal of the lubricating effect of PRP in the mouth) as otherwise known in the industry of wine makers.

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Protein-nucleic acid interactions

Tannin-PRP complex. Three dimers B3 bind to a fragment of the IB7 human saliva protein. As determined from NMR and molecular modeling.

These are collaborative works, not in the main frame of the team and performed by members by taking advantage of the NMR facility. RNA loop-loop complexes (I. Lebars, coll. INSERM U869). An RNA aptamer, as identified by in vitro selection against the TAR element of HIV-1, is capable of inhibition of the activation of the RNA transcription that is mediated by the TAR-tat “kissing” complex. In order to increase the aptamer resistance to nucleases and its affinity to TAR, chemical modifications of the type “Locked Nucleic Acid” (LNA) have been introduced in different positions of the loop anti-TAR. The biological effects of these constructs have been tested on cell cultures. The main objective is to characterize at the atomic level the interactions ruling the formation of such complexes. NMR studies on such complexes afforded demonstrating that positions of LNA residues in the anti-TAR loop induce structural modifications at the interface between TAR and the aptamer. These structural changes are correlated with the observed biological effects. The 3D NMR structure Complex TAR/LR0656 (PDB Id : 2PN9) : of the TAR/LR0656 (modified aptamer inhibitor) afforded Superposition of 10 structures of lowest energy characterization of interactions involved in the complex. It has calculated with CNS, from NMR constrains (Lebars also been evidenced that introduction of 2 additional residues et al., NAR, 2007) brings a stabilization of the loop-loop interaction, when

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comparing to the unmodified aptamer. Our data also showed that the introduction of LNA residues maintains a normal WatsonCrick base pairing with a looploop conformation close to an A-type. The TAR element of HIV-1 thus constitutes a valuable target for developing therapic agents. Viral Ribonuclease (B. Odaert, coll. Ecole polytechnique/ICSN). RegB is a viral ribonuclease involved in the control of the cellular cycle of the T4 phage. It inactivates messenger RNAs that are produced after infection while specifically cutting the GGAG sequence found in initiation sites for translation. The goal was to determine the 3D structure and identify the binding sites to messengers RNAs. The protein has been produced by over-expression under a doubly labeled (15N-13C) form and purified by means of a Histidine tag. Heteronuclear three dimensional NMR has been used to assign all protein resonances. The 3D structure has been determined using molecular modeling under NMR constrains. A comparative analysis of the various mechanisms has been put forward in order to better understand how the endoribonucleases select and cut the RNA targets.

5 significant publications: 1. Sani, M. A., Keech, O., Gardeström, P., Dufourc, E. J., and Gröbner, G. (2009) Magic-angle phosphorus NMR of functional mitochondria: in situ monitoring of lipid response under apoptotic-like stress. Faseb Journal 23, doi: 10.1096/fj.1009-134114 2. Garnier-Lhomme, M., Byrne, R. D., Hobday, T. M. C., Gschmeissner, S., Woscholski, R., Poccia, D. L., Dufourc, E. J., and Larijani, B. (2009) Nuclear Envelope Remnants: Fluid Membranes Enriched in STEROLS and Polyphosphoinositides. PLoS ONE 4, e4255. 3. Jean-Francois, F., Elezgaray, J., Berson, P., Vacher, P., and Dufourc, E. J. (2008) Pore Formation Induced by an Antimicrobial Peptide: Electrostatic Effects. Biophys. J. 95, 5748-5756 4. Loudet, C., Manet, S., Gineste, S., Oda, R., Achard, M. F., and Dufourc, E. J. (2007) Biphenyl bicelle disks align perpendicular to magnetic fields on large temperature scales: A study combining synthesis, solid-state NMR, TEM, and SAXS, Biophysical Journal 92, 3949-3959. 5. Beck, J. G., Mathieu, D., Loudet, C., Buchoux, S., and Dufourc, E. J. (2007) Plant sterols in "rafts": a better way to regulate membrane thermal shocks, Faseb Journal 21, 1714-1723.

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Teaching of Membrane Biophysics and NMR (liquid & solid state). Master 1 & 2 level. Practical laboratories on NMR (Master students working on 300, 400, 500 MHz NMR machines). Practical training of NMR users (UMR, IECB and external people). Co-responsible for the Master “Biochimie Structurale –Biologie-Physique-Chimie”, University Bordeaux Full congress organization: “17th GERLI”, international, Biarritz, October 2010 (200 participants). Full international school organization: “1st EBSA course in membrane Biophysics”, international, Arcachon, June 2010 (100 participants). Partial organization: “7th European Biophysics”, Genoa, Italy, (900 participants) Partial organization: “GFPP congress”, Strasbourg, 200 participants, Partial organization: “GEIMM 13 congress”, Colmar France (200 participants) Full workshop organization: “NMR & Soft Matter”, international, Arcachon, May 2007 (100 participants). Partial organization: “6th European Biophysics”, international, London, July 2007 (1100 participants). Partial organization: “Fête de la science: Des bulles tensioactives” October 2007. Partial organization: “Congrès de Biophysique”, national, Biarritz, October 2006, (150 participants).

Management and running of technological platforms. The team ensures the maintenance and manages the NMR platform for UMS 3033 (IECB). The team brings scientific and engineering competence for running 6 machines (100 MHz WB, 300 MHz NB with sample changer, 300 MHz WB, 400 MHz NB, 500 MHz WB, 700 MHz NB). Approximately 60 people (UMR and UMS) have access to the platform after being properly trained by the team. The team also ensures studies for both academic and industrial external users (TOTAL, SANOFI, etc.) In 2006 the team was at the initiative of setting up the Aquitaine NMR network, whose role is to foster regional scientific exchanges. This networking was very useful in building up the funding (Aquitaine region, Ministry of Research, Europe, CNRS, INRA) and the negotiation of two spectrometers (600 & 700 MHz). Both machines have been successfully installed in July 2007 at IECB and in March 2008 at INRA. The team succeeded also in bringing up a scientific program and a budget to acquire, as part of the national TGIR NMR, a 800 MHz machine. Bordeaux is now part of this national NMR large scale facility with Paris, Lille, Grenoble, Lyon and Orleans. The team ensures the local contact and running for external users.

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TEAM «STRUCTURE AND ACTIVITY OF BIOLOGICAL MACROMOLECULES» Team Leader: Bernard Gallois, Research Director CNRS. Team Composition (2006-2009): Jean Chaudière, Pr Univ Bx 1 Jean - Marc Chevalier, IR2 CNRS Chloé Maugé, Doctorante

Thierry Granier, CR1 CNRS Claude Manigand, IE2 CNRS Nadia Trabelsi, Doctorante

Béatrice Langlois d’Estaintot, CR1 CNRS Pierre Petit, Doctorant Mahmoud Gargouri, Doctorant

B.Sc. and M.Sc. trainees (stay between 2 and 6 months): 6

Team Production (2006-2009) : Publications, peer reviewed (ACL) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI)

12 1

Oral communications in congress (COM) Poster communications in congress (AFF)

1

Grants public & private, k€/year

5 12 35 k€

Scientific Activity: Résumé Notre principal domaine de recherche concerne la biosynthèse des flavonoïdes. Les études entreprises ont pour but de caractériser les mécanismes d’action de diverses enzymes responsables de la formation de deux classes de composés aux propriétés remarquables, les anthocyanes et les tanins condensés. Les enzymes concernées ont été clonées, exprimées, purifiées au laboratoire. Leur instabilité en solution, à laquelle s’ajoute le plus souvent celle de leurs substrats, a nécessité des efforts importants afin que soient menées à bien l’étude de leurs propriétés structurales, la caractérisation de leur activité et la détermination de leurs propriétés enzymatiques. Parmi les principaux résultats, soulignons la première description structurale de trois de ces enzymes : une dihydroflavonol 4-réductase, une leucoanthocyanidine réductase et une anthocyanidine réductase. Ces structures ouvrent la voie à l’étude de la spécificité de substrat au sein de ces enzymes, et par delà même, à la compréhension de la typicité phénolique. Nous avons en outre montré, lors de l’étude du mécanisme réactionnel de l’anthocyanidine réductase, que cette enzyme synthétise, in vitro, la formation de flavan-3-ols, de stéréochimie inattendue, susceptibles de jouer un rôle phytotoxique important chez la plante. Nous avons également montré, l’apport de certaines techniques d’analyse à la résolution de problèmes spécifiques (technique MS/MS pour l’étude de la régiospécificité de transferts de deutérures; méthode d’Hummel & Dreyer afin de déterminer des constantes de dissociation de complexes enzymatiques). Parallèlement à ces études, nous nous sommes intéressés aux interactions entre facteurs cellulaires et viraux (virus de la mosaïque de laitue) et contribuons à l’étude de la reconnaissance d’une surface protéique par les foldamères afin d’inhiber des interactions protéines – protéines. Abstract The main field of research in the group is devoted to the investigation of the mechanistic properties of key enzymes involved in the flavonoid biosynthetic pathway. These enzymes catalyse the production of two important classes of compounds, the anthocyanins and the proanthocyanidins (or condensed tannins), which present remarkable properties, not only in plants but also in human health. Enzymes are cloned, overexpressed and purified in the laboratory. Their known instability in solution, added most often to that of their substrates, required great efforts to encompass the difficulties encountered to investigate their structural, functional and enzymatic properties. Among the main results, let us underline the first structural description of three of these enzymes never obtained so far. These structures are of great interest to investigate the driving forces of the substrate specificity of these enzymes and thus the phenolic composition in plants. Another result of interest, obtained during the mechanistic study of grape anthocyanidin reductase, was to demonstrate the production, in vitro, of flavan-3-ols of unexpected conformations and of phytotoxic activity. We also demonstrated the relevance of several analytical methods to solve specific questions (MS/MS techniques to investigate the regiospecificity of double transfer of “deuterides”; Hummel & Dreyer method to determine dissociation constants of enzyme complexes). In parallel to the previous studies, we investigated the interactions between host and viral factors (lettuce mosaic virus) and contribute to the study of the protein surface recognition by foldamers in order to inhibit protein - protein interactions.

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Enzymes of the flavonoid biosynthetic pathway

Flavonoids constitute a large class of phenolic compounds widely studied, due to their important properties. They are present in most higher plants, and involved in relevant functions such as pigmentation, reproduction and protection against pathogens. When ingested, they may act in disease treatment and prevention, mainly due to their 46uinine46g46t capabilities. Many clinical effects have been ascribed to these compounds, including anticancer, anti-inflammatory and a putative role has been found against coronary heart disease. The goal of our research, initiated since now 4 years, is to bring insights in the functional and structural properties of enzymes which govern the biosynthesis of anthocyanins and proanthocyanidins (or condensed 46uinine), two major classes of flavonoids. Because these compounds are responsible for organoleptic properties of red wine, part of this work is supported (2 PhD research grants) by CIVB (Conseil Interprofessionnel du Vin de Bordeaux). Four enzymes are under active investigation in our group: dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR). The full-length coding sequence for these proteins was PCR amplified with a cDNA library from Vitis vinifera as template. The recombinant proteins have been heterologously expressed in E. coli and successfully purified. Their activity has been demonstrated and their structural and mechanistic properties reported. The crucial point in the study was to optimize the stability conditions of both the enzymes and most of their substrates in solution in order to get crystals or to investigate the enzymatic The flavonoid biosynthetic pathways. properties. ƒ DFR and dihydroflavonols DFR is a pivotal enzyme in the flavonoid biosynthesis pathway as it catalyses the last common step leading to anthocyanins and proanthocyanidins. It catalyses the NADPH-dependent reduction of dihydroflavonols to flavan 3,4-diols. Due to its instability in solution, it was mainly characterised from a molecular genetic point of view. We described the 3D structure of the DFR – NADP+dihydroquercetin ternary complex at 1.8 Å (PDB code: 2c29) and the exact geometry of the active site (Petit P., et al, J. Mol. Biol, 2007, 368, 1345 – 1357). Analysis of the interactions between the cofactor, the substrate and the surrounding atoms of the enzyme reveals the reduction process. The relative positions of the substrate and coenzyme is compatible with a hydride transfer from the nicotinamide ring of NADP+ and the carbon atom of the substrate carbonyl group. We also demonstrated the importance of a protein segment which lines the substrate binding site and enlightens the role of a specific residue at position 133 assumed to control substrate recognition. The 3D structure of the DFR – NADP+ - DHQ activity of the wild type enzyme and its variant N133D has complex. been quantified, using dihydroflavonols of various hydroxylation patterns. Results evidence that position 133 strongly influences the reduction efficiency but cannot be solely responsible for the recognition of the hydroxylation pattern of dihydroflavonols. New mutations within the segment are under investigation to fully elucidate the recognition mechanisms. ƒ Kinetic and mechanistic studies of DFR These studies were designed to provide kinetic and mechanistic information on DFR, and to develop a “kinetic toolbox” which could be systematically used in future comparative studies of DFR mutants. a) Kinetic studies at pH 6.5 and 30°C Initial rates were measured by monitoring the time-course of the 340-nm absorbance decrease resulting from the joint consumption of dihydroquercetin and NADPH. Maximal activities were obtained at pH 6.5 and 50 mM NaCl. In such conditions, the enzyme undergoes thermal activation from 25 to 35°C. Studies of steady-state kinetics were performed at pH 6.5, 50 mM NaCl and 30°C. Substrate inhibition was observed above 35 µM dihydroquercetin, a threshold concentration which is unexpectedly low. At lower concentrations of dihydroquercetin, double-reverse primary plots support a sequential hyperbolic mechanism which is rapidequilibrium ordered with NADPH binding first. Secondary plots of the corresponding intercepts and slopes, and 46

non-linear least-squares analysis, provided reliable estimates of the 6 EB corresponding parameters. In this ordered mechanism, NADP+ is released last since it acts as a competitive inhibitor with respect to K 1 NADPH, with Ki(NADP+) = 30.8 ± 2.8 µM. From the theoretical enzyme E K K concentration deduced from 280 nm absorbance, the apparent turnover K 5 2 K 8 7 -1 EQ EA EAP EBQ number of the enzyme is kcat = Vmax/[ET] ≥ 540 min . Non-linear least-squares fitting suggest that substrate inhibition is due to the K K k formation of at least two unproductive complexes. One of them is 4 3 EPQ EA B clearly the abortive complex E.NADP+.DHQ whose 3D structure had k been mentioned above, whereas the other one may be either an E.DHQ Vv-DFR : Rapid-equilibrium ordered mechanism dead-end complex or an E.(DHQ)2 complex derived thereof. (Trabelsi & al., submitted). b) Binding equilibrium and kinetic studies of DFR at pH 7.5 and 30°C In this work, our goal was to use binding-equilibrium techniques and full consumption curves to get additional information and improve our analysis of initial rates at steady state. At pH 6.5, it turned out that the nonenzymatic degradation of NADPH was too high, so that we worked at pH 7.5. Using the chromatographic method of Hummel and Dreyer, we demonstrated that NADPH, DHQ and NADP+ independently bound to the free enzyme, each with a single hyperbolic binding site. We then collected full consumption curves in the presence of an NADPH-recycling system based on glucose-6-phosphate and glucose6-phosphate dehydrogenase. Along each of these curves, the NADPH concentration was fixed (10 to 260 µM) while the leucocyanidin concentration (not commercially available) could be calculated for each point. Rate curves were calculated. They were markedly concave-up, which demonstrates that leucocyanidin is a strong inhibitor of DFR. A more detailed interpretation of this study is still ongoing. ƒ DFR and flavonols Flavonols constitute another class of flavonoids which is, together with dihydroflavonols, present in plants during the expression of the DFR gene. Flavonol recognition by DFR has been studied. Interestingly, these compounds bind the active site of enzyme. Structures of the so-formed complexes (PDB code: 2nnl, 2iod, 3c1t) show drastic modifications with respect to that of the abortive DFR-NADP+-DHQ complex. Two flavonol molecules bind to the catalytic site in a stacking arrangement and alter its geometry which becomes incompatible with enzymatic activity. This result suggests that flavonols may act as inhibitors. This structural conclusion has been further confirmed by spectroscopic measurements showing that the enzyme activity towards dihydroflavonols is strongly reduced by flavonols (Trabelsi N., et al, Acta Cryst D., 2008, 883 – 891). ƒ ANR and flavan-3-ols. Binding of 2 flavonol molecules The respective role of ANR and LAR, which was still under debate to DFR. in the last few years, has been recently elucidated. These two NADPHdependent enzymes provide separate pathways for the synthesis of catechin and epicatechin, the flavan-3-ol monomers required for the formation of condensed tannins. a) ANR activity: From earlier biochemical studies, ANR was thought to catalyse the double NADPH-dependent reduction of anthocyanidins to 2R,3R-flavan-3-ols, i.e. (-)-epi-catechin when 47uinine47g is used as substrate. We recently expressed and purified ANR from grape (Vitis vinifera). The enzyme activity was only observed at low salt concentration. RP-HPLC, LC-MS and NMR experiments clearly established that the enzyme acts as an epimerase and produces a 50/50 mixture of 2,3-cis and 2,3-trans flavan-3-ols which have been identified by chiral chromatography to be 2S,3S- and 2S,3R-flavan-3-ols, i.e. the naturally rare (+)-epicatechin and (-)-catechin, when cyaniding is used as the substrate of the reaction (Gargouri M., et al, Acta CrystD, 2009; in press). Stereospecifically deuterated NADPD demonstrated the pro-S regioselectivity of the enzyme and the fact that the reaction mechanism involves two hydride transfers from two distinct NADPH molecules. At pH 7.5 and 30°C, the first hydride transfer to anthocyanidin was shown to be irreversible, and the resulting intermediate structure not released during catalysis. The regiospecificity of deuterium incorporation was analysed by LC/ESIMS/MS. This led to the conclusion that deuterium was always incorporated at C2 and C4. ANR reverse activity was assessed in the presence of excess NADP+. In such a case, the enzyme behaves as a flavan-3-ol C3epimerase, but this is only observed with 2R-flavan-3-ols, not with 2S-flavan-3-ols normally produced by the enzyme. The proposed mechanism involves the formation of a quinone methide intermediate serving as C4 target for the second hydride transfer (Gargouri M., et al, J.B.C., submitted). 61

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34

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b) Enzymatic properties: Cyanidin is unstable in solution and yields a mixture of structures with distinct absorbance spectra at equilibrium. To encompass this problem, we needed to look for conditions that stabilized the substrate around neutral pH, thereby enabling direct and accurate spectrophotometric measurements of it consumption rates. Studies of steady-state kinetics were performed by monitoring the 578 nm absorbance of cyaniding in the presence of a glucose/glucose oxydase/catalase system of oxygen depletion which significantly decreased the initial rate of non-enzymatic degradation of cyanidin. Double-reversed plots and non-linear regression analysis of enzyme initial rates support a hyperbolic sequential mechanism which is rapidequilibrium ordered, with NADPH binding first. The chromatographic Vv-ANR : Bi Uni Uni Bi rapidmethod of Hummel and Dreyer was used for binding-equilibrium studies equilibrium ordered of stable enzyme ligands, i.e. NADPH, NADP+ and catechin. This + confirmed hyperbolic binding of NADPH, NADP to the free enzyme, with a single site per enzyme molecule. Dissociation constants were estimated. Conversely, no significant binding of catechin to the free enzyme or the binary NADPH-enzyme complex was observed. These investigations led us to suggest that the most likely mechanism is sequential ordered Bi Uni Uni Bi, with NADPH binding first and NADP+ released last (Gargouri, M., to be published). To the best of our knowledge, the method of Hummel and Dreyer had never been used in the past to study binding equilibria of substrates and products of an enzyme. Our work demonstrates that this method can be a powerful tool in studies of enzyme-ligand binding c) ANR structure: Only crystals of the apoenzyme have been obtained so far. They grew under high salt concentration conditions, that is to say under conditions at which the enzyme is not active. The structure was solved at 2.2 Å resolution (PDB code: 2rh8) and explains the enzyme inactivity. The active site geometry was found to be wide open, with the side chains of the catalytic residues turned away from the nucleotide binding site. To our knowledge, ANR is the only third protein of the short-chain dehydrogenase / reductase family for which a non functional catalytic site is described. In each case, crystals were always obtained in presence of high salt concentrations, a result which led us to consider that high salt concentration is a factor responsible for the alteration of the protein conformation and consequently for the enzyme activity (Gargouri M., et al, Acta CrystD, 2009; in press). ƒ LAR and flavan-3-ols. LAR catalyses the NADPH – dependent reduction of flavan-3,4-diols (such as Vv-ANR 3D structure: Open leucocyanidin) to 2R,3S-flavan-3-ols (such as (+)-catechin). Even though it belongs to conformation of the catalytic the SDR superfamily, the enzyme differs from DFR and ANR. It does not present a triad catalytic triad so that the reduction mechanism might be different. Once more, little was known about the structural and enzymatic properties of the enzyme until we succeeded to clone, overexpress, purify it from Vitis vinifera under an active and stable (though cleaved) form. The LAR natural substrate being described in the literature as unstable, the enzyme-dependent production of catechin was demonstrated by RP-HPLC and LC-MS upon addition of the enzyme and substrate responsible for the biosynthesis of leucoanthocyanidin (i.e. dihydroflavonol 4-reductase and dihydroquercetin), to a solution containing LAR and NADPH. Chiral cochromatography allowed us to further characterise the reaction product as (+)-catechin. Y168 a) LAR structures: Several LAR structures have been determined. We first solved the CAT structure of the holoenzyme at 1.87 Å (PDB code: 3cjv) and that of the I171 ternary abortive complex: LAR – NADPH – catechin at 2.28 Å resolution N170 (PDB code: 3du9). The enzyme being systematically expressed in E. coli with the coenzyme bound to it, we succeeded to unfold the protein prior to refold it by dialysis. The structure of the native protein at 2.72 Å H172 resolution (PDB code: 3fjr) was then described together with that of a E175 reconstructed holoenzyme obtained by co-crystallisation of the apoprotein NADPH with NADPH (resolution: 1.75 Å, PDB code: 3fff). These structures allowed us to describe in details the structural catalytic site of the LAR changes associated to the different steps of the catalytic mechanism ternary complex (Maugé C. & al, submitted). We evidenced the particular role of two residues, K140 and H122, which interact respectively via a water molecule with the catechin C7 and C5 48

hydroxyl groups, and described the possible hydride transfer between the nicotinamide ring of the coenzyme and the C4 atom of the substrate. This led us to suggest that the reduction mechanism implies a dehydration of the substrate leading to the formation of a methylen 49uinine intermediate prior to 2R,3S-flavan-3-ols formation. b) Kinetic study of LAR : We recently succeeded in stabilising the LAR substrate over long enough time to determine initial rates with confidence. We demonstrated that the enzyme utilizes NADH as coenzyme with a lower efficiency than NADPH (KM(NADPH) < 1μM) and used this property to initiate kinetic studies.

•

Virus - host interactions.

In collaboration with the group of O. Gall, UMR 5471, INRA, Villenave d' Ornon, we were interested in the comprehension of the plant-potyvirus interaction mechanisms between cellular and viral factors. The goal was to characterize the interaction between the lettuce mosaic virus (LMV) protein VPg0 and the lettuce translation initiation factor, eIF4E. These proteins were expressed and purified in sufficient quantity to crystallize them, either separately or complexed together. They unfortunately appeared to be unstable in solution, and tend to precipitate even at low concentration (around 1 mg/ml). A high number of different crystallization conditions were tested. Only very thin needles of poor diffraction quality were obtained. Circular dichroism experiments showed that VPg0 displayed a disordered structure ant that the N-terminal part of eIF4E (46 aa) was also fully disordered, preventing us to get better crystals.

•

Protein – Foldamer Interactions

In parallel to the previous studies, we bring our contribution to explore the use of quinoline-derived aromatic amide foldamers (developed in I. Huc’s group) to inhibit protein-protein interactions. To do so, foldamers need to recognize and to bind a specific protein surface. X-ray diffraction appears to be a powerful tool to illustrate binding interactions. The first protein target was human carbonic anhydrase II (HCAII), an enzyme which binds sulfonamide inhibitors in its active site. Several sulfonamide derivatives, ending with a quinoline sub-unit have been designed and co-crystallise with HCAII in order to test their faculty to act as an ideal anchor for potent foldamers. Several structures have been solved. Work is still ongoing to approach the structure assisted design of foldamers, tethered to HCAII, and decorated to bind a specific surface.

5 significant publications: 1. Colloc’h, N., Sopkova de Oliveira Santos, J., Retailleau, P., Vivaré, D., Langlois d’Estaintot, B., Gallois, B., Brisson, A., Risso, J.J., Prangé, T., & Abrani, J.H. (2007) Protein crystallography under Xenon and Nitrous oxide Pressure : comparison with in vivopharmacology studies and Implications for the mechanism of Inhaled Anesthetic Action. Biophysical J., 92,1 -8. 2. Petit, P., Granier, T., Langlois d’Estaintot, B., Manigand, C., Bathany, K., Schmitter, J.-M., Lauvergeat, V., Hamdi, S. and Gallois, B. (2007). Crystal Structure of Grape Dihydroflavonol 4-reductase, a key enzyme in Flavonoid Biosynthesis. J. Mol. Biol., 368, 1345 – 1357. 3. Trabelsi, N., Petit, P., Manigand, C., Langlois d’Estaintot, B., Granier, T., Chaudière, J. and Gallois B. (2008) Structural evidence for the inhibition of grape dihydroflavonol 4-reductase by flavonols. Acta Cryst D64, 883 – 891. 4. N. Buzhynskyy, M. Golczak, J. Lai Kee Him, O. Lambert, B. Tessier, C. Gounou, R. Bérat, A. Simon, T. Granier, J.M. Chevalier, S. Mazères, J. Bandorowicz-Pikula, S. Pikula & A. R. Brisson. Annexin-A6 presents two modes of association with phospholipid membranes. A combined QCM-D, AFM and cryo-TEM study. J. Struct. Biol. (2009), in press 5. Gargouri, M., Manigand, C., Maugé, C., Granier, T., Langlois d’Estaintot, B., Cala, O., Pianet, I., Bathany, K., Chaudière, J. and Gallois, B. (2009) Structure and epimerase activity of Anthocyanidin reductase from Vitis Vinifera. Acta Cryst D, in press.

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Teaching of Methods to solve 3D structures by X-Ray diffraction. (Master 1 & 2 level in 3 different masters). Teaching of Protein Structure (Master 1). Teaching of Biochemistry (Master, CAPET, Agrégation, …) Teaching of Enzymology (Licence & Master) Practical laboratories on Crystallisation of Biological Macromolecules (Master 2, UMR) Practical laboratories on 3D graphics (Master 2 students). Practical training of Protein Structure Determination (Master 2 students, 2 different masters). Partial organisation of the “Journées de l’Association Bordelaise de Cristallographie” (2 days/year)

49

TEAM « MASS SPECTROMETRY OF BIOLOGICAL MOLECULES » Team Leader: Jean-Marie SCHMITTER, Professor, University Bordeaux 1. Team Composition (2006-2009): Katell BATHANY, IE U Bdx1

Corinne BURE, IR CNRS

Luc NEGRONI, IR CNRS (from Sept. 2009) Yorgos PAPASTAMOULIS Doctorant (50%, co-dir. JM Merillon) Armelle BALLADE, Doctorante

Benoît PLET Doctorant Adéline DELCAMBRE, Doctorante Xiaonan KANG Post-doctorante

Stéphane CHAIGNEPAIN, IR CNRS, 50%, Fabien XUEREB, Doctorant Naima SAAD, Doctorante (50%, co-dir. P. Bressollier)

M.Sc. trainees (stay of 6 months): 6, including two ERASMUS students (Spain and Germany)

Team Production (2006-2009): Publications, peer reviewed, (ACL) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI) Oral communications in congress (COM) Poster communications in congress (AFF)

26 1

Books or Book chapters (OS) Patents

1 1

4

Defended PhD Theses or HDR

2

2 9

Grants : public & private k€/year

25 k€

Scientific activity Résumé Les travaux de recherche impliquant la spectrométrie de masse suivent trois axes majeurs : analyse de protéines, microbiologie et analyse lipidomique. L’analyse des protéines inclut l’analyse quantitative de protéines-médicaments dans le plasma humain (erythropoïetine), l’analyse protéomique d’expression différentielle (carcinome hépatocellulaire), diverses méthodologies d’analyse de complexes protéines-protéines (prion, lithostathine), protéines-polyphénols (phénomène d’astringence, protéines de la salive) ou des interactions protéine-lipides (ANR PCV). Pour la partie dédiée à la microbiologie, développée en étroite collaboration avec le groupe de M. Urdaci, il s’agit de caractériser la structure de composés biologiquement actifs produits par des souches de Bacillus. Il s’agit notamment de lipopeptides, d’antibiotiques et de lantibiotiques. Certaines des souches analysées sont des probiotiques, et l’étude inclut l’analyse des protéines de surface. Pour la partie lipidomique, une collaboration a été entamée avec le laboratoire de Biogénèse membranaire (UMR 5200). Ceci concerne plus particulièrement des lipides d’origine végétale (projet ANR PANACEA), et implique la mise en place de méthodologies d’analyse de lipides par couplage entre spectrométrie de masse et nano-chromatographie. Abstract Research activities relying on the use of mass spectrometry follow three major axes: protein analysis, microbiology and lipidomics. Protein analyses include quantitative analysis of protein-drugs in human plasma (Erythropoietin), differential expression proteomics (hepatocellular carcinoma), various methodologies for the analysis of proteinprotein complexes (prion, lithostathine), protein-polyphenols complexes (astringency phenomenon, salivary proteins) or protein-lipid interaction (ANR PCV). The microbiology related part is developed in strong interaction with M. Urdaci’s group. The goals are the characterization of biologically active compounds produced by Bacillus strains, including lipopeptides, antibiotics and lantibiotics. Somes strains are probiotics, and the research work includes the analysis of surface proteins. The work related to lipidomics relies on a strong collaboration with the Laboratoire de Biogénèse membranaire (UMR 5200). This concerns mainly plant lipids (ANR project PANACEA), and implies the development of hyphenated methodologies such as mass spectrometry combined with nano-chromatography.

50

Quantitative analysis of therapeutic proteins (collaboration with D. Breilh, Hospital Haut-Lévêque, University of Bordeaux) Immuno or radio-immuno assays are common techniques for quantitative analysis of therapeutic proteins in human plasma. However, in front of new drugs, or of known drugs bearing chemical modifications such as glycans or PolyEthylene Glycol (PEG), these methodologies may lack reliability, in particular for patients treated by a combination of drugs, as is the case for VIH-VHC co-infected patients. A quantitative methodology relying on tandem mass spectrometry would give access to a high level of selectivity. However, therapeutic concentrations of protein-drugs in plasma might be as low as 10 attomoles/μL, thus requiring the utmost performances of mass spectrometers in terms of sensitivity. The global standard concept The limit of detection and the selectivity requested by this type of analysis cannot be reached with intact proteins. The preferred way is the analysis of peptides obtained by proteolytic cleavage of the therapeutic protein, using isotopically labelled peptides for absolute quantification. Instead of targeted analysis, as used in the AQUA concept, we have developed a global internal standard methodology relying on chemical derivatization of all peptides produced by tryptic cleavage. The isotopic label is introduced by means of a derivatization reagent that targets specifically lysine groups. This reagent, 2-methoxy-4,5-dihydro-1H-imidazole, has been synthesized in the laboratory under a light version (D0) and a heavy version (D4) (coll. : F. Godde). As described below, the analytical flow chart begins with a depletion step that removes by affinity chromatography 90 % of seven major plasmatic proteins (including albumin and IgGs). A further step of purification is achieved by reversed phase chromatography, prior to tryptic cleavage and chemical derivatization with the D0 reagent. In parallel, the pure therapeutic protein is also submitted to proteolytic cleavage and peptides are labeled with the D4 reagent. The mixture of D4 labeled peptides is then used as global source of internal standards for absolute quantitative analysis.

Flow chart for the quantitative analysis of a therapeutic protein in human plasma Multiple reaction monitoring (MRM) with a quadrupole type instrument (4000 QTRAP, in collaboration with Applied Biosystems, Darmstadt, Germany) has been used for the quantification step. Applied to the case of human erythropoietin (EPO), a limit of quantification of about 20 attomoles/μL has been reached by using two peptides, each one providing three transitions. The method is selective and reliable, and its sensitivity is adequate, considering the expected therapeutic concentrations of EPO in plasma that are around 500 attomoles/μL for patients treated for anemia. This

S/N = 21

S/N = 89

MRM analysis of a tryptic EPO peptide labelled with D0 reagent using a single MRM transition (top) and the sum of three transitions (bottom trace). A signal to noise ratio of 89 is obtained in the latter case for a sample load on column of 100 attomoles EPO/μl of plasma. 51

concept of global internal standard opens the way to the quantitative analysis of other therapeutic proteins, and is currently applied to the analysis of pegylated interferon.

Wine astringency approached by energy resolved mass spectrometry Astringency of red wines is related to the fact that polyphenols (tannins) interact strongly with human salivary proteins, and in particular with Proline Rich Proteins (PRPs), leading to a removal of the lubricating effect of PRPs in the mouth. A model system has been built with PRP fragments prepared by solid phase synthesis (14 and 34 amino acids, with several proline rich repeats) and a selection of 21 polyphenols. The interaction between polyphenols and PRP fragments has been investigated by Energy Resolved Mass Spectrometry (ERMS) using electrospray ionization of the complexes: the energy required for a 50% disruption (DE50) of the complexes inside the ion trap of a mass spectrometer has been determined, leading to PRP-polyphenol affinity scale in the gas phase built on the basis of DE50 values an affinity scale in the gas phase. Evidence for the important role of hydrogen bonds was found, with highlighting of hydroxyl groups important for the formation of the complex, and confirmation of their role by molecular modeling (collaboration with M. Laguerre). This work has been supported by the Comité Interprofessionnel du Vin de Bordeaux (PhD grants for B. Plet and A. Delcambre).

Characterization of biologically active compounds produced by Bacillus strains Among bacteria, Bacillus strains are known to produce numerous compounds that display various biological activities. These Grampositive microorganisms are widely distributed in nature, able to produce spores and acquire in this way a resistance to hostile environments and concomitantly synthesize active compounds, including lipopeptides that display strong antimicrobial and antifungal activities. Further, probiotics based on Bacillus bacteria contribute to the prevention and to the treatment of infectious diseases and of gastrointestinal microbial disorders, including acute diarrhea, antibiotic-associated diarrhea, and traveler’s diarrhea. MALDI mass spectrometry has been used for fast screening of these active compounds. However, this approach has some severe limitations, the major one being related to spectral discrimination occurring upon analysis of complex mixtures by MALDI-MS, in particular when compounds such as surfactins and iturins are produced together. In most cases, compounds identified by direct analysis of colonies picked from Petri dishes do not account for the biological activities that can be detected for a given strain.

SURFACTINS

ITURINS

O

O

E

Rn

L

O

X

L

X

Rn

L

Y S

V

D

N

HN

MYCOSUBTILINS

FENGYCINS

X

E

E

Rn

O

aT

P

Y

Q

O

z

I

N

Rn

Y

Y N

HN

aT = AThr Z = Orn X = A Fengycins A X = V Fengycins B

N

O OH

X1

Y N

HN

T

X4 X3

S

Q

P

AMICOUMACINS

BACILLOMYCINS

Rn

P

X = N Iturins A X = D Iturins B

X = I / L Surfactins A / C X = V Surfactins B

OH

N

Q

OH

O

NH2

O O NH

OH

COR

X2

X1 = N X2 = P X3 = E X4 = S Bacillomycins D X1 = N X2 = Q X3 = P X4 = N Bacillomycins F X1 = D X2 = S X3 = Q X4 = S Bacillomycins L

R = NH2 Amicoumacins A R = OH Amicoumacins B

52

Other drawbacks are related to the loss of information for low molecular Surfactins  weight compounds, i.e. below 700 Da, as is the case for amicoumacins, and occurrence of cation adducts due to the composition of culture media. Therefore, biological activities of the compounds revealed by MALDI-MS fingerprinting rarely account for the whole Iturins set of activities displayed by a given strain. We first made attempts to improve this fast characterization method by introducing a cation exchange step involving a lithium salt, in order to reduce the complexity of spectra and improve the reliability of identification by MALDI/MS/MS, but with very limited success. Therefore, we have designed a more reliable methodology for rapid characterization of biologically active compounds produced by Bacillus strains. First, the composition of the solvent used for the extraction from whole bacteria was optimized. Then, the extract was separated by reversed phase liquid chromatography on a micropreparative scale, with post-column effluent splitting: 10% of the effluent was sent to an ion trap mass spectrometer for ESI/MS/MS analysis, and 90% of it was directed to a fraction collector. This step was followed by the characterization of biological activity on collected fractions. Further analysis by S MALDI/MS/MS was applied for unknown CH compounds. ║ Asn Ala Dha The structure of a new lantibiotic with CH S Arg Dhb Leu Trp Phe Tyr Ala │ strong anti-Staphylococcus aureus activity, isolated Phe Lys Ala Phe Gly Ala Abu Ala Ala Ala NH from a probiotic bacillus strain, has been fully Pro Gly characterized (international patent, 2007). Its S S structure is closely related to the structure of mutacin. RT: 0,0 0 - 39, 94

N L: 7,9 8E7

3 0,7 0

1 00

B as e P ea k F : + c ESI F ull ms [ 3 95 ,00 -20 00 ,00] M S

95

0 80 71 7C4 Ne wSou ch e 63 ZCulo t24 HLa nd yM e th8

90

Amicoumacins

85 80 75 70

Rel ative Abu n da nc e

65 60 55 50

30 ,04

45

11 ,74

40 35 30

4 ,94

31 ,39

25 20

5 ,79

33, 01

29 ,48

15

20 ,4 4 2 1,1 4

4 ,35

10

3,79

5

1 0,1 3

1 ,84 2 ,04

6, 1 3

21 ,23

1 1,0 5

2

4

6

8

12 ,25

10

28 ,99

3 5,6 4

2 2 ,6 4

9 ,28

0

0

33 ,52

2 9 ,3 0

12

1 3 ,2 6

14

1 6, 58

2 3, 2 5

17 ,92

16

18

20

22

24

25 ,68 28 ,37 26

28

36, 01 37, 25

30

32

34

36

3 9,2 3

38

Ti me ( min)

O

Lipids Collaborative research with researchers belonging to UMR 5200 has led to the characterization of lipids occurring in Detergent Insoluble Microdomains (DIM, or lipid rafts) in plants, and to the discovery of a new N-acylethanolamine synthase in A. Thaliana. Further work in common has two objectives: the set up of methodologies relying on mass spectrometry for the fast characterization of the whole lipid content of a cell (lipidomics, set up of a service platform) and the characterization of the content and signaling role of lipids from DIMs occurring in tobacco BY2 cells in response to a stress induced by cryptogein (ANR Panacea project, S. Mongrand coordinator).

O

OH

OH H

H OH

(CH2 )13 O H

H

HO

CH3 O

H

A1

CH3

HN

OH

G

OH

Z0

Y0 Collision induced fragmentation scheme of a ceramide occurring in plant DIMs

Collision induced fragmentation scheme of a NAPE from A. Thaliana. occurring in plant DIMs

53

5 significant publications: 1. 2.

3. 4.

5.

Faure, L., Coulon, D., Laroche-Traineau, J., Schmitter, J.M.,, Testet, E., Lessire, R. and Bessoule J.J. (2009). Metabolism of N-Acylethanolamines: Discovery and characterization of an Arabidopsis thaliana NAPE synthase J. Biol. Chem., in press. Aime, C., Plet, B., Manet, S., Schmitter, J.M., Huc, I., Oda, R., Sauers, R. R., Romsted, L. S. (2008) Competing Gas-Phase Substitution and Elimination Reactions of Gemini Surfactants with Anionic Counterions by Mass Spectrometry. Density Functional Theory Correlations with Their Bolaform Halide Salt Models. J. Phys. Chem. B 112, 14435-14445. Petit, P, Granier, T, d'Estaintot, B.L., Manigand, C., Bathany, K., Schmitter J.M., Lauvergeat, V., Hamdi, S., Gallois, B. (2007) Crystal structure of grape dihydroflavonol 4-reductase, a key enzyme in flavonoid biosynthesis. J. Mol. Biol. 368, 1345-1357. Laloi, M., Perret, A. M., Chatre, L., Melser, S., Cantrel, C., Vaultier, M. N., Zachowski, A., Bathany, K., Schmitter, J.M., Vallet, M., Lessire, R., Hartmann, M. A., and Moreau, P. (2007) Insights into the role of specific lipids in the formation and delivery of lipid microdomains to the plasma membrane of plant cells. Plant Physiol. 143, 461-472. Nazabal, A., et Schmitter, J. M. (2006) Hydrogen-Deuterium Exchange analyzed by Matrix Assisted Laser Desorption-Ionisation Mass Spectrometry and the HET-s prion model. Methods in Enzymology, 413, 167-181.

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Analytical chemistry at BSc level (chromatography, mass spectrometry) Mass spectrometry at master level Protein purification and characterization at master level Proteomics at master level Practical experiments in mass spectrometry at master level Practical training in mass spectrometry for students at PhD and post-doc level

Managment and running of technological platforms The team ensures the maintenance and manages the mass spectrometry platform for UMS 3033 (IECB). The team has brought scientific and engineering competence for running 5 instruments (MALDI and ESI ionisation) for several research teams inside and outside the CBMN research unit. At the fall of 2009, the MS team will join Génomique Fonctionnelle de Bordeaux, building a site dedicated to mass spectrometry applied to “omics” (proteomics, lipidomics) with access to a complementary set of six tandem mass spectrometers.

54

DEPARTMENT: BIOMIMETIC AND MEDICINAL CHEMISTRY Two teams constitute the department: Team Name Biomimetic & Bioorganic Supramolecular Chem Medicinal Chemistry

Laboratory Head Ivan Huc, DR CNRS Léon Ghosez, PR UBx1

The department deals with organic synthesis of biologically active molecules and biomimetic supramolecular scaffolds. One of the main objectives is to produce new drugs or new biomimetic scaffolds to deliver molecules. Themes: • • • • • •

Aromatic oligoamide foldamers Synthetic helices with polar channel Helicoidal molecular capsules Protein tertiary structure mimics New drugs Synthesis of anticancer drugs.

Technical approaches: • • • • • •

Organic chemical synthesis Chromatography Mass spectrometry X-ray crystallography Liquid state NMR Circular dichroism.

55

TEAM « BIOMIMETIC SUPRAMOLECULAR CHEMISTRY » Team Leader: Ivan Huc, Research Director, CNRS. Team Composition (2006-2009): Frédéric Godde, Maître de Conférence Kolupula Srinivas, Post-Doc, 24m Chunyan Bao, Post-Doc, 24m Jiang Zhu, Post-Doc, 18m Nicolas Delsuc, PhD student Thomas Delclos, PhD student

Yann Ferrand, CR2 CNRS (since 2007) Katta Laxmi-Reddy, Post-Doc, 24m Jone Iriondo, Post-Doc, 24m Amol Kendhale, Post-Doc, 18m Tracey Marshall, PhD student

David Sanchez, Post-Doc, 18m Emanuela Berni, Post-Doc, 12m C. Douat-Casassus, Post-Doc, 9m Xavier de Hatten, Post-Doc, 24m Marine Stupfel, PhD student

B.Sc. and M.Sc. trainees (stay between 2 and 6 months): 13

Team Production (2006-2009): Publications, peer reviewed, (ACL) Invited conferences congress (INV) Invited seminars/lectures Oral communications in congress (COM) Poster communications in congress (AFF) Books or Book chapters (OS)

38 21 36 16 12 1

Edition of Books (DO) Congress/Workshop organization Prizes/distinction Defended PhD Theses or HDR Grants : public & private k€/year

1 2 2 3 300 k€

Scientific activity Résumé. Les foldamères sont des objets moléculaires artificiels adoptant des conformations repliées. Nos travaux concernent une classe de foldamères préparés par chimie organique de synthèse par étape. Le premier objectif des recherches dans le domaine des foldamères est d’étendre le registre de structures et de fonctions des biopolymères, en d’autres termes, d’étendre l’espace des objets de synthèse vers des architectures plus grandes et plus complexes. Les biopolymères nous servent de source d’inspiration, mais il est important de souligner que le but des recherches sur les foldamères n’est pas de copier les biopolymères mais au contraire d’explorer les motifs structuraux et les fonctions des chaînes artificielles qui ne peuvent être réalisées à l’aide des monomères naturels, peptides ou nucléotides : mimer n’est pas copier ! De ce point de vue, les foldamères oligoamides aromatiques que notre groupe a développés depuis notre premier article en 2000 se sont avérés être des objets remarquables car leurs conformations repliées sont extrêmement stables, modulables et faciles à prédire. Nos efforts au cours des quatre dernières années peuvent être divisés en trois sujets. Dans le premier sujet, nous avons élaborés plusieurs stratégies pour concevoir et synthétiser des protéomimes : des molécules organiques de synthèse dont la taille et la complexité se comparent à celles d’une petite protéine. Nos succès en la matière ont conduit à plusieurs avancées importantes, comme les plus grandes molécules de synthèse caractérisées par la cristallographie des rayons x sur des monocristaux, ou les premières hélices triples et quadruples artificielles. Dans le deuxième sujet, nous avons effectué des études fondamentales du phénomène de repliement et aussi des comportements physiques et chimiques des foldamères oligoamides aromatiques. Ainsi, la stabilité thermodynamique des conformations hélicoïdales a été déterminée, et des comportements chimiques et physiques inhabituels ont été mis en évidence, ouvrant la voie à de nouveaux développements. Par exemple, nous avons montré que les hélices aromatiques véhiculent le transfert d’un électron entre des donneurs et accepteurs à leurs extrémités et aussi que qu’elles subissent des substitutions électrophiles régiosélectives rapides. Dans le troisième sujet, nous explorons les fonctions des foldamères. Un concept unique de conteneur moléculaire hélicoïdal a été proposé qui permet de complètement entourer des molécules invitées. De plus, de nouveaux foldamères hydrosolubles ont montré des activités biologiques très prometteuses, notamment des interactions sélectives avec de l’ADN en tétrade de guanine. Abstract. Foldamers are artificial molecular structures that adopt folded conformations. Our research focuses on a class of foldamers produced by step-wise synthetic organic chemistry. The main objective of foldamer research is to expand the registry of structures and functions of biopolymers, in other words to expand the chemical space towards larger and more complex architectures. Biopolymers serve as inspirational models, but it should be emphasized that the purpose of foldamer research is not to copy biopolymers, but on the contrary to explore the structural patterns and the functions featured by artificial backbones that cannot be reached using natural 56

backbones, peptides or nucleotides: mimicking is not copying! In this respect, the aromatic oligoamide foldamers that our group has been working on since our first paper in 2000 have proven to be remarkable objects in that their folded conformations are extremely stable, tunable and easy to predict. Our efforts in the last four years can be organized in three subjects. In subject 1, we have focused on various design and synthetic strategies to produce proteomimetics: folded artificial organic molecules whose size and complexity resemble that of small proteins. Successes in this subject have led to a number of breakthroughs such as the largest synthetic molecules characterized by single crystal x-ray crystallography and the first artificial organic triple and quadruple helices. In subject 2, we have carried out fundamental investigations on folding phenomena and also on the chemical and physical behavior of aromatic oligoamide foldamers. The thermodynamic stability of helical conformations was determined, and peculiar chemical and physical behaviors were demonstrated paving the way towards new developments. For example, it was shown that aromatic helices mediate electron transfer between donors and acceptors at their extremities and also that they can undergo regioselective enhanced electrophilic substitutions. In subject 3, the functions of aromatic amide foldamers are being explored. A unique design of helical molecular containers that completely surround their guests has been proposed, and water soluble foldamers show highly promising biological properties, in particular selective interactions with G-quadruplex DNA. Foreword. During the 2006-2009 period, synthetic foldamers have become our main, almost exclusive, research topic. Our group now has a strong leadership and is widely recognized internationally in this emerging field. An illustration is the co-edition of the first book on the subject in 2007. An outline of our main results is given below. Our activity covers extensive efforts in synthetic organic chemistry, some modeling studies, extensive spectroscopic and crystallographic characterization, investigation of reactivity, photophysics, as well as hostguest and biological properties. We have also being involved in an ongoing productive collaboration with the group of Reiko Oda at CBMN concerning various aspects of the expression of molecular chirality at a supramolecular level in assemblies of synthetic amphiphiles. This work has already led to 18 publications in peer-reviewed journals including 6 in the 2006-2009 period. For details about this work, please refer to the research outline of Reiko Oda’s group. Finally, collaboration with Prof. N. Metzler-Nolte at the University of Bochum in Germany about mimics of hydrogenase enzymes has led to two papers and is not presented here.

• Design and synthesis of protein-sized synthetic foldamer architectures Over the last decade, a number of synthetic oligomers of various chemical compositions have been shown to adopt folded conformations resembling the secondary motifs of biopolymers. Typically, relatively short sequences (5-15 units) were shown to adopt helically folded conformations. We have largely contributed to these developments and introduced several families of aromatic amide synthetic foldamers. A major challenge for chemists now consists of assembling several of these secondary folded motifs into tertiary structures that would resemble an entire protein. As illustrated below, we have made several important steps in that direction in the last four years. The ultimate goal of this curiosity driven research is the ability to design and synthesize objects that would be structurally as large and complex as proteins and functionally as efficient as enzymes. Covalent helix connection In the context of a project funded by ANR (blanc 2005), we have explored the prospect to use stepwise synthesis to prepare artificial tertiary folded motifs based on aromatic amide sequences (e.g. pyridine or quinoline amino-acids). Convergent schemes allowed us to prepare substantial amounts (> 100 mg) of such “proteomimetics” having FWs up to 15 kDa (Angew. Chem. 2007, JACS 2009). Folded conformations have been Proteomimetics. Left: crystal structure of a large (>8 kDa) quinoline derived characterized in the solid state by x-ray foldamer with a branched architecture. Right : the crystal structure of a small crystallography and in solution by NMR. (65 aa) protein is shown at the same scale for comparison The longest sequence prepared to date in our group comprises 48 monomer in one helix. To the best of our knowledge, these objects represent world records as the largest molecules characterized by crystallography that are not proteins or nucleic acids.

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Metal mediated helix attachment Metal directed dynamic assembly of helically folded aromatic oligoamides has also been explored as an approach towards “proteomimetics”. This research was made possible thanks to a collaborative ERA-chemistry grant obtained together with Dr. Jonathan Nitschke, at Cambridge University, UK. This approach takes advantage of the power of self-assembly, and of self Metal directed dynamic assembly of foldamers. Two crystal structures showing selection that allows one species to how metal coordination allows one to control the relative orientation of helical segments. emerge from a dynamic mixture as a major product if it is stabilized by folding. In the example shown on the right, the folded objects bear three intrinsically chiral subunits – two helices and one metal center – and may potentially exist as a complex mixture of isomers. However, self-selection eventually leads to the prevalence of one (Chem. Eur. J. 2008). Hybridization into multi- stranded helical architectures The ability of helically folded aromatic oligoamides to extend like spring and reciprocally intercalate, eventually forming a double helix was first characterized by us in 2000 (Nature). It provides an efficient means to Multistranded proteomimetics. Formula and crystal structure of the first artificial quadruple prepare large multi-helical helical motif. Water molecules (red sheres) occupy the inner channel. assemblies. This work was thoroughly continued in the last 4 years, leading to 6 publications including 3 in Angewandte Chemie (2006, 2006, 2008). A fruitful collaboration has been established with Prof. Hua Jiang, the first Post-Doc of the group and now a Professor at the Institute of Chemistry of the Chinese Academy of Science in Beijing. An important discovery was made of the first artificial quadruply helical structure based on fluoroquinoline oligoamides. Recent unpublished results show that naphthyridine based oligoamides form the first example of an artificial organic triple helix. .

• Foldamer chemical and physical properties Besides the spectacular designs exemplified above, fundamental physical and chemical investigations have been carried out that might lead to numerous new developments. CHEMICAL BEHAVIOR (reactivity, photochemistry, electron transfer) Three independent studies of the chemical behavior of helically folded aromatic amides have been published. As shown in the illustration on the right, one concerns their enhanced and regioselective reactivity towards electrophilic substitutions (JACS 2008, collaboration with Prof. Léon Ghosez, funding from ANR blanc 2005). Another study concerns the Enhanced regioselective brominations. Scheme of the bromination reaction (left) photochemistry involving anthracene of a quinoline monomer within a helically folded oligoamide sequence. Crystal units within the helical sequences (J. structure (right) of a monobrominated quinoline octaamide resulting from an Org. Chem. 2008). A third and more enhanced regioselective bromination. recent study (JACS 2009) in collaboration with the group of Prof. Schening in Eindhoven allowed us to demonstrate that helical aromatic amides facilitate ps scale photoinduced electron transfer between donors and acceptors at their extremities via a super-exchange mechanism. This work suggests that electrons are actually conveyed through the helix backbone as it is in DNA. 58

PHYSICAL BEHAVIOR (folding, spectroscopy, theory) Studies aiming at understanding and quantifying folding processes in aromatic amide foldamers have been reported. A collaboration with a Prof. Takafuji (Kumamoto University, Japan) allowed us to determine the very high thermodynamic A herringbone helix. Combining amino-quinoline and aminomethyl-pyridine stability (above 100 kJ/mol) of a series of units alternatively in the same sequence results in an unprecedented folded aromatic amide foldamers (ChemPhysChem pattern in solution and in the solid state (crystal structure on the right). 2008). Theoretical and spectroscopic (vibrational circular dichroism) allowed to unambiguously assign absolute helix handedness. Finally, the study of aromatic-aliphatic foldamer hybrids led to the discovery of an unprecendeted folding patter (see above, JACS 2007).

•

Foldamer functions

Biological applications A major turn in our research was achieved through the preparation of fully water soluble aromatic oligoamides foldamers (JOC 2006, Marie Curie FP6 contract), leading to the first series of biological studies of these compounds and now an Cell penetration. Fully water soluble cationic aromatic foldamers spontaneously important component of our penetrate a variety of cell lines. A confocal microscopy image of Hela cell showing projects (and funding). Thus, penetration of a fluorescein labeled foldamer is shown on the right. initial studies showed that helical aromatic oligoamide foldamers bearing cationic groups are non toxic, resist protease degradation and spontaneously enter cells (Angew. 2007). In collaboration with Prof. S. Balasubramanian at the University of Cambridge, we could show that these compounds selectively bind to G-quadruplex DNA with respect to duplex DNA with record high affinities (JACS 2007, JACS 2009). Folded capsules and molecular recognition After almost a decade of work on aromatic amide foldamers, we now have about 6 kinds of monomers at our disposal that we can synthesize on multigram scales. We have thus engaged in combining these monomers in the same sequences in order to elicit specific properties. Our monomers (quinoline, pyridine, naphthyridines, aza-anthracenes…) notably differ in that they code for helices of various diameters. In 2005, we first published the concept of molecular containers built from the folding of an oligomer into a helix whose diameter is large in the center and small at the ends, and thus that might encapsulate guests in their interiors. The cavity of this prototype was small and could only encapsulate one water molecules. Over the last four years, this concept has been developed and several single helical or double helical containers have been produced to selectively encapsulate guests as large as 1,10-decanediol in solution and in the solid state (Chem Eur. 2007, 2009, Angewandte 2008). This work offers a new design strategy for synthetic receptors targeted to polar and chiral guests, and also to control over the timeframe of their capture and release.

A double helical capsule. Programming an aromatic amide foldamer sequence allows one to design a molecular container that encapsulate a guest (here 1,10 decanediol) through the formation of a double helical segment that creates an elongated cavity and two single helical segments that end-cap the cavity.

X-ray crystallography The research outlined above implicitly illustrates the intimate collaborations we have engaged with several crystallographers in Bordeaux. Our group has been the main user of IECB’s crystallographic facility, managed by Dr. Brice Kauffmann (IR CNRS) and has continuously and smoothly collaborated with Prof. Jean-Michel Léger, from the faculty of Pharmacy (Univ. Bordeaux 2). Credit should be given to their contributions here. 59

Given our dependence from crystallography, we wish to express our concern about which resource we will be able to rely on after Prof. Léger retires in 2011.

5 significant publications: 1.

2.

3. 4. 5.

Wolffs, M., Delsuc, N., Veldman, D., Vân Anh, N., Williams, R. M., Meskers, S. C. J., Janssen, R. A. J., Huc, I., Schenning, A. P. H. J. (2009) Helical Aromatic Oligoamide Foldamers as Organizational Scaffolds for Photoinduced Charge Transfer. J. Am. Chem. Soc. 131, 4819. Bao, C., Kauffmann, B., Gan, Q., Srinivas, K., Jiang, H., and Huc, I. (2008) Converting sequences of aromatic amino acid monomers into functional three-dimensional structures: second-generation helical capsules. Angew. Chem. Int. Ed. 47, 4153. Delsuc, N., Léger, J.-M., Massip, S. and Huc, I. (2007) Proteomorphous objects from abiotic backbones. Angew. Chem. Int. Ed. 46, 214. Gillies, E., Deiss, F., Staedel, C., Schmitter, J.-M. and Huc, I. (2007) Development and biological assessment of fully water-soluble helical aromatic amide foldamers. Angew. Chem. Int. Ed., 46, 4081. Shirude, P., Gillies, E. R., Ladame, S., Godde, F., Shin-ya, K., Huc, I. and Balasubramanian, S. (2007) Macrocyclic and helical oligoamides as a new class of G-quadruplex stabilizers. J. Am. Chem. Soc. 129, 11348.

Management of science -

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Ivan Huc serves as co-director of the European Institute of Chemistry and Biology since 2008 Meeting organization: o Aquitaine meeting on polymers, Arcachon 2007 (member of organizing committee, 130 participants) o Aquitaine meeting on polymers, Arcachon 2009 (member of organizing committee, 130 participants) o Workshop on foldamers and protein surface recognition, Bordeaux 2010 (full organization, 100 participants) Ivan Huc and Frédéric Godde have been elected members of various councils at the department and university level. Research contracts over 2006-2009: Project Source Amount Duration

FOLDAPPI FOLDAPSULES 1 year post-doc 2 year post-doc – HELICAL TRANSPORTERS 2 year post-doc FARQUAD Foldaptamers 2 year post-doc PhD fellowship Dynamic Combinatorial Chemistry One-Year post-doc Dynamic Self-assembly of Proteomorphous Objects Abiotic mimics of proteins Dynamic Combinatorial Chemistry Abiotic mimics of proteins Helical Antibiotics PhD fellowship PhD fellowship

European Union -Marie Curie IAPP ANR Blanc 2008 Université Bordeaux 1 European Union -Marie Curie

220 000 euros

02/2009 – 01/2013

225 000 euros 40 000 euros 160 000 euros

09/2009 – 08/2013 11/2008 – 10/2009 03/2009 – 02/2011

European Union -Marie Curie

160 000 euros

04/2009 – 03/2011

ANR – Physics/chemistry – biology interface ARC French Ministry of Research European Union -Marie Curie Research Training Network French Ministry of Research ERA-chemistry (European)

210 000 euros

01/2008 – 12/2011

80 000 euros 65 000 euros 207 000 euros

11/2007 – 10/2009 10/2007 – 09/2010 09/2006 – 08/2010

38 500 euros 205 000 euros

11/2006 – 10/2007 03/2006 – 12/2008

288 000 euros 7 000 euros

01/2006 – 12/2008 01/2005 – 12/2009

5 000 euros 149 000 euros

01/2006 – 12/2006 12/2004 – 06/2006

80 000 euros 60 000 euros

10/2004 – 09/2008 10/2004 – 09/2007

ANR – “programme blanc” CNRS – funding for COST networks University Bordeaux 1 (BQR) European Union -Marie Curie PostDoctoral Fellowship Asian Bank of development French Ministry of Research

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TEAM « ORGANIC AND MEDICINAL CHEMISTRY » Team Leader: Léon Ghosez, Professor Emeritus Team Composition (2006-2009): Tomasz Balakier, Post-Doc Santosh Jangari, Post-Doc De Paolis Michael, Post-Doc Mena Cervignon Marisa, Post-Doc Oosting Peter, Doctorant

Ozanne Aurélie, Post-Doc Commandeur Margaret, Post-Doce Xunwei Wu, Post-Doc Mendoza Oscar, Post-Doc Guillonneau, Doctorant

Commandeur Claude, Post-Doc Wei Zhu, Post-Doc Hess Elisabeth, Post-Doc Na Sun, Post-Doctorant

Team Production (2006-2009): Publications, peer reviewed, (ACL) Publications, peer reviewed, not yet in bases (ACLN) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI) *10 publications are on hold by the industrial partner

7

Oral communications in congress (COM)

14

10

Congress/Workshop organization

15

15

Defended PhD Theses or HDR

11

Grants : public & private k€/year

2 450 k€

Scientific Report Résumé: La synthèse de “petites” molécules sera requise tant qu’elles seront utilisées pour la mise en évidence et l’étude des fonctions de nouvelles macromolécules biologiques ou en temps agents thérapeutiques potentiels. Cette approche « chimique » de la biologie requiert un accès rapide à une grande diversité de molécules complexes ayant des propriétés nouvelles. Toutefois, les macromolécules biologiques n’occupent qu’une fraction très réduite du descripteur de l’« espace chimique » accessible par la synthèse. Le chimiste de synthèse a donc besoin d’un guide lui permettant de choisir à bon escient des structures moléculaires susceptibles d’agir sur les macromolécules de la nature. Des analogues de « petites » molécules naturelles devraient permettre d’accéder plus rapidement à des molécules bioactives que l’utilisation de grandes collections de molécules conçues de manière « aveugle ». Les produits naturels ont un rôle à jouer dans l’organisme qui les produits et ce rôle résulte le plus souvent d’une interaction avec une macromolécule biologique. Leur activité a été optimisée par l’évolution pendant des millions d’années de criblage à haut débit ! L’une des thématiques majeures de notre groupe est donc de développer des procédés efficaces de production d’analogues de produits naturels. Nous avons ainsi développé récemment une réaction à multicomposants conduisant à des dérivés complexes de la pipéridine, un hétérocycle commun à de nombreux alcaloides bioactifs (publié dans Angew. Chem Int Ed. 2009, ASAP, sélectionné pour image de couverture). Nous avons également préparé une collection d’azalactames bicycliques fonctionnalisés conçus par calculs pour l’inhibition de protéases à sérine. Ces pharmacophores seront ultérieurement transformés en inhibiteurs de proprotéine convertases, une classe d’enzymes impliquées dans le déclanchement de phémonènes responsables du développement et de la progression de tumeurs malignes. Notre groupe s’intéresse également à la synthèse totale du GPC004FD4, un produit naturel présentant une activité antitumorale intéressante qui n’est actuellement plus accessible par voie naturelle (extraction). Cette synthèse et celle d’analogues obtenus par la technique DTS (Diverted Total Synthesis) vise a établir des relations structure-activité pour cette classe de substance. Il en est de même pour une nouvelle classe de produits naturels étudiés principalement par Pfizer qui est constituée d’alcaloïdes terpenoïques dérivés de pyrrolobenzoxazine. Nous en avons entrepris la synthèse totale et effectué la synthèse d’analogues simplifiés pour en étudier les propriétés insecticides. Un nouveau projet a été récemment initié qui vise à développer de nouvelles méthodes non-génotoxiques et n’utilisant aucun métal lourd permettant la benzylation ou l’allylation de substrats insaturés : éthers d’énols, acétals de cétènes, allyl silanes, aromatiques activés, iminoéthers. Ce programme ambitieux a déjà conduit à des résultats tout à fait originaux que nous envisageons de publier prochainement. Le chef de groupe est également impliqué dans un projet ANR dont l’investigateur principal est I. Huc. Le projet consiste à évaluer la réactivité d’unités monomériques ou de groupes fonctionnels incorporés dans un foldamère en vue d’évaluer le rôle de la structure tertiaire de ces molécules sur leur réactivité (publié dans J. Am. Chem. Soc. 2008, 130, 13210-13211).

Abstract: Synthesis of « small » molecules will be needed as long as they will be used for the discovery of biological macromolecules, the study of their biological function and their potential for the development of new therapeutic agents. This chemically-driven approach to biology will still gain in importance since the development of the humane genome project is expected to lead to an exponential growth of biological targets. This approach requires the development of synthetic methods which provide a quick access to complex and diverse molecular structures exhibiting properties never seen before. However biological molecules populate only a very small fraction of the multidimensional 61

chemical descriptor space available by synthesis. The synthetic chemist will therefore need guidelines to prepare molecules with a chemical descriptor allowing them to interact with biological macromolecules. Small natural product analogs should allow for entry into the discovery process of bioactive molecules at a much more advanced stage that does the screening of standard diversity libraries: natural products have some purpose for the producer, and their purpose is to interact with a biological target, generally microbial or vegetal. Their activity has been shaped and optimized by evolution during millions of years of environmental high-throughput screening. One of our major endeavours at IECB has thus been the development of efficient synthetic processes for the production of new natural product analogs. We have been able to develop an extremely powerful multi-component reaction to build piperidine derivatives which are common substructures in many bioactive natural alkaloids (published in Angew. Chem Int Ed. 2009, ASAP, selected for cover picture of the volume. We have also prepared a series of functionalized azalactam scaffolds designed to be efficiently cleaved by serine proteases. The design was done in collaboration with Dr. G. Dive (University of Liège) using a theoretical mechanistic model developed in our early studies on inhibitors of bacterial trans-peptidases and β-lactamases. We are presently using these scaffolds to develop inhibitors of proprotein convertases, a class of enzymes which are well-known cancer-associated proteins acting as «masterswitches » at different levels of tumor development and progression (cell proliferation, invasiveness, metastatic ability). We have also progressed in the total synthesis of GPC004FD4, a natural producst showing an interesting antitumoral activity. In this particular case, the natural product was no longer available and there were some uncertainties about the structure. Total syntheses of the two hypothetical structures should solve the problem. The synthesis of one molecule has almost been completed. The synthetic route was designed to allow the easy production of analogs. A major project of our group deals with the synthesis of analogs of a class of complex natural products discovered by Pfizer which are terpenoid pyrrolobenzoxazine alkaloids. These are potential insecticides. Major progresses have been made and several analogs have been delivered for testing. Two new projects have been recently initiated. I shall mention here the development of new methods of alkylation which do not involve genotoxic reagents. Our first results are extremely promising. The group leader is also involved in a collaborative ANR project with Dr. I. Huc. The project aims to study the chemical reactivity of specific monomers or functional groups incorporated in foldamers in order to assess the role of the folding. This project has led to totally unexpected and, therefore, interesting results (published in J. Amer. Chem.Soc 2008, 130, 13210-13211).

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Diversity-oriented synthesis of piperidine scaffolds. Applications to the synthesis of alkaloid-like compounds

The goal was to define short synthetic routes towards a collection of small heterocyclic molecules of enough skeletal, functional and stereochemical diversities so as to create a broad distribution of compounds in chemistry space.We have developed unique 3-6 component reactions which enable to create a variety of heterocyclic scaffolds which can then be transformed in a few steps in a wide variety of complex heterocycles that could modulate biomacromolecular functions in a useful way. These studies were performed in collaboration with an industrial partner who is in charge of the production of chemical libraries by solid phase synthesis. .Figure 1 shows a representative synthesis of an advanced intermediate towards aspidosperma alkaloid analogs. O

 

Cl O

LiHMDS TB SO CHO

O 5

1. Et3N 2. MeOH 3. TBAF 5 5%

O HO

O

HN

1. MsCl 2 . NaH

O

O

O N

O

81%

NTs

NTs

NTs

6

7

4

Scheme 1 Application of a new multicomponent reaction to the synthesis of a complex alkaloid Scaffold

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Total syntheses: GPC004FD4 and CJ-12662 and CJ-12663

Sibiromycin is a natural alkaloid belonging to the class of pyrrolo[1,4]benzodiazepine antibiotics. Some natural analogs of OH   OH OH H sibiromycin (figure H O N Me 2) show some H N Me aglycone promising antitumoral activities N O N O but are unfortunately Me Me O not readily available O O O from the natural MeHN MeHN aminoglycoside sources. We recently OH OH OH OH undertook a research Figure 1. Sibiromycin and analogs 62

programme aiming at developing practical synthesis of this class of interesting antibiotics. The synthetic strategy for the aglycon fragment rests upon a new strategy to construct polysubstituted anilines involving (a) the cycloaddition of Nsilylated ketenimines to acetylene dienophiles, (b) the silicon-directed electrophilic substitution of the resulting cycloadducts. At this point we have already provided an easy access to the natural sugar fragment of the molecule and various analogs. Also the aglycone part has been synthetized and we are presently studying the coupling of the two fragments. CJ-12662 and CJ-12663 (figure 2) belong to a unique class of terpenoid pyrrolobenzoxazine alkaloid. It displays excellent activities against insect pests, acari and helminths. The alkaloid part has been synthesised by the groups of Barrett and Baldwin. The terpenoid part has not yet been synthesised. Our project consisted of (1) effecting a total synthesis of the terpenoid part, (2) the resynthesis of the alkaloid part, (3) the preparation of analogs by replacement of the terpenoid part by other terpene or other terpene derivatives. During the course of these studies, we have Figure 2. Structures of CJ-12662 and CJ-12663 developed a new methodology allowing introducing stereospecifically two vicinal side chains on the olefinic carbons of a cyclic enone (Scheme 2). This has been published as an invited paper for the 50th anniversary issue of Tetrahedron letters. While studying the synthesis of the alkaloid parts, we observed unusual results during the course of the oxidation of the precursor 3-hydroxypyrroloindole. These results will soon be published. Scheme 2. A method for the vicinal alkylation of enone

• Proprotein convertases as tagets for cancer chemotherapy Proprotein convertases are calcium-dependent serine proteases responsible for maturation of protein precursors by sequence-specific cleavage at the carboxy-terminal of a basic amino acid. Many PC substrates are well-known cancerassociated proteins: growth factors, growth factor receptors, integrins, matrix metalloproteases (MMPs). They act as «masterswitches » at different levels of tumor development and progression: cell proliferation, invasiveness, metastatic ability. PC’s are overexpressed in some cancer tissues. Except for furin, very little is known about the tridimensional structures of PC’s and the nature of the active site : thus there is a need to target broad swathes of chemical space known to overlap with a specific area of biology space, here the area of Penicillin Binding Proteins. Our approach is based on the reasonable assumption that all serine proteases react by a unified mechanism. The selectivity of an inhibitor for representatives of this class of enzymes would then be exclusively controlled by recognition elements carried by the “molecular machinery” of the inhibitor. Using a mechanistic model for the inhibition of penicillin binding proteins by β-lactam antibiotics, G. Dive calculated for us the activation energy of the ring opening of a serie of lactams and the related hydrazides by a molecule of methanol mimicking a serine residue. In the aza-lactam serie, both the βand γ- lactams fused to a 5- or 6-membered ring (Figure 1) were particularly well-suited for undergoing ring-opening reactions. N The project which is supported by ARC consists in developing a n n = 1,2 simple synthetic route to bicyclic aza-β- and γ-lactams scaffolds N R: protecting group for building libraries of potential PC’s inhibitors. At this stage O we have already establish a new route to bicyclic lactam CO 2R scaffolds using ring-closing metathesis (RCM) as key reaction. 13 new aza-lactam scaffolds have been prepared. Scheme 3 Figure 3: Aza-analogs of β- and γ-lactams shows two examples of such synthesis. CO2 E t N

CO2 Et

N O

N N

N

53%

N O

O

N N

81% O

Scheme 3. Examples of RCM reactions using Grubbs 2nd generation catalyst.

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• Green Chemistry: benzylation and allylation of nucleophilic substrates using non-genotoxic reagents This part of our programme is still confidential. We have found that, by using new non-metallic catalysts discovered earlier by our group, it was possible to activate cheap and non-toxic allylic and benzylic substrates and react them successfully with nucleophilic substrates such as enol ethers, keteneacetals and electron-rich aromatics.

• Study of the electrophilic substitution in helical aromatic oligoamides ( with Huc’s group) The selective transformation of a molecule often rests on the interplay of functional groups. In contrast, enzymes rely on their folded conformations to recognize their substrate and achieve selective transformations. Thus a well defined folded conformation of a substrate could result in specific environments favouring a reaction at a specific site without the assistance of an enzyme or the use of protecting groups. We were able to show a remarkable rate and regioselectivity enhancement of electrophilic substitutions in helically folded aromatic oligoamides. This selectivity much depended on the presence of substituents remote from the reaction sites. The resuls will be depicted in more details in Huc’s report.

5 Significant Publications 1. Multicomponent reactions for the synthesis of complex piperidine scaffolds; Wei Zhu, Marisa Mena, Eric Jnoff, Na Sun, Patrick Pasau and Léon Ghosez; Angew. Chem. Int. Ed. 2009, ASAP. 2. Synthesis of enantiopure α-chlorocyclobutanones as scaffolds for the diverted total synthesis of serine protease inhibitors; Gaoqiang Yang and Léon Ghosez; Eur. J. Org. Chem. 2009, (special issue, invited paper) ASAP. 3. Studies related to the total synthesis of the sesquiterpene core of the pyrrolobenzoxazine product CJ-12662; Malgorzata Commandeur, Claude Commandeur, Michael De Paolis, Andrew J. Edmunds, Peter Maienfisch and Léon Ghosez; Tetrahedron Lett. 2009, 50, 3359-3362(special issue, invited paper). 4. Remote substituent effects and regioselective enhancement of electrophilic substitutions in helical aromatic oligoamides; Kolupula Srinivas, Brice Kauffmann, Christel Dolain, Jean-Michel Léger, Léon Ghosez and Ivan Huc; J. Am. Chem. Soc. 2008, 130, 13210-13211. 5. Functionalization of [60] fullerene via organometallic reagents; Elise Champeil, Conor Crean, Carlos Larraya, Gennaro Pescitelli, Gloria, Proni and Léon Ghosez; Tetrahedron, 2008, 64, 10319-10330.

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DEPARTMENT: PHYSICAL-CHEMISTRY OF MEMBRANOUS SYSTEMS Three teams constitute the department Team name Study of biochemical systems by Vibrational spectroscopy & optical methods Modeling of Biomolecules & Numerical Imaging Morphology, Dynamic and Function of Self Assemblies of Amphiphilic Molecules

Laboratory Head Bernard Desbat, DR CNRS Juan Elezgaray, DR CNRS Reiko Oda, DR CNRS

The departement deals with physical-chemistry properties and computing of assemblies of lipids, peptides and proteins. Themes: • • • • • • • • • •

Cationic Amphiphiles, Peptido-lipids, nucleo-lipids. Controlling aggregates morphology of amphiphilic molecules with counter ions. Inorganic Nano structures, Scafolds of amphiphilic assemblies. Photosensible Selfassemblies. Protein-mediated membrane fusion. Theoretical calculus of protein topology. Development of mesoscopic force fields. Computation of membrane assemblies made of peptides/proteins/lipids. Molecular Dynamics of membranous peptides, rafts and lipids phases. Membrane fusion and virus penetration. Model membranes under the forme of orientaed layers Cyclodextrin-cholesterol complexes.

Technical Approaches: • • • • •

Optical microscopy, neutrons & X-rays scattering, Light scattering. Intensive computing, mesoscopic dynamics, computing code development. Development of spectroscopic methods: PMIRRAS, ATR, VCD, Raman Vibrational and Optical methods IR, UV-vis Brewster. Langmuir Blodgett

65

TEAM « VIBRATIONAL SPECTROSCOPIES BIOCHEMICALS SYSTEMS »

AND

OPTICAL

PROPERTIES

OF

Team Leader: Bernard Desbat, Research Director, CNRS. Team Composition (2006-2009): Sophie Lecomte CR1 CNRS Zahia Fezoua, Doctorante Enora Prado, Doctorant

Géan Julie MCF UnivBordeaux 4 Nessim Azaram, Doctorant Eloise Lancelot, IR, ADERA

Sabine Castano MCF Univ Bordeaux 1 Ta Ha Phuong, Doctorante

Team Production (2006-2009): Publications, peer reviewed, (ACL) Publications, peer reviewed, not yet in bases (ACLN) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI) Oral communications in congress (COM)

41 1 7

Poster communications in congress (AFF) Books or Book chapters (OS) Patents

14 4 1

4

Defended PhD Theses or HDR

3

10

Grants : public & private k€/year

30

Scientific activity Résumé Le groupe: spectroscopie vibrationnelle et méthodes optiques des systèmes biochimiques est intervenu dans 4 thèmes principaux: interactions peptides ou pseudopeptides/membranes, interactions protéines/membranes, transfert d'électron et protéine, interaction ADN/molécules biologiques. Le thème interactions entre peptide ou pseudopeptide avec les membranes a été l’un des premiers abordés par l’équipe lors de la mise au point de la technique PMIRRAS à la surface de l'eau et concerne essentiellement des peptides (ou pseudo-peptides) antimicrobiens. Nous avons repris l'étude de l'alaméthicine dont le modèle d'interaction avec les membranes ne nous satisfaisait pas. L'autre sujet de ce thème important résulte d'une collaboration avec Gilles Guichard sur l'étude des interactions des oligo-urés vis-à-vis des membranes en relation avec leurs propriétés antimicrobiennes. Cette étude a tout d'abord nécessité un travail d'interprétation vibrationnelle et nous disposons actuellement des indices anisotropes infrarouges de ces oligo-urés en structure hélice et feuillet béta. Ces données nous permettent d'interpréter le spectre infrarouge d'un oligo-uré en interaction avec les monocouches et bicouches phospholipidiques et de déterminer à la fois sa structure et son orientation, suivant la nature des lipides des membranes. Le second thème, voisin du précédent, concerne les interactions protéines/membranes. Nous avons montré que l'adsorption de l'annexineV sous des couches phospholipidiques se faisait via les ions calcium par l'intermédiaire des groupes phosphates et que dans le cas du magnésium, l'ion était trop masqué dans la protéine pour pouvoir interagir avec les lipides. Dans le cas du système SNARE, nous avons révélé le comportement unique de la partie transmembranaire de la protéine VAMP qui possède une transition de phase réversible entre une structure en hélice alpha et une structure en feuillet béta. De plus cette transition est dépendante de la nature des lipides qui constituent la membrane. Nous avons commencé une étude de la croissance d'amyloïde fibrille en fonction des mutations d'un nombre réduit de résidus. Les premiers résultats infrarouges sont prometteurs et montrent que la toxicité variable de ces systèmes vis-à-vis de la levure doit pouvoir se corréler avec les structures secondaires d'auto-assemblage variables au sein de ces fibres. Ce sujet doit grandement tirer parti des techniques de microfluidiques en association avec notre projet d'imagerie infrarouge. Le troisième thème, transfert d'électron et protéine, est le travail exclusif de Sophie Lecomte et résulte de la finalisation des travaux qu’elle a réalisé à Paris avant de rejoindre le CBMN en mai 2007. Le quatrième thème, interaction ADN/molécules biologiques, est devenu, à la suite de collaborations nouvelles et de nouveaux financements, un des axes majeurs du groupe. Tout d’abord, dans le cadre des études de transfection, le travail que nous avons réalisé avec les équipes de P. Lehn et J.M. Lehn sur l’étude des interactions de l’ADN avec un lipide chargé positivement, le BGTC, s’est avéré suffisamment intéressant pour que cette collaboration se poursuive et s‘applique à de nouveaux lipides synthétiques. Nous sommes également impliqués dans l’étude de lipides nucléotides (projet région) pour lesquels nous avons montré que suivant les conditions physicochimiques, l’ADN pouvait se fixer sur ces lipides, par reconnaissance des bases ou par interaction électrostatique via un ion calcium. Finalement dans le cadre d’une thèse nous avons débuté la réalisation d’un couplage microfluidique/Raman SERS pour détecter facilement et rapidement la reconnaissance des brins d’ADN.

Abstract The Team: vibrational spectroscopy and optical methods of biochemical systems has been involved in 4 major themes: peptides (or pseudopeptides)/membranes interactions; proteins/membranes interactions; electron transfer and protein interaction; DNA/biological molecules interactions. 66

The first topics, devoted to the study of interactions between peptide or pseudopeptide with membranes, was one of the first we addressed during the development of technology PMIRRAS on the water surface with essentially antimicrobials peptides. We have included the study of alamethicin model whose interaction with membranes does not comply. Another important work has been done in collaboration with Gilles Guichard on the study of oligo-urea interactions towards membranes in relation to their antimicrobial properties. This study firstly needs a vibrational interpretation and we now have both anisotropic infrared optical data of oligo-urea in the helix and beta sheet structures. These data allow us to interpret the infrared spectrum of an oligo-urea interacting with phospholipid monolayers and bilayers and to determine both its structure and orientation, depending on the nature of lipid membranes. Always in relation with The second theme, close to the previous one, concerns the protein/membranes interactions. We have shown that the adsorption of annexinV under phospholipid monolayers occurs via calcium ions through the phosphate groups. At contrary the magnesium ion was too hidden in the protein to interact with lipids. In the case of the SNARE system, we have revealed the unique behaviour of the transmembrane part of the VAMP protein that has a reversible phase transition between an alpha helix and a beta sheet structure. Furthermore, this transition is dependent on the nature of the lipids constituting the membrane. We started a study of the growth of amyloid fibril based on mutations of a small number of residues on a prion protein. The first infrared results are promising and show that the variable toxicity of these systems on yeast must be able to correlate with the secondary structures of self-assembly organization within these fibbers. This topic should greatly benefit from the microfluidic techniques in conjunction with our future infrared imaging system. The third theme, electron transfer protein, exclusive work of Sophie Lecomte, and follows the completion of the work she has done in Paris before joining the CBMN in May 2007. The fourth theme, DNA/biological molecules interaction, is now a main topic of our group, as a result of new collaborations and new funding. In the field of transfection studies, the work we have done with teams of P. Lehn and JM Lehn on the study of interactions of DNA with positively charged lipid, BGTC, has proved its interest and this collaboration will continue and apply on new synthetic lipids. is the transfection problem, we are also involved in the study of lipids nucleotides (Projet Région Aquitaine) for which we have demonstrated that DNA could be bound to the lipid, depending on the physicochemical conditions by recognition of nucleobases or by electrostatic interaction via a calcium ion. Finally, the last subject is a part of a thesis, and we began the realization of the coupling of a microfluidic cell and Raman SERS to easily detect the recognition of DNA strands.

1. Interactions peptides (or pseudopeptides)/membranes Alamethicin organization in phospholipid model membrane Alamethicin is a 20-residue amino acids antimicrobial peptide produced by the fungus Trichoderma viride. It belongs to the family of peptaibols whose characteristic is to contain a large number of alpha-aminoisobutyric acid (Aib), a non-coded and strongly helix-promoting residue. The helical nature of those peptides is supposed to be the cause of their biological activity. In forming helical bundles in membranes, they would alter membrane permeability. This activity is used by fungi as a defense mechanism against target organisms. As a result, many peptaibols are effective antibiotics as it is the case for alamethicin. Previous studies have shown that alamethicin forms voltagedependent transmembrane channels. However, no consensus exists on the mechanism of action of alamethicin today’s. A first series of experiments leads at the air-water interface lets thinking that alamethicin in interaction with DPhyPC (1,2 DiPhytanoyl-sn-PhosphoCholine) monolayer, adopts a few secondary structures with notably the part structured in -helix rather straightened in the monolayer. To confirm this result, experiments should be done with a deutered aqueous subphase to avoid the perturbation of the amide I band by H2O absorption. In order to go further in the understanding of its mechanism of action, we would like to study alamethicin in interaction with supported bilayers formed on ATR crystal. Thus, ATR polarized spectra could give us information on the orientation in this model system closer of biological membrane. Interaction of oligo-urea compounds with membrane model (collaboration, G. Guichard, IBMC, UPR 9021, D. Cavagnat, ISM, UMR 5250) Oligo-urea synthesized by the team of Gilles Guichard can be treated as pseudo-γ peptides whose amide group is replaced by urea group. These systems have a propensity to form stable helices with two hydrogen bonds on each carbonyl group and some of them have antibacterial properties. We have begun work to understand how this type of molecule could act on membranes. As almost nothing exists on the assignment of IR and Raman spectra of these compounds we have established collaboration with D. Cavagnat to solve this problem. Two calculations were done, to determine the spectral characteristics of the oligo-urea in helix and beta sheet structures. From the Ab-Initio data we obtained the direction of the moments of transition and we were able to establish the anisotropic optical constants of an oligo-urea depending its secondary structures, see figure below. Now, the spectral simulations conducted with these optical data give us the possibility to understand the PMIRRAS spectra of oligo-urea in monolayer and polarized ATR spectra of oligo-urea in supported bilayers. The preliminary results show that oligo-urea seems in equilibrium between the two secondary structures when interacting with membrane models.

67

2) Interacttions proteeins/membrranes Role of phospphate group inn the binding of Annexin A55 to membran ne, via Ca2+ ioon (collaboraation A.Brisson n, CBMN) Annnexin A5 (AnnxA5) is a meember of a faamily of homologous proteins shharing the ability a to bind b to c phosppholipid mem mbranes in a Ca2+negatively charged dependent manner. m Wee used PMIIRRAS, BAM M and ellipsometry to investigatee changes botth in the struccture of Anx5 and phospholipidd head grouups associatedd with b Wee established that the seccondary membrane binding. structure of AnxA5 A is connserved, mainlly in -helicees. 70% of the α-heliices of AnxA A5 are orienteed perpendicuularly to Schheme of orientaation of AnxA55 in presence of o Ca2+, (a) the interfacee and 30% are a oriented in the plane of the undder phospholipid, projection in the (z,y) planne, (b) under phospholipidd monolayer, in agreemennt with the known phoospholipid, prrojection in thhe (x,y) plane (Fezouaorientation of o crystallized AnxA5 on suupported mem mbranes. Boubegtiten et al., (Biophysical JJ., 2009, submitted) We demonsttrated that thhe phosphate group is thhe main group involvved in the binnding of AnxxA5 to phosphholipids via Ca2+ C ions, evven when som me carboxylatte groups aree accessible (P PS). PMIRRA AS spectra alsso indicate a change of caarboxylate oriientation in thhe aspartate and a glutamatee residues implicated in the association off the AnxA5, which could be b linked to thhe 2D crystalliization of prottein under thee phospholipidd monolayer. The selectivitty of the Ca22+ ion was allso demonstraated. Mg2+, ffor example, is i not able too promote the binding of AnnxA5 to mem mbrane. Moleccular dynamicss studies (colllaboration: M M. Laguerre, CBMN) C reveall m and half of them are a completelyy embedded in i the protein. that Mg2+ arre dramaticallly less accessiible to water molecule In this condittion, such ion cannot interaact with the phhosphate group p of the membbrane. MR 5095, Chriistophe Cullin) n) Amyloid: struucture-toxicityy relationshipp (collaboratioon: IBGC, UM Thee link between aggregatedd proteins andd toxic speciees stems from m earlier studdies on neuro odegenerativee diseases. In the previous work, the teaam of Prof. Cullin C has dem monstrated thee capacity forr a harmless amyloid a to bee myloid speciees (reducing coonsiderably th he transformed into a toxic am b changing onnly few residuues of the Prio on growth of thee yeast cell) by Forming Doomain (PFD) of Het-s am myloid (HET--s218–289). In I vitro, we evidenced that thhe fibers form med by WT annd M8 amyloid ds nic differ dramaatically. Wheen observed by transmisssion electron microscopy, WT amyloidds exhibit some typical µm-long µ fiberrs, a asseemble into 800 nm-long fillaments. ATR Rwhilst M8 amyloids FTIR allowed us to confirm m that M8 waas also an amyyloid but with ha nd totally differrent secondaryy structure baased on a mixxed parallel an antiparallel organization o o the β-sheetss. Another asppect of toxicitty of to clarify is how h lipids annd membrane components are a involved in i the process of o amyloid toxxicity. There are a increasingg evidences th hat many amylooids interact with membraanes. Our firrst experimen nts reveal that thhe toxic mutannt (M8) stronggly interacts with w monolayeer of phospholiipid, whereass WT has pooor affinity foor phospholipiid monolayer. In I interactionn with phosphholipid layerss, M8 and WT W In vitro characterisatio c on of Het-s PFD D (WT) and have β-antipaarallel rich strructure but different orientaations of the ββ toxic mutant m (M8) by TEM and ATR-FTIR. sheet are obsserved. Two other o importannt topics are not n presented in i (Berthellot et al. FASEB B J. 2009) this part; onee concerns thee study of systtem SNARE in i collaboratio on with three CB BMN teams, R. R Oda, J. Lanng and M. Lagguerre. This project p has recceived partial support from the ANR andd is presented in i the report of o the laboratoory by Reiko Oda O who brin ngs the projectt. The other prroject involvees the study off retina proteinns in collaboraation with Proofessor Christiian Salesse fro om Laval Univversity in Queebec. t and d protein 3) Electron transfert Metaloproteiins probe by SERS S coupled with electrochemistry In thhe last decadee a performingg methodologyy was applied to probe the electron e transffer process off hemeprotein,, as Cytc-c5522. SERS expeeriments couppled with electtrochemistry allow to idenntify which strructure are in nvolved in thee process of ellectron transfe fer. With deveelopment of tiime-resolved SERR experiiments, the raate constant of the electronn transfer can be b determine for each idenntified structurre. It was esseentially the acctivity of Sophhie Lecomte in i the LADIR R laboratory (U UMR 7075). We W are presentting here the last l results obttained on two hemoproteinss. Electron trannsfer process of o Cytochrom me c552 from thermos t therm mophilus Cytoochrome c5522 (Cyt-c552) and a its redox partner ba3-oxidase from Thermus T therm mophilus posssess structurall differences compared c withh Horse heart cytochrome c(cyt-c)/cytoch c hrome c oxidaase (CcO) sysstem, where th he recognitionn between parttners and the electron transsfer (ET) proccess is initiateed via electroostatic interacttions. We dem monstrated byy surface-enhaanced resonancce Raman (SE ERR) spectrosscopy that rou ughened silverr electrode coaated with unch harged mixedd self-assembleed monolayerrs HS–(CH2)nn–CH3/HS–(C CH2)n + 1–OH H 50/50, n = 5, 5 10 or 15, w was a good mo odel to mimicc the Cyt-c5522 redox partnner. The confformation of the t heme is 6cLS, 6 whatevver the redox state of the iron. All thee 68

adsorbed molecules are well oriented, on such biomimetic electrodes and transfer one electron during the redox process. We established that the electron transfer process is fast and reversible. By varying the chain length of the SAMs, we have also evidenced that adsorbing Cyt-c552 onto a short chain length should improve the rate of the electron transfer. Characterization of immobilized recombinant human neuroglobin (collaboration, LEM, UMR 7591, Véronique Balland) Electrochemical investigation of the electron-transfer (ET) kinetics of chemical or biological redox molecules immobilized as a monolayer on conductive surfaces has been recognized to be a powerful approach for better understanding long-range electron transfer, which is one of the fundamental processes in nature. This subject is also of interest for potential applications, for example, in biomolecular electronics where biological molecules are integrated on the surface of electronic transducers to provide new strategies for biosensing. As a small hemoprotein, we have selected recombinant histidine-tagged human neuroglobin (hNb), which has a size (17 kDa) comparable to that of cyt c. The hemoprotein presents an accessible heme pocket and specific heme iron bis-histidine hexacoordination in its native state with a characteristic resonance Raman spectrum. It is therefore a good candidate for both investigating its direct electron transfer at an NTAmodified gold electrode and characterizing its structure by SERR Model for immobilized Fc-Im2 and hNb (on Ni(II)-NTA-SAM electrode (Balland et al. spectroscopy. The his-tagged hNb on NTA modified electrodes was thus investigated by cyclic voltammetry, potential-dependent SERR Langmuir, 2009) spectroscopy, and time-resolved SERRS spectroscopy. The absence of spectral change in the SERR spectrum of hNb immobilized on the Ni(II)-NTA SAM tends to confirm that the native structure of the immobilized protein is retained on such a modified surface. Direct electronic communication between the electrode and the immobilized protein was evidenced only by SERR spectroscopy but not by cyclic voltammetry. We established, from SERR experiments, that all of the immobilized proteins remain electroactive and non denaturated in both redox states. 4) Interaction DNA/Biological Molecules DNA/BGTC interactions (collaboration P. Lehn, Brest University, et J.M. Lehn, Collège de France) The lipid bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups which displays high transfection efficiency in vitro and in vivo when used alone or formulated as liposomes with the neutral colipid 1,2-di-[cis-9-octadecenoyl]-sn-glycero-3-phosphoethanolamine (DOPE). Since transfection may be related to the structural and physicochemical properties of the self-assembled supramolecular lipid-DNA complexes, we used the Langmuir monolayer technique coupled with Brewster angle microscopy (BAM) and polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) to investigate DNA-BGTC and DNA-BGTC/DOPE interactions at the air/water interface. We show that BGTC forms stable monolayers at the air/water interface. When DNA is injected into the subphase, it adsorbs to BGTC at 20 mN/m. Whathever the (+/-) charge ratio of the complexes used, the DNA interacts with the cationic lipid and forms either an incomplete (no constituent in excess) or a complete (DNA in excess) Detection of DNA or RNA without probe (collaboration L.Servant, ISM, monolayer UMR5250; C. Di Primo, IECB) of oriented double strands parallel to the lipid monolayer plan. We also show that, under a homogeneous BGTC/DOPE (3/2) monolayer at 20 mN/m, DNA adsorbs homogeneously to form an organized but incomplete layer whatever the charge ratio used. Compression beyond the collapse of these mixed DNA-BGTC/DOPE systems leads to the formation of dense DNA Typical set-up displaying the microchannel monolayers under an asymmetric lipid bilayer with a bottom layer of BGTC with the two incoming fluid: colloid (A), in contact with DNA and a top layer mainly constituted of DOPE. These oligonucleotide (B) and the confocal probing results allow a better understanding of the mechanisms underlying the volume of the Raman microscope. formation of the supramolecular BGTC-DNA complexes efficient for gene transfection. Designing fast and efficient analytical tools allowing the detection of DNA or RNA at very low concentration within small volumes, without specific probe, remains a major issue of significance to perform diagnostic or to detect pathogen agents. Various strategies have been suggested and developed to achieve this goal, but they involve various steps of DNA-labeling and/or DNA- immobilization. We demonstrated that Surface Enhancement Raman Scattering (SERS) appears to be a very sensitive technique to obtain qualitative, quantitative and structural information about DNA or RNA. A main advantage of SERS technique is its sensitivity and the capability of DNA or RNA detection using only the vibrational marker bands that are specific of each base. Our strategy is to detect DNA or 69

RNA hybridization by SERS spectroscopy coupling with microfluidic platform. The use of microfluidic platforms allows better control of temporal resolution, as well as consummation of small volumes. Thus, we already shown that using microfluidic device we are able able to detect in 1s the presence of polynucleotide, as polyA and polyU, at low concentration (less than micromolar). However we observed a high aggregation of the colloid in the channel. Thus, we now develop a new approach using digital microfluidic devives. It will prevent from the sedimentation and improve the mixing between colloid and nucleotides. Interaction of anionic nucleolipid (Lipnuc) with DNA (Collaboration L. Navailles, CRPP, Bordeaux; P.Barthélémy, INSERM, U869) Gene therapy is a promising tool for developing new strategies against genetic diseases and cancers. One of the main factors to achieve gene delivery is to create a vector for delivering nucleic acids into targeted cells. Furthermore, for medical applications, the vector must be prepared to allow the greatest transfection efficiency and the lowest cytotoxicity. Recently, DNA delivery systems based on O anionic lipids have emerged as a possible alternative to NH cationic lipids. In collaboration with the teams of P. N O HO Barthelemy and L. Navailles we started a study on O O anionic nucleolipids with the general organization of: O O O P O one or two lipid chains, one phosphate one sugar and one d iC 16-3'-dT O H ON a+ nucleobase. For exemple, here is the diC16-3'-dT. The O main advantages of those systems are their low toxicity and the possibility to modulate the interactions with nucleic acids between ionic and base-pairing interactions. We used the Langmuir method coupled to Brewster angle microscopy and PMIRRAS infrared spectroscopy to investigate interactions between nucleolipids and nucleic acids. We have been able to characterize nucleolipids films and confirm that the different types of interactions with DNA (electrostatic and base-pairing) can be modulated by the accessibility of the lipid base to the DNA and to the presence (or not) of calcium in the solution. Finally, during our collaboration with SANOFI we performed an original study on the adsorption of the active H1N1 influenza virus on a DMPC/GM3 lipid monolayer. The comparizon with the inactive virus behavior show that the inactive process inhibate the fusion step of the virus with the membrane cell

5 significant publications: 1. 2. 3. 4. 5.

Yassine, W.; Taib, N.; Federman, S.; Milochau, A.; Castano, S.; Sbi, W.; Manigand, C.; Laguerre, M.; Desbat, B.; Oda, R.; Lang, J. (2009) Reversible transition between α-helix and ß-sheet conformation of a transmembrane domain, Biochem. Biophys. Acta-Biomembranes, in press. Berthelot, K.; Immel, F.; Gean, J.; Lecomte, S.; Oda, R.; Kauffmann, B.; Desbat, B.; Cullin, C. (2008) Driving amyloid toxicity in a yeast model by structural changes: a molecular approach. Federation of American Societies for Experimental Biology,, in press. Balland, V; Lecomte, S; Limoges, B (2009) Characterization of the Electron Transfer of a Ferrocene Redox Probe and a Histidine-Tagged Hemoprotein Specifically Bound to a Nitrilotriacetic-Terminated SelfAssembled Monolayer, Langmuir, 25, 11, 6532-6542. Castano, S.; Delord, B.; Fevrier, A.; Lehn, J. M.; Lehn, P.; Desbat, B. (2008) Brewster angle microscopy and PMIRRAS study of DNA interactions with BGTC, a cationic lipid used for gene transfer, Langmuir, 24, 95989606. Banc, A.; Desbat, B.; Renard, D.; Popineau, Y.; Mangavel, U.; Navailles, L. (2007) Structure and orientation changes of omega- and gamma-gliadins at the air-water interface: A PM-IRRAS Spectroscopy and Brewster angle microscopy study, Langmuir, 23, 13066-13075.

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Teaching of Raman and SERS spectroscopy. Licence 3 and Master 2 level. CNRS Formation: Raman spectroscopy applied to study of biological molecules, IECB (3 days) CNRS Formation: Ecole thématique Sciences de la miniaturization et biologie, Grenoble 8-12 juin 2009

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TEAM “MODELING OF BIOMOLECULES & NUMERIC IMAGING” Team Leader: Juan Elezgaray, Research Director, CNRS. Team Composition (2006-2009): Juan Elezgaray, DR 2 CNRS Michel Laguerre, DR2 CNRS Jean Dessolin, CR 1 CNRS Nada Taïb, Post-Doctorante AFM

Marc Lamblin, Post-Doctorant ARC Claude Commandeur, Post-Doctorant ARC Julie Gonzalez , Doctorante co-tutelle Jean-Michel Arbona, Doctorant

Guillaume Naturale, Doctorant BDI Julien Lefeuvre, Doctorant Clément Arnarez, Doctorant Judith Elkaïm, Doctorant

B.Sc. and M.Sc. trainees (stay between 2 and 6 months): 11

Team Production (2006-2009): Publications, peer reviewed, (ACL) Publications, peer reviewed, not yet in bases (ACLN) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI) Publications, peer reviewed in published national colloquia (ACTN) Oral communications in congress (COM)

25 1 9

Poster communications in congress (AFF) Books or Book chapters (OS) Patents

13 2 1

3

Defended PhD Theses or HDR

2

2

Grants : public & private k€/year

20

1

Scientific activity Résumé Nous développons plusieurs thématiques, pour la plupart centrées sur l'application et le développement de méthodes de simulation numérique pour la modélisation de molécules d'intérêt biologique, ainsi que leurs interactions. Un second volet de notre activité est dédié à l'imagerie à l'aide de plasmons de surface. De façon plus précise, à l'aide de simulations de dynamique moléculaire, nous avons caractérisé les mécanismes d'activation de la kinase AKT-1 et étudié des récepteurs de type GPCR. Nous avons aussi décrit au niveau atomique la structure d'assemblages lipidiques contenant des Gemini, et montré l'importance des interactions électrostatiques dans les processus d'insertion de certains peptides antibiotiques dans des bicouches lipidiques. Par ailleurs, nous développons une activité importante basée sur des méthodes de « docking » et de « drugdesign » qui nous ont permis de déposer un brevet portant sur un nouvel inhibiteur de XIAP. En parallèle, nous avons développé des méthodes rapides de « docking » et construit une bibliothèque de structures de protéines kinases. En ce qui concerne le second volet, nous avons étudié en détail, de façon analytique, les distributions de champ électromagnétique autour d'une sphère éclairée par un faisceau fortement localisé, en interaction avec les plasmons de surface portés par une couche métallique et montré l'importance de la rugosité de cette couche dans le processus de détection. Abstract Our research is mainly centered on the application and development of numerical simulation methods to model biologically relevant molecules as well as their interactions. We also consider the image formation process in plasmon based devices. More precisely, using molecular dynamics simulations, we have characterized the action mechanism of the AKT-1 kinase and studied GPCR receptors. We have also described at the atomic level crystal structures of Gemini lipids and characterized the role of the electrostatic interactions in the pore formation process with specific antibiotic peptides. We also have a strong activity centered on docking and drug-design methods that led to a patent for a new inhibitor of XIAP. In parallel, we have developed new and fast docking procedures and build up a library of kinase structures. Concerning the activity around numerical imaging, we have studied analytically the electromagnetic field distributions around a spherical bead in the vicinity of a thin metallic layer, in the presence of a tightly focused beam and demonstrated the role played by the roughness of this layer in the imaging process.

•

All-atom Molecular Dynamics

This part of the team's research is developed mainly along three axes: lipidic assemblies, proteins and finally membrane proteins within biomembrane models. The first axis encompasses mainly molecular dynamics of complex lipidic assemblies using an all-atom representation: i.e., spherical or cylindrical micelles of various surfactants, Langmuir films and various bilayers of biologically relevant lipids but also fluorinated Gemini derivatives or bicelles. This work is performed in strong collaboration with several teams involved in experimental Biophysics (Reiko Oda or Erick Dufourc at IECB, Bernard Desbat at CBMN, Shawn Wettig in Waterloo University, Ontario). Very recently we succeeded in determining at the 71

atomic level the global structure of a nano-object containing tartrates of geminis (see below). This is the first structure at an atomic level of such a nano object (paper published in JACS).

Final structure of the gemini tartrate film structure at an atomic level: geminis are in shade of red, tartrates in shade of green and water molecules are in blue. Tubes of aligned water molecules can be easily seen in the right panel. Between the two interdigitated layers a hexagonal lattice of water molecules insures the global cohesion of the structure.

Concerning the protein axis, we have largely focused our work on kinases surexpressed in various cancers and mainly on the mechanism of activation of AKT-1 which is involved in numerous regulation pathways and thus in many cancers (Cancer Institute UK). Two papers have been published in PloS Biology and a review has been published on this subject. The aim of the project was to unravel at an atomic level the very complex activation process of this important kinase. The whole mechanism is a succession of several interdependent events which knowledge will maybe allow intervening at various stages of the activation and then will allow blocking selectively some specialized pathways. The whole work has been highlighted in England on several internet sites like Yahoo England or Channel Four and in France in the Journal du CNRS. This work is now extended to the kinase PDK-1 which is one of the major activating factors of the AKT cascade.

Complete structure of AKT-1: 2 orthogonal views of the Cterm docked on the WT PH/KIN complex : PH in pink, KIN in green, Cterm backbone in orange, W80 in red with translucent surface, F469 & 472 in yellow, S473 in cyan, Y474 in mauve, Q218 in aquablue with translucent surface. The water channel is visible in the center of the left image and on the right it is cut just at its middle by the clipping plane. The interaction of F469 & 472 with W80 is obvious alongwith their location on the walls of the channel. There is an interaction of S473 with Q218.

Finally the third axis involves the interactions of peptides and proteins with biomembrane models. Part of this work deals with the interaction of membrane models with peptidic toxins or with trans-membrane peptides. In collaboration with E. Dufourc's team, we have studied the mechanism of insertion of antibiotic peptides (cateslytin). We have shown that the mechanism is essentially similar to an electroporation phenomenon, the triggering electric field being provided by the peptides themselves. Molecular dynamics simulations show that the insertion of a few peptides into the bilayer is a rather fast process (characteristic time scale: 50 ns), that can be significantly accelerated by the addition of a very small electric field normal to the bilayer. The peptides that form the pore are mostly unstructured, contrarily to all the models previously proposed. The comparison of the conductivity of this pore to patch-clamp measurements yields further confirmation of the validity of the model.

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We also have considered molecular dynamics of membrane receptors in a full lipidic environment and monitoring of the interaction drug/receptor. Actually the main interest lies in the GPCR super-family including human dopamin D2, human leukotrien receptors, but also 5HT1 receptors in a collaboration with Bernard Veyret (Laboratory IMS CNRS/EPHE and contract with EDF) and the opiate receptors in collaboration with Vanderbilt University School of Medicine (a NIH grant application will be deposited in fall for the second time). A paper has been published in Protein Science. One of the main goals of this project is to understand at an atomic level the activation process of GPCR proteins. This knowledge will allow specific interaction by means of drugs with fundamental stages of the functional mechanism of these highly targeted receptors.

• Drug-Design and High-Troughput in-silico screening The activity lies at the frontier between biology and chemistry. Starting from a biological problematic, we are searching for small molecules able to interact with protein targets; virtual screening is performed with pre-filtered chemical databases, or with in-house collections. This approach leads to the discrimination of the best putative ligands which are then synthesized in our group or through collaborations with other teams. Synthesis of pro-apoptotic & anti-cancer compounds The precursor work engaged in 2003 to design inhibitors of IAPs (Inhibitor of Apoptosis Proteins) has evolved in the course of the following years. Work started with the design of small molecules, naphthoquinones to mimic the SMAC protein (Second Mitochondrial Activator of Caspases). While organic syntheses were engaged, a virtual database of nearly a thousand compounds was prepared and screened by docking on XIAP (X-linked Inhibitor of Apoptotic Protein). This collection was essentially constructed with compounds which were attainable from a chemical point of view, and led to the discrimination of individual as well as series of compounds. From this a virtual screening procedure was set up in order to automatize and increase the selection speed. Out of eighty synthesized molecules, nearly a third proved to activate apoptosis and kill cancer cells. While a patent was registered, a first article describing our synthetic approach was published; two more papers should arise from this collaboration with a local start-up company. fermeture de cycle O H2 N

O O

N N H O O

H 2N

NH2

O

N N H O

N H

NH2

N H

O

peptide N-terminal de SMAC (AVPI) O O

R2

R

H2N

H N

N N H

R O O

N H

H2 N

n R1

O O

How to mimic SMAC: N-terminus tetrapeptide AVPI interact with XIAP, cyclisation allow a convenient rigidification of the peptide, the aliphatic (and chiral) cycle is replaced by an aromatic (naphtoquinone).

This experience led us to another class of compounds that is now under synthesis through an ARC fellowship (Association pour la Recherche contre le Cancer) to Dr. M. Lamblin. The chosen scaffold is monocyclic, resulting from the naphthoquinone ring simplification. The biological activity of the new series will be evaluated through collaboration with colleagues from CNRS in Toulouse. An unexpected cyclisation reaction observed during the synthesis of the naphthoquinones presented above has convinced us to prepare another series of small molecules that were virtually screened on kinases and phosphatases. New series of tri-cyclic compounds are prepared by G. Naturale in the course of his PhD (CNRS-Region Aquitaine grant). Both synthetic projects are co-directed with Dr. F.X. Felpin from ISM (UMR 5255, Bordeaux). Development of procedures for virtual screening In order to select potential ligands from a collection, an automatized procedure was set-up in the course of the IAP’s project. This was based on the high-throughput docking of chemical databases with various softwares. In order to speed-up the virtual screening and to increase the accuracy of the ligand discrimination, various procedures are tested combining docking, re-scoring, determination of the best scoring function with a training set and consensus scoring if necessary. A first application is under way to select the best ligands for GSK-3 kinase, in order to support the drug design and organic synthesis projects, as part of the PhD studies of J. Elkaim (MENRT grant).

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Collaboration with Drs. P. Lestienne and J. Rosenbaum (INSERM U889) has been initiated with Dr. M. Laguerre in order to construct a model of the helicase protein. A virtual screening has been realized on this model with a commercial database in order to find original ligands. The discriminated compounds will be tested by our partner while another database will be screened in silico to search for new scaffolds. Parallel to this project, the preparation of chemical databases, commercial or academics, is under way in order to prefilter the molecules to be tested. Several collaborations are under course on the same kind of project aiming at identifying putative ligands from known protein structures in the field of cancer, neurodegenerative diseases, GPCR, or to get a better understanding of the interactions between a hit molecule and its own receptor. Bank of proteins. In 2006, with the help of a german Erasmus student we have designed a kinases data-bank. The strategy was to download all available X-ray structures of kinases (1200 or so X-ray structures present in the PDB) and then filter this base in a stepwise way in order to sort around 300 well resolved structures each with an active site completely detemined at the atomic level. This subset represents around 85-90 different kinases among the 518 actually known for human species. This subset can then be used as a target for High Throughput in-silico screening by docking either with known drugs (new targets or possible side-effects) or with designed molecules to be synthesized. Actually the routine rate of testing is around 2-3000 molecules/protein a day with a maximum theoretical rate around 20-25 000 molecules/protein a day. This year we have extended this axis with a more friendly user approach in order either to be able to select a class of protein Complete model of human helicase with the (kinases, phosphatases, proteases ...) or just to select all targetted ATP binding sites in red volumes biologically relevant targets. This is really feasible as there are actually 50 000 structures within the PDB data-bank but this will very likely result in 5 to 10 000 pertinent targets (thus including a precise active site which can be targeted by docking techniques). In this way it will be possible to test one molecule on all known (X-ray) biological targets in one day!

•

Surface plasmon microscopy

This work is the result of a close collaboration with F. Argoul's team at ENS Lyon. The goal is to develop a non intrusive tool to detect small variations of the dielectric constant in the vicinity of a metal-dielectric interface. Typical applications range from DNA microarray characterization to cell imaging. Experimentally, the Argoul's team has shown that dielectric as well as metallic nanoparticles (R > 10nm) can be detected with this type of microscopy. The technique is based on the interferometric detection of the perturbations induced on the plasmon excitations supported by a thin gold layer (width ~ 50nm). The sample to be imaged is in the vicinity of this layer. Our contribution to this work is to provide with a model that allows a quantitative description of the measurements. We have written and studied a set of exact solutions of the Maxwell equations describing the field distributions in a system made of a spherical bead in front of a planar metallic layer. The agreement between this theory and the experimental data is good excepted for particle sizes (~ 10nm) comparable to the scale where the gold deposit cannot be considered as flat. The model also provides with a set of optimal experimental parameters, such as the gold width and the range of incident angles. Overall, the efficiency of plasmon microscopy can be traced back to the amplification by the plasmon excitations of the evanescent waves scattered by the nanoparticles.

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5 significant publications: 1. 2. 3. 4. 5.

V. Calléja, M. Laguerre, P. J. Parker & B. Larijani (2009) Role of a Novel PH-kinase Domain Interface in PKB/Akt Regulations: Structural Mechanism for Allosteric Inhibition. PloS Biology, 7 (1), 189-200. Oda R., Artzner F., Laguerre M. & Huc I., (2008) Structure of self-assembled chiral nano-ribbons and nanotubules revealed in the hydrated state. J. Amer. Chem. Soc., 130 (44), 14705-14712. Jean-Francois, F., Elezgaray, J., Berson, P., Vacher, P., and Dufourc, E. J. (2008) Pore Formation Induced by an Antimicrobial Peptide: Electrostatic Effects. Biophys. J. 95, 5748-5756 Calleja V., Alcor D., Laguerre M., Park J., Hemmings B., Vojnovic B., Downward J., Parker P. P. & Larijani B., (2007) Intra- and inter-molecular interactions of Kinase B define its activation in vivo. PloS Biology, 5 (4), 780-791. Kuntz A. N., Davioud-Charvet E., Sayed A. A., Califf L. L., Dessolin J., Arnér E. S. J., and Williams D. L., (2007) Thioredoxin glutathione reductase from Schistosoma mansoni: An essential parasite enzyme and a key drug target, PLoS Medicine, 4(6), 1071-1086.

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Teaching duties Michel Laguerre Molecular Modeling courses within the BioInformatics Master of Bordeaux I, 1st year in 2004-2007. Molecular Modeling courses within the Structural Biology Master of Bordeaux II/Bordeaux I, 1st year in 20042009. Molecular Modeling courses within the Module "Apoptose" Master 2 University Bordeaux II Cell Biology and Physiopathology in 2006-2009. Jean Dessolin Module "Apoptose" Master 2 University Bordeaux II Cell Biology and Physiopathology "Pro-apoptotic compounds drug design" (2 hours). Scientific Animation within IECB Jean Dessolin Organic Chemistry Seminars, (twice monthly) for students and tenure researchers in English. Organization of CBMN Scientific Day (Novembre 2008) Administrative responsibility / Committee membership Michel Laguerre • Member of the Scientific Committee of the Regional Computational Center M3PEC • Member of the Internal Scientific Committee of the Bergonié Cancer Institute Research • Member of the Clinical Resarch Council of the Bergonié Cancer Institute Research • Member of the Gestion Committee of the "Bioinformatics Centre of Bordeaux" • Member of the Management Committee of the IECB • Member of the Administration Board of CBMN - UMR 5248 CNRS Jean Dessolin • Member of the Administration Board of CBMN - UMR 5248 CNRS Juan Elezgaray • Member of the Scientific Council of the University of Bordeaux I (2004-2007)

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TEAM « MORPHOLOGY, DYNAMIC AND FUNCTION OF SELF-ASSEMBLIES OF AMPHIPHILIC MOLECULES » Team Leader: Reiko Oda, Research Director, CNRS. Team Composition (2006-2009): Sabine Manet, PhD student Rumi Tamoto, PhD student Thomas Delclos, Postdoc Yevgen Karpichev, Postdoc

Carole Aimé, PhD student Ren-Wei Chang, PhD student Sébastien Buchoux, Postdoc

Wissam Yassine, PhD student Roni Kiagus Ahmad, PhD student Zahia Fezoua, Postdoc

B.Sc., M.Sc. trainees, (between 2 and 6 months): 6 Exchange program (between 2 and 6 months): 1 (Emory University-undergraduate), 1(Amherst Collegeundergraduate), 1(Kumamoto University-PhD)

Team Production (2006-2009): Publications, peer reviewed, (ACL) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI) Oral communications in congress (COM)

14 10

Poster communications in congress (AFF) Congress/Workshop organization

8 2

1

Defended PhD Theses or HDR

4

7

Grants : public & private k€ / year

106k€

Scientific activity Introduction Self-assembly of amphiphilic molecules allows construction of supramolecular structures with their sizes covering from nanometer to micrometer range. These self-assembles imply a great number of molecules (the smallest structure, micelles already require tens to a hundred of molecules) and their assembling mechanism is often very complex where a number of parameters play important roles in cooperative and dynamic manner, thus, it is still difficult to predict the morphologies and dynamics of aggregates from constituent molecular structures. Other than in the more classical domains of utilities such as food science, cosmetics, cleaning or petrol, where they are used as detergents, gelling agents, viscosifiers or emulsifiers, or in biology where they are mainly considered as membrane constituent, recently, renewed interests emerge on these systems with their structural characteristics, particularly because of their capacity to form nano to micrometric structures with well defined geometry in hierarchical manner. More and more reports show the examples of tuning of the morphology and dynamics of such assemblies at atomic level. Such “bottom up” approaches, developed in the fields such as nano-bio-technology, micro-robotic, nanoelectronics, nanoreactors, require very well organized molecular arrangement as well as the arrangements of the assemblies, therefore very good understanding of the assembling mechanism seems inevitable for designing new systems. The understanding of the mechanism of molecular self-assemblies is also directly related to the structural study of biological self-assemblies, another field with strong potentials such as membranes of cytosqueletons on which depend a number of biological functions. We are interested in understanding the mechanism of formation of amphiphilic molecular assemblies in order to design and build new nano-micrometric molecular assembly systems of amphiphilic molecules, for which the morphologies and functions can be finely tuned. This requires first of all understanding the role of different parameters (molecular architecture and various physico-chemical parameters) on the molecular assemblies. The control of the assembly formation at molecular level allows their functionalization with biomolecules (peptides, nucleotides...). The assemblies can therefore serve as the support for the biomolecular structure formation or the induction of interaction between the aggregates via molecular recognition.

Counterions effects One of the very important molecular parameters that determine the way how the charged amphiphiles self-assemble is the nature of their counterions. Depending on their counterions, the amphiphilic molecules can behave in totally different manners. The origin of these differences is very complex: hydrations of ions, ionic volume, pka, nucleophilicity of ions, are only a few of many factors which play important roles. We have studied cationic surfactants with ~40 different types of counteranions. Their solubilities, the cmcs, ionization degrees and the morphologies of aggregates have been investigated in order to understand the effect of interaction between molecules and their counterions. (JACS submitted, PhD thesis, Sabine Manet). The information which we can obtain from such studies is crucial for understanding ion-specific effect which is a fundamental phenomenon observable in any charged molecular assemblies. The effect of the amphiphiles - counterions interaction in the gas phase reaction is being studied by Mass spectrometry and DFT calculation and is compared with their behaviors in solution (Col. L. Romsted (Rutgers University) J. Phys. Chem.B 2008, PhD thesis, Carole Aimé). 76

(Left) Examplles of the resuults showing the t effects of counter-anionss on the self-aassemblies of cationic surfacctants, cmc & solubilisation temperature. (R Right) general tendencies t of thhe relationshipss between the ioon properties & the aggregatio on properties.

Morphology control of nannometric chiraal assemblies:: twisted, heliccal ribbons, annd tubules. me of them shhowed particuularly interestiing behaviors,, Amoong the differrent counterionns that we havve studied, som for examplee, tartrate. They T are di-aanions and chiral, c and when w they arre complexedd with cation nic non-chirall dimeric/dicattionic surfactaants, they form m chiral ribboon which exprress supramoolecular chiraality of the orrder of 10 nm m to microns. The T morpholoogies of these chiral assembblies can be co ontrolled withh a number off parameters. (Nature ( 1999, Angewandte Chemie 19988, JACS 2007). It is importaant to note thatt the amphiphhilic parts of thhese moleculees are achiral. wed us to bettter understandd the mechanism of the chirrality transferr The detailed study of this very originall system allow c to achiral membbranes from molecular m leveel up to mesosscopic level (JJACS 2002, J.. Phys. Chem. from chiral counterions A 2004, JAC CS 2008, Chiraality 2009). (P PhD thesis’, Damien Berthieer, Thomas Laabrot, Aurélie Brizard, Caro ole Aimé). .

(Left) morphollogical variatioon of chiral selff-assemblies as functions f of (A) A) temperature, (B) time, (C) additives. (JACS S 2007 ) (Righht) Chiral molecuular organizatioon induces an asymmetric tennsion on the membrane, m and the tension froom two adjacen nt membranes is crossed to givee rise to twist of the ribbons. The T network of these t twisted ribbons leads to the gelation off solvents.(JACS S 2008)

•

Interacction betweeen cationic membran nes and bio ological (pooly)anions

o Peptide conffinement and structure s induction at cation nic membranee surface, peptido-amphiphiles, a of nucleoamphip philes o Recognition between the aggregates n of a numberr of processes in biological systems. Thee The interactiion peptide-lipid and nucleeic acid-lipid are the origin use of simpllified model system s for whhich we can control variou us parameterss in independdent manners allows betterr understandinng of these coomplex interactions at mollecular level. We have devveloped the syystems of lipo opeptide andd nucleolipid by b using bioloogical polyaniions such as oligopeptides o or nucleotidees complexed to cationic am mphiphiles. Inn both cases, the idea wass to investigaate the interaaction between anions by particularly focusing on the effect off pids: we havee shown that oligopeptidess confinement of these anioons in the viciinity of cationnic membranees. Peptide-lip orly organizedd when they are alone in solution, cann (oligo glycinne or oligo allanine) whichh are well solluble and poo organize them mselves to weell defined β sheet s structurres when they are confined at the surfacee of membran nes (Langmuirr 2006, Chem Commun 20007). (PhD theesis, Aurélie Brizard, B Rony y Kiagus Ahm mad). Nucleollipids: the co onfinement off ognition behavvior between the aggregatees, although itt mononucleottides at membbrane surface can induce innteresting reco 77

is not based on the Watson-Crick mechanism. (J. Colloid Interface. Sci. 2005, Langmuir 2007, Langmuir submitted, PhD thesis, Carole Aimé, Rumi Tamoto).

Chiral ribbons induced by complex cationic surfactant-anionic peptides; confined peptides form β sheet.(ChemCommun 2007) Acetate anion

Non -chiral

Chiral mononucleotide AMP

Vesicle cationic surfactant

Ac

Ion exchange

Helix fiber

Chiral amplification

Acetic acide

vesicules

+ nucleotides N

Acetate GMP

helices N

GMP nucleotides

In-situ vesicles to helices transition induced by nucleotide-cationic surfactant complex. The photos at the bottom show the growing processes of helices.(Langmuir 2009)

Remarkably, for these two systems again, the chirality of peptides and nucleotides led to the expression of supramolecular chirality of the assemblies whereas the cationic amphiphiles were achiral as it was the case with tartrates. Such reciprocal and cooperative effects between membranes and counterions seem to be general in the case of the systems studied here. The membranes serve as the template for the structure formation as well as the intermolecular recognition between counterions which are confined at their surfaces, and reciprocally the chirality of the counterions lead to the expression of supramolecular chirality at membrane level.

Physico-chemical study of the membrane fusion induced by the interaction between membrane proteins The structures and functions of membrane proteins are closely related; therefore the mechanism of protein structuration is a key factor for understanding their function. We have developed a very close and fruitful collaboration between my group, the group of a cell biologist, Jochen Lang (CBMN), the group of a spectroscopist of biomolecules, Bernard Desbat (CBMN) as well as the group of molecular dynamic modeling, Michel Laguerre (CBMN). The project focuses on the structural study of SNAREs proteins which play crucial role for the membrane fusion during exocytosis. We are particularly interested in the structural properties of the transmembrane domains of these proteins. Our data indicate that the transmembrane domains of these proteins are particularly sensitive to the lipid memrane environment such as peptide to lipid ratio, membrane lateral pressure and membrane charge density and show unprecedented reversible transition between α-helix and β-sheets. These findings may shed light on understanding the mechanism of creation of pore on the membrane during membrane fusion (BBA 2009, AC-Interface physique-chimiebiologie (2002), two BQRs, a FRM fellowship and ANR-PCV (2007)

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(Α)

(Β)

(C)

 

(A) reversible a helix to b sheet as a function of TM domains of SNARE (VAMP) proteins to lipid ratio. (BBA.2009) (B) Structures and orientations of SNARE (VAMP) proteins vary as a function of surface pressure (Langmuir Blodgett film) and the charge density of surrounding lipids (yellow lipids (b) are neutral lipids, DMPC, blue lipids(e) are negatively charged lipids, DMPG, (c) and (d) are mixed monolayers with various ratios of DMPC and DMPG) (C) Schematic illustration of the structural transition between α-helical structure and β sheet assemblies as a function of peptide to lipid ratios. The transmembrane domains of VAMP proteins are initially present as an α-helix (a) and upon an increase in the peptide to lipid ratio (b) the peptides reversibly fold into β-sheets (c). The conformational transition may disturb the geometry of the surrounding lipid bilayer and thus promote membrane fusion. (d-f)

• Bicelles Due to their capacity to orient themselves under magnetic field, the disc-like lipid assemblies bicelles (bilayer micelles) are developed as model membrane systems for the structural study of biomolecules in interaction with lipid membranes. In collaboration with the group of E. Dufourc (CBMN) who has been studying these nano-objects by NMR, we have performed structural studies of bicelles (by different lipids, DMPC-DCPC or DBBPC-DCPC) to demonstrate how lipid structures can influence the structure of bicelles and their orientation and dynamics under magnetic field (Biophys. J. 2002, 2007). The contribution of our group in this study focuses on the structural study of bicelles by SAXS and freeze fracture electron microscopy.

Freeze fracture TEM images of bicelles

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(Left) 2D scatttering pattern of o SAXS of nonn oriented bicelles (left) and orriented bicelless from DMPC/D DCPC and TBB BPC/DMPC after 20 minuttes in magneticc field at 35°C.(BioPhys J 20007) (right)Two types of scatterring pattern off bicelles fitted with w a form factor of lipid bilayers; Bicellles a) DMPC/D DCPC, b) TBBP PC/DCPC

5 significaant publicattions: 1. Oda R, Artzner, A F., Huc, H I., Lagueerre, M. Mollecular structu ure of self-assembled chiraal nano-ribbo ons and nano-tubules reevealed in the hydrated statte J. Am. Cheem. Soc (2008 8) 130 (44), 144705–14712 2. Delclos, T. Aimé, C. Pouget, P E. Brrizard, A. Hucc, I. Delville, M.-H. and Oda R. Individdualized Silicaa Nanohelicess N s Using Lipid dic Self-Assem mblies Nanoleetters (2008), 8, (7), 1929-and Nanootubes: Tuning Inorganic Nanostructures 1935. S Milochau, A., Castano, S., Sbi, W., Manigand, M C.,, Laguerre, M., M Desbat, B.,, 3. Yassine, W., Taib, N.,, Federman, S., ween α-helix an nd β-sheet connformation off a transmemb brane domain. Oda, R., and Lang, J., Reversible trransition betw BA) – Biomem mbranes, ASAP 2009 Biochimiica et Biophyysica Acta (BB 4. Loudet, C., C Manet, S.,, Gineste, S., Oda, R., Achhard, M. F., an nd Dufourc, E. E J. (2007) B Biphenyl bicelle disks alignn perpendiccular to magnnetic fields on large temperaature scales: A study combbining synthessis, solid-state NMR, TEM,, and SAX XS, Biophysicaal Journal 922, 3949-3959. 5. Brizard A, A Aime C, Laabrot T, Huc I., I Berthier D.., Artzner F., Desbat B., Odda R. Counterr-ion, temperaature and timee modulatioon of nanomeetric chiral ribbbons from geemini-tartrate amphiphiles. J. Am. Chem m. Soc (2007 7), 129, 3754-3762.

Teaching, research & education n training, diffusion of o science (ccongress, w workshops, scientific open days etc.) Courses at Master M 2 leveel in the deppartment of Chemistry, C mo odule chimie moléculaire de vivants, « Assemblagee lipidique » 144 hours ETD//an. Courses at Master M 2 level in the departm ment of bioloogy, module Biologie B Celluulaire & Physiiopathologie « Assemblagee lipidique » 2 hours ETD/ann. Partial organnization: 1st Jointed Fukuoka-Bordeaux Conference ― New Trendds in Molecullar and Materials Sciences,, June 2006 pan, jan. 20077 (240 particippants) Partial organnization: Japann France Fronttier of Sciencee, Shonan, Jap Full organizaation: “Fête dee la science: Des D bulles tenssioactives” Occtober 2007. Partial organnisation: Systèèmes Avancéss à base de Flluides Organiisés (SAFO) – Workshop, Bordeaux, May 2007 (1500 participants)

c of the t technological platfform Participattion in the context The group has expertise andd ensures thee maintenancce and functiions of severral small to medium-sizee B) such as sm mall angle x-rray scatteringg, light scatteering, circularr equipments of the platforrm for UMS 3033 (IECB dichroism, peptide syntheesizer, but alsoo water purifiier, lyophiliseer, and centriffuge. All of thhese apparatus are used byy e groupps (academicss and industriaals). several groupps of IECB buut also by the external

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TEAMS OR SCIENTIST WILLING TO JOIN CBMN FOR THE NEXT QUADRENNIAL As it will be presented in the project part 4 new teams are proposed to join the UMR. There are also scientists from other laboratories who wish to join our laboratory. One of the teams (Patrick Cottin’s) will be merged into Oliviers Lambert’s team. Their respective scientific reports (shortest versions) appear in the following • Team Gilles GUICHARD: Biomimetic Chemistry o (with Karine GIONNET)

• Team Fernando LEAL-CALDERON: Transfer & Flow of Energetic Fluids o (with Pascale SUBRA-PATERNAULT, Patrick SAUVANT, Chrystel FAURE, Claude ATGIE)

• Team Patrick COTTIN: Proteolysis Growth & Muscle Development (Joining Olivier Lambert’s Team) • Team Jean-Pierre AIME: Molecular Physics & Hertzian Optics • Scientists who join CBMN-labeled teams o Cyril PETIBOIS joining Bernard DESBAT’s Team o Victor MAURIZOT joining Ivan HUC’s Team

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TEAM « BIOMIMETIC CHEMISTRY GROUP – IBMC UPR 9021 CNRS, STRASBOURG » Team Leader: Gilles Guichard, Research Director, CNRS. Team Composition (2006-2009)*: Christophe Pardin, Post-Doctorant CNRS Gersande Léna Lucile Fischer, Doctorante Marie-Charlotte Lechner, doctorante

Nagendar Pendem, Post-doctorant UdS Nathalie Trouche, Doctorante Joel Boeglin, Doctorant Edith Chardon, Doctorante

Aude Violette Sébastien Wieckowski, Doctorant Paul Claudon, Doctorant Karen Lamour, AI CDD

M.Sc. trainees (6 months): 3 *Jean-Paul Briand, DR2 CNRS was also associated to the Biometic Chemistry group in Strasbourg over this period. Because he remains a UPR 9021 member, his research activities have not been included in this report.

Team Production (2006-2009): Publications, peer reviewed, (ACL) Publications, peer reviewed, not yet in bases (ACLN) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI) Oral communications in congress (COM) Poster communications in congress (AFF)

22 4 5

Books or Book chapters (OS) Congress/Workshop organization Patents

1 4 4

8

Defended PhD Theses or HDR

7

5 11

Grants : public & private k€/year

128 k€

Scientific activity Résumé L’équipe de Chimie Biomimétique s’intéresse à la conception et la synthèse de molécules d’intérêt immunologique/pharmacologique ainsi qu’au développement méthodologiques en chimie peptidomimétique (« foldamères », multivalence). Les compétences acquises et développées par l’équipe s’étendent de la chimie organique de synthèse à l’analyse conformationnelle de biomolécules en passant par les techniques de chimie combinatoire. Quatre grandes thématiques ont été / sont développées en parallèle dans l’équipe: i) glycopeptides dérivés du collagène de type II et polyarthrite rhumatoïde, ii) chimie protéinomimétique et architectures multimériques, iii) Hélices biomimétiques : de la structure à la fonction, et iv) exploration de la diversité chimique à partir de dipeptides. En s’appuyant sur des collaborations avec des équipes de l’unité et de l’extérieur, ces recherches ont permis l’identification et la caractérisation de molécules à activité immunopotentiatrice, proapoptotique, antimicrobienne, ou inhibitrice des phospholipases A2 sécrétées (sPLA2), des enzymes importantes pour l’immunité innée et l’inflammation . Abstract In the biomimetic chemistry team, we have been interested in designing and synthesizing organic molecules with functions in biology (e.g. immunology) by integrating multiple approaches in peptidomimetic chemistry (e.g. programmed self-organization, multivalency, combinatorial chemistry). Over this period, four research programs have been developed : i) synthesis of glycopeptides derived from type II collagen and their role in arthritis, ii) proteinomimetic chemistry / multivalent architectures, iii) biomimetic helices : From structure to function, and iv) expanding chemical diversity from simple dipeptides. In collaboration with biologists of the laboratory and from other groups, this work has led to the identification and the characterization of molecules with immunomodulating; proapoptotic, or antibacterial activity as well as to a new class of inhibitors of secreted phospholipases A2 (sPLA2), a family of enzymes important for inflammation.

• Recognition of glycosylated peptide epitopes by T cells in autoimmunity In type II collagen (CII), lysine residues can undergo hydroxylation followed by glycosylation with either a β-Dgalactopyranosyl or an α- D-glucopyranosyl-(1→2)-β- D-galactopyranosyl moiety. Recent results suggest that a T-cell response towards glycosylated peptide fragments of CII could play an important role in the development of rheumatoid arthritis (RA). In particular, the CII-derived peptide encompassing residues 256–270 and containing a galactosylated (2S,5R)-5-hydroxylysyl (Gal-5-Hyl) residue at position 264 (Figure), has been identified as an immunodominant T-cell epitope in collagen-induced arthritis (CIA), a mouse model for RA.

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The development of efficient routes to Gal-5-Hyl building blocks and synthetic CII-derived glycopeptides by us and others has been instrumental in studies aimed at understanding the role of lysine hydroxylation and glycosylation in CIA. We have shown that enantiopure 5hydroxy- and 4-hydroxy-6-oxo-1,2-piperidinedicarboxylates are extremely versatile building blocks for the synthesis of 5and 4-hydroxylysine derivatives, respectively. To further explore the fine specificity of bCII-reactive T cells involved in the initiation and/or regulation of CIA, we have designed and synthesized new series of analogues of CII immunodominant glycopeptide [β-D-Gal-(5R)-5Hyl264]CII(256–270), carrying diverse modifications at the critical hydroxylysine (Hyl) 264 side chain. Responses of anti-CII T-cell hybridomas to this panel of CII256–270 analogues incorporating Gal-Hyl264 with a modified side chain were determined (coll. C. Fournier, Institut Cochin, Paris). Our data provided new experimental evidence that integrity of both galactose HO-4 and hydroxylysine side-chain primary amino groups are mandatory for activation of anti-Gal-Hyl264 TCRs. Although the chemistry side project has now been stopped, there is still some interest to evaluate whether the modified glycopeptides could have a protective/aggravating effect in the CIA model.

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CII-derived glycopeptide immunodominant T-cell epitope and designed analogues. Model of interaction between [β-D-Gal(5R)-5-Hyl264]CII(256–270) with the MHC molecule and T cell receptor (Glatigny et al. Arthritis Res. Ther. 2007, Chaloin et al. Org. Synth. 2008, Marin et al. Eur. J. Org. 2008).

• Multivalency as a tool to target and activate cell-surface receptors of TNFR family. Ligand-induced oligomerization is an important process for activating cell surface receptors such as tumor necrosis factor (TNF) receptor superfamilies. TNF-R family members can transduce a variety of intracellular signals culminating in cell proliferation, differentiation, survival, and death. Ligands of the TNF family self-assemble around a three-fold symmetry axis to form non-covalent homotrimers that can each bind three receptor molecules. The geometry of the resulting hexameric ligand-receptor 3:3 complex is favorable to the formation of the intracellular signaling complex. We were interested to develop Synthetic Multivalent Ligands (SMLs) as tunable probes to modulate biological processes and to dissect signalling pathways associated with TNFRs. In collaboration with S. Fournel (IBMC, Strasbourg) and more recently with H Gronemeyer (IGBMC, Illkirch) and O. Micheau (INSERM U866, Dijon), we have been focusing on the design of trimeric ligands targeting CD40 and TRAIL-R2, two TNFRs playing a major role in immunity and apoptosis, respectively. Early work led to the design of C3-symmetric peptides constructed on macrocyclic plateforms (e.g. cyclic D,L-hexapeptides, cyclic β-peptides) that were shown to mimic at least partially the biological effects of CD40L (activation of dendritic cells,…). We recently extended this approach to a new series of synthetic multimeric molecules incorporating peptide Multivalent display as a strategy to activate TNFRs. sequences reported to bind TRAIL-R2. Covalent dimeric and trimeric Exemples of trimeric platform investigated (Trouche versions were found to bind TRAIL-R2 with increased avidity et al. J. Am. Chem. Soc. 2007). compared to monomers and more importantly, to trigger the TRAIL apoptotic pathway in various cancer cell lines. The agonistic activity observed provide evidence that these peptides are able to recognize TRAIL-R2 in a biologically relevant manner mimicking natural TRAIL.

• Controlling folding and self-assembly with short chain urea-based peptidomimetics H-bonds provide a very versatile way to create intrastrand (either local or remote) connections useful to control folding in new oligomeric materials inspired by biopolymers. Pioneered by the work of Seebach and Gellman, non natural oligoamides based on β- and γ-amino acids now represent the quintescential peptidomimetic foldamers. The pattern of intrastrand H-bond interactions in helix forming oligoamide foldamers can be further manipulated trough insertion of heteroatoms in the backbone to generate non-amide linkages (e.g., N-oxyamide, hydrazide, urea). The urea group which shares a number of features with the amide linkage, i.e. rigidity, planarity, polarity, and hydrogen bonding capacity is an interesting surrogate.

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Local folding induced by the urea fragment in short-chain oligomers Examination of local folding and H-bonding patterns in model compounds can be extremely informative to gain insight into the propensity of longer-chain oligomers to adopt specific folding patterns based on remote interactions. Using a combination of experimental techniques (i.e. X-ray diffraction, FT-IR absorption Nearest neighbour interactions induced by the urea moiety in model ureidopeptides. Compounds and NMR spectroscopy) and A and B are, analogues of β- and γ-peptides, respectively. Whereas the formation of a C8 urea turn theoretical calculations at the stabilized by 1←3 H-bond interaction is favoured in the A series, folding is disfavoured in the B series. Calculated models are shown (Fischer et al. Org. Biomol. Chem. 2008). density functional theory (DFT) level (coll with Romuald Poteau, Univ. Toulouse), we have examined the local folding patterns induced by the urea fragment in short-chain aza analogues of β- and γ-amino acid derivatives (exemplified by molecules A and B in the associated Figure). We found that the urea-turn, a robust C8 conformation based on 1←3 H-bond interaction, is largely populated in model ureidopeptides obtained by replacing the α-carbon of a β-amino acid by a nitrogen (A type). This H-bonding scheme is likely to compete with remote H-bond interactions, thus preventing the formation of secondary structures based on remote intrastrand interactions in longer oligomers. In related oligomers obtained by the addition of a methylene in the main chain, nearest-neighbour H-bonded interactions are unfavorable i.e. the corresponding C9 folding pattern is hardly populated. In this series, folding based on remote intrastrand interactions becomes possible for longer oligomers. We present spectroscopic evidence that tetraureas (B type) are likely to be the smallest unit capable of reproducing the Hbonded motif found in 2.5-helical N,N’-linked oligoureas. NMR Approaches to improve monitoring and description of the helical conformation of short chain oligoureas The development of foldamers for biological and biomedical applications requires both precise structural information and appropriate methods to detect folding. With the aim to speed-up the characterization of helical oligourea foldamers, we have explored the possibility to directly monitor helix formation of oligoureas bound to a solid support at different stages of chain growth by using high-resolution magic-anglespinning (HRMAS) NMR spectroscopy (coll K. Elbayed, Univ Strasbourg and M. Piotto, Bruker). HRMAS NMR spectroscopy is a powerful technique to investigate the structure and mobility of immobilized species including natural oligomers, such as α-peptides. Insight into the conformational preference of oligoureas was gained by monitoring a simple descriptor of conformational Monitoring helical folding of oligoureas during chain length elongation homogeneity (e.g., chemical shift difference by HRMAS NMR. Left : Structure of DEUSS and DEUSS-immobilized oligoureas. Right : Solvant effect on chemical shift differences between between diastereotopic main chain CH2 protons) at diasterotopic αCH2. protons of 6-mer oligourea (Violette et al. Chem Eur. different stages of oligourea chain growth on a J. 2008) solid support swollen in solvents of low to high polarity. One advantage of the method is that only minute amount of material is required. By using DEUSS, a perdeuterated poly(oxyethylene)-based resin, the intensity of the signal arising from the solid support (oxyethylene protons) could be reduced by two orders of magnitude (i.e. to the level of attached molecules) compared to its nondeutarated version. These experiments allow us to determine the influence of factors, such as chain length, side chains, and surrounding environment, on secondary structure On-bead NOESY spectra of high quality and with resolution comparable to that of liquid samples were obtained for longer oligomers (hexaurea), thus allowing detailed structural characterization. Early calculations with NMR constraints extracted from homonuclear spectra provided compelling evidence that oligoureas adopt a stable right-handed helical fold. More recently, NMR at 13C natural abundance was used to further improve the quality of the structure calculations (coll. E. Miclet, UPMC, Paris). Conformation-dependent vicinal couplings 3JHH from diastereotopic proton resonances are generally difficult if not impossible to obtain with standard NMR experiments. A CH2-TROESY derived sequence was introduced to precisely measure the missing 3JHH couplings. Applied to a 9-mer oligourea, this pulse scheme provided 19 previously unobserved scalar coupling measurements. Incorporation of these couplings in the simulation annealing protocol was accompanied by a ca 30% decrease of the root mean square deviation (RMSD) obtained over an ensemble of 20 structures. This approach developed for 84

oligoureas is likely to be of practical value to increase the quality of NMR-based structures when applied to other classes of peptidomimetic folding oligomers bearing backbone methylene groups (e.g. β-, γ- and δ-peptides, peptides as well as β- and γ-aminoxy acids). From Structure to function: mimicking helical host-defense peptides The finding that enantiopure N,N’-linked oligoureas adopt a stable and regular 2.5-helical fold suggested to us that oligoureas could serve to mimic the structure and function of α-helices. Concurrently, we started to investigate membrane disruption properties and antimicrobial activity of oligoureas designed to mimic globally amphiphilic cationic α-helical host-defense peptides (coll. H. Monteil, Univ. Strasbourg). Compared to conventional antibiotics, they possess a low potential for the induction of bacterial resistance, which makes them attractive candidates for the development of antimicrobial agents. Noteworthy, designed amphiphilic oligoureas as short as 8 residues (see Figure) were as active as mellitin (a cytotoxic peptide from been venom with antibacterial properties) against gram-negative and gram-positive bacteria Optimized calculated structure of a (including methicillin-resistant Staphylococcus aureus (MRSA)) and yet displayed urea nonamer using methylene vicinal couplings extracted form the CH2selectivity for prokaryotic versus mammalian red blood cell membrane..Our results TROESY. Superimposition of the 20 suggest that antibacterial oligoureas may be acting by a mechanism involving best structures (Guichard et al. Magn. membrane permeation. Circular dichroism studies in hepes buffer saline (HBS) and Reson. Chem. 2008) in the presence of negatively charged phospholipid vesicles as model membranes are consistent with increased helix population in lipidic environment. The present work is the first attempt to design bioactive oligoureas based on their propensity to adopt helical structures. Studies aimed at improving the potency, the selectivity, as well as gaining further insight into the antibacterial mechanisms of 2.5-helical oligoureas are underway. Overall the strong helix folding propensity, together with the diversity of available side chain appendages and resistance to protease degradation, makes the oligourea backbone a promising candidate for biomedical applications. Self-assembling hybrid amide/urea-based cyclic systems Antibacterial oligoureas. Sequence of 8-mer oligourea designed to form Synthetic peptides, owing to their diversity in an amphipathic helical structure and corresponding CD spectra in size, conformations and appended functionalities, aqueous and lipidic environments showing the increased propensity for helix formation in the contact of phospholipids (Violette et al. Chem. represent highly versatile elements for the Biol. 2006) construction of bio-inspired H-bonded tubular nanostructures. Likewise, we have shown that macrocyclic oligoureas display a strong propensity to form various types of H-bonded columnar and tubular stacks. Self-assembling properties of hybrid urea/amide macrocycles generated by cyclooligomerization of chiral dipeptidederived building blocks have been investigated recently. A high level of hierarchical and directional control can been achieved in these systems.

• . Exploring dipeptide-derived molecular diversity and applications In the field of peptidomimetic chemistry, small and medium rings derived from peptides are of particular interest owing to their facile access, the chemical and stereochemical diversity of amino acids, and enhanced diversity resulting from postcyclization appending operations. In addition, such molecules are potentially useful to constrain intrinsically short linear peptide chains and for mimicking local folded polypeptide structures, such as β-turns. The 1,3,5-triazepan-2,6-dione system We have reported the 1,3,5-triazepan-2,6-dione system as a novel, conformationally restricted, and readily accessible class of dipeptidomimetics. The synthesis of this densely functionalized skeleton is achieved in only four steps from a variety of simple linear dipeptide precursors. To extend the practical

X-ray structures of representative members of the 1,3,5-triazepan-2,6dione family (Muller et al. J. Med. Chem. 2006, Schaffner et al. Chem. Commun. 2006, Lena et al. Chem Eur. J. 2006, Lena & Guichard Curr. Org. Chem. 2006).

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value of 1,3,5-triazepane- 2,6-diones, a general polymer-assisted solution-phase synthesis approach amenable to library production in a multiparallel format has been developed. The conformational preferences of the 1,3,5-triazepan-2,6dione skeleton were investigated in detail (voir Figure) and compared to the related 2,5-diketopiperazines (DKP). Molecular and structural diversity can be increased further through post-cyclization appending operations at urea nitrogens. Towards Inhibitors of secreted Phospholipase A2 (sPLA2s) Because they are structurally diverse and rapidly accessible in a library format from dipeptides, 1,3,5-triazepan-2,6diones have a strong potential for use in biological screens. In collaboration with D. Rognan (Fac. Pharma, Illkirch), we have investigated the application of an inverse docking method to the identification of the most likely targets of a small panel of triazepandiones. A collection of 2,150 druggable active sites from the Protein Data Bank was screened by highthroughput docking to identify putative targets for five representative molecules of a combinatorial library sharing a 1,3,5-triazepan-2,6-dione scaffold. Five targets were prioritized for experimental evaluation by computing enrichment in individual protein entries among the top 2% scoring targets. Out of the five proposed proteins, sPLA2 was shown to be a true target for a panel of 1,3,5-triazepan-2,6-diones which exhibited micromolar affinities toward two human sPLA2 members, i.e. human group V and X sPLA2s. Interestingly, these two sPLA2s and other members of this emerging family of enzymes have become attractive therapeutic targets in a number of inflammatory diseases including asthma, rheumatoid arthritis, and atherosclerosis.

5 significant publications: 1. 2. 3. 4.

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A. Violette, N. Lancelot, A. Poschalko, M. Piotto, J. P. Briand, J. Raya, K. Elbayed. A. Bianco, G. Guichard (2008) Exploring helical folding of oligoureas during chain elongation by HRMAS NMR. Chem. Eur. J. 14, 3874-3882.. Trouche, N., Wieckowski, S., Sun, W., Chaloin, O., Hoebeke, J., Fournel, S., Guichard, G. (2007) Small Multivalent Architectures Mimicking Homotrimers of the TNF Superfamily Member CD40L: Delineating the Relationship between Structure and Effector Function. J. Am. Chem. Soc. 129,13480-92. P. Muller, G. Lena, S. Bezzine, E. Boilard, M. Lazdunski, G. Lambeau, G. Guichard, D. Rognan. In silicoguided target identification of a scaffold-focussed library: 1,3,5-Triazepane-2,6-dione as novel phospholipase A2 inhibitors J. Med. Chem. 49, 6768 – 6778. G. Lena, E. Lallemand, A. C. Gruner, J. Boeglin, S. Roussel, A.-P. Schaffner, A. Aubry, J.-F. Franetich, D. Mazier, I. Landau, J.-P. Briand, C. Didierjean, L. Rénia, G. Guichard. (2006) 1,3,5-Triazepan-2,6-diones as Structurally Diverse and Conformationally Constrained Dipeptide Mimetics. Identification of Malaria Liver Stage Inhibitors From a Small Pilot Library. Chem Eur. J. 12, 8498-512. A. Violette, S. Fournel, K. Lamour, O. Chaloin, B. Frisch, J.-P. Briand, H. Monteil, G. Guichard. (2006) Mimicking helical antibacterial peptides with non-peptidic folding oligomers. Chem. Biol. 13, 531-538..

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Teaching Chemical biology and peptidomimetic chemitry (Master 1 & 2 level), Université de Strasbourg Full congress organization : 16ème Réunion du Groupe Français des Peptides et Protéines (GFPP), Villé, France, may 2009, (~200 delegates) Partial congress organization : 12ème Rencontres en Chimie Organique Biologique (RECOB), Aussois, France, mars 2008, (~120 delegates) Partial congress organization: 15ème Réunion GFPP, Dinard, France, may 2007 (~160 delegates) Partial congress organization: 11ème RECOB, Aussois, France, mars 2006, (~120 delegates).

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Team Member: Dr. Karine GIONNET, Chargée de Recherche INSERM Team Composition (2006-2009): Groupe de Chimie Bio-organique, UMR CNRS 5084 Pr. Gérard Déléris, Pr Université Bordeaux 2 (Team Leader) Dr. Karine Gionnet, CR INSERM Dr. Victor Maurizot, CR CNRS Dr. Cyril Petibois, MCU Univeristé Bordeaux 2

Dr. Sandra Rubio, AI Université Bordeaux 2 Marc-Elias Bakleh (doctorant) Sébastien Lavielle (doctorant)

Katia wehbe (doctorante) Karima Belbachir (doctorante)

El-Farrouck Moustoifa (doctorant)

Razia Noreen (doctorante)

Anis alouini (doctorant)

Personnal Production (2006-2009): Publications, peer reviewed, (ACL) Poster communications in congress (AFF)

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Oral communications in congress (COM) Defended PhD Theses or HDR

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Scientific activity Les travaux présentés ci-après ne concernent que ceux dont j’ai été personnellement impliqué durant les quatre dernières années au sein du groupe de chimie bio-organique de l’UMR CNRS 5084. Scientific activities presented below concern only those by whom I was personally implied during the last four years within the bio-organic chemistry group of the UMR CNRS 5084. Résumé Depuis ces dernières années l’angiogenèse est apparu comme une cible d’un très grand intérêt pour le développement d’agents d’imagerie ou de thérapie des cancers, mais également pour les infarctus. Toutefois, le développement de nouvelles molécules ciblant l’angiogenèse reste à l’ordre du jour. Dans ce contexte, nous avons développé le cyclo-VEGI, un cyclo-peptide de 17 acides aminés, qui inhibe la liaison du Facteur de Croissance de l’Endothélium Vasculaire (VEGF) à son récepteur KDR (IC50 de 1.5 µM). Afin d’utiliser au mieux les potentialités d’une telle molécule, nous avons entrepris diverses fonctionnalisations pouvant conduire à de nouveaux vecteurs sélectifs à des fins diagnostiques ou thérapeutiques. Parmi ces fonctionnalisations, la formation de dimères a montré une nette amélioration dans l’inhibition de la liaison du VEGF à son récepteur. De même, le couplage entre une porphyrine et le peptide a conduit à la formation de photosensibilisateurs hybrides pour une photothérapie dynamique (PDT) sélective des cellules cancéreuses. Encore, l’introduction d’une chaîne polyfluorée sur le peptide, couplée à l’utilisation des analyses par faisceau d’ions, nous ont permis d’imager des zones d’activité angiogénique à l’échelle cellulaire, non permise jusqu’ici par la Tomographie par Emission de Positrons (TEP). Enfin, nous avons débuté la synthèse de nanoparticules de fer fonctionnalisée présentant une très grande stabilité au milieu biologique. Abstract For these last years, angiogenesis appeared as a promising target for the development of imaging or therapeutic agents in cancer, but also for infarcts. However, the development of new molecules targeting angiogenesis is of great interest. In this context, we developed the cyclo-VEGI, a 17 amino acids cyclo-peptide, which inhibits the binding between the vascular Endothelial Growth factor (VEGF) and his receptor KDR (IC50 of 1.5 µM). To use in best the potentialities of such a molecule, we began miscellaneous fonctionalisations which can lead to new selective vectors in diagnostic or therapeutic purposes. Among these fonctionalisations, dimers showed a net improvement in the inhibition of the binding between VEGF and his receptor. Also, the coupling between a porphyrine and the peptide led to the formation of crossed photosensitizers, which can be used for a selective dynamic phototherapy (PDT). Still, the introduction of a polyfluorinated chain on the peptide, coupled with the use of ion beam analyses, allowed us to image angiogenic activity areas at a cellular scale, not reached by Positon EmissionTomography (PET). Finally, we began the synthesis of functionalized iron nanoparticles presenting a very good stability in the biological media.

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Conception of nanovectors for angiogenesis targeting During these last years, it clearly appeared that angiogenesis constitutes a particularly relevant therapeutic target in cancer. Its inhibition is crucial in cancerology where it was proved that a tumor could not exceed a restricted size without angiogenesis; the blood flow then ensure the tumor supply of the nutrients and metabolic waste disposal. Tumors, primary as well as metastases, are the seat of great neo-angiogenic activity. However, interest of this phenomenon is much broader such as in infarcts, cerebral or of the myocardium. These data opened the door to an active pharmaceutical development based on angiogenesis principles. Nevertheless, the development of new molecules targeting angiogenesis is not only at its

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Scheme 1 : Inhibition test of our dimers to the binding between VEGF and its receptor KDR

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beginnings but will have doubtless a promising future. In this area, we have developed cyclo-VEGI, a peptide which inhibits binding between Vascular Endiothelial Growth Factor (VEGF) and its receptor KDR(IC50 of 1.5 µM). In order to use it as a selective nanovector for therapeutics or diagnostics, we functionalized cyclo-VEGI. In this field, first results have highlighted that the presence of an additional basic function, introduced by an arm, increased by a factor 10 the affinity of peptide with respect to KDR (IC50 of 0.16 µM). We thus initiated the synthesis of cyclo-VEGI derivatives presenting polyamine arm. Two strategies were considered: one on support and one in solution. Unfortunately, the two ways don’t lead to the desired product. In addition, various studies showed that the connection between a ligand and its receptor was increased when ligand was synthesized in polymeric form, thus increasing affinity by cooperativ effect. Based on these results, we wished to couple cyclo-VEGI with various families of multivalents ligands. Indeed, we showed during a first test, that the distance between two antiangiogenic monomers (in our case the cyclo-VEGI) is of great importance (scheme 1). It was then necessary to design and synthesize ligands of variable size and rigidity to obtain an optimum affinity of the multimeric peptide. We thus synthesized a family of dimers starting from polyethylene glycol family, presenting various sizes. The structural analyses are currently in hand and after confirmation, the tests of affinity will be initiated. In addition, tumoral vascularization imaging is important for diagnosis and therapeutic follow-up. Today, one of the most sensitive and noninvasive imaging method is Positron Emission Tomography (TEP). TEP has a relatively poor spatial resolution (2-3 mm for 18F) which doesn’t allow imaging of very small tumors. On the other hand, the principal marker is a glucose derivative, namely 18-2-fluoro2-déoxyglucose (18FDG). This last, like glucose (natural substrate of the cells), is nonspecific of tumoral cells, although these last have an increased need of FDG, compared to normal cells. Hence indirect analytical techniques were required to assess the localization of more selective fluorinated compounds at a micrometric scale. This multimodality imaging approach should provide accurate Scheme 2: fluorine distribution in CAM treated with F13information on the metabolic activity of the target tissue. So, cyclo-VEGI we proposed to use ion beam analysis to image fluorine 19 of synthesized fluorinated biomarkers, to determine their distribution with a tissue, cellular, or subcellular scale. These analysis will allow to validate the relevance of the biological target selected and asserted by TEP. This work is completed in close cooperation with R. Ortega team, in CNRS U5084 unit, which develops ion beam analysis techniques for the quantitative determination of the chemical elements distributions in cells and tissues. We had already confirmed that we could detect fluorine 19 at the cellular level by this technique, using FDG as model. As detection

Scheme 3: Hybrid photosensitizers threshold of fluorine 19 was high, we had to synthesize a polyfluorinated derivative of glucose (G-polyF) to improve its detection. We also synthesized F13-cyclo-VEGI, a polyfluorinated derivative of cyclo-VEGI, in order to propose a specific marker of the blood capillaries present in angiogenic tissues. These three fluorinated derivatives were then injected into the vascular network of chicken embryos to study their distribution on the chorioallantoid membrane (CAM) or within U87 glioma implanted onto the CAM. Results obtained for both glucose derivatives showed that fluorine is well detected in tissues, but their distribution isn’t specific. Results obtained for F13-cyclo-VEGI are much more promising, showing specificity 2 to 3 times higher for blood capillaries versus surrounding tissues (scheme 2). We have also developed, for the first time, photosensitizing hybrid molecules constituted by meso-tritolylporphyrines carrying cyclo-VEGI peptide. These photosensitizers could be used for cancer diagnosis or tumor treatment using photodynamic therapy (PDT). Syntheses were realized with a convergent approach, coupling porphyrins and peptides by the formation of an amide bond, or by the formation of a 1,4-disubstituted triazole with a click-chemistry (scheme 3). This work was completed in collaboration with the chemistry laboratory of natural substances, University of Limoges, qualified in the development of porphyrins. The Fe3 O4 @SiO2 @TESD Fe3 O4 @TESDTEG and first tests of affinity are currently in progress. Lastly, TEG,Bar : 20 nm Fe3 O4 @APTS stability in HCl studies showed that the covalent grafting of biomolecules Scheme 4 : MET of decoratied nanoparticules (A) and their stability on iron oxide nanoparticules Fe3O4@SiO2 was possible in HCl 1M (B) without deteriorating the accessibility and the molecular

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recognition of enzymes (7), but also that they could be internalized in cells (8). So, if such particles are coupled with the cycloVEGI, they could then be uptaken by tumoral endothelial cells and thus be employed as nanovectors for drugs to target angiogenisis. However, several synthesis methods of these nanoparticules were described but the size and the physical properties are generally not controlled. We thus applied the sonochimistry, which enabled us to develop a method for the synthesis of monodisperse nanoparticules Fe3O4, superparamagnetic, and slightly aggregate when recover by the silica. These particles were then functionalized by an arm organized in Self Assembled Monolayer (SAM) on our nanoparticules surface and having at its end a triethylene glycol which would prevent opsonisation of iron oxides particules in blood. Decorated particles were then obtained, with a diameter lower than 20 nm, thus sufficiently small to circulate in the blood-vessels and to penetrate in the cells, and which are protected by SAMs, stabilizing them and making them furtive in the biological environment during 24 hours (scheme 4).

5 Significant publications: 1. 2. 3. 4. 5.

Gonçalves, M., Estieu-Gionnet, K., Berthelot, T., Laïn, G., Bayle, M., Canron, X., Betz, N., Bikfalvi, A., and Déléris, G. (2005) Design, synthesis and evaluation of original carriers for targeting VEGF receptor interactions. Pharm. Res 22, 1411-1421 Gonçalves, M., Estieu-Gionnet, K., Laïn, G., Bayle, M., Betz, N., and Déléris, G. (2005) On-resin cyclization of e head-to-tail cyclopeptide using an allyldimethylsilyl polystyrene resin pre-loaded by metathesis. Tetrahedron 61, 7789-7795 Clochard, M.-C., Cuscito, O., Berthelot, T., Betz, N., Bittencourt, C., Pireaux, J.-J., Goncalves, M., Gionnet, K., and Deleris, G. (2008) Surface specific peptide immobilization on radiografted polymers as potential screening assays for antiangiogenic immunotherapy. Reactive & Functional Polymers 68, 77-908. Devès G., Roudeau S., Carmona A., Lavielle S., Gionnet K., Déléris G., Ortega R. (2009) Fluorine microimaging and quantification using nuclear reaction analysis: A tool for validating tissue distribution of positron emission tomography tracers. Applied Physics letters 95, in press. Bakleh M.E., Sol V., Estieu-Gionnet K., Granet R., Déléris G., Krausz P. (2009) An efficient route to VEGF-like peptide porphyrin conjugates via microwave-assisted “Click-Chemistry”.tetrahedron in press.

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Every year, teaching intervention in master1 and 2 in “Master de Biochimie Interface Chimie Biologie Physique Chimie”, université Bordeaux1/Bordeaux2.

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Dispersed Media Laboratory [JE n° 2389 from 2003 to 2008. Incorporated TREFLE laboratory (UMR CNRS 8508) in January 2008] Team Leader: F. Leal Calderon, Professor, Polytechnic Institute of Bordeaux (IPB). Team Composition (2006-2009): Maud Cansell, Professeur, IPB Fernando Leal Calderon, Professeur, IPB Julien Monteil, IGE, IPB Emilie Vaique, Doctorante

Nicolas Drelon, Doctorant Marie Bonnet, Doctorante Céline Villatte, Doctorante Samy Chadel, Doctorant

Honda Thongdeng, Doctorant Carolina Garcia Darras, Doctorante Leslie Couëdelo, Doctorante Damien Habouzit, Doctorant

B.Sc. and M.Sc. trainees (stay between 2 and 6 months): 8

Team Production (2006-2009): Publications, peer reviewed, (ACL) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI) Oral communications in congress (COM) Poster communications in congress (AFF)

17 1

Books or Book chapters (OS) Congress/Workshop organization

2 2

8

Patents

1

18 4

Defended PhD Theses or HDR Grants : public & private k€/year

2 35 k€

Scientific activity Abstract We studied the stability and functionality of soft colloids like simple emulsions, multiple emulsions, and phospholipid vesicles. Our general strategy consisted of : (i) optimizing the formulation and fabrication process so that to obtain materials with reproducible properties and a well defined colloidal size (monodisperse colloids) (ii) studying the kinetic stability under storage conditions and (iii) probing the functionality of the colloids under well defined conditions (gastrointestinal conditions, for example). The colloids are stabilized by low-molecular weight surfactants, naturally occurring polymers (proteins) or solid colloidal particles. Emulsions comprising partially crystallized droplets The design of materials with tunable properties represents a major challenge of current physical research. Methods which exploit self-assembly under thermal equilibrium are certainly viable, but non-equilibrium processes may provide better control and versatility. Typical examples can be found in emulsion science where a very large set of materials, from low viscosity fluids to highly elastic pastes, can be obtained and preserved for a very long period of time. We examined a novel route to obtain gels based on the unrelaxed coalescence of crystallized droplets in a primary oil-in-water emulsion.. Under the effect of a gentle thermal treatment, fluid emulsions turned into hard gels whose evolution was arrested by the solid nature of the droplets. The kinetic evolution of the bulk elastic modulus G’ during the thermal treatment was interpreted within the frame of percolation theory. The experimental data supported the conclusion that coalescence involves irreversible bridging between crystallized and melted patches in the thin liquid films. Stimulus responsive solid stabilized emulsions Pickering or solid-stabilised emulsions are surfactant-free metastable dispersions of two non-miscible fluids kinetically stabilised by colloidal particles. Such emulsions exhibit remarkable kinetic stability and allow reducing the use of hazardous surfactants. The design of emulsions which can evolve under the effect of an external stimulus is a real challenge, and the possibility of destabilizing them “on demand”, by simply modifying a compositional variable in the system is a topic of current investigation. An abundant literature is especially devoted to pH-sensitive particulate emulsifiers, where a moderate pH variation may induce a transition between the initially dispersed state and partially or total macroscopic separation of the oil and water phases because of particle desorption. The aim of our studies was somewhat different: we used pH- and salt-responsive particles that remained adsorbed as a mean to trigger the average droplet size and to control the relative kinetic stability of the emulsions. We studied oil-in-water emulsions stabilised by pH-sensitive colloidal silica or latex particles. Depending on the composition of the continuous phase, the same type of particles and the same emulsification process led to emulsions characterised either by large drops densely covered by the particles, or to small droplets weakly covered. The two kinetically stable states could be tuned reversibly by using pH or salinity as compositional stimuli. We characterized the emulsions behaviour in these two limiting cases and we examined the possible mechanisms allowing stabilisation, especially in the case of low surface coverage. Encapsulation and nutritional targeting Recently, there has been increasing interest in the development of compartmented colloidal structures or microcapsules. For example, W/O/W emulsions consist of small water droplets dispersed in larger oil globules, which are themselves dispersed in an aqueous continuous phase. Double emulsions present many interesting possibilities for the controlled release of chemical substances initially entrapped in the internal droplets. They also have a number of 90

potential benefits over conventional emulsions, such as reduction of fat content, taste masking and protection of labile ingredients or sensitive probiotics. Nevertheless, there have been many difficulties associated with preparing this type of multiple emulsions with sufficient stability for utilization within the food industry, due to coalescence or due to diffusion of water molecules from the internal aqueous phase to the bulk aqueous phase or vice-versa. We formulated water-in-oil-in-water (W/O/W) emulsions based on different triglyceride oils and food-type surface active species. Magnesium was encapsulated in the inner aqueous droplets. Emulsion stability was assayed through particle sizing and magnesium release. Irrespective of the oil nature, both the primary W/O and W/O/W emulsions were quite stable regarding the droplet size parameters (no coalescence). Magnesium release was due to diffusion and/or permeation mechanisms. When W/O/W emulsions were placed in the presence of pancreatic lipase, the emulsion triglycerides were hydrolysed by the enzyme. These results indicated a possible use of W/O/W emulsions loaded with magnesium ions in food applications Formulation of colloidal systems for in vitro and in vivo studies In order to claim functional properties for food presenting a specific nutritional composition (omega 3 fatty acid, phospholipid, high vitamin content….), formulation and characterization of these foods is required. In particular, food rich in polyunsaturated fatty acids are subjected to oxidation which must be prevented. Besides the physicochemical characterization of the colloidal systems under various storage conditions, we tested these lipid systems so that to increase the bioavailability of the nutritional compounds. Thus, we studied the different physicochemical and enzymatic processes that may affect the bioavailability of lipid nutrients. We use different colloidal systems that vary in: (1) the type of oil tested (including the chemical nature of the fatty acid present and the physical state of the lipids used); (2) the type of the lipid vector (emulsion, liposome); (3) the properties of the lipid vector (emulsifiers used to stabilize the emulsion, particle size). The different systems were tested in conditions that mimic the gastrointestinal conditions, on cell cultures (Caco 2) and in vivo in rats. Interesting results have already been obtained on liposomes. We are currently extending our investigations to oil-in-water emulsions.

5 significant publications: 1. 2. 3. 4. 5.

M. Bonnet, M. Cansell, A. Berkaoui, M.H. Ropers, M. Anton, F. Leal-Calderon. (2009) Release rate profiles of magnesium from multiple W/O/W emulsions. Food Hydrocolloids, 23, 92-101 M. Cansell, A. Battin, P. Degrace, J. Gresti, P. Clouet, N. Combe (2009). Early dissimilar fate of liver EPA in rats fed liposomes or fish oil and gene expression related to lipid metabolism. Lipids, 44, 237-247. F. Thivilliers, E. Laurichesse, H. Saadaoui, F. Leal-Calderon, V. Schmitt. (2008) Thermally-Induced Gelation of Oil-inWater Emulsions Comprising Partially Crystallized Droplets: the Impact of Interfacial Oil Crystals, Langmuir, 24, 1336413375 F. Gautier, M. Destribats, R. Perrier-Cornet, J-F. Dechezelles, J. Giermanska, V. Héroguez, S. Ravaine, F. Leal-Calderon, V. Schmitt. (2007) Pickering emulsions with stimulable particles: from highly to weakly covered interfaces, Physical Chemistry Chemical Physics, 9, 6455-6462 M. Cansell, N. Moussaoui, M. Mancini (2007). Prostaglandin E2 and interleukin-8 production in human epidermal keratinocytes exposed to marine lipid-based liposomes. International Journal of Pharmaceutics, 343, 277-280.

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Maud Cansell Teaching of Lipids (definition, extraction, characterization, use in food products) (L3 & 4 level) Teaching Encapsulation (Master 1, L4 & 5 level). Partial organization: “Fête de la science: titre : Fats and oils in food (since 2006) Fernando Leal Calderon Teaching of Surface thermodynamics, Colloidal Science, Formulation (Master 1 & 2 level). Teaching of Thermodynamics (L3 level) Teaching of Mass Spectrometry and Optical Spectroscopies (L3 level)

Management. Maud Cansell - Teaching manager of the food and science department of « Institut des Sciences et Techniques des aliments de Bordeaux (ISTAB, Engineering School) » (since 2008, www.istab.u-bordeaux1.fr/). - Quality manager of « Institut des Sciences et Techniques des aliments de Bordeaux (ISTAB, Engineering School) » (2004-2008, www.istab.u-bordeaux1.fr/). F. Leal Calderon - Director of « Laboratoire des Milieux Dispersés Alimentaires (LMDA) » (JE n° 2389, Bordeaux 1 University, www.lmda.u-bordeaux1.fr/) (2003-2008). - Assistant director of « Institut des Sciences et Techniques des aliments de Bordeaux (ISTAB, Engineering School) » (2004-2006, www.istab.u-bordeaux1.fr/). - Director of « Institut des Sciences et Techniques des aliments de Bordeaux (ISTAB, Engineering School) » (from 2006, www.istab.u-bordeaux1.fr/). - Associate Director of the Carnot Institute “LISA” : Lipids for Industry SAfety & health (from 2007, www.lisacarnot.eu) - Treasurer of the French cluster “PROD’INNOV”: Innovating products and processes for nutrition and health (from 2007, www.prodinnov.fr/).

91

TEAM « MILIEUX FLUIDES ET REACTIFS » Team Member: P. Subra-Paternault, Directeur de recherche INST2I, section 10 P. Subra-Paternault, a CNRS scientist of the INST2I department, moved to Bordeaux in october 2006, after being promoted Directeur de Recherche (DR2). She left her unit LIMHP (UPR1311, 93430 Villetaneuse) and joigned the TREFLE unit (UMR 8508) on a full-time basis in September 2008, after the completion of an European project where herself and her LIMHP team was strongly involved. The moving at TREFLE was accompanied by the transfer of four supercritical pilots and several analytical set-ups, offering at Bordeaux new opportunities for product processing. The report hereby concern thus activities mostly undertaken at LIMHP, and since 2008, at TREFLE.

Team Production (2005-2009): Publications, peer reviewed, (ACL) Publications, peer reviewed in published international colloquia (ACTI) Oral communications in congress (COM) Poster communications in congress (AFF)

12

Books or Book chapters (OS)

1

1

Edition of Books (DO) (coordination)

1

5 12

Congress/Workshop organization Defended PhD Theses or HDR Grants : public & private k€/year

1 1 65

Scientific activity Topics concern crystallization, precipitation and formulation assisted by supercritical fluids, with the objective of an improved understanding of the process mecanisms that control the produced material characteristics and consequently, their properties. Materials are mostly Active Pharmaceutical Ingredient, pure or with excipient, whose characteristics as mean size, particle size distribution, crystal form or habit are susceptible to impact their dissolution rate and lately, their biodisponibility. The product characteristics are strongly affected by the process since the generation of the solid phase involves three coupled phenomena of phase equilibria, hydrodynamic and nucleation/growth. On crystallization topic, my activities at LIMHP focused on the incidence of phase equilibria on product properties and were supported by an extensive experimental approach; at TREFLE, a simulation approach is currently developed with an assistant professor via a collaboration with the ‘computational fluid mechanics’ team. The aim of such modeling of pure API crystallization is to take in account the three contributions in order to fully describe the crystallization process and, finally, to propose more efficient reactors, conditions or process that go along with sustainable chemistry, including a scale-down to microreactor (coll. LOF Bordeaux). Up to now, a model of mixing a solvent pseudo-droplet with a dense CO2-phase, accounting for isotherm or non-isotherm conditions, is developed. Regarding precipitation and formulation topics, activities were focused on fabrication of more complex systems of specific functionalities : API-biopolymer(s) to enhance the dissolution rate, to sustain the release or to deliver specifically the API (Coll. Y.Pentchev, UTCM Sofia Bulgarie, X.Saurina, Univ. Barcelone, Nunes da Ponte, ITQB Lisbonne), modifier-oxide or oxide-biopolymer to improve dispersibility in lipophilic media, microfibers of polymer blends as scaffolds for tissue engineering (Coll. C.Domingo, ICMAB, Barcelone). Since 2008, and with financial support of Region Aquitaine, the selective extraction and formulation of lipids are investigated (coll. M. Cansell), widening the topic of complex systems produced via supercritical techniques to colloid-based formulations.

5 significant publications: 1-P. Subra-Paternault, C. Roy, A. Vega-Gonzalez, C. Domingo, Solvent effect on tolbutamide crystallization induced by compressed CO2 as antisolvent, Journal of Crystal Growth, 309 ( 2007) 76-85 2- C. Roy, A. Vega-González, P. Subra-Paternault, Theophylline formulation by supercritical antisolvents, Int. J. Pharmaceutics 343 (2007) 79-89 3- P. Subra-Paternault, Ch. Roy, A. Vega-Gonzalez, P. Jestin, Crystallization of drugs using supercritical CO2 as antisolvent : from phase equilibria to products Intern. J. Chem. Reactor Eng. Vol 5 (2007) A45 4- A. Duarte, A. Simplicio, A.Vega-González, P. Subra-Paternault, P.Coimbra, M.H. Gil, H. de Sousa, C. Duarte, Impregnation of an intraocular lens for ophthalmic drug delivery , Current Drug Delivery, 5 (2008) 102-107 5- A. Vega-Gonzalez, P. Subra-Paternault, A. Lopez-Periago, C. Garcia-Gonzalez, C. Domingo, Supercritical CO2 antisolvent precipitation of polymer networks of l-PLA, PMMA and PMMA/PCL blends for biomedical applications, European Polymer Journal 44 (2008) 1081-1094

92

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) •

• • • • • •

•

Teaching Class on Supercritical Fluids and Materials, Speciality course (9h) Master 2 level, Université Paris 13. Training on supercritical equipments (Ph’D, Master, Engineer Schools students, from France, Bulgaria, India, Portugal, Spain). Member of organizing committee of the 9th international symposium on supercritical fluids, Arcachon, 17-20 may 2009. Coordination of the book ‘Microencapsulation : des sciences aux technologies’, Lavoisier ISBN 978-2-74300976-2, 2007) Settling and coordination of a national network on supercritical fluids, scf-PILA (12 laboratories) Review of european projects STREP at the European Commission (2004, 2005) Inter-universities actions with Portugal (2005) and Bulgaria (2006) Project leader and grants: Université Bordeaux1/ BQR [2008-2011], Aquitaine Project [2008-2011], European STREP project [2005-2008]

Managment and running of technological platforms. • •

Team manager ‘ Fluides et procédés polyphasiques’ at LIMHP [2000- 2007] (7 scientists), Team manager ‘Fluides Interfaces et Particules’ at TREFLE since november 2007 Elected member at Conseils Scientifiques, Administration of ‘Institut Galilée’, Université Paris 13, until october 2006

93

TEAM « UNITE MICRONUTRIMENTS, REPRODUCTION, SANTE » Team Member: Dr. Patrick Sauvant, Maître de Conférences, ENITA de Bordeaux. Team Composition (2006-2009): Catherine Bennetau-Pelissero, PR ENITAB (Head) Dr. Svitlana Poix-Shinkaruk, IR ENITAB Sébastien Vergne (doctorant)

Patrick Sauvant, MC ENITAB Valérie Lamothe, AI, ENITAB

Mylène Potier-Georges, IR ENITAB Charlotte Carreau, doctorante

Personnal Production (2006-2009): Publications, peer reviewed, (ACL) Publications, peer reviewed, not yet in bases (ACLN) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI)

7

Poster communications in congress (AFF)

6

1

Books or Book chapters (OS)

2

5

Defended PhD Theses or HDR

1

2

Grants : public & private k€/year

202 k€

Scientific activity Résumé Les isoflavones, contenues en quantité substantielle dans le soja, sont actuellement considérées comme une alternative naturelle aux traitements hormonaux substitutifs de la ménopause. En effet, certains effets bénéfiques leur ont été attribués par l’observation des populations asiatiques. Les résultats des études, ayant trait aux isoflavones, sont contradictoires, en particulier lorsque leurs effets cliniques sont recherchés. Il apparaît donc important d’étudier la biodisponibilité des isoflavones, nécessaire préalable à leur efficacité biologique. L’objectif de ces quatre dernières années de recherche était donc de caractériser les facteurs influant sur la biodisponibilité des isoflavones de soja : la formulation des compléments alimentaires, la matrice alimentaire, le contexte alimentaire, la chronicité d’ingestion et l’appartenance à une ethnie ont été étudiés. Enfin, l’effet des isoflavones de soja sur l’ostéoporose a été étudié au travers d’une étude in vitro sur une lignée ostéoblastique humaine et suggère l’intérêt d’une prévention nutritionnelle de l’ostéoporose par les isoflavones de soja. Abstract Soy isoflavones are currently considered as an alternative to menopausal hormone replacement therapy. Indeed, some of their beneficial effects are known with data resulting from Asian epidemiological studies. However results obtained from clinical studies are discrepant. As a consequence, it appears important to study the bioavailability of isoflavones, a prerequisite of their biological activity. The aim of this four years research activity has been to study the effect of factors influencing their bioavailability, such as industrial processes used in the making of soy-based supplements, dietary matrix, dietary context, chronic ingestion of soy-based products, ethnic appurtenance. The effect of isoflavones on osteoporosis was suggested with an in vitro study, with a human osteoblast line being used. In conclusion, soy isoflavones may be taken into consideration for the nutritional prevention of osteoporosis.

5 significant publications: 1.

2.

3.

4.

Vergne Sébastien, Sauvant Patrick, Lamothe Valérie, Chantre Philippe, Asselineau Julien, Pérez Paul, Potier Mylène, Durand Marlène, Moore Nicholas, Bennetau-Pelissero Catherine. Influence of ethnic origin: Asian vs Caucasian, and background dietary habits on the bioavailability of isoflavones. Br. J. Nutr. (2009, in press) Vergne Sébastien, Bennetau-Pelissero Catherine, Lamothe Valérie, Chantre Philippe, Potier Mylène, Asselineau Julien, Durand Marlène, Jean-James Garreau, Moore Nicholas, and Sauvant Patrick. Higher bioavailability of isoflavones after a single ingestion of a soy based supplement than a soy based food in young healthy males. Br. J. Nutr. (2008), 99, 333-344. Vergne Sébastien, Titier Karine, Bernard Virginie, Asselineau Julien, Durand Marlène, Lamothe Valérie, Potier Mylène, Pérez Paul, Demotes-Mainard J, Chantre Philippe, Moore Nicholas, Bennetau-Pelissero Catherine and Sauvant Patrick. Soy flour don not affect the bioavailability of isoflavones: a human study. J. Pharm. Biomed. Anal. (2007), 43, 1488-1494. Abergel Armand, Sapin Vincent, Dif Nicolas, Chassard Christophe, Darcha Claude, Marcand-Sauvant Julie, Gaillard-Martinie Brigitte, Rock Edmond, Dechelotte Pierre, Sauvant Patrick. Growth arrest and decrease of alpha SMA and type I collagen expression by palmitic acid in the rat hepatic stellate cell line PAV-1. Dig Dis Sci (2006), 51, 986-995.

94

5.

Mathey Jacinthe, Lamothe Valérie, Coxam Véronique, Sauvant Patrick and Bennetau-Pelissero Catherine. Plasma and urinary concentrations of soy isoflavones as biomarkers of intake and predictors of clinical effects. J. Pharm. Biomed Anal (2006), 41, 957-965.

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Lecturer in “human nutrition” at the ENITA of Bordeaux. Lecturer in “animal nutrition” at the ENITA of Bordeaux In charge of the management of the third year of engineer formation specialized in “Human Nutrition and Health”. In charge of a lecture on isoflavones (3h) in a professional Master formation at Vichy entitled “project leader in pharmacy and nutrition” a delocalized formation from University of Clermont Ferrand I. Every year a total of around 240h Eq TD has been performed for the ENITA of Bordeaux. Reviewer for Journal of Agricultural and Food Chemistry; International Journal of Environmental Analytical Chemistry; Journal of Pharmacy and Pharmacology ; European Journal of Clinical Nutrition ; British Journal of Nutrition

95

Chrystel Faure, Professor, Polytechnic Institute of Bordeaux (IPB). Personal Production (2006-2009): Publications, peer reviewed, (ACL) Oral communications in congress (COM) Poster communications in congress (AFF)

9 10 7

Congress/Workshop organization Defended PhD Theses

2 1

Scientific activity Résumé Nous avons poursuivi nos travaux entamés en 2003 portant sur la formulation et l’utilisation de vésicules multilamellaires de type « oignons » dans un triple but : (i) la vectorisation, (ii) la synthèse de nanoparticules inorganiques de morphologie contrôlée, (iii) la nano-structuration de surface. Nous avons travaillé sur la synthèse de nanoparticules (Nps) d’or au sein des oignons, et montré que, grâce à ce milieu confiné, nous pouvions : contrôler la taille des Nps via la composition de la vésicule, la voie d’insertion du précurseur d’or dans les oignons et le mécanisme de synthèse choisie contrôler la forme des Nps via la composition de la vésicule induire l’assemblage des Nps formées Nous avons également mis en évidence les paramètres clés pour la stabilité de l’hybride oignon/nanoparticule, à savoir la taille et le nombre des Nps. Nous avons également incorporé des nanoparticules d’oxydes de fer magnétiques dans les oignons dans le double but de permettre la visualisation par Imagerie par Résonance Magnétique du vecteur oignon et la mort de la cellule malade ciblée par hyperthermie magnétique. Deux voies ont été employées : la synthèse intra-vésiculaire par précipitation d’ions fer, et l’incorporation d’un ferrofluide (dispersion de Nps magnétiques) dans les vésicules. Les oignons magnétiques réalisés à partir du ferrofluide possèdent des propriétés d’agents de contraste T2 remarquables, meilleures que ceux reportés jusqu’à aujourd’hui, tandis que les propriétés d’hyperthermie magnétique mesurées sur les oignons obtenus par synthèse intra-vésiculaire se révèlent prometteuses pour des fins thérapeutiques. Nous avons enfin développé une nouvelle technique de dépôt de nano-plots inorganiques sur des surfaces basée sur la vectorisation, sous champ électrique, de capsules contenant un précurseur rédox. Nous avons validé ce procédé, en utilisant les oignons, pour produire des surfaces recouvertes de nanoplots de cuivre dont la taille et la densité peuvent être aisément contrôlées. Cette technique est sur de nombreux points (coût, simplicité, rapidité) plus performante que celles existantes. Abstract We continued our work started in 2003 bearing on the formulation and the use of “onion-type” multilamellar vesicles in triple drank: (i) vectorization, (ii) the synthesis of inorganic nanoparticles of controlled morphology and (iii) the nano-structuring of surface. We worked on the synthesis of gold nanoparticles (Nps) within onions, and showed that, thanks to these confined systems, we could control the size of Nps via the vesicle composition, the introduction pathway of gold precursor and the synthesis mechanism. The Nps shape as well as their assembly could also be controlled via the vesicle composition. We also highlighted the key parameters for the stability of the hybrid onion/nanoparticle, namely the size and the number of the nps. We also incorporated magnetic iron oxide Nps in onions with a double aim of allowing visualization by Resonance Magnetic Imaging of the vector onion and the death of the sick targeted cell by magnetic hyperthermia. Two ways were employed: the intravesicular synthesis by iron ions precipitation, and the incorporation of a ferrofluid (magnetic Nps dispersion) in the vesicles. The magnetic onions formed from the ferrofluid have remarkable properties as T2 contrast agents, better than those deferred until today, while the magnetic onions obtained by intra-vesicular synthesis prove promising magnetic hyperthermia properties. We finally developed a new technique of deposit of inorganic nano-dots on surfaces based on vectorization, under electric field, of capsules containing a redox precursor. We validated this process, by using onions, to produce surfaces covered with copper nano-dots of which the size and the density can be easily controlled. This technique is on many points (cost, simplicity, speed) more powerful than those existing.

5 significant publications: 1. 2. 3. 4.

Faure, C., Debecker, D. P., Meyre, ME, Derré, A. and Gaigneaux, E. M. (2008) A new bio-inspired route to metal nanoparticlebased heterogeneous catalysts Small 4,10, 1806-1813 Meyre, M.E., Tréguer-Delapierre, M. and Faure, C. (2008) Radiation-induced synthesis of gold nanoparticles within lamellar phases. Formation of aligned colloidal gold by radiolysis. Langmuir 24, 9, 4421-4425 Olea, D., Viratelle O. and Faure, C. (2008) Polypyrrole-glucose oxidase biosensors. Effect of enzyme encapsulation in multilamellar vesicles on analytical properties. Biosensors and Bioelectronics 23, 788-794 Faure, C. Guillot, S., Weissbecker, P. and Saadaoui, H. (2006) Multilamellar vesicle-assisted electrodeposition of inorganic nanodots. Adv. Mat. 18, 91141-1146. 96

5.

Faure, C., Nallet, F., Roux, D., Milner, S., Gauffre, F., Olea, D. and Lambert, O. (2006) Modeling leakage kinetics from multilamellar vesicles for membrane permeability determination: application to glucose. Biophys. J. 91, 4340-4349

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Co-responsible for the Master “Chimie” Specialty “Polymères et Colloïdes”, University Bordeaux, 2007/09 Co-responsible for the Licence “Science Physique et Chimie”, University Bordeaux, 2004/07 Responsible of the specialty “Colloïds” for the European Master “FAME” (Functionalised Advanced Materials Engineering of Hybrids and Ceramics). Responsible of 8 « Teaching Units »: - Electrochimie Fluides complexes, Stages en laboratoires, Chimie des solutions, Licence Level - Physico-chimie des milieux colloïdaux, Physico-chimie des polymères en solution, Dynamique des fluides complexes, Tensioactifs : de la solution aux interfaces Master 1&2 level Partial organization: “11th International Conference on Electroanalysis”, Bordeaux, France, July 2006 (900 participants) Partial organization: “FAME Congress”, Alméria, Espagne, September 2008 (150 participants) Partial organization: “Fête de la science: liposomes pour le vivant” October 2006,2007 Coordinator of the team « Physico-Chimie des Systèmes Biologiques » (15 persons) of the Centre de Recherche Paul Pascal, 2005/06 Responsible of 2 scientific projects for 2 industries (ADISSEO and BAKEMARK)

97

TEAM « LABORATORY OF FOOD BIOLOGICAL VALUE ANALYSIS » Team Leader: ATGIE, Claude MCF – HDR, Bordeaux 1 University Team Composition (2006-2009): Claude Atgié, MCU, Univ Bordeaux 1- HDR

Carine Ferrand –MCU, Univ Bordeaux 1-

Michel Baccaunaud - PAST

Team Production (2006 - 2009): Publications, peer reviewed, (ACL) Poster communications in congress (AFF)

5 2

Books or Book chapters (OS) Congress/Workshop organization Grants : public & private k€/year

1 2 14 k €

Scientific activity Résumé Le thème de mes activités de recherche est centré autour de la compréhension des mécanismes d’actions cellulaires de la vitamine A dans le contrôle du développement du tissu adipeux, et des perturbations de ces voies de signalisation par des surcharges lipidiques d’origines nutritionnelles. Ce thème s’inscrit dans une nouvelle approche de la nutrition qui a pour but de mieux comprendre le rôle des nutriments dans les voies de signalisation cellulaire. L’ensemble des travaux porte sur l’action cellulaire de la vitamine A dans différentes situations nutritionnelles et hormonales et plus précisément sur l’expression des récepteurs nucléaires de l’acide rétinoïque (métabolite actif de la vitamine A) et de certains facteurs de transcription régulant des gènes cibles dans les tissus adipeux. La mise en évidence des interactions entre les lipides et l'action intracellulaire des rétinoïdes, ainsi qu'un certain nombre de travaux montrant que les acides gras sont capables de moduler l'expression de gènes impliqués dans leur propre métabolisme et dans la plasticité des tissus adipeux, ont orienté nos études sur l’influence d’un apport déséquilibré en lipides sur les voies de signalisation des rétinoïdes dans ces tissus. Des résultats antérieurs nous ont suggérés que des liens existaient entre des modifications de ces voies de signalisation, induites par des régimes riches en graisse, et les prises de poids observées suite à un régime hyperlipidique. Les résultats de nos études expérimentales démontrent clairement qu’un régime inducteur d’obésité (régime hyper lipidique – hyper énergétique) active les voies de signalisation des acides gras (augmentation de l’expression de PPARϕ et de l’expression d’aP2; marqueur de différenciation) aboutissant à un développement excessif de l’ensemble des tissus adipeux. Il apparaît par ailleurs, qu’un apport supplémentaire de vitamine A par la ration alimentaire, semble potentialiser ce phénomène (Redonnet et al. 2008) par une action directe sur les précurseurs cellulaires dans ces tissus. Les résultats de nos études biomédicales (Bairras et al. , soumis) nous révèlent que dans des états d’obésité humaine, nous retrouvons des modifications des profils de récepteurs nucléaires (PPAR et RAR) dans le tissu adipeux qui vont dans le même sens que les variations observées dans les études expérimentales. Parallèlement à ces études, et dans l’objectif de valider des modèles d’obésité génétique, nous avons caractérisé d’un point de vue de la régulation du métabolisme des tissus adipeux, deux modèles de rats génétiquement obèses : le rat Zucker (porteur du gène fa/fa) et le rat SHR/N-cp, obèse et diabétique de type II, porteur du gène « corpulent » (cp/cp). Ces deux modèles de rats génétiquement obèses présentent une activité lipolytique excessive de l’ensemble des territoires adipeux, avec un défaut massif dans la régulation du métabolisme lipidique aboutissant à une dyslipidémie exacerbée et un diabète non insulinodépendant chez le rat SHR/N-cp. (Mauriège et al., 2008, Atgié et al., 2009). Ces modèles pourront servir de cibles privilégiées pour des études expérimentales dans des tests d’efficacité d’agents bioactifs pour des traitements de l’obésité et du diabète.

Abstract My research activities were developed to understand the relation between the cellular action of the vitamin A and the normal regulation of the white adipose tissue growing in situations of excessive caloric intakes. This is a new approach of experimental nutrition studies in order to better understand the roles of the nutrients in the regulation of cellular signaling processes. We particularly studied the cellular action of vitamin A and the expression of their nuclear receptor (RAR and RXR) in the white adipose tissue (WAT) of obese rats after exposure to a high fat diet. The evidence of interactions between the intracellular action of lipids and retinoids, associated to the observation that fatty acids are able to modulate the expression of many genes involved in the metabolism and the plasticity of the adipose tissues, lead us to investigate the effect of a imbalance diets in the expression of the retinoid receptors in WAT. The results of ours experimental studies clearly showed that a diet induced obesity (by a “cafeteria” type of diet) stimulated the fatty acids signaling pathway (by increase in PPARϕ and increase in aP2, a marker of differentiation) leading to an excessive growing of the WAT. It seems also that an increase of the vitamin A level in the diet, concomitantly with the “cafeteria diet” has a synergic positive effect (Redonnet et al. 2008), by directly affecting the stem cells recruitment in the WAT. On the other hand, the results of our biomedical studies (Bairras et al. soumis), 98

showed that in human obesity, the same changes in the nuclear receptor profiles (PPAR, TR and RAR) were observed in the adipose tissues, but also in a tissue much more accessible: the peripheral mononuclear blood cells (PBMC). In others parallel experiments, in order to characterize some genetic models of animal obesity, we studied the morphologic and metabolic regulations of many subcutaneous and intra abdominal deposits of two types of obese rats: the first one, the Zucker rats (homozygote for the “fatty’ gene), and the second, the SHR/N-cp (homozygote for the “corpulent” gene). We reported that these two genetic models of obese rats showed an excessive development of all their fat depots (Bairras et al. 2008), and particularly the subcutaneous ones in the SHR/N-cp (Atgié et al., 2009). This increase in the fat mass leaded to a deregulation of the lipid metabolism, as attested by a severe dyslipidemia and a type 2 diabetes, observed in the SHR/N-cp model (Atgié et al., 2009). These genetics models will be interesting for further studies in order to validate bioactive molecules for the treatments of obesity and/or diabetes.

5 significant publications: 1. 2.

3.

4.

Bairras C., Mauriège P., Bukowiecki L. and C. Atgié. Regulation of lypolysis in white adipose tissues of lean and obese Zucker rats. Journal of Physiology and Biochemistry. 2007.63 (4): 01-10. Redonnet A., Ferrand C., Bairras C., Higueret P., Noël-Suberville C., Cassand P., and C. Atgié. Synergic effect of vitamin A and high-fat diet in adipose tissue development and nuclear receptor expression in young rats. British Journal of Nutrition. 2008, 100, 722-730. Atgié C., Hadj-Sassi A., Bukowiecki LJ and P. Mauriège. High lipolytic activity and dyslipidemia in the Spontaneous Hypertensive/NIH Corpulent (SHR/N-cp) rat: a genetic model of obesity and type II diabetes. Journal of Physiology and Biochemistry 2009, 65 (1) 33-42. Céline Bairras, Anabelle Redonnet, Henri Dabadie, Henri Gin, Claude Atgie, Véronique Pallet, Paul Higueret and Catherine Noël-Suberville. RARj and TRb expressions are decreased in PBMC and SWAT of obese subjects in weight gain. (soumise à Journal of Physiology and Biochemistry).

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Teaching (196 H) : Teaching of Biochemical Molecules in Food. Influence of Food Processing Teaching of Nutrition (Recommended nutrient intakes, Nutritional and Safety Quality of Food). Teaching of Food Toxicology, Evaluation of hazard in food (assessment and risk management, French and European legislation) Teaching of toxicological and technological aspects of food additives (Conference in a Master of Animal Experimental Biology, Toulouse Veterinary School and Toulouse University) Administrative responsibility: Responsible for the Master “Biologie, Santé”, Professional Speciality: “Transformations Agroindustrielles” from Bordeaux 1 University. Responsible of a Research Program (CTP « Communauté de Travail des Pyrénées ») « Potential roles of the Mediterranean Diet in the treatment of obesity and diabetes” 2007 – 2009 (Inter regional program for a thematic Network). Articles reviewing activity: Occasional Scientific Reviewer for: British Journal of Pharmacology, Molecular Cell Endocrinology Expertise activity: Scientific expertises for The French Agency for Food Safety (Afssa) in the “Additifs, Arôme et Auxilliaires Technologiques” Scientific Expert Comitte. 2006-2009. Membership in Scientific Societies or institutes: - Member of the French Nutrition Society (SFN) - Member of the French Institute of Nutrition

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TEAM « PROTEOLYSIS AND MUSCLE DEVELOPMENT » Team Leader: Patrick Cottin, Professor, University Bordeaux 1. Team Composition (2006-2009): Elise Dargelos, MCU Laetitia Daury, MCU Sylvie Poussard, IR2 Université Cédric Brulé, Doctorant Sébastien Goudenège, Doctorant Ludovic Leloup, Doctorant Pascal Stuelsatz, Doctorant B.Sc. and M.Sc. trainees (stay between 2 and 6 months): 15

Abdessatar Hadj Sassi, MCU Ilam Chehl, Post-doctorante Hélène Roumes, Doctorante

Team Production (2006-2009): Publications, peer reviewed, (ACL)

10

Invited conferences congress (INV) Oral communications in congress (COM)

2 8

Poster communications in congress (AFF) Defended PhD Theses or HDR Grants : public & private k€/year

8 3 150 k€

Scientific activity Ce bilan de l'Unité ne fait état que des activités de recherche menées par les enseignants-chercheurs, chercheusr et techniciens sollicitant une intégration dans l'Unité CNRS-Université Bordeaux 1-IPB-ENITAB, Chimie et Biologie des Membranes et Nano objets, directeur E. DUFOURC.

Résumé : Le système protéolytique calcium-dépendante considéré comme un des systèmes protéolytiques intracellulaires majeurs est constitué de cystéines protéases appelées calpaïnes. Ce sont des protéases soit ubiquitaires soit tissus spécifiques : les deux mieux caractérisées sont à ce jour deux isoformes ubiquitaires connues sous le nom de µ-calpaïne et de m-calpaïne. Ces calpaïnes sont responsables de très nombreux phénomènes protéolytiques, la protéolyse étant ellemême très limitée. Toute dérégulation de ce système entraîne des pathologies très graves : les calpaïnopathies. En dépit de l'abondance des nouvelles informations, de nombreuses questions concernant le système calpaïne dans le développement du tissu musculaire reste sans réponse, à titre d'exemple comment l'activité de ces enzyme est-elle régulée, quelles sont les réelles fonctions physiologiques ainsi que les substrats potentiels dans la cellule musculaire saine et la cellule musculaire pathologique etc… Afin de répondre à ces questions, à partir de modèles cellulaires très appropriés et de techniques très adaptées, nos principaux résultats montrent que : - le système protéolytique calcium-dépendant est très impliqué dans les phénomènes d'atrophies ainsi que le vieillissement musculaire (& I) - le système protéolytique calcium-dépendant est très impliqué dans les phénomènes d'adhésion et de migration des myoblastes ainsi que dans les phénomènes métastatiques (& II) - le système protéolytique calcium-dépendant est très impliqué dans l'équilibre musculaire atrophie/hypertrophie suite à une forte régulation par un ou plusieurs facteurs musculaires de croissance et de différenciation : myostatine (& III). Tous ces résultats devraient à moyen terme être finalisés dans divers domaines touchant à la fois le domaine médical (cancer, atrophie et dystrophies musculaires, sarcopénies) et le domaine agroalimentaire (meilleure maîtrise de la croissance musculaire chez les ovins et bovins).

Abstract The calcium-dependent proteolytic system, one of the major intracellular proteolytic systems, is composed of cysteine proteases named calpains. They are ubiquitous or tissue-specific enzymes. The two best characterised isoforms are the ubiquitously expressed µ- and m-calpains. Calpains are responsible for limited proteolytic events. Deregulations of this system induce very serious pathologies named calpainopathies. Despite a wealth of new informations, many primary questions concerning the calpain system in muscle development remain unanswered: for example, how calpain activity is regulated, what are the real physiological functions and physiological substrates in normal and pathological muscular cells etc… To try to answer to these questions, using very appropriated cellular models and adapted technics, our main results show that - The calcium-dependent proteolytic system is very involved in muscular atrophies and muscle aging (& I)

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- The calcium-dependent proteolytic system is very involved in myoblast adhesion and migration, and cell invasion (& II). - The calcium-dependent proteolytic system is very involved in the balance muscular atrophy/hypertrophy through a tight regulation by growth and differenciation factors: myostatin (& III). All these results will be able to have applications in various domains as well as the medical level (cancer, muscular atrophy and dystrophies, sarcopenia) as the agrofood one (better control of muscular growth for sheeps and bovines). I) CALCIUM-DEPENDENT PROTEOLYTIC SYSTEM FUNCTION IN MUSCULAR ATROPHIES: DYSTROPHIES AND MUSCLE AGING Muscle degenerative process associated with aging could be compared with the phenotype observed in some myopathies. Loss of muscle mass results from an imbalance between regeneration events and catabolism rate linked with cell death (apoptosis or necrosis). Proteolytic activity is largely involved in the balance between muscle regeneration and cell death since proteases are well known to be regulated by environmental signals but also involved in the transduction of these signals. Among these proteases, calpains (calcium-dependent proteolytic proteases) are strongly involved in proliferation and apoptosis, two processes responsible for the impairment of myogenic stem cells regenerative potential. Calpains implication in muscle cell differentiation has been clearly demonstrated and modifications of their expression levels have been associated with muscle atrophy and myopathies. Calpains and signal transduction associated with caveolae Caveolae, intracellular micro-domains, have been shown to be concerned by muscle aging, skeletal muscle regeneration. Because caveolae contain a large number of signalling proteins, they are thought to play a key role in signal transduction pathways. The major components of these vesicular structures are caveolins. Interestingly, mutations affecting the caveolin 3 gene (isoform highly expressed in differentiated muscle) have been shown to lead to limb girdle muscular dystrophy type 1C (LGMD1C). In a previous work we demonstrated the presence of active mcalpain in myoblast caveolae and its involvement in MARCKS (Myristoylated Alanine Rich C-Kinase Substrate) translocation from caveolae to cytosol in a PKCα-dependent way. This translocation may lead to cytoskeletal dynamic changes involved in myoblast fusion. Furthermore, eight differentially expressed caveolae associated proteins were identified by 2-DE and LCMS/MS analyses using an m-calpain antisense strategy. Under these conditions, we identified cytoskeletal (vinculin, desmin, and vimentin) and mitochondrial proteins (Hsp60, 75 kDa NADH-ubiquinone subunit, and ATP synthase regulatory subunit α). These two last components are part of the oxidation-reduction respiratory chain complexes I and V, which were recently localised in detergent-resistant lipid RAFTs. This study corroborates the cleavage by m-calpain of cytoskeletal proteins essential for myofibrillar assembling, myoblast fusion, and cell motility. It is also the first time that the action of m-calpain on these cytoskeletal proteins has been demonstrated in caveolae. This study also implicates m-calpain in several mitochondrial pathways, particularly in the respiratory chain. The relationship between caveolae and mitochondria is still poorly understood. Perhaps these vesicles intervene in mitochondrial protein transport or turnover. Further investigations are necessary to define more precisely the role of mcalpain in a putative regulation of identified mitochondrial proteins. All these results were described by Goudenege et al. Proteomics 2007. Calcium-dependent proteolysis, oxidative stress and muscle aging (Project supported by AFM: Association Française contre les Mypathies) Aging is associated with a progressive and involuntary loss of muscle mass also known as sarcopenia. This condition has been reported even among healthy, physically active subjects. Although sarcopenia is now well documented, the aetiology of this condition still remains poorly understood and could well be a multifactorial disorder: -Impairment in the regenerative potential of myogenic stem cells has been shown to contribute to the low regeneration process associated with sarcopenia. -Deregulation of intracellular proteolytic activities has been associated with muscle aging, and calpain activity in particular has been linked with aging in other tissues. -Reactive oxygen species (ROS) accumulation and increase of intracellular calcium level have been demonstrated. -Disruptions in several signalisation pathways have been observed in sarcopenic muscle. For a review, see Dargelos et al. Biochimie 2008. In that context, our first goal was to make an inventory of pro-sarcopenic signals, especially those involving calpains, in rat skeletal muscle. In a second set of experiments we aimed at inducing an oxidative stress in human satellite cells in order to characterize the effects on calcium‐dependent proteolysis.     1-Calcium-dependent proteolytic system and skeletal muscle aging We were the first to provide evidence for a global increase of the calcium-dependent proteolysis during muscle aging (Dargelos et al. Exp. Gerontol. 2007). To identify the key proteins (substrates or regulators) interacting with calpains during muscle aging, immunoprecipitations coupled with proteomic analyses and protein identification by nanoLC-MS/MS have been undertaken. Among the identified proteins, several are localized in mitochondria (ATPsynthase), others are implicated in calcium homeostasis (calcium ATPase, ryanodine receptor and calsequestrin) or linked with myofibre integrity (nebulin, alpha actinin). These results have been confirmed by reverse co-immunoprecipitation and cellular co101

localisation by confocal microscopy. Ryanodine receptor and ATPsynthase were also identified as calpain substrates. Such results underline an important role of calpains in cellular homeostasis and mitochondrial metabolism during aging and suggest a possible link between calpains and oxidative stress. This part of our work will be soon submitted to Biogerontology. 2-Calcium-dependent proteolytic system and oxidative stress in human muscle cells Cellular events associated with muscle regeneration are related to satellite cells functionality such as proliferation, migration and then fusion with the altered mature fibbers. The age-related depletion of satellite cells associated with aging is well characterized and we have shown in precedent works that calpain activity play a critical role in cellular events associated with regeneration: satellite cells proliferation, migration and fusion. Apoptosis due to oxidative stress is one of the mechanisms involved in the age-related depletion of satellite cells. Calpains could be implicated at several levels in this process and the objective of the described study is to precise the incidence of oxidative stress on calpain function. LHCNM2 human cell lines (kindly provided by Vincent Mouly, Institute of Myology) were used as a model. After stress induction with H2O2, we first studied indicators of oxyidative stress: PP38 MAPK was well activated, carbonyl groups increased significantly and the level of survival cells was approximately of 40%. In these conditions, calpain activity and expression were significantly increased. A differential proteomic approach is currently being carried out in our laboratory to dissect the molecular events linking oxidative stress to calcium-dependent proteolysis. The enhancement of the antioxidative potential of satellites cells could help to compensate the age-dependent imbalance of the redox system observed in these cells and then improve muscle ability to regenerate and replace damaged fibres in old muscle. So the effect of a natural antioxidant (Oligopin®) on H2O2induced oxidative damages in LHCN-M2 cells was tested (Industrial partnership: Dérivés Résiniques et Terpeniques 40105 DAX France). Oligopin® is a natural antioxidant extracted from French maritime pine bark. Human satellite cells LHCN-M2 were pre-incubated with Oligopin® (0.05 mg/mL) before H2O2 treatment. The pine bark extract was able to partially restore cell viability, while the H2O2-induced apoptotic cell death was completely abolished. Similarly, calpain activity was not affected by H2O2 treatment when the cells were pre-incubated with the pine bark. Here we show that oxidative stress is able to activate the calcium-dependent proteolytic system in human myoblasts (Dargelos et al. submitted to Exp. Cell Res.). These data, together with those previously published on differentiated rat skeletal muscle fibres, where we demonstrated calpain activation during ageing, strongly support the involvement of oxidative stress in the aetiology of sarcopenia through calpain-dependent pathways. II) INVOLVEMENT OF THE CALCIUM-DEPENDENT PROTEOLYTIC SYSTEM IN MYOBLAST ADHESION AND MIGRATION, AND CELL INVASION. Myoblast adhesion and migration Previous works of our laboratory demonstrated that ubiquitous calpains regulate murine myoblast migration. Since 2006, we addressed the question how calpains played a pivotal role during myoblast migration, a crucial step required to align myoblast to permit them to fuse. Like, migration is a succession of adhesion and de-adhesion steps, we first investigated the role of each calpain isoform in attachment and spreading of murine C2C12 myoblasts. Inhibition of each ubiquitous calpain isoform by antisense strategy induces a drastic decrease in C2C12 myoblast attachment and spreading (Mazères et al., Cell Motil. Cytoskeleton, 2006). The cells were characterized by the loss of membrane extensions and the disorganization of stress fibers. These experiments argue for the involvement of these proteinases during adhesion of C2C12 myogenic cells. During myoblast migration significant modifications in calpain expression was noted: µ-calpain expression decreased while m-calpain expression increased (Leloup et al., Int. J. Biochem. Cell Biol., 2006). These results, together with those previously obtained in our laboratory; confirm that m-calpain is implicated in the myoblast migration process. After addition of growth factors such as IGF1, TGFβ1 and insulin myoblast motility was enhanced. Inhibition of calpain activity leads to inhibition of this growth factor-mediated migration (Leloup et al., Int. J. Biochem. Cell Biol., 2006). Correlatively, in contrast to µ-calpain, significant increases of m-calpain expression and activity were measured after treatment by these different growth factors, suggesting that m-calpain plays a crucial role in growth factor-mediated migration. In addition, it has been demonstrated that the ERK/MAPK pathway was involved in this process resulting in the increased myoblast migration (Leloup et al., Int. J. Biochem. Cell Biol., 2007). A proteomic analysis has been conducted to identify new potential substrates (Mazères et al., Cell Motil. Cytoskeleton, 2006). In comparison with control cells, numerous proteins accumulate in calpain inhibited cells. Some of them appear to act as calpain substrates during myoblast migration. Experiments are currently in progress to explain the role of calpain activity (via the cleavage of these substrates) at the molecular level during myoblast migration. Very recent data using a new human cell line, LHCN-M2, showed that, at time of myoblast migration, ubiquitous calpains might play a pivotal role in the organization of actin cytoskeleton (Roumes et al., submitted to Int. J. Biochem. Cell Biol.). Indeed, the inhibition of calpain activity, in this human cell line, reduced, at once, cell adhesion, velocity, dispersion capacity and invasive behavior. Moreover, myoblasts had a rounded morphology, failed to form membrane protrusions, and presented a reorganization of focal adhesions associated with an increase of vinculin expression and a disorganized actin cytoskeleton. Because of its cell localization, µ-calpain seems to be more particularly involved in the actin stress fiber remodeling. Cell invasion Alveolar and embryonal rhabdomyosarcoma (ARMS and ERMS, respectively) are a soft-tissue sarcoma commonly encountered in childhood and adolescence. These rhabdomyosarcoma (RMS)arise from immature cells that are destined 102

to form striated skeletal muscle. RMS can acquire invasive behavior and can form metastasis in lung, bone, marrow, and lymphatic nodules. This metastasis decrease less than 20% the patient healing. Among the many proteases implicated in cell motility, the calpains play an essential role in regulating migration and invasion which are both phenomena implied in metastasis development. Targeting calpains may present a novel approach toward restraining metastases and development of ARMS or ERMS cancers. The aim of this research is to study the calpain implication in RMS potential metastatic.According to our results, it seems at present that calpains play a crucial role in the rounded morphology and the weak adhesiveness of RMS cells compared to LHCN-M2 cells (Roumes et al., in preparation). Moreover, calpains may be implicated in the loss of stress fibers in ARMS and ERMS and a deregulation of focal adhesion turnover leading to an increase of migration and invasion of these cells (Roumes et al., in preparation). In this way, targeting calpain activity may represent an interesting strategy for limiting development of ARMS and ERMS tumors as well as their metastatic behaviors.III) ROLE OF MYOSTATIN IN MUSCLE ATROPHY: IDENTIFICATION OF MECHANISMS INVOLVED IN MYOSTATIN INDUCED ATROPHY Project supported by ANR: Agence Nationale de la Recherche (2009-2011) Interactions myostatin/calcium-dependent proteolytic system The development of the muscular mass results from a balance between proliferation, differentiation and cellular death, protein synthesis and degradation. The muscular atrophy is characterized primarily by impaired protein synthesis and an increase in protein degradation. Protein degradation is the result of several proteolytic systems among which the calcium dependent proteolytic system calpains and calpastatin (an endogenous specific inhibitor) plays a very important role. The preliminary results obtained concerning the interactions myostatin/calcium dependent proteolysis open new prospects for studies targeting the influence of myostatin on the expression, regulation and consequently the activity of the calcium-dependent proteolytic system in direct relation to meat quality. Indeed, some authors suggested that m and µ-calpains could be very implied in the development of meat tenderization. Others highlighted that the origin meat hardness observed in certain animals having a muscular hypertrophy (Callipyge sheep for example) would be due to a strong expression of the calpastatin, which means a very low calcium dependent activity. Likewise, it was also shown on the one hand that the postnatal development of skeletal muscle was controlled by the activation of the satellite cells and on the other hand that muscular hypertrophy was due in part to an increase recruitment of the satellite cells. Myostatin is able to modify the expression levels of certain myogenic transcription factors such as MyoD, myogenin and the expression of some cycline kinases (p21, Rb) inhibitors. Furthermore, myostatin gene expression has been shown to be induced by MyoD which preferentially binds and up regulates the myostatin promoter activity. The preliminary results obtained suggest that there was an important interaction between myostatin and the proteolytic calcium dependent system. Accordingly, the study will present two actions: In collaboration with INRA, Montpellier, we have shown (from C2C12 muscle cells stably transfected with a vector for overexpression of myostatin) that overexpression of this molecule induced an increased share of expression of calpastatine of about 50% and the other a decrease in the expression of μ-calpain from around 60%. In these conditions no change in the expression of m-calpain was observed. These results show that myostatin overexpression induces an overall significant influence of the protein expression of two of the calcium-dependent proteolytic. Furthermore, in our experimental conditions, a total inhibition of the process of myoblast differentiation / myotube was noted. 5 significant publications: 1. 2. 3. 4. 5.

Dargelos E., Poussard S., Brulé C., Daury L., Cottin P. (2008) Calcium-dependent proteolytic system and muscle dysfunctions: A possible role of calpains in sarcopenia. Biochimie 90, 359-368. Dargelos E., Brulé C., Combaret L., Hadj-Sassi A., Dulong S., Poussard S., Cottin P. (2007) Involvement of the calcium-dependent proteolytic system in skeletal muscle aging. Experimental Gerontology 42, 1088-1098. Leloup L., Daury L., Mazeres G., Cottin P., Brustis JJ. (2007) Involvement of the ERK/MAP Kinase signalling pathway in milli-calpain activation and myogenic cell migration. Int. J. Biochem. Cell Biol. 39, 1177-89. Goudenège S., Dargelos E., Claverol S., Bonneu M., Cottin P., Poussard S. (2007) Comparative proteomic analysis of skeletal muscle cell caveolae after milli-calpain deregulation. Proteomics 7, 3289-3298. Leloup L., Mazeres G., Daury L., Cottin P., Brustis JJ. (2006) Involvement of calpains in growth factor-mediated migration. Int. J. Biochem. Cell Biol. 38, 2049-2063.

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Teaching of food biochemistry, cell and molecular biology. Institut des Sciences et Techniques des Aliments de Bordeaux, Licence 3 and Master 1 & 2 levels.

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TEAM « NANOMECHANICS ON SOFT MATERIALS » Team Leader: Jean-Pierre Aimé, Research Director, CNRS. Team Composition (2006-2009): Rodolphe Boisgard MC Charlotte Bernard, Doctorante Jean-Michel Arbona, Doctorant

Gérard Couturier Pr Julien Buchoux, Doctorant Mahamadou Seydou (post doctorant)

Sophie Marsaudon MC Cédric Jai, Doctorant Sébastien Vatinel (post doctorant)

Team Production (2006-2009): Publications, peer reviewed, (ACL) Publications, peer reviewed, not yet in bases (ACLN) Invited conferences congress (INV) Séminaires Oral communications in congress (COM)

11 1 8 13 8

Poster communications in congress (AFF) Books or Book chapters (OS) Congress/Workshop organization Defended PhD Theses or HDR Grants : public & private k€/year

5 6 6 3 100 k€

Scientific activity Abstract As part of the team works, focus on studies of mechanical properties at nanometer scale of soft materials and fluids, we opened several routes to investigate biological systems in liquid or in air with an atomic force microscope. In close collaboration with several groups, Microsystems at IEMN, multiwalled carbon nanotubes (MWNT) AFM tips at NASA Ames research center, Single Walled Carbon Nanotubes (SWNT) AFM tips at Institute Néel and physics department at LETI, we are involved in the development of a Nano ElectroMechanical System, the use of nanoneedles and carbon nanotubes as AFM tips and the study of air-liquid interfaces. Within that frame, the team has concentrated his efforts on the mechanical properties of carbon nanotubes and nanofluidics. The tip is the central part of any near field technique which has always retained much attention. This is an old story that started more than two decades ago with the merging of the near field techniques tunneling microscopy (STM, AFM and SNOM). STM and AFM need to have well defined tip apex, size and shape, as its properties determine the physical origin of the contrast of the image. Ideally, the tip apex should match the experiment desired scale with a stable and robust chemical composition. This is barely the case for commercial Silicon or Silicon Nitride tips. Treatments can be done to diminish the apex size with acid attack or growth of carbonaceous apex under ion beam. However, the tip usually becomes fragile and breaks rapidly. Other requirements are a stable tip at the molecular level, i.e., ideally chemically inert at the molecular scale, with weak adhesion forces. To face these very challenging tasks Carbon Nanotubes appear as the appropriate nano objects solving many of those problems. Carbon nanotubes are rolled graphene sheets into nanometer diameter size cylinders. Their small diameter (from 1 to some 10 nm), controlled cylinder geometry, hydrophobic properties, exceptional mechanical properties make them robust with reduced strength of the interaction with soft sample. The other key point is to image biological samples in liquid environment, but the cantilever large hydrodynamic forces restrict the use of dynamic AFM hindering a quantitative analysis. A way to reduce the hydrodynamic forces is just to keep everything out of the liquid except the very end of the tip apex. The image of air – liquid interfaces has leaded us to investigate the mechanical properties of a nanomeniscus. Over the last decade, a new field emerges where materials from life systems invade the nanotechnology domain. In that field, DNA material occupies a central part. Based on the Watson-Crick interaction, short nucleotide sequences are used as bit of information from which can be built multi scale structures and design new functional materials of high level of complexity. Within this frame, we have started work to conceive a foldable nanosensor DNA Origami.

• Near field probes based on Carbon Nanotubes: Collaboration: IEMN (Lille), Institut Néel (Grenoble), Grant ANR PNano Improve LM, PICS S. Marsaudon. The aim of this program is to design a new AFM resonator to increase the resonance frequency towards the MHz and to the decrease the size of the mechanical device in order to reduce the level of hydrodynamic forces in fluids. The conception and the fabrication of the electromechanical system is done by the MEMS group at the IEMN (Lille) (fig 1). In this project, our team does work on the development of Carbon Nanotube as a tip. Within a close collaboration with the Institut Néel, a large number of tips with single walled CNT are available (fig. 2a). During that period, with the help of the PICS program, the group has also developed the capability to fix multi walled CNT at the tip apex (fig. 1b). The tip as a probe to scan a surface is the central part of any near field technique which has always retained much attention. This is a relative old story that started more than two decades ago with the merging of the near field techniques tunneling microscopy -STM-, AFM and SNOM. Near field techniques need to have well defined tip apex 104

with controllled size and shape. The ideeal probe shouuld have an attomic size or at least a mollecular size. An A even moree important reqquirement shoould be a stable tip at the molecular m leveel, i.e., ideallyy chemically innert at the mo olecular scale. These strongg requirementss make the design of an apppropriate tip a very challengging task. Thiis is the main reason why a flexible beam m made of a CNT is comm monly consideered as the beetter choice too get highly eefficient AFM M nanoprobes. However, beecause it is oftten difficult too avoid high flexibility, f man ny situations describing thee contact betw ween the CNT T and the surfaace can be envisioned. Theerefore the unnderstanding of o the mechannical behavior of CNT tip is i demanding.. We have deeveloped a sim mple model showing the competition between elasticity and addhesion, it giv ves analyticall expressions for f the frequenncy shift that matches well the experimen ntal data. (Figg. 3) Fig 1: Integratted electronic circuitry to be F b inserted in a modified AF FM head (l (left). Scheme of the NEMS S force nanosennsor made off a radial vibra ating d disk.(right). Deevice designed d at the IEMN N.

Fig. 2: (Left) Scanning eleectron microsccopy (SEM) iimage of a siingle wall F c carbon nanotuube directly grown g on a commercial tip apex. (rig ght) SEM im mage of a muulti wall carbo on nanotube fixed f on a com mmercial tip apex. a Note thhe scale 10 tim mes larger tha an in Fig.2 lefft. Fig. 3 Experrimental (collor point) F fr frequency shhifts with normalized d distance for a single walleed (a) and m multi walledd (b) nano otube for d different osscillation amplitudes a c compared too theoretical curves ( (black)

a

a Knt poutre en appui élastique (N/m)

8

ks (k n L) 3 (1 + cos(k nl ) cosh(k nl ) ) = n( k n l ) cosh(k nl ) ) eequation: kl 3(sinh( k n l ) cos(k nl ) − sin

7 6 5 4 3 2 1 0

b Fig 4: (a) Moodeling of thee ensemble caantilever and carbon nano F otube. The c cantilever of spring s constan nt kl is endedd with a carbbon nanotube at the tip a apex. The CN NT is describeed as a sprinng with consstant ks. The boundary c condition is an a elastic onee. The shift of the resonannt frequency takes into a account the booundary cond dition and the variation of tthe spring con nstants kc. T The calculatedd resonant frequency f shif ift is given bby the solutio on of the

0

1

2

3

4

5

6

7

Knnt oscillateur équivalent (N/m)

8

b

(b) Solving thee equation ab (b bove with as an a input the eexperimental frequency f shifts gives thee associate spring constants ts (square sym mbols). The strraight line iss the usual deerivation of th he spring consstant with a fr free boundary condition a the cantileveer end. Experiimental data are at a issued from m 4 carbon na anotubes.

In order o to accurrately understaand and contrrolled how the tip apex touuches the surfface, we havee developed a new approach based on thee use of a therrmal noise forrcing. As therrmal noise forrcing induces a oscillation amplitude a lesss a the surfacce can be studdied with the help h of a veryy than the angsstrom, the conntact betweenn the carbon naotube apex and weak external perturbatioon. In figuress 4a and 4b we present a short descriiption of the theoretical modelling m andd experimentall results. The interaction between b a Carrbon nanotubee (CNT) and a Graphene shheet is calculaated using MM M+ molecularr m d describe the contact c properties between n a CNT Atom mic Force Microscope (AF FM) tip and a mechanical modelling.to graphite surfface. The slidiing motion of the CNT: the graphene sheeet either at the CNT apex oor with a given n CNT lengthh contacting thhe surface is innvestigated. Fig 5 : The aim of the Mollecular Mechhanics investigaations of Carrbon nanotubbe and Grapphene sheet interaction is to mimic thhe AFM CN NT tip scanningg a graphite surface: top: left, l binding energy e variationn of (9,0) CNT T without torsiion, right, adhhseive energy inn function of torsion t angle. Bottom, strucctures of CNT(99,0) without torsion t (a), with 30° (b), 60° 6 (c) and 90° (d) of torssion. Structurres are builtt with ftware. nanotubee Modeler soft 105

• Air-liquid interface properties and proteins in membrane: 1- Imaging an air liquid interface: Nanomechanical properties of a nanomeniscus. Collaboration Amal Chabli, F. Bertin, D. Mariolle LETI (Grenoble), T. Ondarçuhu (CEMES, Toulouse). The idea driving this work is rather simple: because an oscillating microcantilever of 100 micrometers length experiences large hydrodynamic forces hindering quantitative investigation of biological systems in liquid, our approach just keeps everything out of the liquid and only the very end of the tip is in the liquid. Therefore, this approach allows investigating interface properties of complex fluid systems with an oscillating microcantilever still a high force gradient sensitivity. The dynamical behaviour of a nanomeniscus is investigated with a oscillating nanoneedle recording information on the change of the shape and viscous contribution. At the air-glycerol interface, height images of the liquid interface with resolutions at nanometer scale are recorded (fig.7) Fig 7: Image at the air-liquid interface of glycerol. The height image is recorded by setting a given (here positive) resonant frequency shift of the AFM oscillator.25 The tip slightly dips into the liquid, and we expect that the meniscus has a height of no more than a few tens of nanometers. The positive resonant frequency shift is due to the elastic response of the contact line. The crosssection shows the height fluctuations: some of them are small (1nm), while others reach magnitudes as large as up to 10 nm, but are slowly varying.

The viscous dissipation inside a water nanomeniscus is the result of a competition between the capillary flow, due to evaporation, enhancing the dissipation and the boundary conditions at the air-liquid interface reducing the influence of the shear motion in the meniscus. Simple hydrodynamic arguments show how macroscopic concepts can apply to explain the dynamical behavior of a very small quantity of an oscillating liquid. Fig 8: Force gradient and dissipation driven by thinning a water nanomeniscus: variation of the resonant frequency shift (filled circles) as a function of time when the nanoneedle oscillates in water. The damping coefficient γ int (empty circles) is simultaneously recorded. The sharp increase in the damping corresponds to the abrupt decrease in the resonant frequency shift. Intricate phenomena happen, leading to opposite effects, induced by the thinning of a nanomeniscus. 2- Proteins in membrane: R. Oda, B. Desbat, J. Lang (CBMN-Bordeaux): ANR PCV Exodynamics. In order to get insight on the structure and conformation of part of SNARE proteins in membrane, we investigate the insertion of Syx70 (see R. Oda report) in DPPC multi layers using the Langmuir Blodgett technique. Multi layers are transferred to reduce the influence of the mica surface on the Syx70- DPPC organization. We have faced difficulty to successfully transfer layer including Syx70, and only transferred multi layers with high dilution were obtained with ratio Syx70/DPPC smaller than 1/30. Below we show results indicating two types of peptide arrangement in DPPC. Fig 9: Tapping images of DPPC/Syx70 transferred on mica at 40 mN/m, height (left) phase (right) with peptide ratio 1/50 (images a and b) and 1/100 (images c and d). The phase β and the Phase α indicate two different types of dissipative behavior due to the Syx70 aggregates. The phase images (b and d) show lower values (dark zone) for the phase β indicating a more pronounced viscous behavior than that of the phase α (bright zone). This result suggests two different types of Syx70 aggregation with a less ordered one in phase β . These two different structures are observed at the two concentrations. The next step is to investigate the membrane filled of Syx70 at the liquid air interface to mimic situations close to that of a cell membrane. To do so a Langmuir micro cuve has been designed and set in a D3000 AFM (fig. 7). 106

Fig 10: Lanngmuir micro cuve inside an a AFM. Witth the help from NIMA, a new Langm muir micro cuuve have beenn built that M. Using the micro m cuve, can be placeed in a Veeco-D3000 AFM we envisioneed to study thhe wetting prroperties of SWNT S and MWNT and to perform study on phoopholipid layyers at the liquid interfaace.

• Cell adhesion prob bed with a quartz miccro balancee.(Collaborration CEA A program,, A. Chabli LET TI). With a CEA prograam providing funds for a tw wo years postt doc and equiipment, one uuses a Quartz microbalancee with dissipattion (QCMD) to probe the cell mechaniical propertiess. QCMD geenerates in fluuids an evanesscent velocityy field, thus applies a a sheaar perturbatioon at the cell-substrate in nterface over a length thaat depends on n the QCMD D frequency: a few hundred nanometres foor frequenciess ranging betw ween 5 to 75 MHz. M Fig 11: Change of the resonance frequencyy (blue) annd dissipationn (red) when the trypsinee EDTA is added. The decrease d of the t resonancee frequencyy shift indicaates the reduuction of thee mechaniccal coupling between the cells and thee substrate. Accordinglyy, the dissipation decreasess m roundedd and imagges show that the cells are more in phase 3, 3 while in phhase 1 the cellss are flat withh an extendded cytoskeletoon structure.

• Foldablle Bio Nan nosensor based b on DNA D Origa ami. ANR PIR 2008--2009 (J. Elezgaray), E , Collaaboration J. Elezgaray CBMN,, C. Di Priimo (IECB B/U869 Insserm), D. Gasparutto G o (INA AC, CEA Grenoble). G The project uses u a recentlly developed method for folding fo long siingle strands of DNA into arbitrary two o-dimensionall shapes using a raster fill teechnique ‘scafffolded DNA origami. The single strand folds are creaated with specific crossoverr 1 nanometeers are obtaineed. The first aim a is to builld molecular, foldable, bioo strands called stapples. Shhapes up to 100 3 structures. captors. A seecond objectivve is to build 3D

Fig 13: Top: numerical simulation off foldable m: AFM dy ynamical DNA origgami Bottom height im mage of firstt folded recctangular origami and a the coompanion caalculated structure. (JM Arboona CBMN N). The rectangularr origamis are circled in the AFMimagee.

5 significaant publicattions: 1. 2. 3. 4. 5.

“Wettingg an oscillatingg Nanoneedle too image an air--liquid interfacee at the nm scalle: dynamical bbehavior of a Nanomeniscus.” N ” C. Jai, J.P. J Aimé, D. Mariolle, M R. Boiisgard, A. Chabbli Nanoletters, V 6, issue 11, 2006 2 pge 2554--2560. “Dynam mic operation modes m of AFM: Non-linear behhavior and theo oretical analysis of the stabilitty of the AFM oscillator.” R.. Boisgard, JP. Aimé, G. G Couturier Inn International Journal J of Non-Linear Mechanics. Elsevier E Edt. 42, 673-680 0, (2007) “Compeetition of elasticc and adhesive properties p of caarbon nanotubees anchored to atomic a force miicroscopy tips “C. “ Bernard, S.. Marsau udon, R. Boisgaard, J.P. Aimé Nanotechnologgy, 19, 35709-3 35718 (2008). “Dynam mical behavior of o an evaporatinng nanomeniscuus: a Boundary y Condition prooblem at the Loocal Scale”. C. Jai, J J.P. Aimé,, R. Boisggard Eur. Phys. Let. 81, 340033, (2008). “Carbonn nanotubes as SPM Tips: undderstanding CNT T tips mechaniical properties and a imaging” S S. Marsaudon , C. Bernard , C.V. Ngguyen , A.M. Boonnot, J.P. Aim mé, R. Boisgard d : in “Applied Scanning S Probee Methods”. Jann 2009.

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Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Teaching: (only higher grades are shown) 1. 2. 3. 4.

Summer School « Second European AFM BioMed Summer School on Theory and Practice of AFM in Life Sciences and Medicine, Marcoule – France September 14th-19th 2009 : S. Marsaudon Summer School « Second European AFM BioMed Summer School on Theory and Practice of AFM in Life Sciences and Medicine, Marcoule – France September 2008: JP Aimé “Nanosondes” , Master 2 Lasers, Matières et Nanosciences : J.P. Aimé (2005-2006), R. Boisgard 2007-2008, R. Boisgard & S. Marsaudon 2009. : 16h cours. « Local force probe.methods : an introduction », Engineering school : Ecole Nationale Scientifique de Chimie et de Physique de Bordeaux : 7h30 cours, J.P. Aimé (2005-2007), S. Marsaudon (2008-2009)

Invitation to scientific careers for young scholars, especially for females : S. Marsaudon, « Les nanotubes de carbone et leur fixation sur des pointes de microscopie à force atomique », poster et témoignage, Café Sciences et Mixité, 13 novembre 2007, Lycée « Les Iris » Lormont (33). Direction of a teaching program: -R. Boisgard is in charge of the “ Preparation au concours de l’agrégation de physique , option Physique » de Bordeaux since 2006 -S. Marsaudon is in charge of the « Master 1 de Physique de Bordeaux » : partially from 2006 to 2009, and totally of the professionnal part in 2009-2010. Other Scientific experiences , responsibilities : • Chairman of NanoSWEC, International Conference created in 2008 and part of the C’Nano GSO. (http://nanoswec.cnanogso.org/) JP Aimé • Co-chairman of the NanoSpain conference, JP Aimé. • Member of the « Comité de Pilotage » ANR P3N, JP Aimé. • Expert of the french « Observatoire des Micro et NanoTechnologies » (OMNT). Survey on the development of Nanosciences and Nanotechnologies, JP Aimé: • 2005-2007 Chairman of the « Nanoconstruction » group. • 2007Member of the « BioInspired Nanosciences and Nanotechnologies». • Partial delegation (1/2) at CNRS for S Marsaudon for 2008-2009 and 2009-2010.

Management and running of technological platforms. - Direction of the CNRS PICS (Projet International de Coopération Scientifique) N° BIOTIP between 4 french laboratoires : CPMOH UMR 5798 (porteur du projet), IEMN UMR 8520, CEA-LETI, MINATEC, Institut Néel, CNRS/UJF and 3 American partners : CV Nguyen from Nasa Ames Research Center, Mountain View, CA, B. Bhushan from Nanotribology Laboratory for Information Storage and MEMS/NEMS (NLIM, now NLB²), Columbus, Oh,and Arvind Raman, Birck Nanotechnology Center, School of Mechanical Engineering. S. Marsaudon. - Chairman of the CPER Pôle Nanosciences Aquitain. Ten laboratories are involved in the CPER. The objective is to support and enhance the development of nanosciences and nanotechnology. J.P. Aimé - Chairman of the french network C’nano Grand Sud Ouest (GSO) (http://www.cnanogso.org/). The network includes three districts : Aquitaine, Midi-Pyrénées et Languedoc Roussillon. GSO is part of a six C’nano networks dedicated to the development of nanosciences and nanotechnology covering the french country. J.P. Aimé

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AS PART OF CNRS UMR 5084 CNAB, TEAM « BIO-ORGANIC CHEMISTRY » Team Leader: Gérard Déléris, Prof. Univ. Bordeaux 2. Team Composition (2006-2009) with C. Petibois students: Cyril Petibois, MCU Bordeaux 2

Karine Gionnet, CR1 INSERM

Mireille Bayle, MCU Bordeaux 2

Sandra Rubio, AI Bordeaux 2

Katia Wehbe, PhD 2005-2008 Seydou Yao, M.Sc 2009

Karima Belbachir, PhD 2008-

Victor Maurizot, CR2 CNRS Annie Perromat, AI Bordeaux 2 (up to 2007) Razia Noreen, PhD 2008-

B.Sc. and M.Sc. trainees (stay between 2 and 6 months): 10

Production C. Petibois (2006-2009): Publications, peer reviewed, (ACL) Publications, peer reviewed, not yet in bases (ACLN) Invited conferences congress (INV) Publications, peer reviewed in published international colloquia (ACTI) Oral communications in congress (COM)

13 2 6

Poster communications in congress (AFF) Congress/Workshop organization Defended PhD Theses or HDR

1

Grants : public & private k€/year

1 2 1 30 k€

6

Scientific activity C.Petibois Résumé Les méthodologies analytiques qui ont été développées sont basées sur la spectrométrie et la spectro-imagerie infrarouge à transformée de Fourier (IRTF) et ont pour finalité l’étude des échantillons biologiques, fluides, cellules et tissus. Les travaux menés jusqu’à présent ont eu pour but de montrer quelles performances analytiques pouvaient être atteintes avec cette technique de spectroscopie vibrationnelle. Par exemple, il a été démontré que la spectrométrie IRTF permettait de déterminer la concentration d’un marqueur chimique actif dans le moyen-IR (-N3) dans le sérum sanguin avec une précision utile à la biochimie clinique jusqu’à 10 nMol.L-1. L’analyse de lysats cellulaires combinée à celle de milieux de culture a aussi permis de quantifier très précisément le métabolisme cellulaire (concentrations en glucose, acide-lactique, glutamine…etc.), ce qui montre que cette technique peut-être utilisée pour des études physiologiques complexes. La même approche a été appliquée à la spectro-imagerie IRTF, nécessitant néanmoins le développement de solutions avancées de traitement des données spectrales (chimiométrie). L’imagerie IRTF a été développée aussi bien pour l’analyse de cellules cryofixées, selon une procédure de culture cellulaire et de fixation développée à façon, que pour l’étude des tissus. Pour ces derniers, il a été démontré que l’on peut avoir accès à des informations moléculaires caractéristiques d’états physiologiques (sain, pathologique…), notamment via l’étude de la structure secondaire des protéines, l’analyse de paramètres métaboliques, ou encore l’état de peroxidation des chaines d’acides gras. L’étude de cellules individuelles a nécessité le recours au rayonnement synchrotron comme source plus brillante de photons IR du fait de la très faible quantité de matériel organique à analyser. Cela a permis l’obtention des toutes premières images IRTF de cellules au monde. Abstract The analytical methods developed are based on FTIR spectrometry and spectro-imaging and aim at studying biosamples, fluids, cells and tissues. The work performed up to now had the objective of showing which analytical performances could be reached with this vibrational spectroscopy technique. As an example, it was demonstrated that FTIR spectrometry was able to determine the concentration of a chemical marker active in the mid-IR (-N3) within blood serum with an accuracy sufficient for the clinical biochemistry down to 10 nMol.L-1. The analysis of cell lysates combined to that of cell culture media also allowed to quantify with high precision the cell metabolism (concentrations for glucose, lactic-acid, glutamine…etc.), thus showing that this technique may be used for complex physiological studies. The same approach was applied to FTIR spectro-imaging, but requiring the development of advances solutions in spectral data treatments (chemometrics). FTIR imaging has been developed for the analysis of cryofixed cells, using a cell culture method and cell fixation protocol specifically developed, as well as for studying tissue contents. For these ones, it was demonstrated that we can have access to molecular information characteristic of a given physiological state (healthy, pathologic…), notably through the study of the secondary structure of proteins, the analysis of metabolic parameters, or even the peroxidation state of fatty acyl chains. The study of individual cell required the use of synchrotron radiation as a brighter source of IR photons due to the very small amount of organic matter found in cells. This development allowed obtaining the very first FTIR images of cells worldwide.

• FTIR spectroscopy of cell lysates Analytical performances of FTIR spectroscopy FT-IR spectrometry has proved to be a useful tool for determining series of plasma molecular concentrations. Dedicated experiments were first performed to test the analytical performance that could be obtained by FT-IR spectrometry using a synthesized N3-peptide exhibiting a -N3 absorption centered at 2110 cm-1, a spectral region where no organic material 109

FT-IR N 3-peptide (nMol/L)

of biologicaal samples absorbs. Furrther, we innvestigated whether w this technology was w able to alllow quantificcation of metaabolic parameeters (glucose 100 C and lactic-acid) within plaasma, cells, annd tissues as ann alternative method m to the 80 b a approaches, w which requiree sophisticateed biological “classical” biochemical 60 material treattment and exppensive reagennts. For this purppose we used a series of plaasma sampless to determinee glucose and 40 lactic-acid cooncentrations,, which are common markkers of cancerr growth. We 20 Y = 0.9 9479 X + 3.046 compared the results of thhe main specttral data treatm ments commo only achieved R = 0.94; S = 11 nMol/L 0 d analysis, such as univvariate (Beer--Lambert) or multivariate for FT-IR data 0 2 20 40 60 80 100 (PLS) calibrrations, as weell as the decconvolution of o the spectraal interval of ( Reference N -peptide (nMol/L) interest (12000-900 cm-1). No significannt differences were found regarding r the analytical performances off these methodds. FTIR sttudy of cell meetabolism Although tumor ischhemia participates to anggiogenesis and cancer proogression, thee contribution n of nutrientt t this processs remains pooorly characteriized. Using seerum-free cultture media, w we used FTIR spectrometryy deprivation to for testing thhe impact of glutamine g depprivation on metabolic m and angiogenic reesponses in A A549/8 cells. Although A thiss led to HIF-inndependent inncrease of VE EGF-A mRNA A, VEGF-A protein p levelss remained loow and correlated with thee induction of GADD34 andd CHOP mRN NAs and apopptosis. This deemonstrates thhat amino acidd deprivation may have noo mpared to glu ucose or oxyggen deprivatioon but may in n contrast bee direct effect on VEGF-deependent angiiogenesis com t cancer proggression by prooviding more selective enviironments. detrimental to 2

x/y

3

• Synchrotron radiaation FTIR R imaging of o cells FTIR micrroscopy with a FPA of deteectors allows routine r chemical imaging on o individual ccells in a few minutes only. The brilliancce of synchrottron radiation (SR) IR sourrces may enhaance the signaal obtained frrom such smaall biosampless containing reeduced amounnt of organic matter. m We innvestigated ind dividual cells obtained from m a cell culturre specificallyy developed foor transmissioon FTIR imagging using eithher a Globar or a SR sourcce coupled to the same insstrumentation.. SR-IR sourcee focalization was optimizeed to control thhe energy disttribution ontoo the FPA of ddetectors. We demonstratedd that accessinng to IR absorpption distributtion from all organic o conten nts of cells at 1x1 µm pixell resolution is possible onlyy with high cirrculating curreent (≥ 1.2 Am mp) illuminatinng a limited number n of thee FPA’s detecctors to increaase the signal-to-noise ratioo of IR imagees. Finally, hiigh current SR R ring is man ndatory to colllect FTIR im mages of biosaamples with a high contrastt in minutes (to ( the opticall image of thee cell presenteed here corressponds its FTIIR image showing amide I distribution.  

•

FTIR R imaging of tissues

Fourier-transform inffrared (FT-IR R) spectro-imaaging enabless a global anaalysis of sam mples h shown thhat FTIR imaaging with a resoluution close too the cellular level. Our reccent studies have allows determ mination of biodistribution b n for several molecules m of interest (carbbohydrates, lippids, proteins) for tissues analyysis without prre-analytical modification m of o the sample,, such as stainning. mation is alsso available from fr the samee analysis, nootably for prootein Molecular sttructure inform secondary sttructure and fatty f acyl chaain peroxidatiion level. Thu us, several caancer markerss are available from FTIR tissuue images, alloowing accuratte discriminatiion between healthy h and tuumor n able to provide p uniqu ue chemical annd morphologgical areas. FTIR imaging appllications are now a tissue sttatus. With thhe fast image acquisition a pro ovided by moodern mid-infrrared information about imaging systtems, it is now w envisaged to analyze ceerebral tumor exereses in delays d compaatible with the neurrosurgery. Acccordingly, wee propose to taake FT-IR imaaging into connsideration for the developmentt of new moleccular histopatthology tools.

5 significaant publicattions: 1. 2. 3. 4. 5.

Petibois C, Piccinini M, M Cestelli-Guiddi M, Déléris G, G Marcelli A. A bright future for synchrotroon imaging. Nature Photonics,, 3(4), 1799, 2009. Wehbe K. K Pinneau R. Moenner M M. Déléris G. Petiboois C. FT-IR sp pectral imagingg of protein conntent changes in n blood vesselss during tuumor growth. Anal. A Bioanal Chem. C 392:129-335, 2008. Petibois C, Drogat B, Déléris D G, Moeenner M. Histollogical mapping g of biochemicaal changes in soolid tumors by FT-IR spectrall imaging. FEBS Let. 581 :5469-74, 20007. Drogat B, B Bouchecareilh M, North-Chhassande S, Pettibois C, Déléris G, Chevet E, Bikfalvi A, Mooenner M. Acutte L-Glutaminee Deprivattion Compromiises VEGF-A Up-regulation U inn A549/8 Humaan Carcinoma Cells. C J. Cell. Phhysiol. 212, 463 3-72, 2007. Petibois C, Déléris G. Chemical mappping of tumor progression p by FT-IR imagingg: towards molecular histopath hology. Trendss Biotechnnol. 24(10), 4555-62, 2006.

Teaching, research & education n training, diffusion of o science (ccongress, w workshops, scientific open days etc.) Teaching: 1992h/year at thee University of o Bordeaux 2,, Faculty of Sp port Sciences (biochemistryy, cell biology y, physiology,, immunologyy, endocrinologgy). Education traaining: Head of a CNRS thematic t schoool “Chimiom métrie des Speectroscopies ett Imageries Moléculaires M CSIM” (http://www.csim.ccnrs.fr/), natioonal level, 22--26 October 2007, Anglet, France F (50 parrticipants).

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Team Member: Dr. Victor Maurizot, Chargé de Recherche CNRS Team Composition (2006-2009): Groupe de Chimie Bio-organique, UMR CNRS 5084 Gérard Déléris, PR Univ Bordeaux 2 (Team Leader) Dr. Karine Gionnet, CR1 INSERM Anis alouini (doctorant) Dr. Sandra Rubio, AI Université Bordeaux 2

Cyril Petibois, MCU Univ Bordeaux 2

Victor Maurizot, CR2 CNRS

Marc-Elias Bakleh (doctorant) Sébastien Lavielle (doctorant) El-Farrouck Moustoifa (doctorant)

Katia wehbe (doctorante) Karima Belbachir (doctorante) Razia Noreen (doctorante)

Production V. Maurizot (2006-2009): Publications, peer reviewed, (ACL)

3

Poster communications in congress (AFF)

3

Scientific activity Victor Maurizot Résumé (1) Une des thématiques de l’équipe «Chimie pour le Vivant» de l'UMR 5084 est l’inhibition de l’angiogenèse tumorale. Dans ce but, un inhibiteur peptidique (VEGFI) a été développé et breveté au laboratoire. Ce peptide se fixe sur les récepteurs du VEGF, surexprimés au niveau des zones tumorales. Une de mes activités de recherche visait d'une part à valoriser cette découverte et utiliser ce peptide comme agent de ciblage des régions tumorales en le fixant sur des nanoparticules de PVDF possédant une écorce de PAA. Ces nano-objets seront utilisés pour le transport d'agents d'imagerie ou de traitement vers les zones tumorales. Ce travail a été effectué en collaboration avec l'équipe du Dr. Clochard au sein du laboratoire des Solides Irradiés, UMR CNRS 7642, Ecole Polytechnique, CEA, par l'intermédiaire d'une thèse en codirection entre les deux laboratoires. Le VEGFI est aussi couplé sur des plateformes oligosaccharides solubles multivalentes de façon à accroître son affinité pour ces récepteurs. Ce travail fait l'objet d'une "ANR jeune chercheur" portée par le Dr. Sébastien Gouin au Laboratoire des Glucides, UMR 6219 à l'Université de Picardie Jules Verne à Amiens et en collaboration avec es Dr. Eric Benoist et Chantal Galaup au Laboratoire de Synthèse et Physicochimie des Molécules d’Intérêt Biologique de Toulouse, UMR 5068. (2) Un deuxième aspect de mon travail est la construction par "design Fig a) Plateformes pseudopeptidiques cycliques, b) foldamères oligo-m-aniline pour la reconnaissance de protéines moléculaire" de nouveaux composés antiangiogeniques synthétiques dont l'architecture sera contrôlée de façon précise par liaisons hydrogène intramoléculaires afin de permettre une orientation optimale des groupements fonctionnels responsables de la reconnaissance pour les récepteurs. Deux approches différentes sont envisagées pour la construction de ces édifices. La première approche se base sur la construction de plateformes cycliques constituées alternativement d'unités aromatique et d’unités acides aminées «naturelles", la rigidité de ces édifices provenant de la structure cyclique ainsi que de la présence de liaison hydrogène intramoléculaire. Les groupements fonctionnels responsables de la reconnaissance pour le récepteur, ou plus simplement pour un substrat, proviendront des chaînes latérales des aa naturels. La deuxième approche est basée sur la construction de foldamères oligo-méta-aniline. Il s'agit d'une nouvelle classe de foldamère oligoamine dont la structuration provient de liaisons hydrogène intramoléculaire. L'étude de ces composés dans le solide montre une structuration originale cofaciale où l'on observe une rupture du réseau de liaisons hydrogène et la formation d'un coude. L'étude structurale en solution par RMN confirme quant à elle le repliement de ces composés. Abstract (1) One of the aims of the UMR 5084 chemistry research team is the inhibition of pathological angiogenesis phenomena. In this field, a peptidic inhibitor (VEGFI or vascular endothelial growth factor inhibitor) has been developed in the laboratory and protected by an international patent. This 17 amino acid peptide binds to VEGFreceptors which are over-expressed in tumors, preventing their association with their natural partner (VEGF). One of my research projects was to develop the use of this peptide as a targeting agent to tumoral regions. The VEGFI was 111

grafted onto polymeric nanoparticles for their use as vectors in therapy and as imaging agents. This work was carried out in collaboration with the team of Dr. Clochard in the "laboratoire des Solides Irradiés", UMR CNRS 7642, Ecole Polytechnique, CEA. VEGFI was also coupled to a water soluble multivalent oligosaccharide platform in order to enhance affinity for VEGF receptors. These multivalent systems may also be used for the coupling of other molecular entities for imaging and therapy. This work is supported by a "young researcher ANR fellowship" driven by Dr. Sebastien Gouin in the "Laboratoire des Glucides", UMR 6219 at the University of Picardie Jules Verne in Amiens and in collaboration with Dr. Eric Benoist and Dr. Chantal Galaup in the "Laboratoire de Synthèse et Physicochimie des Molécules d’Intérêt Biologique" in Toulouse, UMR 5068. (2) A second aspect of my research is the rational design of new synthetic anti-angiogenesis compounds. Within this purpose, I am currently developing new molecular platforms with predefined architectures in order to optimize orientation of functional groups involved in the recognition of VEGF receptors. Two different approaches have been investigated. The first design is based on cyclic oligoamide compounds comprised of alternating aromatic units and "natural" amino acid units (Fig a). The cyclic nature of these platforms and the presence of intramolecular hydrogen bonds are expected to rigidify the overall architecture. The functional groups of the amino acid unit side chains will be involved in the recognition process with substrates and later with natural receptors. The second approach is based on the construction of oligo-meta-aniline foldamers (Fig b). These compounds represent a new class of foldamers that adopt stable compact structure in solution through intramolecular hydrogen bonds. In the solid state, these foldamers show a non conventional cofacial architecture that can be compared to β-sheets structures. 5 significant publications: 1. 2. 3. 4. 5.

V. Maurizot, S. Massip, J.-M. Léger and G. Déléris, "Cylindrical sheet formation of oligo-meta-aniline foldamers", Chem. Comm., accepté. S. Deshayes, V. Maurizot, M.−C. Clochard, T. Berthelot, C. Baudin, G. Déléris, "Synthesis of specific nanoparticles for targeting and imaging tumor angiogenesis using electron−beam irradiation", Radiation physics and Chemistry, accepté. V. Maurizot, M. Yoshizawa, M. Kawano, M. Fujita, "Control of molecular interactions by the hollow of coordination cages" Dalton Trans., 2006, 2750-2756. V. Maurizot, C. Dolain, I. Huc, "Intramolecular vs. Intermolecular Induction of Helical Handedness in Pyridinedicarboxamide Oligomers", Eur. J. Org. Chem., 2005, 1293-1301. V. Maurizot, G.Linti, I. Huc "Solid state characterization of oligopyridine dicarboxamide helicates" Chem. Comm., 2004, 924-925.

Teaching, research & education training, diffusion of science (congress, workshops, scientific open days etc.) Teaching of combinatorial chemistry in the "Master Spécialité Biochimie−interface biologie physique chimie" at the University of Bordeaux 2.

112

Journals (ACL) & average (2003-2008) Impact factors Source WOS 2008

PUBLICATIONS & PRODUCTIONS 2006-2009

Nb

SCIENCE

30,268

2

Applied Physics Letters

2

PLOS Biology

14,662

1

European Journal of Neuroscience

1

Progress in Lipid Research PLOS Medicine

13,358 13,180

1

Gold Bulletin

4,000

2

Journal of Structural Biology

3,964

1

Publications, peer reviewed, (ACL) Publications, peer reviewed, not yet in bases (ACLN) Invited conferences (INV) Publications, peer reviewed in published international colloquia (ACTI) Publications, peer reviewed in published national colloquia (ACTN) Oral communications in congress (COM) Poster communications in congress (AFF) Books or Book chapters (OS) Edition of Books (DO) Congress/Workshop organization Patents Defended PhD Theses or HDR Research Grants International activities

376 1 145 50 1 129 169 23 3 26 12 54 72 33

Articles in journals with IF > 10 Articles in journals with 10 > IF > 5 Articles in journals with 5 > IF > 2 Articles in journals with 2 > IF Average impact factor per article

21 60 223 48 4,3

113

4,096 4,001

Coordination Chemistry Reviews

12,228

1

Current Gene Therapy

3,860

Nano Letters Current Biology

12,189 11,142

1

1

1

Epl Optics Letters

3,816 3,803

Angewandte Chemie-International Edition

11,025

2

Journal of Organic Chemistry

3,777

1

Advanced Materials

10,231

2

Journal of Chromatography A

1

Proceedings of the National Academy of Sciences of the USA

10,228

1

Journal of Bacteriology

3,758 3,748

1

Molecular & Cellular Proteomics Journal of the American Chemical Society

9,391 8,256

1

Biology of the Cell

3,740

11

1

1

Trends in Biotechnology

8,212

2

Chemphyschem NANOTECHNOLOGY

3,739 3,727

1

Journal of Neuroscience

8,122

2

Chembiochem

3,684 3,672

1

Cancer Research

7,980

1

Current Organic Chemistry

2

Biomaterials

7,325

1

1

Small

7,292

1

American Journal of Physiology-Gastrointestinal and Liver Physiology 3,645 3,592 Journal of Cellular Biochemistry

2

Embo Reports

7,265

1

Current Protein & Peptide Science

1

7,134 7,128

1

Protein Science

3,484

4

Physical Review Letters FASEB Journal

1

Analyst

3,472

3

NUCLEIC ACIDS RES

6,968

1

Experimental Gerontology

3,435

1

Current Opinion in Colloid & Interface Science Plant Physiology

6,700 6,650

3

International Journal of Pharmaceutics

2

2

FEBS J

3,431 3,421

1

Journal of Controlled Release

Biochemistry

3,402

Analytical Chemistry

6,116 5,918

3

1

1

Journal of Gene Medicine

3,387

1

Chemistry & Biology Journal of Biological Chemistry

5,743 5,575

1

Journal of Immunology

3,373

5

2

Biochemical Society Transactions

3,298

1

Acs Nano

5,472

1

Journal of Mass Spectrometry

2

Biochimica Et Biophysica Acta-Molecular Cell Research

5,383

1

FEBS Letters

3,261

9

Chemistry-a European Journal

5,335

3

British Journal of Nutrition

3,233

5,330 5,255

1

Microbiology-Sgm

3,200

2

Food Chemistry

5,191

2

Analytica CHIM ACTA

3,183 3,170

3 3

Biochimie Journal of Agricultural and Food Chemistry

3,168 3,165

1

Analytical Biochemistry

3,145

2

Biosensors & Bioelectronics

2 2

ENDOCRINOLOGY Proteomics

2

Journal of Medicinal Chemistry

7

Biophysical Journal

5,079 5,036

7

Chemical Communications

4,997

3,549

3,263

1

Molecular Pharmacology

4,921

1

Microbial Ecology

3,145

1

Soft Matter

Physical Chemistry Chemical Physics

3,139

Journal of Materials Chemistry

4,890 4,862

1

2

3

1

Bioconjugate Chemistry Journal of Physical Chemistry B

4,802 4,688

4

Analytical and Bioanalytical Chemistry Organic & Biomolecular Chemistry

3,107 3,025

Biogerontology

2,962

2

BIOMACROMOLECULES

4,658

2

Food Hydrocolloids

2,940

1

Journal of Physiology-London

4,646

1

New Journal of Chemistry

2,937

1

Note: Although the UMR CBMN has been created in January 2007 publications are given from 2006 to give a 4-year period for evaluation

Journals in which the UMR publishes

2

4

Journals in which the UMR publishes are summarized in the next 2 pages with their Impact Factor (IF), from the Web of Science (Average overs 5 years, 2003-2008).

Journals in which the UMR publishes

1

11

Journals & Impact Factor (ACL)

IF(20032008)

IF(20032008)

1

Nb

Quantitative Results

1

Arthritis Research & Therapy

4,645

1

3

International Journal of Biochemistry & Cell Biology

Bioorganic & Medicinal Chemistry Journal of Raman Spectroscopy

2,913 2,893

Applied ENVIRON MICROB

4,543 4,535

2

1

1

Journal of Physical Chemistry A

2,889

1

MOL BIOSYST Journal of Molecular Biology

4,527

2

4,521

8

Cell Motility and the Cytoskeleton TETRAHEDRON

2,880

4

2,869

1

Macromolecules

4,431

6

European Journal of Organic Chemistry

2,853

21

Langmuir

4,347

3

International Dairy Journal

2,850

1

Cellular Signalling

1

European Journal of Inorganic Chemistry

2,787

6

2

Journal of Colloid and Interface Science

2,755

1

Biochimica Et Biophysica Acta-Biomembranes Acta Biomaterialia

4,321 4,305 4,255

2

Journal of Electroanalytical Chemistry

2,724

1

Biochemical Journal

4,251

1

Journal of the Electrochemical Society

2,702

1

Inorganic Chemistry

4,215

1

Journal of Chromatography B

2,676

1

Journal of Cellular Physiology

4,096

1

Metabolism-Clinical and Experimental

2,645

114

Publication & production list (2006-2009) Publications, peer reviewed, (ACL) Nb

Journals in which the UMR publishes

IF(20032008)

Nb

Journals in which the UMR publishes

IF(20032008)

4

BIOPOLYMERS

2,631

1

Protein and Peptide Letters

1,135

1

Enzyme and Microbial Technology

2

Bulletin Du Cancer

1,044

3

Journal of Applied Microbiology

2,629 2,601

2

Journal of Supercritical Fluids

2

Journal of Microbiology and Biotechnology

0,945

1

Journal of Food Biochemistry

0,937

1

BLOOD CELLS MOL DIS

2,587 2,564

1

Radiation physics and Chemistry

0,931

1

Journal of Physiology-Paris

2,529

1

2

Journal of Pharmaceutical and Biomedical Analysis

2,525

1

High Pressure Research Pharmaceutical Biology

0,866 0,707

2

Diabetes & Metabolism

2,517

1

Journal of Rapid Methods and Automation in Microbiology

0,624

1

European Polymer Journal TETRAHEDRON-ASYMMETRY

2,514 2,512

1

Journal of Sports Science and Medicine

0,564

1

1

International Journal of Chemical Reactor Engineering

0,531

1

Journal of Magnetic Resonance

2,512

3

Acta Cryst E

0,367

1

BioTechniques

2,499

1

Acta Physica Polonica A

0,324

2

Journal of Applied Physics Research in Microbiology

2,479 2,451

2 1

Journal of Molecular Microbiology and Biotechnology

2,421

1

Biointerphases* Biomol NMR Assign*

1

Immunology Letters

2,409 2,390

1

2

Current Drug Delivery

1

Histochemistry and Cell Biology

2

Tetrahedron Letters

2,382

1

Internatioanl Food Research Journal*

1

Aiche Journal

3

Journal of Chemical Biology*

5

European Biophysics Journal with Biophysics Letters

2,334 2,324

1

Médecine Nucléaire*

1

Biotech. Prog

2,292

1

Microbiology and Biotechnology*

1

International Journal of Oncology

2,270

1

Microbiology and Technology*

1

Chirality

Organic Syntheses*

Fems Microbiology Letters Sar and Qsar in Environmental Research

2,232 2,223

1

1

1

Pharmacie Clinique*

1

2,212

1

1

2,184

1

PLoS One*

Reactive & Functional Polymers

2,183

1

Proceedings ParCo, NIC Series*

1

Cell Biochemistry and Biophysics

2,176

1

1

Fems Immunology and Medical Microbiology

2,155

1

Sensors Sign Transduc*

1

Terra Nova Journal of Nanoscience and Nanotechnology

2,140 2,100

1

The Open Biochemistry*

1

Colloids and Surfaces a-Physicochemical and Engineering Aspects

2,097

1

Planta Medica

2,089

1

Oligonucleotides

1

Computer Physics Communications

2,053 2,038

APPLIED SPECTROSCOPY

3. 4. 5. 6. 7. 8. 9. 10.

*non referenced in 2008 WOS

11. 12.

2,013

2

Materials Science & Engineering C-Biomimetic and Supramolecular S 1,969

1

Letters in Applied Microbiology

1,935

1

Journal of Crystal Growth

1,929

1

Micron

1,870

2

Acta Cryst D

1,827

1

Chemistry & Biodiversity

1,811

1

Surface Science Journal of Peptide Science

1,787 1,782

5

2.

Plant Signaling Behavior*

Lipids

1

1

1.

Europhysics Letters*

2

1

2006

1

Nanoscale Research Letters

2

Digestive Diseases and Sciences

1,731 1,685

1

International Journal of Non-Linear Mechanics

1,684

1

Journal of the Science of Food and Agriculture

1

Journal of Enzyme Inhibition and Medicinal Chemistry

1,585

1

European Journal of Lipid Science and Technology

4 3

Comptes Rendus Chimie Magnetic Resonance in Chemistry

1,582 1,521

1

Helvetica Chimica Acta

1,456

3

Journal of Physiology and Biochemistry

1,397

1

Applied Biochemistry and Biotechnology

1,374

1

Journal of Materials Science

1,346

13. 14. 15.

1,614

16. 17.

1,471

18. 19. 20.

21. 22.

115

Abergel, A., Sapin, V., Dif, N., Chassard, C., Darcha, C., Marcand-Sauvant, J., Gaillard-Martinie, B., Rock, E., Dechelotte, P., and Sauvant, P. (2006) Growth arrest and decrease of alpha-SMA and type I collagen expression by palmitic acid in the rat hepatic stellate cell line PAV-1, Digestive Diseases and Sciences 51, 986-995. Begaud-Grimaud, G., Battu, S., Liagre, B., Leger, D. Y., Beneytout, J. L., and Cardot, P. J. P. (2006) Preapoptotic sub-population cell sorting from diosgenin apoptosis induced 1547 cells by Sedimentation Field-Flow Fractionation - The effect of channel thickness on sorting performance, Journal of Chromatography A 1128, 194202. Belin, C., de Franco, P. O., Bourbousse, C., Chaignepain, S., Schmitter, J. M., Vavasseur, A., Giraudat, J., Barbier-Brygoo, H., and Thomine, S. (2006) Identification of features regulating OST1 kinase activity and OST1 function in guard cells, Plant Physiology 141, 1316-1327. Bernad, S., Soulimane, T., Mehkalif, Z., and Lecomte, S. (2006) Characterization and redox properties of cytochrome c(552) from Thermus thermophilus adsorbed on different self-assembled thiol monolayers, used to model the chemical environment of the redox partner, Biopolymers 81, 407-418. Berthelot, T., Goncalves, M., Lain, G., Estieu-Gionnet, K., and Deleris, G. (2006) New strategy towards the efficient solid phase synthesis of cyclopeptides, Tetrahedron 62, 1124-1130. Bezin, S., Charpentier, G., Fossier, P., and Cancela, J. (2006) The Ca2+-releasing messenger NAADP, a new player in the nervous system, Journal of Physiology-Paris 99, 111-118. Bianco, A., Fournel, S., Wieckowski, S., Hoebeke, J., and Guichard, G. (2006) Solid-phase synthesis of CD40L mimetics, Organic & Biomolecular Chemistry 4, 1461-1463. Brizard, A., Dolain, C., Huc, I., and Oda, R. (2006) Asp-Gly based peptides confined at the surface of cationic gemini surfactant aggregates, Langmuir 22, 3591-3600. Buffeteau, T., Ducasse, L., Poniman, L., Delsuc, N., and Huc, I. (2006) Vibrational circular dichroism and ab initio structure elucidation of an aromatic foldamer, Chemical Communications, 2714-2716. Burckbuchler, V., Wintgens, V., Lecomte, S., Percot, A., Leborgne, C., Danos, O., Kichler, A., and Amiel, C. (2006) DNA compaction into new DNA vectors based on cyclodextrin polymer: Surface enhanced Raman spectroscopy characterization, Biopolymers 81, 360-370. Busetta, B., Picard, P., and Precigoux, G. (2006) Simulation of oligopeptide dynamics and folding. The use of NMR chemical shifts to analyse the MD trajectories, Journal of Peptide Science 12, 33-42. Busson, M., Daury, L., Seyer, P., Grandemange, S., Pessemesse, L., Casas, F., Wrutniak-Cabello, C., and Cabello, G. (2006) Avian MyoD and c-Jun coordinately induce transcriptional activity of the 3,5,3 'triiodothyronine nuclear receptor c-ErbA alpha 1 in proliferating myoblasts, Endocrinology 147, 3408-3418. Cansell, M., Moussaoui, N., Petit, A. P., Denizot, A., and Combe, N. (2006) Feeding rats with liposomes or fish oil differently affects their lipid metabolism, European Journal of Lipid Science and Technology 108, 459-467. Carvalho, E., Mateus, N., Plet, B., Pianet, I., Dufourc, E., and De Freitas, V. (2006) Influence of wine pectic polysaccharides on the interactions between condensed tannins and salivary proteins, Journal of Agricultural and Food Chemistry 54, 8936-8944. Caubet, R., Pedarros-Caubet, F., Quataert, Y., Lescure, A., Moreau, J. M., and Ellison, W. J. (2006) Assessing the contamination potential of freshly extracted Escherichia coli biofilm cells by impedancemetry, Microbial Ecology 52, 239-243. Cazenave, C., Bathany, K., and Rayner, B. (2006) Formation of N-branched oligonucleotides as by-products in solid-phase oligonucleotide synthesis, Oligonucleotides 16, 181-185. Ceriani, A., Di Giulio, A., Fantoni, R., and Scotti, P. (2006) Cooling in rifting sequences during increasing burial depth due to heat flow decrease, Terra Nova 18, 365-371. Chevallier, S., Nagy, F., and Cabelguen, J. M. (2006) Cholinergic control of excitability of spinal motoneurones in the salamander, Journal of Physiology-London 570, 525-540. Daury, L., Chailleux, C., Bonvallet, J., and Trouche, D. (2006) Histone H3.3 deposition at E2F-regulated genes is linked to transcription, Embo Reports 7, 66-71. Douat-Casassus, C., Marchand-Geneste, N., Diez, E., Aznar, C., Picard, P., Geoffre, S., Huet, A., BourguetKondracki, M. L., Gervois, N., Jotereau, F., and Quideau, S. (2006) Covalent modification of a melanoma-derived antigenic peptide with a natural quinone methide. Preliminary chemical, molecular modelling and immunological evaluation studies, Molecular Biosystems 2, 240-249. Drelon, N., Gravier, E., Daheron, L., Boisserie, L., Omari, A., and Leal-Calderon, F. (2006) Influence of tempering on the mechanical properties of whipped dairy creams, International Dairy Journal 16, 1454-1463. Dufaitre-Patouraux, L., Riveline, J. P., Renard, E., Melki, V., Belicar-Schaepelynck, P., Selam, J. L., Guerci, B., Millot, L., Brun, J. M., Fermon, C., Catargi, B., Gin, H., Jeandidier, N., Leieune, P. J., and Lassmann-Vague, V. (2006) Continuous intraperitoneal insulin infusion does not increase the risk of organ-specific autoimmune disease in type 1 diabetic patients: results of a multicentric, comparative study, Diabetes & Metabolism 32, 427-432. 116

23. Dumetz, F., Duchaud, E., LaPatra, S. E., Le Marrec, C., Claverol, S., Urdaci, M. C., and Le Henaff, M. (2006) A protective immune response is generated in rainbow trout by an OmpH-like surface antigen (P18) of Flavobacterium psychrophilum, Applied and Environmental Microbiology 72, 4845-4852. 24. Elezgaray, J., and Laguerre, M. (2006) A systematic method to derive force fields for coarse-grained simulations of phospholipids, Computer Physics Communications 175, 264-268. 25. Faure, C., Guillot, S., Weisbecker, P., and Saadaoui, H. (2006) Multilamellar-vesicle-assisted electrodeposition of inorganic nanodots, Advanced Materials 18, 1141-+. 26. Faure, C., Nallet, F., Roux, D., Milner, S. T., Gauffre, F., Olea, D., and Lambert, O. (2006) Modeling leakage kinetics from multilamellar vesicles for membrane permeability determination: Application to glucose, Biophysical Journal 91, 4340-4349. 27. Fedorova, O. A., Andryukhina, E. N., Fedorov, Y. V., Panfilov, M. A., Alfimov, M. V., Jonusauskas, G., Grelard, A., and Dufourc, E. (2006) Supramolecular assemblies of crown-containing 2-styrylbenzothiazole with amino acids, Organic & Biomolecular Chemistry 4, 1007-1013. 28. Ferrand, C., Redonnet, A., Prevot, D., Carpene, C., and Atgie, C. (2006) Prolonged treatment with the beta(3)adrenergic agonist CL 316243 induces adipose tissue remodeling in rat but not in guinea pig: 1) fat store depletion and desensitization of beta-adrenergic responses, Journal of Physiology and Biochemistry 62, 89-99. 29. Ferrand, Y., Daviaud, R., Le Maux, P., and Simonneaux, G. (2006) Catalytic asymmetric oxidation of sulfide and styrene derivatives using macroporous resins containing chiral metalloporphyrins (Fe, Ru), TetrahedronAsymmetry 17, 952-960. 30. Freville, F., Richard, T., Bathany, K., and Morean, S. (2006) Targeting of single-stranded oligonucleotides through metal-induced cyclization of short complementary strands, Helvetica Chimica Acta 89, 2958-2974. 31. Garnier, M., Dufourc, E. J., and Larijani, B. (2006) Characterisation of lipids in cell signalling and membrane dynamics by nuclear magnetic resonance spectroscopy and mass spectrometry, Signal Transduction 6, 133-143. 32. Giacomelli, C., Le Men, L., Borsali, R., Lai-Kee-Him, J., Brisson, A., Armes, S. P., and Lewis, A. L. (2006) Phosphorylcholine-based pH-responsive diblock copolymer micelles as drug delivery vehicles: Light scattering, electron microscopy, and fluorescence experiments, Biomacromolecules 7, 817-828. 33. Giamarchi, A., Padilla, F., Coste, B., Raoux, M., Crest, M., Honore, E., and Delmas, P. (2006) The versatile nature of the calcium-permeable cation channel TRPP2, Embo Reports 7, 787-793. 34. Gillies, E. R., Dolain, C., Leger, J. M., and Huc, I. (2006) Amphipathic helices from aromatic amino acid oligomers, Journal of Organic Chemistry 71, 7931-7939. 35. Grajcar, L., El Amri, C., Ghomi, M., Fermandjian, S., Huteau, V., Mandel, R., Lecomte, S., and Baron, M. N. 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J., and Larijani, B. (2009) Role of a Novel PH-Kinase Domain Interface in PKB/Akt Regulation: Structural Mechanism for Allosteric Inhibition, Plos Biology 7, 189-200. 320. Campbell, V. E., de Hatten, X., Delsuc, N., Kauffmann, B., Huc, I., and Nitschke, J. R. (2009) Interplay of Interactions Governing the Dynamic Conversions of Acyclic and Macrocyclic Helicates, Chem.-Eur. J. 15, 61386142. 321. Cansell, M. S., Battin, A., Degrace, P., Gresti, J., Clouet, P., and Combe, N. (2009) Early Dissimilar Fates of Liver Eicosapentaenoic Acid in Rats Fed Liposomes or Fish Oil and Gene Expression Related to Lipid Metabolism, Lipids 44, 237-247. 322. Castano, S., Delord, B., Fevrier, A., Lehn, J. M., Lehn, P., and Desbat, B. (2009) Asymmetric lipid bilayer formation stabilized by DNA at the air/water interface, Biochimie 91, 765-773. 323. Chisholm, E. J., Vassaux, G., Martin-Duque, P., Chevre, R., Lambert, O., Pitard, B., Merron, A., Weeks, M., Burnet, J., Peerlinck, I., Dai, M. S., Alusi, G., Mather, S. J., Bolton, K., Uchegbu, I. F., Schatzlein, A. G., and Baril, P. (2009) Cancer-Specific Transgene Expression Mediated by Systemic Injection of Nanoparticles, Cancer Research 69, 2655-2662. 324. Commandeur, M., Commandeur, C., De Paolis, M., Edmund, J. A., Maienfisch, P., and Ghosez, L. (2009) Studies related to the total synthesis of the sesquiterpene core of the pyrrolobenzoxazine natural product CJ-12662, Tetrahedron In Press. 325. Deshayes, S., Maurizot, V., Clochard, M. C., Baudin, C., and Déléris, G. (2009) Synthesis of specific nanoparticles for targeting and imaging tumor angiogenesis using electron−beam irradiation, Radiation physics and Chemistry in press. 326. Deves, G., Roudeau, S., Carmona, A., Lavielle, S., Gionnet, K., Deleris, G., and Ortega, R. (2009) Fluorine microimaging and quantification using nuclear reaction analysis: A tool for validating tissue distribution of positron emission tomography tracers, Appl. Phys. Lett. 95, 3. 327. Diller, A., Loudet, C., Aussenac, F., Raffard, G., Fournier, S., Laguerre, M., Grelard, A., Opella, S. J., Marassi, F. M., and Dufourc, E. J. (2009) Bicelles: A natural 'molecular goniometer' for structural, dynamical and topological studies of molecules in membranes, Biochimie 91, 744-751. 328. Dinh, D. H., Vellutini, L., Bennetau, B., Dejous, C., Rebiere, D., Pascal, E., Moynet, D., Belin, C., Desbat, B., Labrugere, C., and Pillot, J. P. (2009) Route to Smooth Silica-Based Surfaces Decorated with Novel SelfAssembled Monolayers (SAMs) Containing Glycidyl-Terminated Very Long Hydrocarbon Chains, Langmuir 25, 5526-5535. 329. Faure, C., Meyre, M. E., Trepout, S., Lambert, O., and Lebraud, E. (2009) Intravesicular synthesis of magnetic nanoparticles into onion-type multilamellar vesicles: magnetonions, J. Phys. Chem. B 13, 8552-8559.

330. Faure, L., Coulon, D., Laroche-Traineau, J., Schmitter, J. M., Testet, E., Lessire, R., and Bessoule, J. J. (2009) Metabolism of N-Acylethanolamines: Discovery and characterization of an Arabidopsis thaliana NAPE synthase, Journal of Biological Chemistry In Press. 331. Ferrand, Y., Klein, E., Barwell, N. P., Crump, M. P., Jimenez-Barbero, J., Vicent, C., Boons, G. J., Ingale, S., and Davis, A. P. (2009) A Synthetic Lectin for O-Linked beta-N-Acetylglucosamine, Angewandte ChemieInternational Edition 48, 1775-1779. 332. Fischer, L., Decossas, M., Briand, J. P., Didierjean, C., and Guichard, G. (2009) Control of Duplex Formation and Columnar Self-Assembly with Heterogeneous Amide/Urea Macrocycles, Angewandte Chemie-International Edition 48, 1625-1628. 333. Gaoqiang, Y., and Ghosez, L. (2009) Synthesis of enantiopure α-chlorocyclobutanones and cyclobutanols as scaffolds for the diverted synthesis of serine protease inhibitors, European Journal of Organic Chemistry In Press. 334. Garcia-Gonzalez, C. A., Vega-Gonzalez, A., Lopez-Periago, A. M., Subra-Paternault, P., and Domingo, C. (2009) Composite fibrous biomaterials for tissue engineering obtained using a supercritical CO2 antisolvent process, Acta Biomaterialia 5, 1094-1103. 335. Gargouri, M., Manigand, C., Mauge, C., Granier, T., Langlois-d'Estaintot, B., Cala, O., Pianet, I., Bathany, K., Chaudiere, J., and Gallois, B. (2009) Structure and epimerase activity of Anthocyanidin reductase from Vitis vinifera, Acta Cryst D. In Press. 336. Garnier-Lhomme, M., Byrne, R. D., Hobday, T. M. C., Gschmeissner, S., Woscholski, R., Poccia, D. L., Dufourc, E. J., and Larijani, B. (2009) Nuclear envelope remnants: fluid membranes enriched in sterols and polyphosphoinositides, PLoS One 4, e4255. 337. Georgieva, D., Schmitt, V., Leal-Calderon, F., and Langevin, D. (2009) On the Possible Role of Surface Elasticity in Emulsion Stability, Langmuir 25, 5565-5573. 338. Ho, T. N. T., Nguyen, N. T., Deschamps, A., Sassi, H., Urdaci, M. C., and Caubet, R. (2009) The impact of Lactobacillus brevis and Pediococcus pentaosaceus on the sensorial quality of "nem chua" - a Vietnamese fermented meat product, Int. Food Res. J. 16, 71-81. 339. Hong, H. A., Khaneja, R., Tam, N. M. K., Cazzato, A., Tan, S., Urdaci, M., Brisson, A., Gasbarrini, A., Barnes, I., and Cutting, S. M. (2009) Bacillus subtilis isolated from the human gastrointestinal tract, Res. Microbiol. 160, 134-143. 340. Jaziri, I., Ben Slama, M., Mhadhbi, H., Urdaci, M. C., and Hamdi, M. (2009) Effect of green and black teas (Camellia sinensis L.) on the characteristic microflora of yogurt during fermentation and refrigerated storage, Food Chemistry 112, 614-620. 341. Jean-François, F., Desbat, B., and Dufourc, E. J. (2009) Selectivity of Cateslytin for fungi: the role of acidic lipid-ergosterol membrane fluidity in antimicrobial action. FASEB J doi:10.1096/fj.09-135574. 342. Jena, P., Shirude, P. S., Okumus, B., Laxmi-Reddy, K., Godde, F., Huc, I., Balasubramanian, S., and Ha, T. (2009) G-quadruplex DNA bound by a synthetic ligand is highly dynamic. Journal of the American Chemical Society 131, in press. 343. Karaca, M., Castel, J., Tourrel-Cuzin, C., Brun, M., Geant, A., Dubois, M., Catesson, S., Rodriguez, M., Luquet, S., Cattan, P., Lockhart, B., Lang, J., Ktorza, A., Magnan, C., and Cargar, C. (2009) Exploring functional Betacell heterogeneity in vivo using PSA-NCAM as a specific marker Plos ONE 4:e5555. 344. Laras, Y., Garino, C., Dessolin, J., Weck, C., Moret, V., Rolland, A., and Kraus, J. L. (2009) New N(4)substituted piperazine naphthamide derivatives as BACE-1 inhibitors J. Enzym. Inhib. Med. Ch. In Press. 345. Le Bihan, O., Bonnafous, P., Marak, L., Bickel, T., Trepout, S., Mornet, S., De Haas, F., Talbot, H., Taveau, J. C., and Lambert, O. (2009) Cryo Electron tomography of nanoparticle transmigration into liposome, J. Struct. Biol. PII:S1047-8477(09)00176-2 DOI:10.1016/j.jsb. 346. Le Floch-Fouere, C., Pezennec, S., Lechevalier, V., Beaufils, S., Desbat, B., Pezolet, M., and Renault, A. (2009) Synergy between ovalbumin and lysozyme leads to non-additive interfacial and foaming properties of mixtures, Food Hydrocolloids 23, 352-365. 347. Loudet, C., Diller, A., Oda, R., and Dufourc, E. J. (2009) Biphenyl phosphatidylcholine: a promoter of liposome deformation and bicelle collective orientation by magnetic fields, Progress in Lipid Research in press. 348. Martin, A., Damian, M., Laguerre, M., Parello, J., Pucci, B., Serre, L., Mary, S., Marie, J., and Baneres, J. L. (2009) Engineering a G protein-coupled receptor for structural studies: Stabilization of the BLT1 receptor ground state, Protein Sci. 18, 727-734. 349. Massias, B., and Urdaci, M. C. (2009) ProReg XL Tool: an easy-to-use computer tool suite for rapidly regrouping a high number of identical electrophoretic profiles, BioTechniques. In Press. 350. Maurizot, V., Massip, S., Léger, J. M., and Déléris, G. (2009) Cylindrical sheet formation of oligo-meta-aniline foldamers, Chem. Comm. in press. 351. Percot, A., Lecomte, S., Vergne, J., and Maurel, M. C. (2009) Hairpin Ribozyme Catalysis: A Surface-Enhanced Raman Spectroscopy Study, Biopolymers 91, 384-390. 352. Perro, A., Duguet, E., Lambert, O., Taveau, J. C., Bourgeat-Lami, E., and Ravaine, S. (2009) Chemical Synthetic Route towards "Colloidal Molecules", Angewandte Chemie-International Edition 48, 361-365. 353. Perro, A., Nguyen, D., Ravaine, S., Bourgeat-Lami, E., Lambert, O., Taveau, J. C., and Duguet, E. (2009) Planar submicronic silica-polystyrene particles obtained by substrate-directed shaping, Journal of Materials Chemistry 19, 4225-4230. 129

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354. Petibois, C., Wehbe, K., Belbachir, K., Noreen, R., and Deleris, G. (2009) Current Trends in the Development of FTIR Imaging for the Quantitative Analysis of Biological Samples, Acta Physica Polonica A 115, 507-512. 355. Saad, N., Bressollier, P., Chaignepain, S., Schmitter, J. M., and Urdaci, M. C. (2009) GAPDH and L. Plantarum surface proteins, Journal of Microbiology and Biotechnology In Press. 356. Saad, N., Urdaci, M. C., Vignoles, C., Chaignepain, S., Tallon, R., Schmitter, J. M., and Bressollier, P. (2009) Lactobacillus plantarum 299v surface-bound GAPDH: a new insight into enzyme cell walls location., Microbiol and Biotechnol. In press. 357. Sanchez, B., Arias, S., Chaignepain, S., Denayrolles, M., Schmitter, J. M., Bressollier, P., and Urdaci, M. C. (2009) Identification of surface proteins involved in the adhesion of a probiotic Bacillus cereus strain to mucin and fibronectin, Microbiology-Sgm 155, 1708-1716. 358. Sanchez, B., Bressollier, P., Chaignepain, S., Schmitter, J. M., and Urdaci, M. C. (2009) Identification of surface-associated proteins in the probiotic bacterium Lactobacillus rhamnosus GG, International Dairy Journal 19, 85-88. 359. Sanchez, B., Chaignepain, S., Schmitter, J. M., and Urdaci, M. C. (2009) A method for the identification of proteins secreted by lactic acid bacteria grown in complex media, Fems Microbiology Letters 295, 226-229. 360. Sanchez, B., Schmitter, J. M., and Urdaci, M. C. (2009) Identification of novel proteins secreted by Lactobacillus rhamnosus GG grown in de Mann-Rogosa-Sharpe broth, Letters in Applied Microbiology 48, 618622. 361. Sanchez, B., Schmitter, J. M., and Urdaci, M. C. (2009) Identification of novel proteins secreted by Lactobacillus plantarium that bind to mucin and fibronectin, J. Mol. Microbiology and Biotechnology Ms No. : 200810003. 362. Sanchez-Garcia, D., Kauffmann, B., Kawanami, T., Ihara, H., Takafuji, M., Delville, M. H., and Huc, I. (2009) Nanosized hybrid oligoamide foldamers: Aromatic templates for the Folding of Multiple Aliphatic Units., Journal of the American Chemical Society 131, 8642. 363. Sani, M. A., Dufourc, E. J., and Grobner, G. (2009) How does the Bax-alpha 1 targeting sequence interact with mitochondrial membranes? The role of cardiolipin, Biochimica Et Biophysica Acta-Biomembranes 1788, 623631. 364. Sani, M. A., Keech, O., Gardeström, P., Dufourc, E. J., and Gröbner, G. (2009) Magic-angle phosphorus NMR of functional mitochondria: in situ monitoring of lipid response under apoptotic-like stress., Faseb Journal 23, DOI:10.1096/fj.1009-134114. 365. Shinkaruk, S., Lamothe, V., Schmitter, J. M., Manach, C., Morand, C., Berard, A., Bennetau, B., and BennetauPelissero, C. (2009) Development and validation of two new sensitive ELISAs for hesperitin and naringenin in biological fluids, Food Chem. In Press. 366. Soenen, S. J.-H., Brisson, A. R., and De Cuyper, M. (2009) Addressing the problem of cationic lipidmediated toxicity ; the magnetoliposome model., Biomaterials In Press. 367. Tabbene, O., Ben Slimene, I., Djeball, K., Mangoni, M. L., Urdaci, M. C., and Limam, F. (2009) Optimization of medium composition for the production of antimicrobial activity by Bacillus subtilis B38 against methicillinresistant Staphylococcus species, Biotech. Prog. In Press. 368. Urdaci, M. C., and Sánchez, B. (2009) Some immunomodulatory effects of probiotic bacteria might be due to porcine neutrophil elastase inhibitor, a serpin present in MRS broth, Immunol. Lett. In Press. 369. Ventimilla, N., Dupont, P. Y., Laguerre, M., and Dessolin, J. (2009) Description and assessment of a model for GSK-3 database virtual screening, J. Enz. Inhib. Med. Chem. In Press. 370. Vergne, S., Sauvant, P., Lamothe, V., Chantre, P., Asselineau, J., Perez, P., Potier, M., Durand, M., Moore, N., and Bennetau-Pelissero, C. (2009) Influence of ethnic origin: Asian vs Caucasian, and background dietary habits on the biovailability of isaflavones, British Journal of Nutrition In Press. 371. Verret, C., El Moueffak, A., Largeteau, A., Frimigacci, M., Demazeau, G., Leal-Calderon, F., and Cansell, M. (2009) Effects of high pressure on anhydrous milk fat crystallization in emulsion, High Pressure Research 29, 5760. 372. Vonarbourg, A., Passirani, C., Desigaux, L., Allard, E., Saulnier, P., Lambert, O., Benoit, J. P., and Pitard, B. (2009) The encapsulation of DNA molecules within biomimetic lipid nanocapsules, Biomaterials 30, 3197-3204. 373. Wolffs, M., Delsuc, N., Veldman, D., Van Anh, N., Williams, R. M., Meskers, S. C. J., Janssen, R. A. J., Huc, I., and Schenning, A. (2009) Helical Aromatic Oligoamide Foldamers as Organizational Scaffolds for Photoinduced Charge Transfer, Journal of the American Chemical Society 131, 4819-4829. 374. Yassine, W., Taib, N., Federman, S., Milochau, A., Castano, S., Sbi, W., Manigand, C., Laguerre, M., Desbat, B., Oda, R., and Lang, J. (2009) Reversible transition between alpha-helix and beta-sheet conformation of a transmembrane domain Biochimica and Biophysica Acta (BBA) Biomembranes DOI:10.1016J/j.bbamem.2009.05.014. 375. Zhu, W., Mena, M., Jnoff, E., Sun, N., Pasau, P., and Ghosez, L. (2009) Multicomponent reactions for the synthesis of complex piperidine scaffolds, Angew. Chem. Int. Ed. In Press. 376. Gargouri, M., Gallois, B., and Chaudière, J., (2009) Binding-equilibrium and kinetic studies of anthocyanidin reductase from Vitis Vinifera. Archives of Biochemistry and Biophysics, In Press.

Publications, peer reviewed, not yet in bases (ACLN) 2006-2009 1.

Vergne Sébastien and Sauvant Patrick. Les isoflavones de soja et leurs effets sur la santé des femmes ménopausées. Phytothérapie (2006), 4, 172-180

Invited conferences (INV) 2006 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 131

Sani, M. A., Gröbner, G., and Dufourc, E. J. (2006) Regulation of apoptosis at the mitochondrial level: Cardiolipin and peptide domain. In Symposium on Lipids, Prague, Czech Republic Pianet, I., Tarascou, I., André, Y., Barathieu, K., Simon, C., Ducasse, A., Moskau, D., De Freitas, V., Fouquet, E., Dufourc, E. J., and Laguerre, M. (2006) Structure et dynamiques des tannins du Vin. In 20ème Journées de Bruker 2006, Paris, France Garnier-Lhomme, M., Byrne, R. D., Grélard, A., Loudet, C., Gschmeissner, S., Woscholski, R., Poccia, D. L., Dufourc, E. J., and Larijani, B. (2006) Natural Membrane Domains as Key Players in Nuclear Envelope Assembly. In Lipid Rafts and Cell Function, Keystone symposia, Keystone, Colorado, USA Beck, J., Loudet, C., Mathieu, D., and Dufourc, E. J. (2006) The role of plant sterols in the rafts concept. In European Lipidomics, Noordwijkerhout, The Netherlands F. Leal Calderon. Summer School SOCON, Laboratoire de Physique du Solide, Orsay (France), June 12-16, 2006. Coarsening of emulsions. Sani, M. A., Gröbner, G., and Dufourc, E. J. (2006) Regulation of apoptosis at the mitochondrial level: Cardiolipin and peptide domain. In Symposium on Lipids, Prague, Czech Republic Pianet, I., Tarascou, I., André, Y., Barathieu, K., Simon, C., Ducasse, A., Moskau, D., De Freitas, V., Fouquet, E., Dufourc, E. J., and Laguerre, M. (2006) Structure et dynamiques des tannins du Vin. In 20ème Journées de Bruker 2006, Paris, France Garnier-Lhomme, M., Byrne, R. D., Grélard, A., Loudet, C., Gschmeissner, S., Woscholski, R., Poccia, D. L., Dufourc, E. J., and Larijani, B. (2006) Natural Membrane Domains as Key Players in Nuclear Envelope Assembly. In Lipid Rafts and Cell Function, Keystone symposia, Keystone, Colorado, USA Beck, J., Loudet, C., Mathieu, D., and Dufourc, E. J. (2006) The role of plant sterols in the rafts concept. In European Lipidomics, Noordwijkerhout, The Netherlands Laguerre M., (2006) "Les Petites Molécules". Séminaire de Formation dans le cadre du Cycle de formation organisé par la Société française du Cancer. Paris. Dessolin J., (2006) "Drug design de composés à visée pro-apoptotique - Vers la conception d'un médicament". Colloques de l'Institut Bergonié, Institut Bergonié (Bordeaux). Dessolin J., (2006) "Drug design : what you need, what you can". Bergonié Cancer Institute Research Day. Ghosez, L. (2006) Tohoku University, Japan, January 2006 Ghosez, L. (2006) Tokyo University, Japan, January 2006 Ghosez, L. (2006) Tokyo Institute of Technology, Japan, January 2006 Ghosez, L. (2006) Nagoya University, Japan, 21st Century COE-RCMS Conference, January 2006 Ghosez, L. (2006) European Workshop on Drug Design,Sienna, Italy, May 2006 Ghosez, L. (2006) Kyoto University, Kyoto, Jpan, May 2006 Ghosez, L. (2006) Okayama University of Sciences, Japan, June 2006 Ghosez, L. (2006) Riken Institute , Tokyo, Japan, June 2006 Ghosez, L. (2006) ACS Tetrahedron Prize Symposium, San Francisco, USA, September 2006 Ghosez, L. (2006) CEDNETS symposium, Warsaw,October 2006 Ghosez, L. (2006) University of Pensylvania, Philadelphia, USA, February 2006 Guichard, G. (2006) Hélices Biomimétiques Antimicrobiennes: de la Structure à la Fonction, « 27e Réunion interdisciplinaire de chimiothérapie anti-infectieuse » (RICAI), Palais des Congrès, Paris, France Huc, I. (2006) Artificial double helical architectures, Workshop on “Recent advances in supramolecular assemblies with nucleic acids”, Bordeaux; France Huc, I. (2006) Dynamic approaches to foldamer structure and assembly, COST workshop on Dynamic Combinatorial Chemistry, Bordeaux, France Huc, I. (2006) Folding, dynamics and assembly of helical biomimetic architectures, ACS-CSIR-AOCCB Conference, Indian Institute of Chemical Techology, Hyderabad, India Huc, I. (2006) Folding, dynamics and assembly of helical biomimetic architectures, Symposium on “Organic Chemistry – Today and Tomorrow”, Indian Institute of Science, Bangalore, India. Huc, I. (2006) Folding, dynamics and assembly of helical biomimetic architectures, RSC Macrocyclic and Supramolecular Chemistry Discussion Group Annual Meeting, University of Leeds, UK Oda, R. (2006) Controlling supramolecular chirality induced by counteranions in the self assemblies of amphiphilc molecules Club Exo-Endocytosis, Anglet 132

31. 32. 33. 34.

35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

Oda, R. (2006) Investigation of gemini surfactant-counterion interactions 1st Jointed Fukuoka-Bordeaux Conference ― New Trends in Molecular and Materials Sciences, Oda, R. (2006) Controlling supramolecular chirality induced by counteranions in the self assemblies of amphiphilc molecules International Symposium on "Organic Chemistry - today and tomorrow” Indian Institute of Science, Bangalore Sauvant P. (2006) Phyto-oestrogènes et ménopause : Dernières avancées scientifiques. Ingrédients Ménopause 2006 conférences organisées par la Société Française des Antioxydants (SFA), Paris, Plet B. and Schmitter J.M. (2006) Analysis of peptide tannins supramolecular complexes by tandem mass spectrometry. 54th American Society for Mass Spectrometry Conference, Seattle, USA Urdaci MC. Baliarda A. (2006) Disease Interactions and Pathogen exchange between wild and farmed fish - a European NETwork. Prague. Brisson A.R. (2006) 6th France-Japan meeting on Nanomaterials, Hokkaido. Controlling the assembly of biomolecules on solid supports at the nanoscale. Brisson A.R. (2006) Réunion France-Inde LAFICS, Bordeaux, Controlling the assembly of biomolecules on solid supports, at the nanoscale. Brisson A.R. (2006) E-Materials Research Society, Nice. Controlling the assembly of biomolecules on solid supports at the nanoscale. Brisson A.R. (2006) 7th France-Japan Symposium on Drug Delivery Systems, Kyoto, 2006. Functionalization of supports with proteins: from biosensors to nanovectors. Brisson A.R. (2006) France-Sweden workshop on Nanobiotechnology, Stockholm, 2006. Controlling the assembly of biomolecules on solid supports at the nanoscale. Brisson A.R. (2006) QCM-D world conference, Boston. Controlling the assembly of biomolecules on solid supports at the nanoscale. Brisson A.R. (2006) University of Freiburg, Prof. K. Aktories. 2D assemblies of proteins on membrane surfaces: structure, mechanism of assembly and nanobiotehnological applications. Brisson A.R. (2006) ENS Cachan, Prof. A. Bentley. Développement d’une plate-forme d’ancrage universelle pour anticorps, protéines. Brisson A.R. (2006) CEA Marcoule. Développement d’une plate-forme d’ancrage pour anticorps, protéines, cellules. Brisson A.R. (2006) Symposium Nanobiotechnologie. LETI-CEA Grenoble. Contrôle de l’assemblage de biomolécules sur supports solides, à l’échelle nano.

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2007 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56.

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Pianet, I., André, Y., Dufourc, E. J., Fouquet, E., Tarascou, I., and Laguerre, M. (2007) 3D Structure, Colloidal self-association and protein recognition of procyanidins determined by NMR and Molecular Modeling. In FAVHealth 2007, Houston, USA Pianet, I., André, Y., Dufourc, E. J., Fouquet, E., Tarascou, I., Carteau, D., and Bassani, D. (2007) The contribution of DOSY NMR spectroscopy to the study of colloidal mixtures. In NMR of Soft Matter, Arcachon, France Odaert, B., Bressolier, P., Urdaci, M., and Dufourc, E. J. (2007) Liquid state NMR study of an antimicrobial peptide from the lantibiotic family. In 2nd Workshop on membrane active peptides, Lisbone, Portugal Dufourc, E. J. (2007) Towards long-lived magnetically orientable membrane systems for the study of peptides and proteins. In Minisymposium on membrane active peptides, Strasbourg, France. Dufourc, E. J., Loudet, C. (2007) Biphenyl Bicelles. In NMR of Soft Matter, Arcachon, France Dargelos E., Poussard S., Cottin P. Calcium-dependent proteolysis and muscle ageing. Conférence plénière, 5ème colloque du groupe thématique « Protéolyse Cellulaire » de la Société Française de Biochimie et Biologie Moléculaire, 27-29 Mars 2007, Seillac, France Daury L., Leloup L., Mazères G., Dedieu S., Cottin P., Brustis JJ. Involvement of calpains during myoblast migration. International Workshop on cell migration, February 1-2, 2007, Evry, France S. Castano, B. Delors, A. Fevrier, J.M. Lehn, P. Lehn, B. Desbat ,"Interaction de l'ADN avec un lipide cationique utilisé pour le transfert de gène, le BGTC: Etude à l'interface air/eau par BAM et PMIRRAS", CFCL 2007, Systèmes anisotropes Auto organisés, 10-13 septembre 2007, Pessac, S. Lecomte, “Le transfert d’électrons dans les hémoprotéines : Méthodes couplées spectrométries vibrationnelles / électrochimie” 12th International Conference on organized molecular films (LB-12) Kraków, July 1-5, 2007 A. Banc, L. Navailles, B. Desbat, Y. Popineau, C. Mangavel, D. Renard, "Interfacial phenomena of wheat storage proteins: towards a biomimetic approach of wheat prolamins assembly in grains", 12th International Conference on organized molecular films (LB-12) Kraków, July 1-5, 2007 Z. Fezoua, S. Lecomte, A. Brisson, B. Desbat, "Study of the Annexin A5 Interactions with Lipid Monolayer at the Air-Water Interface using PM-IRRAS and Brewster Angle Microscopy", 12th International Conference on organized molecular films (LB-12) Kraków, July 1-5, 2007

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S. Bussières, E. Trudel, P. Desmeules, B. Desbat, R. Breton, C. Salesse,"Binding of Peripheral Membrane Proteins to Phospholipid Monolayers" 12th International Conference on organized molecular films (LB12) Kraków, July 1-5, 2007 Pianet, I., André, Y., Dufourc, E. J., Fouquet, E., Tarascou, I., and Laguerre, M. (2007) 3D Structure, Colloidal self-association and protein recognition of procyanidins determined by NMR and Molecular Modeling. In FAVHealth 2007, Houston, USA Pianet, I., André, Y., Dufourc, E. J., Fouquet, E., Tarascou, I., Carteau, D., and Bassani, D. (2007) The contribution of DOSY NMR spectroscopy to the study of colloidal mixtures. In NMR of Soft Matter, Arcachon, France Odaert, B., Bressolier, P., Urdaci, M., and Dufourc, E. J. (2007) Liquid state NMR study of an antimicrobial peptide from the lantibiotic family. In 2nd Workshop on membrane active peptides, Lisbone, Portugal Dufourc, E. J. (2007) Towards long-lived magnetically orientable membrane systems for the study of peptides and proteins. In Minisymposium on membrane active peptides, Strasbourg, France. Dufourc, E. J., Loudet, C. (2007) Biphenyl Bicelles. In NMR of Soft Matter, Arcachon, France Parello J., Banères J.-L. & Laguerre M., (207) "Hydration in GPCR-mediated signal transduction". Communication orale présentée à l'ESF-FWF Conférence "Water interfaces in Physics, Chemistry and Biology: a multi-disciplinary approach" Innsbruck (Autriche). Laguerre M., Dessolin J. (2007) "Apport de la modélisation moléculaire à la conception de médicaments et à la dynamique des protéines". Conférence présentée à Limoges dans le cadre du Cancéropôle Grand Sud-Ouest. Commandeur C.; Laguerre, M.; Dessolin J. (2007) "Drug design and in silico screening of pro-apoptotic compounds" 16èmes Conférences Européennes du Groupement des Pharmacochimistes de l'Arc Atlantique, Bordeaux Ghosez, L. (2007) 7th Southampton Symposium on Organic Synthesis, UK, January 2007 Ghosez, L. (2007) International Symposium “Organic chemistry, Present and Future”, Louvain-la-Neuve, April 2007 Ghosez, L. (2007) 3rd Euchem Conference on Pericyclic Reactions, Syracuse, Italy, June 2007 Ghosez, L. (2007) Queen Mary College,London, UK, Postgraduate Symposium , July 2007 Ghosez, L. (2007) Symposium in honour of Prof. Sauer, München, Germany, July 2007 Guichard, G. (2007) Folding and self assembly of N,N’-linked oligoureas. Improving on (oligo)amides? Conference invitée à l’ “International meeting on frontiers in chemistry” organisé par le Department of Chemistry du “Korea Advanced Institute of Science and Technology (KAIST)”, Daejeon (Republic of Korea), 22-24 février 2007, hôte Prof. Hee-Seung Lee Huc, I. (2007) Protein-sized Synthetic Folded Architectures, 3rd STIPOMAT conference, Les Diablerets, Switzerland Huc, I. (2007) Protein-sized Synthetic Folded Architectures, IV French-Russian International Symposium “Supramolecular systems in chemiustry and biology”, Moscow, Russia. Huc, I. (2007) Chirality in helical nanomolecules, International conference on “Chirality at the Nanoscale”, Sitges, Spain Huc, I. (2007) Dynamic equilibria and folding processes, DCC Marie Curie Network Meeting, Stockholm, Sweden. Huc, I. (2007) Protein-sized Synthetic Folded Architectures, International Symposium “Organic Chemistry Present and Future”, Louvain-La-Neuve, Belgium. Taveau J.C., Dubois M., Le Bihan O., Trépout S., Almagro S., Hewat E., Durmort C., Heyraud S., GulinoDebrac D., Lambert O. (2007) Structure of artificial VE-cadherin based junctions In Biochemical Society Annual Symposium-Structure and function in cell adhesion. Manchester, UK Lambert O. (2007) Développement de nouveaux outils d’imagerie pour le transfert de gènes Giens, Congres de la Société Francophone de Thérapie Cellulaire et Génique. Dubois, M., P. Vacher, B. Roger, D. Huyghe, B. Vandewalle, J. Kerr-Conte, F. Pattou, N.M. Moussa, and J. Lang, (2007) Glucotoxicity inhibits late steps of insulin exocytosis, in EASD - Islet Study Group: Brussels, Belgium Oda, R. (2007) ICMR summer school, the University of California in Santa Barbara,”Polymeric and SelfAssembled Gels”. a Tutorial on Circular Dichroism Oda, R. (2007) ICMR summer school, the University of California in Santa Barbara,”Polymeric and SelfAssembled Gels”. Seminar on the research topic Oda, R. (2007) Controlling supramolecular chirality induced by counteranions in the self assemblies of amphiphilc molecules The 17th Iketani Conference: The Doyama Symposium, University of Tokyo, Japan Oda, R. (2007) Controlling supramolecular chirality induced by counteranions in the self assemblies of amphiphilc molecules Japan France Frontier of Science, Shonan, Japan Sauvant P. (2007) Les phyto-oestrogènes : études pharmacologiques, cliniques et épidémiologiques. Conférences intitulées « Les plantes chez la femme ménopausée, une alternative aux traitements hormonaux de substitution ? » et organisées par la Société Française d’Ethnopharmacologie (SFE), Metz Odaert, B., Bressolier, P., Urdaci, M., and Dufourc, E. J. (2007) Liquid state NMR study of an antimicrobial peptide from the lantibiotic family. In 2nd Workshop on membrane active peptides, Lisbone, Portugal 134

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Brisson A.R. (2007) International meeting on the Annexins, Irvine, USA. Biotechnological applications of molecular tools derived from Annexin A5. Brisson A.R. (2007) International meeting on the Annexins, Irvine, USA. Biotechnological applications of molecular tools derived from Annexin A5”. Brisson A.R. (2007) CIC-Biomagune, San Sebastian, Dr.. I. Reviakine. 2D self-assembly of Annexin-A5 on lipid surfaces : basic mechanism and biotechnological applications. Brisson A.R. (2007) INSERM-U 828, Prof. T. Couffinhal. Assemblages 2D de protéines au niveau de surfaces membranaires : le système de l’Annexine-5 : aspects fondamentaux et applications biotechnologiques. Brisson A.R. (2007) Centre de Recherche sur les Pathologies des Plaquettes. Complexes entre Annexine-5 et membranes lipidiques : Etudes fondamentales et développement d’outils pour le marquage membranaire.

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2008 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119.

Dufourc, E. J. (2008) Biphenyl Bicelles: a long lasting magnetically oriented biomembrane. In GERLI, Compiègne, France Dufourc, E. J. (2008) Magnetic camemberts. In 1st Meeting of the Aquitaine NMR Network, Bordeaux, France Dufourc, E. J. (2008) The role of phytosterols in plant adaptation to temperature. A solid state NMR Study. In International Symposium on Plant Lipids, Bordeaux S. Castano, B. Delors, A. Fevrier, J.M. Lehn, P. Lehn, B. Desbat DNA interactions with BGTC, a cationic lipid used for gene transfer GERLI2008, 5ième congrès de lipidomique , 21-23 octobre 2008, Compiègne Dufourc, E. J. (2008) Biphenyl Bicelles: a long lasting magnetically oriented biomembrane. In GERLI congress, Compiègne, France Dufourc, E. J. (2008) Magnetic camemberts. In 1st Meeting of the Aquitaine NMR Network, Bordeaux, France Dufourc, E. J. (2008) The role of phytosterols in plant adaptation to temperature. A solid state NMR Study. In International Symposium on Plant Lipids, Bordeaux Laguerre M., "Interaction of dopamine with its own D2 receptor as viewed by molecular dynamics". Communication orale présentée au Congrès: "Structure-function relationships of G-protein coupled receptors in the spotlight" organisé à la Grande-Motte les 12 & 13 juin 2008 Gallois, B. (2008) De la structure à la fonction des macromolécules biologiques. Importance de la biologie structurale. XIXèmes journées de l’Association Tunisienne des Sciences Biologiques. Hammamet, Tunisie First international conference on drug design and discovery, Dubai UE Ghosez, L. (2008) “A cocktail of new synthetic methods”, Seminar at Syngenta , Goa, India Ghosez, L. (2008) “Efficient syntheses of natural products-like scaffolds as tools for chemical biology”, ESFCOST High-Level Research Conference on Natural Products Chemistry, Biology and Medecine Acquafredda, Italy, Ghosez, L. (2008) Seminar at University of Burgos (Spain) June 2008 Ghosez, L. (2008) Seminar at University of Vigo (Spain) June 2008 Ghosez, L. (2008) Seminar at Vernalis, Cambridge, UK June 2008 Ghosez, L. (2008) Seminar at University of Manchester, UK, June 2008 Ghosez, L. (2008) Seminar at Astra-Zeneca, Manchester, UK, June 2008 Ghosez, L. (2008) Seminar at University of Florence, Italy, September 2008 Ghosez, L. (2008) Seminar at Servier Institute , Croissy, France November 2008 Guichard, G. (2008) Building Molecules with Protein–like Structures and Functions: the Foldamer Approach, “1st Annual Symposium French-American Kavli Frontiers Of Science”, Roscoff, France. Guichard, G. (2008) Architectures Protéomimetiques pour la reconnaissance (Bio)moléculaire. Symposium SFC Grand-Est.5, Vandoeuvre les Nancy, France. Huc, I. (2008) Bioinspired folded aromatic oligoamides, VIII jornadas de quimica farmaceutica, Vittoria, Spain Huc, I. (2008) Protein-sized Synthetic Folded Architectures, Summer school on “Supramolecular systems in chemistry and biology”, Tuapse, Russia. Huc, I. (2008) Folding, dynamics and assembly of helical biomimetic architectures, Sycocal, La Bourboule, France Huc, I. (2008) Helical nanomolecules: structure and functions, International Symposium on Macrocyclic and Supramolecular Chemistry, Las Vegas, USA. Huc, I. (2008) Synthetic helical nanomolecules, invited lecture, International Symposium on Chirality, Geneva, Switzerland Oda, R. (2008) Controlling supramolecular chirality induced by counteranions in the self assemblies of amphiphilc molecules at Kyushu Univ. Global-COE/Bordeaux University IECB Joint Seminar, Fukuoka, Japan Oda, R. (2008) Counterion Effects on the Self-Assembly of Gemini Surfactants , Surfactant in Solution Berlin, Germany Sauvant P. (2008) La xérophtalmie : marqueurs et stratégies nutritionnelles. 7ème Journées Francophones de Nutrition (JFN), Brest.

Brisson A.R. (2008) INSERM Workshop on Molecular interactions: theory and biophysical tools for quantitative measurements. La Lande des Maures (France). Binding and 2D organization of proteins on membrane surfaces, monitored by QCM-D and AFM. Brisson A.R. (2008) Franco-Brazilian Workshop on Polymers – FBPLO3- Florianopolis (Brazil). Functionalization of solid supports and nanoparticles with lipid and protein layers. Brisson A.R. (2008) GEIMM-13- Mittelwihr (France). 2D self-assembly of Annexin-A5 at membrane surfaces : basic processes and biotechnological applications. Brisson A.R. (2008) CIMTEC 2008 – 3rd International Conference on Smart Materials, Structures and Systems. Acireale (Italy). 2D crystals of proteins on lipid surfaces. Brisson A.R. (2008) AVS Meeting – Boston (USA). 2D self-assembly of annexin-A5 on lipid surfaces: biological function, mechanism of assembly and biotechnological applications. Brisson A.R. (2008) NanoSWEC – Bordeaux. 2D self-assembly of annexin-A5 on lipid surfaces: biological function, mechanism of assembly and biotechnological applications.

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Zhendre, V., Larijani, B., Dufourc, E.J. (2009) The role of phosphoinositides in nuclear membrane assembly. 6th European Biophysics Congress, Genoa, Italy H.P. Ta; K. Berthelot; C.Cullin; L.Servant; B. Desbat; J.Gean; S;Lecomte, (2009) Analyse des relations structure/toxicité de prions par spectroscopies vibrationnelles (PM-IRRAS, ATR). GFPP congress, Albé, France Jean-Francois, F., Castano, S., Desbat, B., Odaert, B., Roux, M., Metz-Boutigue, M. H., and Dufourc, E. J. (2009) A new mode of action for antimicrobial peptides. In 1st Meeting of the Aquitaine NMR Network, Bordeaux, France. Jean-Francois, F., and Dufourc, E. J. (2009) A new mode of action for antimicrobial peptides. In GERLI congress, Rennes, France. Odaert, B. (2009) Etude biophysique de peptides antimicrobiens en interaction avec des systèmes modèles membranaires. Biologie structurale et phénomènes transitoires. In Congress of NMR and structural biology, Dourdan. Elezgaray J., (2009) "Optical response of decorated nanostructures". Invited conference, “Physics of complexity”. Ghosez L. (2009) Seminar University of York, UK Ghosez L. (2009) Bellus Symposium, Basel Huc, I. (2009) Foldamers - Expanding the Chemical Space, International symposium on medicinal chemistry, Brussels, Belgium Huc, I. (2009) Aromatic mimics of biomolecular folding, International symposium on novel aromatic compounds, Luxemburg. Huc, I. (2009) Folding and assembly of organic nanomolecules, French-Japanese workshop on Nanomaterials, Tsukuba, Japan Huc, I. (2009) Aromatic mimics of biomolecular folding, NIH-INRIA workshop modeling in structural biology, Rocquencourt, France. Huc, I. (2009) Abiotic mimics of proteins: design, synthesis, structure and chemical reactivity, Journée de bilan ANR blanc 2005, Paris, France. Huc, I. (2009) Aromatic mimics of biomolecular folding, Symposium on Magnetic Resonance & Biomolecular Mimetics, 15th NMRS Meeting, Hyderabad, India Oda, R. (2009) The role of counterions on the self-assembling behaviors of cationic amphiphiles. at RBPGO4, ile de Berder, France Sauvant P. (2009) Ostéoporose et ménopause : quelle étude clinique menée pour prouver une efficacité dans le cadre de la ménopause et de l’ostéoporose. 2ème conférence « Etude Clinique et allégation 2009 », organisée par la Société Française des Antioxydants (SFA), Paris, France. Sauvant P. (2009) Aspects nutritionnels et physiologiques des rétinoïdes. Xème Congrès de la Société Francophone Vitamines et Biofacteurs (SFVB), Grenoble, France Brisson A.R. (2009) Delphe, Workshop on Infectious diseases – Hanoi (Vietnam) – Nanotechnological tools in the study of spore coat proteins. Brisson A.R. (2009) Workshop on Diagnosis of infectious diseases - HoChiMinh City (Vietnam) – Brisson A.R. (2009) 5th International Conference on Annexins “Annexin2009” – Lacanau – Annexin 5: mechanism of membrane binding, function and applications.

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Schmidt, V., Giacomelli, C., Brisson, A. R. and Borsali, R. (2006). Towards an easy access to Annexin-A5 protein binding block copolymer micelles. In “Symposium on Nanostructured Biological Materials; 5th Meeting of the Brazilian-Materials-Research-Society ed.” . pp. 479-488, Elsevier Science Bv, Florianopolis, Brazil. C. Romero, B. Bazin, A. Zaitoun, F. Leal-Calderon. (2006) Behavior of a biodegradable scale inhibitor invert emulsion for squeeze application, SPE 98275, SPE International Symposium and Exhibition on Formation Damage Control, Lafayette. Los Angeles (USA). F. Thivilliers, N. Drelon, J. Giermanska-Kahn, J-C. Loudet, F. Leal-Calderon, V. Schmitt. (2006) Gels of emulsion comprising crystallised oil: formation and mechanical behaviour. Proceedings of the 4th world congress on emulsions. Lyon (France), Paper n° 3.3-269. Schmitt, F. Leal-Calderon. (2006) Measurement of the coalescence frequency in concentrated emulsions. Proceedings of the 4th world congress on emulsions. Lyon (France) Paper n° 3.3-279 (2006). V Schmitt, S. Arditty, J. Giermanska-Kahn, F. Leal-Calderon. Proceedings of the 4th world congress on emulsions. Lyon (France), October 3-6, 2006. Paper n° 4.3-280 (2006). Pickering emulsions: new materials with original properties. C. Granger, P. Barey, J.P. Da Costa, M. Cansell (2006). Influence de la formulation sur les propriétés des crèmes glacées. Revue Générale des Routes et des Aérodromes, 851, 33-36. C. Granger, P. Barey, M. Cansell (2006). Impact of the formulation on ice cream air phase structure and stability. Proceedings of the 4th International Symposium on Food Rheology and Structure, Ed. by P. Fisher, P. Erni and E.J. Windhab, ETH Zurich (Suisse), 149-153. C. Granger, P. Barey, J.P. Da Costa, J. Toutain, M. Cansell (2006). Characterization of colloidal products by front-face fluorescence: Application to oil-in-water emulsions and food systems. Proceedings of the 4th International Symposium on Food Rheology and Structure, Ed. by P. Fisher, P. Erni and E.J. Windhab, ETH Zurich (Suisse), 317-321. M. Cansell, N. Combe, P. Clouet (2006). Comparison of the metabolic fate of n-3 PUFA in plasma and liver of rats supplemented with marine lipid-based liposomes or fish oil. Chemistry and Physics of Lipids, 143(1-2), 56. Bernad S., Leygue N., Korri-Youssoufi H., Lecomte S. (2006) Kinetics of the electron transfer reaction of Cytochrome c(552) adsorbed on biomimetic electrode studied by time-resolved surface-enhanced resonance Raman spectroscopy and electrochemistry, 20th Joint Biannual Meeting of the Societe-Francaise-deBiophysiques/Groupe-d'Etudes-des-Interactions-Molecules-Membranes/Groupe-de-Recherche-en-Ingenieriedes-Proteines, Anglet Biarritz, FRANCE, 2006, pp. 1039-1048 Jean-Francois F., Khemtemourian L., Odaert B., Castano S., Grelard A., Manigand C., Bathany K., MetzBoutigue M.H., Dufourc E.J., (2009) Variability in secondary structure of the antimicrobial peptide Cateslytin in powder, solution, DPC micelles and at the air-water interface, Eur Biophys J., 36 pp. 1019-1027 Violette A, Averlant-Petit MC, Rognan D, et al. (2006) Parameters influencing helix stability of oligourea foldamers, 19th American Peptide Symposium, San Diego, CA, In Understanding Biology Using Peptides, pp 716-717. Fournel S, Wieckowski S, Sun W, et al. (2006) Functional mimic of CD40L homotrimers by C3-symetric peptide scaffolds. 6th Annual Meeting of the Federation-of-Clinical-Immunology-Societies, San Francisco, CA. Clin. Immunol. 119, S47 Chevallier S., Ijspeert A.J., Ryczko D., Nagy F., Cabelguen J.M. (2006) Organisation of the spinal central. pattern generators for locomotion in the salamander: Biology and modelling, Meeting on Networks in Motion, Elsevier Science Bv, Stockholm, SWEDEN, pp. 147-161 Kang, X., Schmitter, J.M., Chaignepain S. et Liu, Y. (2006) Quantitative differential analysis between HCC and dysplasic nodule using isotopic-coded lysine tag J. Gastroenterol. Hepatol., 21, A85 Kang, X., Schmitter, J.M., Chaignepain S. et Liu, Y. (2006) Evaluation of isotopic labelling to lysine residues for quantitative proteomics and de novo sequencing J. Gastroenterol. Hepatol., 21, A196 Massias, B. Arturo-Schaan, M. Elie, A. M. Bebin, K. Hocde, V. Denayrolles, M. Urdaci, MC (2006) Effects of non-antibiotic additives on the microbial equilibrium of broiler chicken intestine. Reprod. Nutr., 2006, 46, S105. Maurizot, V., Yoshizawa, M., Kawano, M., and Fujita, M. (2006) Control of molecular interactions by the hollow of coordination cages, in 9th Dalton Discussion Meeting, pp 2750-2756, Royal Soc Chemistry, Manchester, ENGLAND Lebars I., Richard T., Di Primo C., Toulme J.J., (2006) LNA derivatives of a kissing aptamer targeted to the trans-activating responsive RNA element of HIV-1, Workshop on RNS Chemistry meets Biology, Academic Press Inc Elsevier Science, Lund, SWEDEN, pp. 204-209

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Jean-François, F., Berson, P., Castano, S., Metz-Boutigue, M. H., Vacher, P., Elezgaray, J., Roux, M., and Dufourc, E. J. (2007) Antimicrobial Cateslytin promotes rigid domains when interacting as beta-sheet on negative membranes. European Biophysics Journal 36, S67-S67 Methot M., Demers E., Bussieres S., Desbat B., Breton R., Salesse C., (2007) Phospholipid monolayer hydrolysis by cytosolic phospholipase A2 gamma and lecithin retinol acyl transferase, 12th International Conference on Organized Molecular Films, Elsevier Science Bv, Cracow, POLAND, pp. 147-150 Sani, M. A., Gröbner, G., and Dufourc, E. J. (2007) Investigation of apoptosis regulation in mitochondrial membrane: from model to in vivo studies. European Biophysics Journal 36, S179-S179 Jean-François, F., Berson, P., Castano, S., Metz-Boutigue, M. H., Vacher, P., Elezgaray, J., Roux, M., and Dufourc, E. J. (2007) Antimicrobial Cateslytin promotes rigid domains when interacting as beta-sheet on negative membranes. European Biophysics Journal 36, S67-S67 Buchoux, S., Garnier, M., and Dufourc, E. J. (2007) A new membrane destabilization mechanism: electrostatic repulsion promoted by lipopeptide Surfactin. European Biophysics Journal 36, S67-S67 Sani, M. A., Dufourc, E. J., and Grobner, G. (2007) Regulation of apoptosis at the mitochondrial level: Cardiolipin and peptide domain. Biophys. J., 568A-568A Elezgaray, J., Jean-Francois, F., Vacher, P., Berson, P., and Dufourc, E. (2007) Electroporation as an alternative action mechanism of antimicrobial peptides. Biophys. J., 612A-612A Carteau D., Bassani D., Pianet I. (2007) The "Ouzo effect": Following the spontaneous emulsification of transanethole in water by NMR, 20th GERM Congress, Elsevier France-Editions Scientifiques Medicales Elsevier, Alenya, FRANCE, pp. 493-498 Cameron A.J., De Rycker M., Calleja V., Alcor D., Kjaer S., Kostelecky B., Saurin A., Faisal A., Laguerre M., Hemmings B.A., McDonald N., Larijani B., Parker P.J., (2007) Protein kinases, from B to C, Focus Topic at Life Sciences 2007 Conference, Portland Press Ltd, Glasgow, SCOTLAND, pp. 1013-1017 D. Alcor, V. Calleja, M. Laguerre, M. Katan, P. P. Parker & B. Larijani. (2007) Revealing protein regulation in single-cells: From protein/protein interaction to protein conformation change. Poster présenté au 51st Biophysical Society Annual Meeting organisé les 3-7-III-2007 à Baltimore, USA. Biophys. J., 69A. Jean-Francois F., Khemtemourian L., Odaert B., Castano S., Grelard A., Manigand C., Bathany K., MetzBoutigue M.H., Dufourc E.J., (2007) Variability in secondary structure of the antimicrobial peptide Cateslytin in powder, solution, DPC micelles and at the air-water interface, European Biophysics Journal with Biophysics Letters 36, 1019-1027 Vergne S., Lamothe V., Chantre P., Potier M., Asselineau J., Perez P., Durand M., Moore N., BennetauPelissero C., Sauvant P.. (2007) Influence of ethnicity on bioavailability of isoflavones in Human: caucasian Vs Asian. 10th European Nutrition Conférence. Paris, France, [Ann. Nutr. Metab 2007 ;51(Suppl 1). P316, page 208]. Vergne S., Lamothe V., Chantre P., Potier M., Asselineau J., Perez P., Durand M., Moore N., BennetauPelissero C., Sauvant P.. (2007) Influence of food matrix on bioavailability of isoflavones after a single ingestion in Human. 10th European Nutrition Conférence. Paris, France, [Ann. Nutr. Metab 2007 ;51(Suppl 1). P315, page 208]. Schmitter, J.M., Bressollier, P., Verneuil, B., Ballade, A., Urdaci, M. (2007) Sample preparation for structure characterization of novel antibiotics produced by bacillus strains Mol. Cell. Proteomics, 6, 29-29. P. Subra-Paternault, Ch. Roy, A. Vega-Gonzalez, P. Jestin, (2007) Crystallization of drugs using supercritical CO2 as antisolvent : from phase equilibria to products, Proceedings 1st International Congress on Green Process Engineering, Toulouse, (CD media) JM. Schmitter, P. Bressollier, B. Verneuil, A. Chobelet, A. Ballade, MC. Urdaci. (2007). Search for New Antibiotics Produced by Bacillus Strains. 55th ASMS Conference on Mass Spectrometry and Allied Topics, June 3-8, Indianapolis, IN, USA. (Actes du congrès publiés par le Journal of the American Society for Mass Spectrometry.) Krieger E., Leger L., Durrieu M.-P., Taib N., Bond P., Laguerre M., Lavery R., Sansom M. S. P. and Baaden M. (2007) Atomistic modeling of the membrane-embedded synaptic fusion complex: a grand challenge project on the DEISA HPC infrastructure. Proceedings ParCo, NIC Series, 38, 729-736.

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Wallgren, M., Sani, M. A., Vestin, H., Dufourc, E. J., and Gröbner, G. (2009) The Anti-apoptotic Mitochondrial Membrane Protein Bcl-2; An Achilles Heel Of Cancer Cells? Biophys. J. 96, 424a-425a Jean-François, F., Elezgaray, J., Metz-Boutigue, M. H., and Dufourc, E. J. (2009) Disordered Pore Formation At Rigid/Fluid Boundary Zones As A New Mechanism For Peptide-Membrane Interaction With The Beta-sheeted Antimicrobial Peptide Cateslytin. Biophys. J. 96, 390A-390A Guichard G, Trouche N, Wieckowski S, Sun W, Chaloin O, Bianco A, Hoebeke J, Schneider P, Fournel S. (2009) Rationally-designed multivalent architectures for mimicking homotrimers of CD40L, a member of the TNF superfamily. Adv Exp Med Biol. 611:355-7 (Proceedings of the 20th American Peptide Symposium, Montreal Canada. Guichard G, Léna G, Boeglin J, Muller P, Boilard E, Lambeau G, Rognan D, Didierjean C. (2009) 1,3,5Triazepan-2,6-diones as conformationally constrained dipeptide mimetics. In silico guided identification of sPLA2 inhibitors. Adv Exp Med Biol. 611:201-2. (Proceedings of the 20th American Peptide Symposium, Montreal Canada. Fischer L, Lena G, Briand JP, Didierjean C, Guichard G. (2009) Mixing urea and amide bonds: synthesis and self-organization of new hybrid oligomers. Adv Exp Med Biol. 611:39-40. (Proceedings of the 20th American Peptide Symposium, Montreal Canada). Bijani C., Sice, S., Elezgaray, J., Degert, C., Broussaud, O. Dufourc, E.J. (2009) Use of lipoaminoacids for nasal delivery of therapeutic proteins. Eur. Biophysics J. 38 S85 (Proc. 7th EBSA European Biophysics congress, Genoa, Italy). Diller A., Loudet, C., Grelard, A., Dufourc, E.J. (2009) Deformation of phospholipids vesicles in high magnetic fields studeied by 31P solid state NMR. Eur. Biophysics J. 38 S184 (Proc. 7th EBSA European Biophysics congress, Genoa, Italy). Zhendre V. Larijani, B., Dufourc, E.J. (2009) Phosphoinositides in nuclear membrane assembly. Eur. Biophysics J. 38 S198 (Proc. 7th EBSA European Biophysics congress, Genoa, Italy).

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C. Commandeur, M. Laguerre & J. Dessolin. (2007) La modélisation moléculaire, un outil de choix pour la conception de molécules contre le cancer. Proc. 3ème Journée du Canceropôle Grand Sud-Ouest, à Bordeaux, Bull. du Cancer, 94 S286.

21.

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22. 2006

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V. Schmitt, S. Arditty, J. Kahn, F. Leal-Calderon. (2006) 4th World Congress on Emulsions. Lyon (France). Pickering emulsions: new materials with original properties C. Granger, P. Barey, J.P. Da Costa, M. Cansell. (2006) 4th World Congress on Emulsions. Lyon (France), October 3-6, 2006. Influence of the formulation on ice cream properties M. Cansell, P Méléard, N. Combe. (2006) 4th World Congress on Emulsions. Lyon (France), October 3-6, 2006. Use of marine lipid-based liposomes for lipid oral delivery. In vitro and in vivo studies V. Schmitt, S. Arditty, J. Kahn, F. Leal-Calderon. (2006) 12th International Association of Colloids and Interface Scientists (IACIS). Beijing (China), October 15-20, 2006. Pickering emulsions: origin of their outstanding elasticity F. Leal-Calderon, V. Schmitt, J. Kahn . (2006) 12th International Association of Colloids and Interface Scientists (IACIS). Beijing (China), October 15-20, 2006. Gels of emulsions comprising crystallisable oils: formation and mechanical behaviour M. Bonnet, A. Berkaoui, M. Cansell, M.H. Ropers, M. Anton, F. Leal-Calderon. (2006) 13th World Congress of Food Science and Technology Congress, ‘Food is Life’ (IUFoST), Nantes (France), September 17-21 2006. Encapsulation of magnesium in W/O/W emulsions: characterization and stability evaluation

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M. Cansell, N. Combe, P. Clouet. (2006) 9th ILPS International Phospholipid Congress, "Biochemistry and Nutrition of Phospholipids". Pécs (Hungary), September 9-10, 2006. Comparison of the metabolic fate of n-3 PUFA in plasma and liver of rats supplemented with marine lipid-based liposomes or fish oil M. Cansell, C. Granger, P. Barey, J.P. Da Costa, J. Toutain. (2006) 4th International Symposium on Food Rheology and Structure (ISFRS). Zurich (Switzerland), February 19-23, 2006. Characterization of colloidal products by front-face fluorescence: Application to oil-in-water emulsions and food systems C. Granger, P. Barey, M. Cansell. (2006) 4th International Symposium on Food Rheology and Structure (ISFRS). Zurich (Switzerland), February 19-23, 2006. Impact of the formulation on ice cream air phase structure and stability Goudenège S., Dargelos E., Cottin P., Poussard S. (2006) Study of m-calpain functions in caveolae of fusing muscle cells using a differential proteomic approach. Congrès de la Société Française d’Electrophorèse et d’Analyse Protéomique. 16-18 octobre 2006, Saint Malo, France. Cottin P., Hadj Sassi A., Cassar-Malek I., Picard B. (2006) Facteur de croissance myostatine et système protéolytique calcium-dépendant : implication dans l'hypertrophie musculaire. Journées de restitution des projets financés sur crédits incitatifs, Département Phase, 14-15 septembre 2006, Tours, France. Daury L., Mazères G., Leloup L., Cottin P., Brustis JJ. (2006) Les calpaïnes ubiquitaires régulent l’adhésion et l’étalement des myoblastes. Colloque Myogenèse V, 10-11 janvier 2006, Institut de Myologie, Paris, France. Leloup L., Mazères G., Daury L., Cottin P., Brustis JJ. (2006) Implication des calpaïnes ubiquitaires dans la migration des myoblastes induite par les facteurs de croissance.Colloque Myogenèse V, 10-11 janvier 2006, Institut de Myologie, Paris, France. Lecomte, S. Bernad, S. Korri-Youssoufi, H. (2006) Le transfert d’électrons dans les hémoprotéines : méthodes couplées spectrométries vibrationnelles/électrochimie 20ème congrès de la Société Française de Biophysique, 14-19 Octobre Anglet, France. Lecomte, S., (2006) Dynamique du transfert d'électrons du cytochrome c552 adsorbé sur des surfaces biomimétiques: analyses in-situ électrochimie / SERS résolue en temps 10ème colloque Groupe Français de Spectroscopie Vibrationnelle, 25-27 janvier Chaumont sur Tharonne, France Jean-François, F., Elezgaray, J., Castano, S., Desbat, B., Khemtémourian, L., Manigand, C., Bathany, K., Roux, M., Metz-boutigue, M. H., Vacher, P., Bergson, P., and Dufourc, E. J. (2006) Etude multitechnique du mode d'action de la Cateslytine: Un nouveau peptide antimicrobien structuré en feuillets beta. In Congres SFB, Communication Orale, Anglet, France Sani, M. A., Gröbner, G., and Dufourc, E. J. (2006) Régulation de l’apoptose au niveau de la mitochondrie : Cardiolipine et domaine peptidique. In 20ème Congrès de la SFB, Communication Orale, Anglet, France Odaert, B., Bressolier, P., Urdaci, M., and Dufourc, E. J. (2006) Liquid state NMR study of an antimicrobial peptide from the lantibiotic family. In Congrès SFB-GEIMM-GRIP, Communication Orale, Anglet, France Jean-François, F., Elezgaray, J., Castano, S., Desbat, B., Khemtémourian, L., Manigand, C., Bathany, K., Roux, M., Metz-boutigue, M. H., Vacher, P., Bergson, P., and Dufourc, E. J. (2006) Etude multitechnique du mode d'action de la Cateslytine: Un nouveau peptide antimicrobien structuré en feuillets beta. In Congres SFB, Communication Orale, Anglet, France Garnier-Lhomme, M., Byrne, R. D., Han, K., Dowicki, M., Michael, N., Gschmeissner, S., Totty, N., Zhendre, V., Cho, A., Pettitt, T. R., Wakelam, M. J., Woscholski, R., Dufourc, E. J., Poccia, D. L., and Larijani, B. (2006) Role of Phosphoinositide Signalling in Nuclear Envelope Assembly. In London Biological Mass Spectrometry Discussion Group, Communication Orale, London, UK Garnier-Lhomme, M., Byrne, R. D., Grélard, A., Loudet, C., Gschmeissner, S., Woscholski, R., Poccia, D. L., Dufourc, E. J., and Larijani, B. (2006) Natural Membrane Domains as Key Players in Nuclear Envelope Assembly. In The cell Biology of Inositol Lipids and Phosphates, Communication Orale, Birmingham, UK Buchoux, S., Garnier, M., Tsan, P., Besson, F., and Dufourc, E. J. (2006) Vers un nouveau modèle de lyse de membranes: exemple de la Surfactine. In 20ème Congrès de la Société Française de Biophysique, Communication Orale, Anglet, France Loudet, C., Manet, S., Gineste, S., Reiko, O., Achard, M. F., and Dufourc, E. J. (2006) Bicelle with the normal oriented parallel to the magnetic field : a study by NMR, electron microscopy and SAXS. In Congrès de la SFB. Oral presentation, Anglet, France. Buchoux, S., Garnier, M., Tsan, P., Besson, F., and Dufourc, E. J. (2006) A new approach to model membrane lysis: example of Surfactin. In French-Benelux meeting on Magnetic Resonance, Communication Orale, Blankenberge, Belgium Faure, C., Guillot, S., Weisbecker, P. and Saadaoui, H. (2006) Multilamellar vesicle-assisted electrodeposition of inorganic nanodots. In Congrès international du réseau européen FAME, Tenerife Meyre, M.E. Duguet, E. Franconi, J.M. and Faure, C. (2006) Magnetic onions for MRI and hyperthermia. In Congrès international du réseau européen FAME, Tenerife Guichard, G., Trouche, N., Wieckowski, S. Sun, W., Chaloin, O., Bianco, A., Toes, R., Melief, C.J.M., Hoebeke, J., Schneider, P., Fournel, S. (2006) Rationally-designed multivalent architectures for mimicking homotrimers of the TNF superfamily. Oral communication, 1st European Chemistry Congress, Budapest, Hungaria. Bonnafous P, Marak L, Bickel T, Trépout S, Mornet S, De Haas F, Talbot H, Taveau J-C, Lambert O (2006) Biofonctionnalisation de nanoparticules: outil pour des études structurales de protéines membranaires par 140

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cryotomographie électronique. 20 ième congres de la Société française de Biophysique, Anglet14-19 Octobre 2006 Lambert O. Taveau J.C . (2006) Imagerie 3D pour les nano-objets.2ième Colloque d’Imagerie 3D en Aquitaine. Bordeaux 11-12 Décembre 2006 Lajus, S., P. Vacher, D. Huber, M. Dubois, M.N. Benassy, Y. Ushkaryov, and J. Lang, (2006) α-latrotoxin induces exocytosis by inhibition of voltage-dependent K+ channels and by stimulation of L-type Ca2+ channels via latrophilin in β-cells, in Club Exo-Endocytose, Anglet, France. Grise, F. and J. Lang. (2006) Localisation et mécanismes moléculaires de senseurs calciques de la sécrétion de l’insuline. in ALFEDIAM. Lyon, France. Schmitter, J.M., Bressollier, P., Verneuil, B., Chobelet, A. Ballade A., Urdaci, M. (2006) Characterization of biologically active compounds produced by Bacillus strains. 19th Tandem Mass Spectrometry Workshop, Lake Louise, Canada Schmitter, J.M., Bressollier, P., Verneuil, B., Chobelet, A. Ballade A., Urdaci, M. (2006) Characterization of biologically active compounds produced by Bacillus strains. 19th Tandem Mass Spectrometry Workshop, Lake Louise, Canada C. Roy, C. Laudani, A. Vega-González, P. Subra-Paternault, (2006) “Coprecipitation of theophylline and polymer using supercritical carbon dioxide, Proceedings of 8th Conference on Supercritical Fluids and Their Applications” May 28-31, 2006, Ischia (Napoli), Italy (CD media)  A. Vega-González, P. Subra-Paternault, D. Vrel, (2006) Aminosalicylate formulation using compressed CO2, Proceedings of 8th International Symposium on Supercritical Fluids November 5-8 2006, Kyoto Japan (CD media) Odaert, B., Bressolier, P., Urdaci, M., and Dufourc, E. J. (2006) Liquid state NMR study of an antimicrobial peptide from the lantibiotic family. In Congrès SFB-GEIMM-GRIP, Communication Orale, Anglet, France Estieu-Gionnet, K., Gonçalves, M., Berthelot, T., Clochard, M-C., Cuscito, O., Bikfalvi, A., Déléris, G, (2006) Conception, synthèse et évaluation de nano-vecteurs originaux pour la thérapie et/ou l’imagerie de l’angiogenèse tumorale Deuxième rencontre du Club nanomatériaux pour les sciences du vivant – Lyon – France.

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C. Granger, P. Barey, J.P. Da Costa, M. Cansell. (2007) 3rd International Symposium on Ice Cream, Inter-Ice 2007 (IDF). Cologne (Germany), December 4-6, 2007. Influence of the interaction between fat and emulsifier on ice cream properties M. Bonnet, A. Berkaoui, M. Cansell, M.H. Ropers, M. Anton, F. Leal-Calderon. (2007) Delivery of functionality in complex food systems: physically-inspired approaches from nanoscale to microscale. Amerst (MA, USA), October 8-10, 2007. Magnesium release from W/O/W emulsions made with different edible oils V. Schmitt, S. Arditty, J. Kahn, F. Leal-Calderon. (2007) 21st Conference of the European Colloid & Interface Society (ECIS). Geneva (Switzerland), September 10-14, 2007. Pickering emulsions: The role of surface coverage on their original properties Stuelsatz P., Veschambre P., Cottin P. (2007) La calpaïne 3 est impliquée dans la régulation de l'activité transcriptionnelle de MyoD au cours de la myogenèse. 2èmes Journées d'Animation Scientifique du Département Phase, 22-24 Octobre 2007, Tours, France. Poussard S., Goudenège S., Dargelos E., Cottin P. (2007) Study of milli-calpain biological functions at the level of skeletal muscle caveolae. 5ème Colloque du groupe thématique « Protéolyse Cellulaire » de la Société Française de Biochimie et Biologie Moléculaire, 27-29 mars 2007, Seillac, France. Daury L., Leloup L., Mazères G., Dedieu S., Cottin P., Brustis JJ. (2007) Implication de la Protéolyse calciumDépendante lors de l'adhésion et de la migration des cellules myogéniques 5ème Colloque du groupe thématique « Protéolyse Cellulaire » de la Société Française de Biochimie et Biologie Moléculaire, 27-29 Mars 2007, Seillac, France. Fezoua,Z., Brisson, A., Desbat, B., Lecomte, S. (2007) Study of the Annexin A5 with Lipid monolayer at the Air-Water interface using PMIRRAS and Brewster Angle Microscopy) XII International Conference on Organized Molecular Films, July1-5 Krakow, Pologne (2007). Jean-François, F., Elezgaray, J., Castano, S., Roux, M., Metz-boutigue, M. H., Vacher, P., and Dufourc, E. J. (2007) Antimicrobial Cateslytin promotes rigid domains fomation when interacting as beta-sheets on negative membranes. In GFPP Congress, Communication Orale, Dinard, France Jean-François, F., Elezgaray, J., Castano, S., Roux, M., Metz-boutigue, M. H., Vacher, P., and Dufourc, E. J. (2007) Enlightenments into the action of Cateslytin, a new antimicrobial peptide promoting rigid domains when interacting as beta-sheets on negatively charged membranes. In NMR of Soft Matter, Communication Orale, Arcachon, France Jean-François, F., Elezgaray, J., Castano, S., Roux, M., Metz-boutigue, M. H., Vacher, P., and Dufourc, E. J. (2007) Enlightements into the action of Cateslytin, a new antimicrobial peptide. In Chemistry and Biology on membranes, Communication orale, Plentzia, Spain Sani, M. A., Gröbner, G., and Dufourc, E. J. (2007) Investigation of apoptosis regulation in mitochondrial membrane: from model to in vivo studies. In NMR & Soft Matter, Communication Orale, Arcachon, France

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Odaert, B., Bressolier, P., Urdaci, M., and Dufourc, E. J. (2007) Liquid state NMR study of an antimicrobial peptide from the lantibiotic family. In 2nd Workshop on membrane active peptides, Communication orale, Lisbone, Portugal Jean-François, F., Elezgaray, J., Castano, S., Roux, M., Metz-boutigue, M. H., Vacher, P., and Dufourc, E. J. (2007) Antimicrobial Cateslytin promotes rigid domains fomation when interacting as beta-sheets on negative membranes. In GFPP Congress, Communication Orale, Dinard, France Jean-François, F., Elezgaray, J., Castano, S., Roux, M., Metz-boutigue, M. H., Vacher, P., and Dufourc, E. J. (2007) Enlightenments into the action of Cateslytin, a new antimicrobial peptide promoting rigid domains when interacting as beta-sheets on negatively charged membranes. In NMR of Soft Matter, Communication Orale, Arcachon, France Jean-François, F., Elezgaray, J., Castano, S., Roux, M., Metz-boutigue, M. H., Vacher, P., and Dufourc, E. J. (2007) Enlightements into the action of Cateslytin, a new antimicrobial peptide. In Chemistry and Biology on membranes, Communication orale, Plentzia, Spain Garnier-Lhomme, M., Byrne, R. D., Grelard, A., Loudet, C., Gschmeissner, S., Woscholski, R., Poccia, D. L., Dufourc, E. J., and Larijani, B. (2007) Role of membrane domains in nuclear envelope assembly: structure, composition and dynamics of nuclear envelope remnants. In Workshop on Membrane Structure/Dynamics in Development and Cell Signalling, Bilbao, Espagne Buchoux, S., M., G., and Dufourc, E. J. (2007) A new membrane destabilization mechanism: electrostatic repulsion promoted by lipopeptide Surfactin. In NMR & Soft Matter, Communication Orale, Arcachon, France Buchoux, S., Garnier, M., Tsan, P., Besson, F., and Dufourc, E. J. (2007) Nouveau modèle de lyse de membranes: exemple de la Surfactine. In 15ème Réunion du Groupe Français des Peptides et des Protéines, Communication Orale, Dinard, France Buchoux, S., Garnier, M., Tsan, P., Besson, F., and Dufourc, E. J. (2007) Vers un nouveau modèle de lyse de membranes: Exemple de la Surfactine. Oral presentation. Workshop annuel SAFO, Pessac, France. Faure, C., Guillot, S., Weisbecker, P. and Saadaoui, H. (2007) Nanoplots inorganiques obtenus par voie électrocolloïdale. In Journées d’électrochimie JE07, Lyon, France Debecker, D.P., Meyre, M.E., Gaigneaux, E.M. and Faure C. (2007) Onion-type multi-lamellar vesicles as an innovative tool for the preparation of metal-based heterogeneous catalysts. In Congrès international du réseau européen FAME, Madère Guichard, G., Trouche, N., Wieckowski, S. Sun, W., Chaloin, O., Bianco, A., Toes, R., Melief, C.J.M., Hoebeke, J., Schneider, P., Fournel, S. (2007) Rationally-designed multivalent architectures for mimicking homotrimers of the tnf superfamily. Oral communication, 20th American Peptide Symposium, Montreal, Canada. Fischer, L., Léna, G., Briand, J. P., Didierjean, C., Guichard, G. Oligomères (2007) Peptidomimétiques à Base d’Urées : Propriétés d’Auto-assemblage en Série Macrocyclique. Oral communication 15ème Réunion du Groupe Français des Peptides et Protéines (GFPP) , Dinard, France. Lambert O, Taveau J.C, Bonnafous P, Trépout S (2007) La cryo-microscopie électronique à l’interface ChimieBiologie. Journées de formation à la tomographie électronique en biologie Toulouse 2-3 avril 2008 Taveau J.C., Dubois M., Le Bihan O., Trépout S., Almagro S., Durmort C., Gulino-Debrac D., Lambert O. (2007) Etude des jonctions adhérentes interendothéliales. 10 ième Colloque de la société française des microscopies (Sfµ) Grenoble 5-8 juin 2007 Lambert O. (2007) Cryotomographie électronique. Ecole thématique du CNRS Nano-objets aux interfaces. Lacanau 28 mai-1 juin 2007 Le Bihan O., Mornet S., Pitard B. Lambert O. (2007) Study of gene transfer using electron microscopy approach. 1er Colloque Européen des Jeunes Chercheurs de la Mucoviscidose, Lille, 29-31 août 2007 Aimé, C., Manet, S., Oda, R., (2007) Studying morphology and structure of micrometric helices of nucleoamphiphiles. 20th Conference of the European Colloid and Interface Society Geneve Suisse sep. 2007 Yassine, W., Desbat, B., Oda, R., (2007) SNAREs PROTEIN A COMPLEXE OF MEMBRANE FUSION : Physico-chemical study of the transmembrane peptides, 9em Journée Francophone de Jeune Chercheurs, Maubuisson, sep 2007 Delville, M.-H., Aimé, C., Delclos T., Brizard, A., Huc, I., Oda, R. (2007) Tunable helical amphiphile assemblies as templates for chiral inorganic sol-gel transcription: How to obtain nanostructured individual inorganic objects, Madeira, oct. 2007 A. Lopez-Periago, A. Vega, P. Subra, C. Domingo, (2007) Supercritical processing of polymers for the production of scaffolds and impregnated drug delivery systems, Proceeding of Ist Iberoamerican Conference on Supercritical Fluids, Iguassu Falls, Brazil, 10-13 April, 2007 Odaert, B., Bressolier, P., Urdaci, M., and Dufourc, E. J. (2007) Liquid state NMR study of an antimicrobial peptide from the lantibiotic family. In 2nd Workshop on membrane active peptides, Communication orale, Lisbone, Portugal Odaert, B., Bressolier, P., Urdaci, M., and Dufourc, E. J. (2007) Liquid state NMR study of an antimicrobial peptide from the lantibiotic family. In NMR and soft matter, Arcachon, France Denayrolles M, Arturo-Schaan M, Massias B, Bebin K, Elie AM, Panheleux, Urdaci MC. 2007. Effect of diets with different fibrous contents on broiler gut microflora and short-chain fatty acid (SFCA) production. European WPSA. 2007. France. 142

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J. Parello, J.-L. Banères & M. Laguerre. (2007) Hydration in GPCR-mediated signal transduction. Communication orale présentée à l'ESF-FWF Conférence "Water interfaces in Physics, Chemistry and Biology: a multi-disciplinary approach" les 8-13-XII-2007 à Innsbruck (Autriche).

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E. Vaique, M. Cansell, S. Pinet. (2008) 2nd EuChemMS Chemistry Congress. Turin (Italia), September 16-20, 2008. Synthesis of fluorescent triglycerides as tools for studying lipid metabolic fate M. Bonnet, A. Berkaoui, M. Cansell, M.H. Ropers, M. Anton, F. Leal-Calderon. (2008) Food colloids 2008: creating structure, delivering functionality. Le Mans (France), April 6-9, 2008. Improvement of magnesium retention in W/O/W emulsions by ion complexation Roumes H., Daury L., Leloup L., Cottin P., Brustis J.-J. (2008) Rôle du système calpaïnes-calpastatine lors de l'adhésion et la migration des cellules myogéniques. Colloque Myogenèse IX, 21-22 janvier 2008, Institut de Myologie, Paris, France. Delhaye, C., Bruneel, J.L., Talaga, D., Guirardel, M., Lecomte, S. Servant L. (2008) Spectroscopie Raman et Microfluidique Colloque Groupe Français de Spectroscopie Vibrationnelle 25-27 juin Toulouse, France. de Lima, V. R., Creczynski-Pasa, T., Caro, M. B., and Dufourc, E. J. (2008) Disordering effects induced by melatonin on phospholipid membranes correlated with its antioxydant properties: a NMR and fluorescence study. In Congrès du GEIMM 13 p. Oral presentation, Colmar, France Debecker, D.P., Meyre, M.E., Derré, A., Gaigneaux, E.M. and Faure C. (2008) Metal nanoparticles formed via onion-type multi-lamellar vesicles and involved in an innovative preparation of heterogeneous catalysts: application in benzene abatement. In 5th International Conference on Environmental Catalysis, Belfast, Ireland Debecker, D.P., Meyre, M.E., Derré, A., Gaigneaux, E.M. and Faure C. (2008) Onion-type vesicular microreactors for a new bio-inspired route to metal nanoparticle-based heterogeneous catalysts. In E-MRS Fall Meeting (European Material Research Society), Varsovie, Pologne Gallois, B. (2008) Cristallographie et Sciences du Vivant. IIèmes journées de l’Association Bordelaise de Cristallographie. Bordeaux, France. Maugé, C., Granier, T., Langlois d’Estaintot, B. and Gallois B. (2008). Expression, Purification et Etudes structurales d’enzymes impliqués dans la biosynthèse des flavonoïdes. IIèmes journées de l’ABC. Bordeaux, France. Gargouri, M., Maugé, C., Granier, T., Langlois d’Estaintot, B., Manigand, C., Bathany, K., Schmitter, J.M., Chaudière, J. and Gallois. B. (2008). Structure et Propriétés Fonctionnelles de l’Anthocyanidine Réductase, enzyme clef de la voie de biosynthèse des tannins condensés. Association Française de Cristallographie, GtBio. Lille, France. Claudon, P. Violette, A. Lamour, K. Fournel, S., Heurtault, B., Godet, J., Jamart-Grégoire, B., Averlant-Petit, M.C., Briand, J. P., Duportail, G., Monteil, H., Guichard, G. (2008) Modifications isostructurales de foldamères antibactériens et conséquences pour l’interaction avec les membranes lipidiques. Oral communication Congrès du Groupe d'Etudes des Interactions Molecules-Membranes (GEIMM), Mittelwihr, France. B. Baptiste, I. Huc, J.-M. Léger (2008). Applications des foldamères d’oligoamides aromatiques. Journée Association Bordelaise de Cristallographie, oral communication, Bordeaux, July. B. Baptiste, I. Huc, J.-M. Léger (2008). Designing aromatic amide Foldamer ligands for Quadruplex recognition. Journée Chimie et Biologie des Membranes et des Nano-objets. IECB, oral communication, Pessac, November Taveau J.C., Nguyen D., Perro A., Ravaine S., Duguet E, Lambert O. (2008) 3D cryo-electron tomography of nucleated and growing PS nodules on silica nanoparticles. 1st Conference on 3D-Imaging ofMaterials and Systems Acquisition - Analysis – Applications. 8-12 Septembre 2008 Carcans-Maubuisson (Bordeaux). Perro, E. Duguet, O. Lambert, Taveau J.C. , E. Bourgeat-Lami and S. Ravaine, (2008). A chemical synthetic route to colloidal molecules. Particles 2008, Orlando/Etats-Unis, 10-13 mai 2008 Perro A., D. Nguyen, S. Ravaine, E. Bourgeat-Lami, O. Lambert, Taveau J.C. and E. Duguet. (2008). Contrôle de la morphologie de nanoparticules biphasées silice/polystyrène : vers des molécules colloïdales., Colloque Nanohybrides 5, Porquerolles/France, 16-19 juin 2008 Trépout S., Taveau, J.C., Benabdelhak, H., Ducruix, A., Lambert, O., (2008) Structure of multidrug efflux pump in Cryo Electron Tomography: OprM-MexA interacting complex. 14ième Congrès Européen de Microscopie (EMC), Aix-la-Chapelle, Allemagne, 1-5 octobre 2008 Trépout S., Taveau, J.C., Benabdelhak, H., Ducruix, A., Frangakis A., Lambert, O. (2008) Structure of OprMMexA complex revealed by cryo electron tomography. Gordon conference 3D cryoelectron microscopy (poster sélectionné), Barga, Italie, 15-20 Juin 2008. Le Bihan O., Mornet S., Pitard B. Lambert O. (2008) Imaging tools to study gene transfer using synthetic vectors. 2eme Colloque Européen des Jeunes Chercheurs de la Mucoviscidose, Lille, 27-29 août 2008. Le Bihan O., Chèvre R., Mornet S., Pitard B. Lambert O. 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F. Salpin, F. Trivier, C. Coupry, S. Lecomte, (2006) “A new quantitative method : non destructive study by Raman spectroscopy of dyes fixed on wool fibres”. XXth International Conference On Raman Spectroscopy, August 21-35, Yokohama, Japan, p. 209 Garnier-Lhomme, M., Byrne, R. D., Grélard, A., Loudet, C., Gschmeissner, S., Woscholski, R., Poccia, D. L., Dufourc, E. J., and Larijani, B. (2006) Natural Membrane Domains as Key Players in Nuclear Envelope Assembly. In Lipid Rafts and Cell Function, Keystone symposia, Affiche, Keystone, Colorado, USA Odaert, B., Bressollier, P., Urdaci, M. Dufourc, E.J. (2006) Liquid state NMR study of an antimicrobial peptide from the lantibiotic family. Congress Société Française de Biophysique-GEIMM-GRIP, Affiche, Anglet, France Garnier-Lhomme, M., Byrne, R. D., Grélard, A., Loudet, C., Gschmeissner, S., Woscholski, R., Poccia, D. L., Dufourc, E. J., and Larijani, B. (2006) Natural Membrane Domains as Key Players in Nuclear Envelope Assembly. In New concepts in Lipidology: from Lipidomics to Disease, Affiche, Noordwijkerhout, Netherlands Buchoux, S., Garnier, M., Tsan, P., Besson, F., and Dufourc, E. J. (2006) A new approach to model membrane lysis : Example of Surfacin. Poster presentation. French/Benelux/Netherlands XXth GERM conference (Blankenberge, Belgium). Commandeur, C.; Dessolin, J.; Laguerre, M. (2006), Synthèse d'inhibiteurs de XIAP pour une chimiothérapie anti-cancer. 20 Octobre 2006, Journée des Chercheurs ARC, Hôpital Européen Georges Pompidou, Paris (France). J. Parello, J.-L. Baneres, J.-P. Girard & M. Laguerre. (2006), Positioning of the agonist leukotriene B4 in the ligand-binding pocket of its G protein-coupled receptor BLT1. Poster présenté au RECOB XI organisé à Aussois du 26 au 30-III-2006. Tarascou, E. Fouquet, D. Moskau, M.A. Ducasse, M. Laguerre & I. Pianet, (2006), Conformational study and 3D structure of two procyanidin trimers. Poster présenté au XXème GERM organisé les 19-22 mars 2006 à Blankenberge (Belgique). Faure, C., Guillot, S., Weisbecker, P. and Saadaoui, H. (2006) Multilamellar vesicle-assisted electrodeposition of inorganic nanodots. In ESEAC2006, Bordeaux, France. Faure, C., Guillot, S., Weisbecker, P. and Saadaoui, H. (2006) Fabrication de nanoplots métalliques par voie électro-colloïdale. In Colloque Electrochimie dans les nanosciences, Paris, France Petit, P., Granier, T., Langlois d'Estaintot, B., Lauvergeat, V., Hamdi S. and Gallois B. (2006). Crystal structure of dihydroflavonol 4-reductase, a key enzyme of the flavonoid biosynthetic pathway. Association Française de Cristallographie. Toulouse. France Petit, P., Granier, T., Langlois d'Estaintot, B., Lauvergeat, V., Hamdi, S. and Gallois B. (2006). Crystal structure of dihydroflavonol 4-reductase, a key enzyme of the flavonoid biosynthetic pathway in grapes. xxiiird International Conference on Polyphenols. Winipeg. Canada Trouche, N.; Wieckowski, S.; Chaloin, O.; Sun, W.; Bianco, A.; Hoebeke, J.; Fournel, S. Guichard, G. (2006) Architectures Trimeriques de Synthèse pour Mimer des Homotrimères de la Superfamille du TNF, Poster, « 11ème Rencontres de Chimie Organique Biologique, RECOB », Aussois, France Trépout S., Taveau J.-C., Benabdelhak H., Ducruix A., Brisson A., Lambert O. (2006) Structure of Multidrug efflux pump studied by Cryo-EM and cryo-tomography. Gordon Conference on Three dimensional electron microscopy, Barga, Italie, 25-30 juin 2006. Trépout S., Taveau J.C., Benabdelhak H., Ducruix A., Lambert O. (2006) Structure of multidrug Efflux Pump Studied by CryoMET and CryoTE. Congrès annuel de la Société Française de Biophysique, Anglet, France, 1419 octobre 2006 Taveau J.-C., Bonnafous P., Mornet S., DeHaas F., A. Brisson, Lambert O. (2006) Silica-nanoparticle encapsulation into liposomes studied by Cryo-EM and cryo-tomography. 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(2006) The effects of physiological concentrations of soy isoflavones on bone cell differentiation, an in vitro study. 1st International Soy and Health Conference. Düsseldorf, Germany, 12-13 October 2006. Vergne Sébastien, Chantre Philippe, Lamothe Valérie, Asselineau Julien, Durand Marlène, Potier Mylène, Bennetau-Pelissero Catherine, Moore Nicholas, Sauvant Patrick. (2006) Isoflavones contained in soy based 146

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Stuelsatz P., Veschambre P., Cottin P. (2007) Calpain 3 is implicated in the regulation of the transcriptional activity of MyoD during myogenesis. (selected poster) 12th International Congress of The World Muscle Society, October 17-20, 2007, Giardini Naxos-Taormina, Italy. Poussard S., Goudenège S., Dargelos E., Cottin P. (2007) Study of milli-calpain biological functions at the level of skeletal muscle caveolae. 5ème Colloque du groupe thématique « Protéolyse Cellulaire » de la Société Française de Biochimie et Biologie Moléculaire, 27-29 mars 2007, Seillac, France. Daury L., Leloup L., Mazères G., Dedieu S., Cottin P., Brustis JJ. (2007) Implication de la Protéolyse calciumDépendante lors de l'adhésion et de la migration des cellules myogéniques 5ème Colloque du groupe thématique « Protéolyse Cellulaire » de la Société Française de Biochimie et Biologie Moléculaire, 27-29 Mars 2007, Seillac, France. Jean-François, F., Elezgaray, J., Castano, S., Roux, M., Metz-boutigue, M. H., Vacher, P., and Dufourc, E. J. (2007) Antimicrobial Cateslytin promotes rigid domains fomation when interacting as beta-sheets on negative membranes. In SAFO Workshop, Affcihe, Pessac, France Jean-François, F., Elezgaray, J., Castano, S., Roux, M., Metz-boutigue, M. H., Vacher, P., and Dufourc, E. J. (2007) Antimicrobial Cateslytin promotes rigid domains fomation when interacting as beta-sheets on negative membranes. In EBSA congress, Affiche, London, UK Elezgaray, J., Jean-Francois, F., Vacher, P., Berson, P., and Dufourc, E. J. (2007) Electroporation as an alternative action mechanism of antimicrobial peptides. In Biophysical Society Conference pp. 612A-612A, Poster presentation, Baltimore, USA Odaert, B., Bressollier, P., Urdaci, M. Dufourc, E.J. (2007) Liquid state NMR study of an antimicrobial peptide from the lantibiotic family. Workshop on NMR and soft matter, Arcachon, France Odaert, B., Bressollier, P., Urdaci, M. Dufourc, E.J. (2007) Liquid state NMR study of an antimicrobial peptide from the lantibiotic family. Journée Scientifique de l'IFR 66 « Pathologies Infectieuses et Cancers” Pessac, France Buchoux, S., Garnier, M., and Dufourc, E. J. (2007) A new membrane destabilization mechanism: electrostatic repulsion promoted by lipopeptide Surfactin. In 6th EBSA & British Biophysical Society Congress, Affiche, Imperial College London, U.K. Jean-François, F., Elezgaray, J., Castano, S., Roux, M., Metz-boutigue, M. H., Vacher, P., and Dufourc, E. J. (2007) Antimicrobial Cateslytin promotes rigid domains fomation when interacting as beta-sheets on negative membranes. In SAFO Workshop, Affcihe, Pessac, France Jean-François, F., Elezgaray, J., Castano, S., Roux, M., Metz-boutigue, M. H., Vacher, P., and Dufourc, E. J. (2007) Antimicrobial Cateslytin promotes rigid domains fomation when interacting as beta-sheets on negative membranes. In EBSA congress, Affiche, London, UK

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(2008) Surfactin triggered small vesicle formation of negatively charged membranes. A novel membrane lysis mechanism. In Congrès du GEIMM 13 p. Poster presentation, Colmar, France Oosting P.; Ghosez L.; Laguerre M.; Dessolin J. (2008) Discrimination of µ-receptor ligands by in silico docking : toward the identification of new pain treatment, Two Days with GPCRs, La Grande-Motte (France). Ventimila N.; Dupont P.-Y.; Laguerre M.; Dessolin J. (2008), Database virtual screening of protein kinases: GSK-3β a case study, 17èmes Conférences Européennes du Groupement des Pharmacochimistes de l'Arc Atlantique-22èmes Journées Franco-Belges de Pharmacochimie, Caen (France) J. Elezgaray, F. Jean-François, P. Vacher, P. Berson and E. Dufourc, (2008), Electroporation as an alternative action mechanism of antimicrobial peptides. Poster présenté à ISQBP, Ascona, 8-15 juin 2008. J. Elezgaray, T. Roland, L. Berguiga, A. Arneodo and F. Argoul, (2008), High resolution surface plasmon scanning microscopy: imaging nano-colloids and single biomolecules, Nanoswec 3-5 novembre 2008, Pessac. Debecker, D.P., Meyre, M.E., Gaigneaux, E.M. and Faure C. (2008) An innovative preparation of heterogeneous metal nanoparticles catalysts for VOC abatement: the onion-type multilamellar vesicles route. In 5th International Conference on Environmental Catalysis, Belfast, Ireland Meyre, M.E., Raffard, G., Franconi, J.M., Clérac, R., Mornet, S., Duguet, E. and Faure, C. (2008) Vésicules multilamellaires magnétiques. In Club Nanomatériaux pour les Sciences du Vivant, vectorisation de molécules actives et ciblage biologique, Bordeaux, France Gargouri, M., Maugé, C., Granier, T., Langlois d’Estaintot, B., Manigand, C., Bathany, K., Schmitter, J.M., Chaudière, J. and Gallois, B. (2008), Crystal Structure and Functional Properties of Anthocyanidin Reductase. XXIVth International Conference on Polyphenols. 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VIIIth International Symposium on Grapevine Physiology and Biotechnology. Adelaïde, Australie. Trabelsi, N., Petit, P., Langlois d’Estaintot, B., Manigand, C., Granier, T., Mliki, A. and Gallois, B. (2008). Structural and spectroscopic evidences of the dihydroflavonol 4-reductase inhibition by flavonols. XIXèmes Journées de l’Association Tunisienne des Sciences Biologiques. Hammamet. Tunisie. Fischer L, Briand JP, Didierjean C, Guichard G. (2008) Control of duplex formation and columnar self-assembly with heterogeneous amide/urea macrocycles. Poster, 30th European Peptide Symposium (EPS), Helsinki, Finlande, France. P. Claudon, A. Violette, K. Lamour, S. Fournel, B. Heurtault, J. Godet, B. Jamart-Grégoire, MC. Averlant-Petit, JP Briand, G. Duportail, H. Monteil, and G. Guichard (2008). Fraternal twins ! γ-peptides and oligoureas are isosteric isostructural foldamers with yet disctinct biomolecular recognition properties. 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Congrès de l’Association Française de Cristallographie, poster, Rennes, October. Marshall, T; Baptiste, B; Laxmi-Reddy, K; Shirude, P.S; Gilles, E.R.; Léger, J-M; Godde, F; Balasubramanian, S; Huc, I; (2008) Designing Aromatic Amide Foldamer Ligands for Nucleic Acid Recognition. Poster Presentation at Chemistry & Biology of Membranes & Nanoobjects (CBMN) Workshop, IECB, Pessac, France Marshall, T; Huc, I; Godde, F (2008) A New Adenine Receptor: Design and Synthesis. Poster Presentation at the Journee Jeunes Chercheurs, IECB, Pessac, France Lambert O., Bonnafous P., Le Bihan O., Pecheur E. Visualizing the molecular details of HCV membrane fusion by cryo-electron microscopy. Poster présenté au 15th International Symposium on Hepatitis C Virus and Related Viruses, San Antonio, TX, 5-9 octobre 2008. Taveau J.C., Nguyen D., Perro A., Ravaine S., Duguet E, Lambert O. 3D cryo-electron tomography of nucleated and growing PS nodules on silica nanoparticles. 1st Conference on 3D-Imaging ofMaterials and Systems Acquisition - Analysis – Applications. 8-12 Septembre 2008 Carcans-Maubuisson (Bordeaux). Perrault M., Lambert O., Aventin S., Bonnafous P., Malbouyres M., Ruggiero F., Pecheur E. Unraveling the molecular details of the entry of the hepatitis C virus into its host cells : biochemical analysis and (cryo)-TEM imaging. 7ième Congrés international de l’ELSO Nice 30 aout -2 septembre 2008. Tamoto, R., Aimé, C., Oda, R., Kinetic formation of micrometric helical structure with self-assembled nucleoamphiphiles in aqueous solution Surfactant in Solution Berlin, Germany, Aug. 2008 Schmitter, J.M., Bressollier, P., Odaert, B., Urdaci, M. (2008) Structure Elucidation of Clausin A, a Novel Lantibiotic Produced by Bacillus clausii Probiotic Strain 56th American Society for Mass Spectrometry Conference, Denver USA. Xuereb, F., Schmitter, J.M., Chaignepain, S., M.C. Saux, M.C. Breilh, D. (2008) La spectrométrie de masse appliquée à l’optimisation des traitements par epoetin beta de l’anémie de patients atteints de tumeurs malignes non myéloïdes. Congrès de la Société Française de Pharmacie clinique, Saint-Malo. Bathany, K., Marchal, S., Verdier, J.M. Nazabal, A., Schmitter, J.M. (2008) Caractérisation de formes fibrillaires de protéines par échange isotopique hydrogène/deutérium analysé par spectrométrie de masse (HXMS) : les limites mises en évidence dans le cas de la lithostathine. Congrès de la Société Française de Spectrométrie de Masse, Grenoble. Bure, C., Schmitter, J.M. (2008) Comparaison de méthodes d’enrichissement de phosphopeptides en vue de leur analyse par spectrométrie de masse. Congrès de la Société Française d’Electrophorèse et d’Analyse Protéomique, Tours. Vega-Gonzalez, C. Domingo, J-M. Adamson, S. Kazarian, P. Subra-Paternault, Microfibres de polymers produites par CO2 comprimé, Congrès francais des Polymères GFP 2008, Lyon, 25-27 novembre 2008 S. Diankov, P. Subra-Paternault, Imprégnation assistée par fluides supercritiques, Congrès francais des Polymères GFP 2008, Lyon, 25-27 novembre 2008 Massias, B., M. Arturo-Schaan, A.M. Elie, G. Rocaboy, M. Denayrolles, M.C. Urdaci (2008) An experimental approach to determine a putative pathogen of turkey hen non specific enteritis associate pathology. XXIII World’s Poultry Congress, Brisbane, Australie. Massias B., Urdaci MC. (2008) CSbyDG : a new clone screening strategy -Application to the exploration of chicken gut microbiota. 6th Joint Symposium INRA-RRI. Gut Microbiome: Functionality, Interaction with the host and impact on the environment. Clermont-Ferrand, France. Clair G, Buré C, Schmitter JM, Urdaci MC, Denayrolles M. (2008) Proteome variation of Vibrio anguillarum bacterium in culturable and viable but non-culturable states. 25ème Congrès de la Société Françcaise d’electrophorèse et d’Analyse Protéomique. SFEAP, Tours, France. Schmitter JM, Bressollier P, Odaert B, Urdaci MC. (2008) Structure Elucidation of Clausin A, a Novel Lantibiotic Produced by Bacillus clausii Probiotic Strain. the 56th ASMS Conference on Mass Spectrometry. USA. K. Gionnet, S. Nikitenko, A.L. Morel, G.Déléris (2008) Sonochemical approach to the synthesis of selfassembled monolayer nanoparticules for angiogenesis imaging - Rencontres internationales de chimie thérapeutique – Angers – France. S. Lavielle, K. Gionnet, R. Ortega, G. 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Iffiú-Soltész Z, Turpin D, Schaak S, Wanecq E, Prévot D, Atgié C., Carpéné C. (2009) Influenca de la tiramina en administration cronica sobre el desarrollo del tejido adiposo y la torerencia a la glucosa en rodeores normoglicemicos. Juin 2009 : XI Congrés National de la Société Espagnole de Nutrition, Sitges. 138. E. Vaique, S. Pinet, L. Couëdelo, C. Boue-Vaysse, L. Fonseca, N. Combe, M. Cansell. (2009) Synthesis of structured triacylglycerols rich in alpha linolenic acid for the study of their metabolic fate in rats. 7th euro fed lipid congress, gratz (autriche), 18-21 octobre 2009. 139. Dargelos E., Brulé C., Cottin P., Poussard S. (2009) Oxidative stress consequences on age-related loss of muscle mass could be mediated by calcium-dependent proteolysis. Gordon Research Conference, Oxidative Stress & Disease Role of Oxidative Stress in Aging and Age-Related Disease, March 8-13, 2009, Lucca (Barga), Italy. 140. S. Castano, B. Delors, A. Février, J-M Lehn, P Lehn and B. Desbat (2009) Assymetric lipid bilayer stabilized by DNA at the air/water interface,16ième congrès du GFPP, 10-15 mai 2009, Albévillé, France 141. F., Géan J., Lecomte S., Oda R., Kauffmann B. and C. Cullin. (2009) Driving amyloid toxicity in a yeast model by structural changes: a molecular approach. Jacques Monod Conferences “Protein folds in infectious and neurodegenerative diseases », Aussois, France  142. Bijani, C., Degert, C., Broussaud, O., Dufourc, E.J. (2009) “Lipoaminoacids as drug carriers”, 7th EBSA International Biophysics Congress Poster presentation, Genoa, Italy. 143. Toupe J, Bressolier P, Schmitter J.M., Urdaci M, Dufourc E.J., Odaert B. (2009) “Structural and dynamical study of a new antimicrobial peptide of the lantibiotic family”, 7th EBSA International Biophysics Congress Poster presentation, Genoa, Italy. 144. Buchoux, S. and Dufourc, E. J. (2009) "Electrostatic wedge": a new membrane destabilization model. Poster presentation. XXI° GERM congress, Fréjus, France. 145. Toupe J, Bressolier P, Schmitter J.M., Urdaci M, Dufourc E.J., Odaert B. (2009) Etude structurale et dynamique d'un nouveau peptide antimicrobien de la famille des lantibiotiques. Congress GFPP, Poster presentation, Albé, France. 146. Cala O., Fabre S., Fouquet E., Dufourc E., Pianet I. (2009) Approche Moléculaire de l’Astringence par l’études des interactions entre Tannins et protéines de la salive, présentation Orale, Journée ISM , Bordeaux, France. 147. Debecker, D.P., Meyre, M.E., Gaigneaux, E.M. and Faure C. (2009) A novel route involving organic vesicles for the preparation of supported silver nanoparticle catalysts. In EuropaCat, Salamanque, Espagne 148. Gargoury, M., Chaudière, J., Manigand, C., Maugé, C., Schmitter, J.M. and Gallois, B. (2009). The epimerase activity of anthocyanidin reductase and its regospecific hydride transfers. Congrès Annuel de la Société Française de Biochimie et Biologie moléculaire. Nancy. France. 149. Gargouri, M., Gallois, B. and Chaudière, J. (2009) Binding-equilibrium and kinetic studies of anthocyanidin reductase. Congrès Annuel de la Société Française de Biochimie et Biologie moléculaire. Nancy. France. 150. Maugé, C., Granier, T., Chaudière, J., Langlois d’Estaintot, B., Manigand ; C. and Gallois, B. (2009). Structural and mechanistic properties of grape leucoanthocyanidin reductase. XXVth European Crystallographic Meeting. Istanbul. Turquie. 151. Beyrath, J., Lechner, M. C. Morizot, A., Pardin C., Micheau, O., Fournel, S., Guichard G. (2009) Synthèse et développement de biomimétiques de la molécule pro-apoptotique « TRAIL » pour de nouvelles stratégies anticancéreuses. Poster, 16ème Réunion du Groupe Français des Peptides et Protéines (GFPP) , Dinard, France. 152. Lechner, M. C. Beyrath, J., Fournel, S. Guichard, G. (2009) Synthèse et développement de sondes fluorescentes et biotinylées pour l’étude du mécanisme d’action de molécules multimériques immunostimulantes.(2009) Poster, 16ème Réunion du Groupe Français des Peptides et Protéines (GFPP) , Dinard, France. 153. L. Fischer, P. Claudon, N. Pendem, J.P. Briand, C. Didierjean, E. Ennifar, G. Guichard. (2009) The Canonical Helix of N,N ’-linked Oligourea Foldamers at Atomic Resolution. Poster, 16ème Réunion du Groupe Français des Peptides et Protéines (GFPP) , Dinard, France. 154. P. Claudon, A. Violette, K. Lamour, S. Fournel, B. Heurtault, J. Godet, B. Jamart-Grégoire, MC. Averlant-Petit, JP Briand, G. Duportail, H. Monteil, and G. Guichard. (2009) Isostructural modifications of antibacterial foldamers ; toward an activity modulation using amide/urea hybrids. Poster, 16ème Réunion du Groupe Français des Peptides et Protéines (GFPP) , Dinard, France. 155. B. Baptiste, I. Huc, Debasish Haldar, J.-M. Léger (2009). Hybridization of long pyridine-dicarboxamide oligomers into double helices: solid state structures, slow strand association and dissociation, and solvent dependence. Journée jeunes chercheurs, IECB, poster, Pessac, May. 156. J.M. Schmitter, F. Xuereb, S. Chaignepain, F. Godde, D. Breilh, M.C. Saux, C. Lenz, M. Glückmann (2009) A widely applicable methodology for quantitative analysis of therapeutic proteins in human plasma. In American Society of Mass Spectroscopy, poster, Philadelphie, USA 157. Ferrand, Y., Kendhale, A., Kauffmann, B., Marie, C., Blot, V., Dubreuil, D., Huc, I. (2009) Helically folded capsules based on aromatic oligoamide foldamers. In ISMSC Conference. Poster presentation, Maastricht, The Netherlands 158. Bonnafous P., Perrault M., Le Bihan O., Bartosch B., Cosset F-L, Penin F Lambert O. Pecheur E (2009) Molecular characterization of HCV pseudoparticles by cryo-electron microscopy using functionalized magnetic nanobeads. 11 ième Colloque de la société française des microscopies (Sfµ) Paris 23-26 juin 2009 151

152

159. 160. 161. 162. 163. 164. 165.

166. 167. 168. 169.

Taveau J.C., Desert A.,Nguyen D., Perro A., Ravaine S., Duguet E, Lambert O. (2009) 3D cryo-electron tomography of complex architectures. 11 ième Colloque de la société française des microscopies (Sfµ) Paris 2326 juin 2009. Raoux, M., S. Chevallier, G. Charpentier, and J. Lang, (2009) Recording of electrical activity of pancreatic betacells by micro-electrode arrays, in 9e Colloque de la Société des Neurosciences. 2009: Bordeaux, France. Papin, J., B. Roger, P. Vacher, A. Mulot, G. Charpentier, F. Pattou, B. Vandewalle, J.C. Jonas, N. Moustaid Moussa, and J. Lang, (2009) Adenylyl cyclase 8 is central to GLP-1 signaling and glucotoxicity in pancreatic cells, in EASD. 2009: Vienna, Austria. Schmitter, J.M., Xuereb, F., Chaignepain, S., Godde, F., Breilh, D., Saux, M.C., Lenz, C., Glueckmann, M. (2009) A Widely Applicable Methodology for Quantitative analysis of Therapeutic Proteins in Human Plasma. 57th American Society for Mass Spectrometry Conference, Philadelphia, USA. T. Fadli, A. Erriguible, S. Laugier, P. Subra-Paternault, (2009) Simulation of heat and mass transfer of a CO2 binary mixture, in miscible conditions of a supercitical antisolvent process Proceedings of 9th International Symposium on Supercritical Fluids May 18-20, 2009, Arcachon ISBN Roy, A. Vega-Gonzalez, C. Domingo, P. Subra-Paternault, (2009) Surface modification of particles assisted by CO2, Proceedings of 9th International Symposium on Supercritical Fluids May 18-20, 2009, Arcachon ISBN Garcia-Gonzalez, A. Lopez-Periago, A. Vega-Gonzalez, P. Subra-Paternault, C. Domingo, C. Roy, A. VegaGonzalez, C. Domingo, P. Subra-Paternault, (2009) Fibrous biopolymer composites obtained using a supercritical antisolvent process, Proceedings of 9th International Symposium on Supercritical Fluids May 1820, 2009, Arcachon ISBN S. Marre, P. Subra-Paternault, C. Aymonier, E. Mignard, (2009) High Pressure High Temperature Microfluidics : a new tool for studying supercritical fluids Proceedings of 9th International Symposium on Supercritical Fluids May 18-20, 2009, Arcachon ISBN N. Saad, M. Urdaci, C. Vignoles, S. Chaignepain, R. Tallon, JM. Schmitter, P. Bressollier (2009) Lactobacillus plantarum 299v cell surface proteins: a new insight into the origin of cell wall associated GAPD. FEMS, Copenhagen, Denmark. Saad N, MC. Urdaci, G. Bigaud, P. Krausz, JM. Schmitter, P. Bressollier (2009) Cell wall-associated lipoteichoic acids are involved in Lactobacillus plantarum 299v adhesion to mucin and Caco-2 cells. CBL, Toulouse, France. Urdaci MC. (2009). Meeting editorial broad “Probiotics and antimicrobial proteins”. International AMP2 2009. St Malo, France.

2008 12. 13. 14.

15.

2009 16. 17. 18.

19. 20. 21.

Book or Book chapters (OS) 22.

2006 1. 2.

Dufourc, E. J. (2006) Solid state NMR in biomembranes, in Chemical Biology (Larijani, B., Woscholski, R., and Rosser, C. A., Eds.), pp 113-131, J. Wiley & Sons Ltd., London. Laguerre, M. (2006) Molecular Dynamics. , in Chemical Biology, Techniques and Applications (B.Larijani, R. W. C. A. R., J.Wiley & Sons, Ltd, Ed.), pp p. 133-150.

4. 5. 6. 7. 8. 9. 10. 11.

Atgié, C., and Ferrand, C. (2009) Additifs et auxiliaires de fabrication dans les industries agro-alimentaires, in Sciences et Technologies Agro-alimentaires (Lavoisier, Ed.), Tec.et Doc. Les colorants alimentaires In Press, Paris. Dufourc, E. J. (2009) NMR for Lipids and Biomembranes, in Wiley Encyclopedia of Chemical Biology (DOI10.1002/9780470048672wecb389p, Ed.), John Wiley & Sons, Inc.,In Press, Chichester, England Garnier-Lhomme, M., Dufourc, E. J., Larijani, B., and Poccia, D. L. (2009) Lipid quantification and structure determination of nuclear envelope precursor membranes in the sea urchin., in Methods in Molecular Biology : Lipid Signaling Protocols (Larijani, B., and Woscholski, R., Eds.), pp 89-110, Humana Press (Springer), Totowa, USA. Grelard, A., Couvreux, A., Loudet, C., and Dufourc, E. J. (2009) Solution and solid state NMR of lipids, in Methods in Molecular Biology : Lipid Signaling Protocols (Larijani, B., and Woscholski, R., Eds.), pp 111-133, Humana Press (Springer), Totowa, USA. Sauvant, P., Graulet, B., Martin, B., Grolier, P., and Azaïs-Braesco, V. (2009. ) Vitamins A, Nutritional significance in Encyclopedia of Dairy Sciences (Second Edition, A. P., Ed.), London. In Press. Plet, B., and Schmitter, J. M. (2009) Wine astringency approached by energy resolved mass spectrometry. , in Practical aspects of ion trap mass spectrometry (R. March, J.E. Todd, editors, and John Wiley & Sons, I., Eds.), Chichester, England. Guillouard, I., Grossiord, B., and Maguin, E. (2009) Génétique des Bactéries Lactiques, in Bactérie Lactique (editors;Economica, Ed.), D. Drider and H. Prévost.

Edition of Books (DO) 2006 - 2009 1.

2007 3.

Khedher, I. B. A., Bressollier, P., Urdaci, M. C., Limam, F., and Marzouki, M. N. (2008) Production and biochemical characterization of Sclerotinia sclerotiorum alpha-amylase ScAmy(1): Assay in starch liquefaction treatments, in Journal of Food Biochemistry, pp 597-614. Watrin, M., Dausse, E., Lebars, I., Rayner, B., Bugaut, A., and Toulme, J. J. (2008) "Aptamers targeting RNA molecules", in Methods in Molecular Biology, Human Press, Mayer. Hao, J., Raoux, M., Azorin, N., Giamarchi, A., Rodat-Despoix, L., Maingret, F., Coste, B., Crest, M., and Delmas, P. (2008) Mechanosensitive cation currents and their molecular counterparts in mammalian sensory neurons, in Mechanosensitivity in Cells and Tissues : Mechanosensitivity of Nervous Systems (Kamkin, A., and Kiseleva, I., Eds.), pp 51-67, Springer Verlag, London, UK. Vergne, S., Bennetau, C., and Sauvant, P. (2008) "Les phyto-oestrogènes et plus particulièrement les isoflavones de soja dans le traitement de la ménopause" in "Les plantes chez la femme ménopausée : Une alternative aux traitements hormonaux de substitution". in Société Française d'Ethnopharmacologie (SFE) (Eds., p.-. Ed.).

Agnès, P., Girard-Egrot, L., Blum, J., Richter, R. P., and Brisson, A. R. (2007) Assemblages protéo-lipidiques et nanostructures biomimétiques, in Les Nanosciences : Nanobiotechnologies et nanobiologie (Belin, Ed.). Calderon-Leal, F., Schmitt, V., and Bibette, J. (2007) Emulsion Science. Basic Principles. 2nd version, in ISBN (Springer, Ed.), 978-0-387-39682-8. Cansell, F. (2007) Use of egg compounds for cosmetics and pharmaceutics.In "Bioactive Egg Compounds", in ISBN, Springer-Verlag, ISBN : 978-3-540-37883-9, ch29, 249-258. Castano, S. (2007) Spectroscopie Raman, Biophysique pour les sciences de la vie et de la santé, in Omniscience. Laguerre, M. (2007) Modélisation moléculaire et conception nouvelles molécules in Bulletin du Cancer Le Grel, P., and Guichard, G. (2007) Foldamers based on remote instrastand interactions In Foldamers, in WileyVCH (Huc, I. a. H., S. Eds Wiley-VCH. Verlag. :Weinheim, Ed.), pp pp.35-74. Huc, I. (2007) Foldamers based on local conbformational preferences. In Foldamers : Structure, Properties and applications, in Wyley-VCH (Hecht, S., and Huc, I., Eds.), pp 35-74, Weinhein. Rigalleau, V., Lang, J., and Gin, H. (2007) Etiologie et physiopathologie du diabète de type 2, in Encyclopédie Médico-chirurgicale [10-366-D-10]. Subra-Paternault, P., Vega-Gonzalez, A., and Roy, C. (2007) Encapsulation assistée par fluides supercritiques, , in Microencapsulation (Lavoisier, Ed.), Coodinateurs: Vandamme, Poncelet, Subra.

2. 3.

Ebel, C., and Dufourc, E. J. , eds (2007) Proceedings of the joint biannual meeting of the "Societe Francaise de Biophysique" (SFB), "Groupe d'Etudes des Interactions Molecules-Membranes" (GEIMM), "Groupe de Recherche en Ingenierie des proteines" (GRIP), Anglet France, 14-19 October 2006, Eur Biophys J. Vol. 36, Springer, London. Hecht, S., and Huc, I., eds (2007) Foldamers: Structure, Properties, and applications. Wyley-VCH, Weinheim Vandamme, Poncelet, Subra, Edt (2007) “ Microencapsulation : des sciences aux technologies’, Coordinateurs:. Lavoisier TEC&DOC, 2007, ISBN 978-2-7430-0976-2 (349p)

Congress, workshops organization 2006 1. 2. 3. 4. 5. 6.

153

4th World Congress on Emulsions, Lyon, France (3-6 Oct.1000 participants), F. Leal-Calderon, member of the Scientific Committee. Congrès de Biophysique, Biarritz, (150 participants, E. Dufourc, vice-president). 11th International Conference on Electroanalysis, Bordeaux (900 participants), C. Faure, member organizing committee 11ème RECOB , Aussois, France, (~120 delegates) G.Guichard, member organizing committee. 6th Club Exo-Endocytose (120 persons), Anglet,. J Lang President 1st Jointed Fukuoka-Bordeaux Conference ― New Trends in Molecular and Materials Sciences, (100 participants), R. Oda Partial organization 154

10.

2007 7. 8. 9. 10. 11. 12.

NMR & Soft Matter, workshop, Arcachon, France, E. Dufourc President, team in organizing committee. 6th EBSA Congress, London, UK, (1100 participants), E. Dufourc, President. 15ème Réunion GFPP, Dinard, France, (~160 delegates) G.Guichard, member organizing committee. 1st Aquitaine Conference on Polymers, Arcachon, France, (130 participants) I. Huc, member of organizing committee. Japan France Frontier of Science, Shonan, Japan, (240 participants), R. Oda Partial organization Systèmes Avancés à base de Fluides Organisés (SAFO) – Workshop, Bordeaux, (150 participants) R. Oda Partial organization

11. 12.

Theses, PhD & HDR

2008 13. 14. 15. 16.

2006

GEIMM 13 congress, Colmar France (200 participants) E. Dufourc, member organizing committee. FAME Congress, Alméria, Espagne, (150 participants), C. Faure, member organizing committee 12ème Rencontres en Chimie Organique Biologique (RECOB), Aussois, France, (~120 delegates) G.Guichard, member organizing committee. 1st International Workshop on Spectral Imaging Techniques with Synchrotron Radiation. Beijing, China,– C. Petibois Scientific Board

1. 2. 3.

2009 17. 18. 19. 20. 21. 22.

4.

Annexin2009, 5th International Conference on Annexins, Lacanau-Océan. A Brisson President 7th EBSA Congress, Genoa, Italy, (900 participants), E. Dufourc, Past-President. 16ème Réunion du Groupe Français des Peptides et Protéines (GFPP), Villé, France, (~200 delegates) G.Guichard, GFPP president. 2nd Aquitaine Conference on Polymers, Arcachon, France, 130 participants, I. Huc, member of organizing committee. 2nd International Workshop on Spectral Imaging Techniques with Synchrotron Radiation. Sania, China, C. Petibois Co-Chair and Scientific Board 9th International Symposium on Supercritical Fluids, Arcachon, 350 participants, P. Subra, committee member.

5. 6. 7.

2010 23. 24. 25. 26.

8.

th

2010: 5 World Congress on Emulsions, Lyon, France (12-14 Oct, 1000 participants), F. Leal-Calderon, member of the Scientific Committee. 2010: 17th GERLI congress, Biarritz, France (200 participants), E. Dufourc President, team in organizing committee. 2010: 1st EBSA course in Biophysics, Arcachon, France, 100 participants, E. Dufourc President, team in organizing committee. 2010: COST Workshp on Foldamers, Bordeaux, France (100 participants), I. Huc President.

9. 10. 11. 12.

Patents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Saulnier P, Benoît JP, Passirani C, Vonarbourg A, Lambert O, Pitard B. Nanocapsules of lipophilic complexes of nucleic acids, International patent. PCT/IB2008/050431, 2007 Bressollier, P., Brugo, M.A., Robineau, P., Schmitter, J.M., Sofier, M., Urdaci, M.C., Verneuil, B. Peptide compound with biological activity, its preparation and its application. PCT Int. Appl., 2007, 13pp, PIXXD2 WO 2007113691 A2 Urdaci MC, Bressolier P, Schmitter JM, Robineau P, Sofier M, Brugo A. Composé peptidique à activité biologique, sa préparation et ses applications. N° IT : MI2006A 000678. PCT in 2007. (brevet SANOFIAVENTIS).

Nick Buzhynskyy, 28 Juin 2006. Etude de la formation d’assemblages protéo-lipidiques sur supports solides, par Microscopie à Force Atomique et Microbalance à Cristal de Quartz et mesure de Dissipation. In école doctorale des Sciences du Vivant, Université Bordeaux 1&2, Bordeaux, France Vaique E. (2006, Oct.) Synthèse de triglycérides structurés et/ou fluorescents pour l’étude du métabolisme lipidique. In école doctorale des Sciences chimiques, Université Bordeaux 1, Bordeaux, France Drelon, N. (defended on Nov, 13, 2006) Elaboration et consolidation thermique de gels à base de substances émulsionnées cristallisables. In école doctorale des Sciences de la vie, Géociences, Sciences de l’Environnement, Université Bordeaux 1, Bordeaux, France Loudet, C. (2006, Octobre) Les bicelles Biphényles : un nouveau modèle de biomembrane pour l'étude pour l'étude de protéines membranires par RMN des solides. In Ecole doctorale des sciences chimiques p. 309, Université Bordeaux 1, Bordeaux, France Violette A. (2006, februrary) Hélices Biomimétiques antimicrobiennes à base d’oligomères d’urées. In Ecole Doctorale des Sciences Chimiques, Université Louis Pasteur, Strasbourg, France. Léna G. (2006, janvier) Développement d’une nouvelle plate-forme hétérocyclique dérivée de dipeptide : la 1,3,5-triazépan-2,6-dione. In Ecole Doctorale des Sciences Chimiques, Université Louis Pasteur, Strasbourg, France. de Hatten, X. (Jun 2006) Hydrogenase mimics. In Ecole doctorale des sciences chimiques, Université Bordeaux 1, Bordeaux, France, jointly with the Department of Pharmacy, University of Heidelberg (Prof. N. MetzlerNolte). Fréderic Boal (Mai 2006) In école doctorale des Sciences du Vivant, Université Bordeaux 1&2, Bordeaux, France Florence Grise (Dec 2006) In école doctorale des Sciences du Vivant, Université Bordeaux 1&2, Bordeaux, France Bouzaine, T. (2006, Décembre) Recherche, caractérisation et sélection de souches lactiques probiotiques pour l'aviculture. In the University of Tunis. Mata, JA. (2006, Juin) Caractérisation et analyse fonctionnelle des exopolysaccharides produits par 4 nouvelles espèces d'Halomonas. In the University of Granada. Bressollier P. (2006), Habilitation à diriger les recherches, Université de Limoges. 2007

Stéphane Mornet, Alain Brisson. Functionalization of gold nanoparticles with oriented proteins. Application to the high-density labeling of cell membranes. WO2007122259 C. Romero, B. Bazin, F. Leal Calderon, Méthode de traitement des puits pétroliers par émulsions de petite taille contenant des additifs, Institut Français du Pétrole, WO 2007093708. Deleris, G.; Rubio-Albenque, S.; Bennetau, B.; Desbat, B.; Buffiere, F.; Chagnaud, J. L.Procédé de préparation d’un support pour l’immobilisation d’une cellule, le dit support et ses utilisations, déposé sous le N° 07 54424, Université Victor Segalen Bordeaux 2, CNRS. J. Dessolin, G. Depierre & M. Laguerre, "Composés de type 1,4-naphtoquinones, compositions les comprenant et utilisation de ces composés en tant qu’agents anti-cancéreux". Brevet FLUOFARMA-CNRS-Université Bordeaux I, French Patent n° 06/51461 (25 april 2006). International extension PCT/FR 2007/000703 (25 avril 2007). Inventors : Dessolin J., Commandeur C., Depierre G., Laguerre M. G. Guichard, S. Fournel, J., Wieckoswki, Trouche N. Nouveaux Ligands de CD40, Leur Procédé de Préparation et leur utilisation pour la préparation de Médicaments. Brevet Français : 07/00809, déposant CNRS 15/02/2007 J. Courty, A. Hovanessian, J.P. Briand, G. Guichard, Y. Hamma. Pseudopeptide HB-19 et traitement du cancer. Brevet Fraçais : 06/03813, déposant CNRS, 27/04/2006 G. Léna, P. Muller, E. Boilard, D. Rognan, G. Lambeau, G. Guichard, Compositions And Methods For The Inhibition of phospholipase A2, Provisional US60/755,626 déposant ImmuPharma 18/01/2006 G. Léna, L. Rénia, E. Lallemand, G. Guichard, Compositions and methods for the treatment and prevention of disease - Provisional US60/755,631 : déposant ImmuPharma : 19/01/2006

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Rémi Bérat, 21 Novembre 2007. Assemblages 2D de l’Annexine A5 : Applications biotechnologiques et Aspects fonctionnels. In école doctorale des Sciences du Vivant, Université Bordeaux 1&2, Bordeaux, France Villatte, C. (2007, Oct.) Etude des mécanismes de relargage de principes actifs nanodispersés : application au traitement de puits. In école doctorale des Sciences chimiques, Université Bordeaux 1, Bordeaux, France Banc, A. (2007, Novembre) Approches biomimétiques de l'assemblage de protéines de réserve de blé, Ecole doctorale des sciences chimiques, Université Bordeaux 1, Bordeaux, France. Garnier-Lhomme, M. (2007, Octobre) Characterization of Membrane Domains in Nuclear Envelope Assembly: Composition, Structure and Dynamics of Nuclear Envelope Remnants. In Ecole doctorale des sciences chimiques & Biochemistry school of graduate studies p. 232, Université Bordeaux 1 & Imperial College of London, Bordeaux & London, France & UK Nada Taïb (2007, novembre). Etude par dynamique moléculaire de protéines membranaires dans un milieu lipidique cohérent. In Ecole doctorale des sciences chimiques p. 308. Université Bordeaux 1, Bordeaux, France. Meyre, ME (2007, Décembre) Incorporation de nanoparticules inorganiques dans des vésicules multilamellaires lipidiques de type « oignon ».. In Ecole doctorale des sciences chimiques p. 250, Université Bordeaux 1, Bordeaux, France. Petit, P. (2007, Février) Etude structurale de la dihydroflavonol 4-réductase, enzyme clé de la biosynthèse des flavonoïdes chez Vitis vinifera. p. 195, Université Bordeaux 1, Bordeaux, France. Wieckowski S. (2007, may) Synthetic CD40L mimetics : biological effects and applications. In Ecole Doctorale des Sciences de la vie et de la santé. 156

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Trouche N. (2007, februrary) Mimétisme fonctionnel des homotrimères de CD40L et TRAIL à l’aide de molécules de symétrie C3. In Ecole Doctorale des Sciences Chimiques, Université Louis Pasteur, Strasbourg, France. Delsuc, N. (Oct. 2007). Synthetic mimics of protein tertiary folded conformation. In Ecole doctorale des sciences chimiques, Université Bordeaux 1, Bordeaux, France Delclos, T. (Oct. 2007) Towards organic-inorganic nanorod hybrids. In Ecole doctorale des sciences chimiques, Université Bordeaux 1, Bordeaux, France Sabine Manet (Oct. 2007) Effet de contreions sur assemblage moléculaire, In Ecole doctorale des sciences chimiques, Université Bordeaux 1, Bordeaux, France Carole Aimé (Oct. 2007) Interaction inter-assemblage moléculaire induite par reconnaissance moléculaire entre nucléobases In Ecole doctorale des sciences chimiques, Université Bordeaux 1, Bordeaux, France Vergne Sébastien (Université de Bordeaux I, Juin 2007). Les isoflavones de soja : leur biodisponibilité chez l’Homme et leurs effets sur la différenciation d’une lignée ostéoblastique humaine. Plet B. (2007, avril) Study of the human saliva proteins/polyphenols interaction by mass spectrometry: an analytical step to understand wine astringency. Ecole doctorale des sciences chimiques p. 242, Université Bordeaux 1, France

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2009 46. 47. 48. 49.

2008 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

Ben Abdelmalek, I. (2008, Décembre) Production, Caractérisation Biochimique, Analyse Moléculaire et Protéomique d’Enzymes α-Amylases de Sclerotinia sclerotiorum. In the University of Tunis. Ho Thi Nguyet Thu (2008, Décembre) Etude de la flore lactique du Nem chua, produit carné fermenté cru traditionnel du Sud Vietnam et maîtrise du processus de fermentation par ajout de souches lactiques sélectionnées spécifiques du produit. In Ecole doctorale Université Bordeaux 1, France & Université de Ho Chi Minh, Vietnam.

50.

Bonnet, M. (defended on Nov. 26, 2008) Libération contrôlée du magnesium par des émulsions doubles: impact des paramètres de formulation. In école doctorale des Sciences chimiques, Université Bordeaux 1, Bordeaux, France Thongdeng, H. (2008, oct.) Lipides et procédé supercritique : de la valorisation de sous-produits agricoles ou aquacoles à la formulation de liposomes pour des applications nutrition et santé. In école doctorale des Sciences chimiques, Université Bordeaux 1, Bordeaux, France Couëdelo, L. (2008, Jan.) Biodisponibilité nutritionnelle de systèmes colloïdaux riches en acides gras polyinsaturés : études in vitro et in vivo. In école doctorale des Sciences chimiques, Université Bordeaux 1, Bordeaux, France Chadel, S. (2008, Nov.) Synthèse et caractérisation d’émulsion fluorées dans le cadre d’une application en tant qu’agent de contraste pour l’imagerie médicale. In école doctorale des Sciences chimiques, Université Bordeaux 1, Bordeaux, France Fezoua-Boubegtiten, Z. (2008, Octobre) Etude Physico-Chimique de l’Annexine A5 en interaction avec des membrabnes phospholipidiques modèles. Ecole Doctorale Chimie Physique et Chimie Analytique, p191, Université Pierre et Marie Curie. Yacine, W. (2008, Octobre) Etude structurale des protéines SNARES dans le complexe protéine lipide impliqué dans la fusion membranaire,Ecole doctorale des sciences chimiques, Université Bordeaux 1, Bordeaux, France. Jean-François, F. (2008, Octobre) Etude de peptides antibiotiques de la famille des cateslytines en interaction avec des membranes. In Ecole doctorale des sciences chimiques p. 200, Université Bordeaux 1, Bordeaux, France BEST PHD THESIS, GERLI PRIZE, 2009 Sani, M.-A. (2008, Novembre) Apoptosis Regulation via the Mitochondrial Pathway: Membrane Response upon Apoptotic Stimuli. In School of graduate studies Umea & Ecole doctorale des sciences chimiques Bordeaux p. 200, University Umea & Université Bordeaux 1, Umea, Sweden & Bordeaux, France Buchoux, S. (2008, Février) Vers un nouveau modèle de déstabilisation des membranes biologiques par les lipopeptides: Apport de la RMN à travers l'exemple de la surfactine. In Ecole doctorale des sciences chimiques p. 237, Université Bordeaux 1, Bordeaux, France Jean Dessolin (2008, juillet). Habilitation à Diriger des Recherches. De la synthèse organique au criblage virtuel par modélisation moléculaire. In Ecole doctorale des sciences chimiques p. 128. Université Bordeaux 1, Bordeaux, France. Bakleh M.E.(2008, Novembre) Méthodologie de synthèse pour l’élaboration de photo-sensibilisateurs sélectifs à visée anti-cancéreuse.- Université de Limoges – France. Fischer L.. (2008, may) Synthèse et propriétés de structuration et d’autoassemblage de mimes peptidiques à base d’urées et leur utilisation en reconnaissance moléculaire. In Ecole Doctorale des Sciences Chimiques, Université Louis Pasteur, Strasbourg, France. Wehbe K. (2008, November) FTIR imaging of gliomas. In Ecole doctorale des sciences chimiques p. 200, Université Bordeaux 1, Bordeaux, France Xuereb F. (2008, décembre) La spectrométrie de masse appliquée à la quantification des protéines médicaments dans le plasma. Application à l’érythropoïétine et à l’interféron. Ecole doctorale des sciences chimiques, p. 251, Université Bordeaux 1, France C. Roy, (juin 2008) ‘Generation de particules par procédé assisté par CO2 comprimé : cristallisation et formulation’, Université Paris 13 Massias B. (2008, Décembre) Développement et mise au point d’outils moléculaires pour l'identification de flores complexes bactériennes. Application à l’étude des flores digestives de galliformes. In Ecole doctorale ABIES, Paris.

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Boris Garnier (Décembre 2009). Développement de nanovecteurs fonctionnalisés par des protéines. In école doctorale des Sciences du Vivant, Université Bordeaux 1&2, Bordeaux, France Garcia Darras, C. (2009, Feb.) Formulation and characterization of colloidal systems rich in polyunsaturated fatty acids for nutritional use. In école doctorale des Sciences chimiques, Université Bordeaux 1, Bordeaux, France Habouzit, D. (2009, March) Rhéophysique, texture de surface et vieillissement du chocolat. In école doctorale des Sciences chimiques, Université Bordeaux 1, Bordeaux, France Bijani, C. (2009, Octobre) Colloides de la santé. In Ecole doctorale des sciences chimiques p. 200, Université Bordeaux 1, Bordeaux, France Zhendre, V. (2009, Décembre) Phosphoinositides. In Ecole doctorale des sciences chimiques p. 200, Université Bordeaux 1, Bordeaux, France Gargouri, M. (2009, Octobre) Biosynthèse des flavan-3-ols: Etudes structurale et fonctionnelle de l’Anthocyanidine réductase de Vitis vinifera. p. 210. Université de Bordeaux 1 et Université de Tunis. Claudon, P. (2009, november) Synthèse, structure, et propriétés d’interaction de foldamères d’urée à repliement hélicoïdal contrôlé, Ecole Doctorale des Sciences Chimiques, Université de Strasbourg, Strasbourg, France Boeglin, J.. (2009, October) Diversification du chassis 1,3,5-triazépan-2,6-dione par synthèse combinatoire – Application à la recherche d’inhibiteurs de phospholipases A2 sécrétées. In Ecole doctorale des sciences chimiques p. 200, Université Bordeaux 1, Bordeaux, France Mouli-Talbi, B. (2009, décembre) Optimisation des conditions de conservation à l'état vivant et de stockage de la crevette royale Penaus kerathurus. Etude de l'impact des bactéries probiotiques sélectionnées. In Ecole doctorale ABIES, Paris & Université de Tunis.

Research Grants : Scientist

Source

Amount

Nanocues

Project Name

Brisson A.

EU FP7

310 k€

MPAuA5 Nanobioimaging Imathabio

Brisson A. Brisson A. Brisson A. Brisson A.

ANR-EMPB ANR-TecSan ANR-PCV Projet Européen FP7 European project FP7

190 k€

Région Aquitaine

216 k€

Europe MENRES

229 k€ 66 k€

MENRES

66 k€

MENRES

11 k€

Desbat B.

Région Aquitaine

56 k€

Desbat B.

ANR Ligue contre le cancer

36 k€

ASMENA Nanother Imagerie de l’organisation des membranes biologiques NAS-SAPNMP4CT Nanosciences Organiation de jonctions adherents VE Cadhérine Biologie cellulaire moléculaire et strcuturale Etude des protéines membranaires Pseudo-peptides for transfection : Synthèse d'hétérocycles originaux ligands de kinases Structure function relationship in Rafts

157

Brisson A. Brisson A. Brisson A. Brisson A. Brisson A. Brisson A.

Dessolin J. Dufourc E.

MENRES

Lipoaminoacides/protéines

Dufourc E.

Physica Pharma

Approche moléculaire de l’astringence

Dufourc E.

CIVB

300 k€ 170 k€

Duration 02/2004 – 01/2007 12/ 2007-11/ 2009 12/ 2006-11/ 2009 01/ 2008-12/ 2011 09/2008 – 08/ 2011 11/ 2008 – 10/ 2012

6 k€ 32 k€ 27 k€ + PhD 28 k€ + PhD

02/2005-01/2007 04/2007-03/2009 09/2005-08/2007 158

NMR of Soft Matter Plateau RMN Aquitain (700+600 MHz)

Dufourc E.

CNRS

25 k€

01/2007-12/2008

Dufourc E.

Région Aquitaine

2780 k€

01/2006-12/2007

Opération 800 MHz

Dufourc E.

365 k€

01/2009-12/2009

Running NMR Platform TGIR

Dufourc E.

CNRS+Région aquitaine + UBx CNRS

Les clés moléculaires du gout

Dufourc E.

CIVB

Interaction tannins et proteines

Dufourc/Schmitter

CIVB

PhD fellowship PhD fellowship PhD fellowship PhD fellowship Post-Doc Felloship Enzymes des flavonoides Biosynthèse des anthocyanes Protéines d’un virus de plante Medicinal Chemistry

Dufourc E Dufourc E Dufourc E Dufourc E Dufourc E Gallois B. Gallois B. Gallois B. Ghosez L.

MENRES MENRES MENRES MENRES CNRS CIVB CIVB ANR Big Pharmas European Union Marie Curie IAPP ANR Blanc 2008 U Bordeaux 1

20 k€ 81 k€ + PhD 47 k€ +2PhD 60 k€ 60 k€ 60 k€ 60 k€ 50 k€ 38 k€ 42 k€ 62 k€ 400 k€

09/2005-08/2007 10/2007-09/2010 10/2006-09/2009 10/2005-09/2008 10/2004-09/2007 10/2008-09/2009

FOLDAPSULES 1 year post-doc 2 year post-doc – HELICAL TRANSPORTERS 2 year post-doc - FARQUAD

Huc I. Huc I. Huc I.

EU -Marie Curie

160 k€

03/2009-02/2011

Huc I.

160 k€

04/2009-03/2011

Foldaptamers

Huc I.

210 k€

01/2008-12/2011

2 year post-doc PhD fellowship Dynamic Combinatorial Chemistry One-Year post-doc Dynamic Self-assembly of Proteomorphous Objects Abiotic mimics of proteins Dynamic Combinatorial Chemistry Abiotic mimics of proteins

Huc I. Huc I.

80 k€ 65 k€

11/2007-10/2009 10/2007-09/2010

207 k€

09/2006-08/2010

Helical Antibiotics

Huc I.

02/2009-01/2013

225 k€ 40 k€

09/2009-08/2013 11/2008-10/2009

PhD fellowship

Huc I.

PhD fellowship Visualisation Microscopie Electronique Transfert de genes VLM7 Silaca based nanovectors Cryotomographie Jonctions Adhérentes interendothéliales TERS imagerie Raman Electromechanical nanodevices Exodynamique Fusion Membranaire : lipides proteines Protéines salive/tannins Encéphalopathies spongiformes Cancéropole Grand Sud-Ouest

Huc I. Lambert O.

ANRS

80 k€

Lambert O. Lambert O. Lambert O.

VLM AFM Région Aquitaine

66 k€ 50 k€ 108 k€

Huc I. Huc I. Huc I. Huc I. Huc I.

38 k€

11/2006-10/2007

205 k€

03/2006-12/2008

288 k€

01/2006-12/2008

7 k€

01/2005-12/2009

5 k€

01/2006-12/2006

149 k€

12/2004-06/2006

80 k€

10/2004-09/2008

60 k€

10/2004-09/2007

Lambert O.

ANR

154 k€

Lecomte S. Oda R. Oda R.

Région Aquitaine CNANO GSO ANR

262 k€ 22 k€

Oda R.

U Bx BQR

Schmitter JM Schmitter JM Schmitter JM

CIVB CEA INSERM

•

Caubet R. 9. Microbiologie alimentaire (enseignement et aussi recherche liés au monde industriel) 10. Collaboration AUF Vietnam 11. Coopération décentralisée Régions Aquitaine-Agadir

•

01/2007-12/2010

220 k€

Brisson A.

Projet Européen Nanocues 1. Université Chalmers (Suède) 2. ETH Zurich (Suisse) 3. Université Heidelberg (Allemagne) 4. Stocholm (Suède) Projet Européen NASSAP 5. Université de Londres 6. Université de Vienne (Autriche) 7. Université de Linz (Autriche) 8. Helsinki (Finlande)

07/2009-12/2009

Huc I.

Huc I.

•

09/2008-08/2010

FOLDAPPI

EU -Marie Curie ANR – PCB interface ARC MENRES EU -Marie Curie Training Network MENRES ERA-chemistry (European) ANR –blanc CNRS – funding for COST U Bx (BQR) EU -Marie Curie Post-Doc Asian Bank of development MENRES

International Activities

Dufourc E. 12. 13. 14. 15. 16. 17. 18.

•

Initiative for Science in Europe (ISE), représentant de l’EBSA, Bruxelles & Lisbonne. Président de l’EBSA European Biophysical Societies Association Organisateur Congrès EBSA, Londres 2007, Gennes, 2009. The future of Science in Europe, Lisbonne, (Assemblée UE), représentant de l’EBSA Cotutelle Thèse Université Bordeaux/Imperial College London (UK) Cotutelle Thèse Université Bordeaux/Universiy Umea (Suède) Cotutelle Thèse Université Santa Catalina, Brésil

Charpentier G. 19. Programme Alliance (Collaboration avec le Dept of Physiology, OCDEM, U Oxford, UK) sur les mécanismes moléculaires de l’exocytose

•

Ghosez L. 20. Dr G. Dive (Université de Liège), Prof. K. Houk (UCLA, USA), laboratoires de biologie et de pharmacologie des partenaires industriels 21. Président du Conseil Scientifique du CiRFC (Centre international de recherches à la frontière de la chimie) de Strasbourg

•

Huc I. 22. Collaboration Européenne financée par ERA-CHemistry avec le groupe du Dr. Jonahtan Nitschke, lecturer à l'université de Cambridge. Thème: "assemblage dynamique dirigé par les métaux de foldamères aromatiques" 23. Membre du réseau européen Marie Curie "Dynamic Combinatorial Chemistry" ( http://wwwdcc.ch.cam.ac.uk/) Membre du réseau européen COST du même nom 24. Membre du GDRE franco-russe "Suprachem" ( http://infochim.ustrasbg.fr/recherche/GDRE/SupraChem.php)

•

Lang Jochen 25. Responsable d’un programme Alliance (Collaboration avec le Dept of Physiology, OCDEM, U Oxford, UK) sur les mécanismes moléculaires de l’exocytose.

•

Odaert Benoît

•

Oda Reiko

•

Urdaci Maria

26. RUG. UNIVERSITY Pays-Bas 27. Tokyo, Riken, UNIVERSITY Japan 28. 29. 30. 31.

01/2007-12/2008 01/2007-12/2009 01/2005-12/2007

Responsable de l’action intégrée Alliance (UK) Responsable de l’action intégrée Alliance CMCU (Tunis) Responsable du programme transfortalier Navarre-Aquitaine TEXAS UNIVERSITY USA

13 k€ 22 k€ 9 k€

159

160

ANNEXE 1 HYGIENE ET SECURITE L’unité CBMN dispose de 3 ACMO répartis sur les 3 sites : Anne-Marie Elie (ENITA), Claude Manigand (Bâtiment B8) et Axelle Grélard (IECB). Comité Hygiène et Sécurité Le directeur de l’unité a mis en place en juillet 2007 un Comité Hygiène et Sécurité pour l’unité. Les représentants du personnel au sein du comité sont au nombre de 7 (3 statutaires et 1 post-doctorant/doctorant). Le comité a eu l’occasion de se réunir une fois au cours de l’année 2007 suite à l’incendie survenu dans un laboratoire de chimie à l’IECB (équipe de l’UMR) en novembre 2007.

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Comité Hygiène et Sécurité Nom Prénom ELIE Anne -Marie GRELARD Axelle MANIGAND Claude BONNAFOUS Pierre DESSOLIN Jean De HATTEN Xavier TREPOUT Sylvain DUFOURC Erick

Qualité IE, ACMO site ENITAB IE, ACMO site IECB IE, ACMO site B8 MdC CR Post-Doctorant Doctorant Directeur Médecin du travail CNRS Resp H &S DR15 CNRS Médecin du travail UBx1 Resp H & S UBx1 Médecin du travail ENITAB Resp H & S ENITAB

Accueil des nouveaux entrants L’accueil et la formation des nouveaux entrants sont mis en place au sein de l’unité. Ce parcours se présente sous différentes formes : Une fiche synthétique : le renseignement de cette « fiche nouvel entrant » permet de vérifier la couverture sociale de l’entrant, sa localisation, les autorisations d’accès au laboratoire. Elle est disponible sur l’intranet du laboratoire.

161

 

Exemple fiche nouvel entrant (page ½) Un règlement intérieur : le règlement intérieur de l’unité est disponible sur le site internet. Il est transmis systématiquement aux nouveaux entrants

162

 

Document d’Accueil : un manuel pratique d’Hygiène et Sécurité (15 pages, disponible depuis septembre 2008) commun aux 3 sites, et disponible en ligne est remis à chaque nouvel entrant.

Journée d’Accueil : une demi-journée, spécifique au fonctionnement de l’unité (mise en place en 2008) vient en complément du document d’accueil. Le directeur de l’unité, les chefs de groupes sont sollicités pour intervenir lors de cette demi- journée. Chaque nouvel entrant effectue, à l’occasion de cette journée, une visite du site qui le concerne avec son ACMO. Les personnels arrivant en cours d’année recevront une formation plus courte et centrée sur le ou les risques qu’ils rencontreront. Site Internet : création d’une page Hygiène et Sécurité sur le site web de l’Unité (mise en ligne des documents d’accueil, diffusion d’informations….)

163

 

Bilan des Actions et des Evènements survenus depuis 2008 dans l’Unité Rédaction du document unique Dans le cadre de la rédaction du document unique, la cartographie des sources de danger pour l’ensemble du bâtiment IECB a été réalisée (7 équipes CBMN localisées à l’IECB). La phase d’inventaire des risques est aujourd’hui la dernière étape avant la mise en forme du document final pour le site IECB. Réhabilitation du laboratoire incendié le 19 novembre 2007 L’aménagement du laboratoire a été modifié avec la suppression d’une paillasse basse. En revanche la séparation plus efficace labo-bureau, ainsi que le raccordement à la ventilation des armoires sous sorbonnes n’ont pu être réalisé pour des raisons techniques et des questions de coûts. L’installation de nouveaux extincteurs à poudre, de couvertures anti-feux et de seaux de sable a permis de compléter les équipements déjà présents et donc de renforcer la sécurité. Le laboratoire a pu être réinvesti en mai 2008. Formation à la manipulation des extincteurs Pour répondre à une demande des personnels de l’unité, une formation à la manipulation d’extincteurs a été organisée en Mai 2008. Cette formation théorique et pratique d’une demijournée (Société ChronoFeu) a été suivie par 24 personnels permanents et non-permanents de l’Unité. Cette formation doit être reconduite tous les 2 ans. Visite du service médical de prévention Les sites du B8 et du B13 (IECB) ont été visités par le médecin de prévention, le Docteur Christine Calas en avril et juin 2009. Le site de l’ENITAB n’a quand à lui pas été visité dans la mesure où un médecin est affecté à l’école. Ces visites ont été l’occasion d’évoquer le mode de fonctionnement des laboratoires et les consignes de sécurités mises en place. Aucun disfonctionnement grave n’a été relevé lors de ces visites. Dans ses conclusions, le Docteur Calas a pointé : -le niveau sonore élevé dans l’ensemble du bâtiment IECB et en particulier dans la salle de spectrométrie de masse -des améliorations à apporter dans la ventilation d’une pièce de stockage des produits chimiques (site IECB) -un problème de sécurisation du sous-sol du B8 Bilan des accidents et incidents survenus dans l’Unité Accidents et Incidents AT Mesures Prises Chute dans un couloir provoqué par un sol 0 humide glissant Projection d’un bout de verre dans l’œil (site 1 IECB) Inhalation de vapeur de Bromure de Propargyl (site IECB)

Personnel de ménage consulté pour modification des heures de passage Sensibilisation au port des lunettes de protection présentes dans les laboratoires. Ajout du numéro d’urgence de la Clinique Ophtalmologique à la liste de numéros présent sur les boites à pharmacie Rappels sur les bonnes pratiques de laboratoire (usage de la hotte dans de bonnes conditions, rebouchage des flacons). Le positionnement d’un rotavapor sous une hotte est à l’étude

Incendie le 19 novembre 2007 Un incendie survenu le lundi 19 novembre 2007 a nécessité l’intervention des pompiers sans toutefois engendrer de blessés. Bien que les flammes n’aient détruit qu’une hotte (4m2), le laboratoire noirci par les fumés a dû être vidé et condamné dans l’attente d’une réhabilitation. L’incident a mis en évidence l’absence de couvertures anti-feux et d’extincteurs à poudre dans 164

 

les laboratoires ainsi que l’insuffisance du nombre de seaux de sable. Tous ces défauts constatés sont en voie d’être corrigés. Par ailleurs à l’occasion de la réhabilitation du laboratoire incendié nous proposons quelques modifications dans l’aménagement : 9 Raccordement de 2 placards ventilés sous sorbonne 9 Achat d’une armoire dédiée aux hydrures 9 Suppression d’une paillasse basse 9 Séparation plus efficace labo-bureau La faisabilité de ces nouveaux aménagements doit être validée par le bureau d’étude chargé des travaux par la Division du Patrimoine et des Infrastructures de l’Université de Bordeaux1. Identification et gestion des risques dans l’Unité Risque Chimique Pour les sites IECB et B8 les déchets chimiques sont évacués régulièrement par la société SIAP. Les déchets sont stockés dans un bunker extérieur dans l’attente de l’enlèvement. Sur le site de l’ENITAB, l’évacuation est effectuée par l’entreprise « Labo-Services », contrat n° 33/2744/2006. Trois armoires ventilées ont été installées dans les laboratoires du bâtiment B8, pour le stockage des solvants et produits chimiques. Risque Radioactif Isabelle Lebars (CR CNRS) est la personne compétente en radioprotection (PCR), pour les sources non scellées, depuis décembre 2004 au titre de l’autorisation N°T330486 (Titulaire Jean-Jacques Toulmé, directeur UMS 3033). Le renouvellement de l’autorisation est demandé pour juillet 2008. Le tri et l’élimination des déchets à vie courte (32P et 35S) sont gérés en décroissance, 6 mois pour 32P et 2 ans pour 35S. L’évacuation des déchets à vie longue (3H) est assurée par la société ANDRA (convention avec Université Bordeaux1). Brice Kauffmann (IR CNRS) est PCR (UMS 3033) pour les sources scellées de rayonnements ionisants. A ce titre, il est responsable de l’organisation de l’activité liée à l’utilisation des 2 générateurs de rayons X de l’IECB (demande d’autorisation en cours). Il forme et encadre les personnels de l’Unité qui utilisent les Rayons X. Risque Biologique Pour le site IECB la société PROCINER est en charge de l’élimination des déchets biologiques. L’enlèvement des déchets est fait de façon hebdomadaire. Problèmes de sécurité Dangers ou Mesures de prévention facteurs de Techniques, risques Organisationnelles et identifiés humaines Isolés les produits CMR et très toxiques pour les stocker dans des Risque emplacements dédiés sous chimique – clés (Site IECB) Risque lié à Déplacement des 5 frigos la qualité de contenant les produits l’air chimiques dans une pièce ventilée (lieu de stockage actuel non adapté) Bruits

Ordre Délais de d’exécution priorité

Personnes ou service chargée de la réalisation

1

1er trimestre ACMO 2010

1

ACMO + I.Huc 1 trimestre (DR – CNRS) 2010 Financement CBMN/IECB

Ambiance sonore élevée à 2 vérifier dans certains locaux

er

courant 2010

ACMO soutien

+ cellule 165

 

ACMO Installation de badges Vol avec et d’entrée personnalisés pour sans l’équipe Urdaci (site effraction ENITA) Installation d’indicateur de présence lumineux à l’entrée Travail isolé de salle de laboratoire non vitrée (équipe Urdaci (site ENITA) Actions réalisées Risque d’asphyxie (Liquide cryogénique)

En cours

et

A prévoir

salle de stockage des 2 containers azote à redéfinir

Chute de morceaux de béton dans Reprendre le béton les circulations

HS CNRS Univ Bdx 1 A. Antoine

1

En 2008 avec déplacements des containers dans une pièce ventilée et mieux dimensionnée En juillet 2008

Alexandra Milocheau

Univ Bdx 1 Division du Patrimoine et des infrastructures

166

 

ANNEXE 2 : PLAN DE FORMATION DE L’UNITE Bernard Desbat est le correspondant formation de l’Unité, il informe et conseille les personnels pour leurs besoins et demandes de formation. Il participe, auprès du directeur d’Unité, à l’élaboration du plan de formation de l’Unité. Il propose aussi des formations, assurées par l'Unité, à destination de l'extérieur (écoles thématiques, stages techniques, etc.). Le plan de formation de l’Unité est soumis pour avis au conseil d’Unité. Rappel du Projet Scientifique du laboratoire Le laboratoire, créé en janvier 2007, est à l'interface entre la biologie, la chimie et la physique. Sa mission est d'apporter une connaissance fondamentale de phénomènes biologiques complexes en les analysants à plusieurs échelles, allant de la molécule à la cellule et à l’organisme. A côté des aspects de recherche fondamentaux, l’UMR développe aussi des aspects très appliqués vers l’adhésion cellulaire, les nanopuces, la vectorisation des principes actifs, la valorisation des bactéries probiotiques, le diabète et les colloïdes alimentaires. La modélisation moléculaire, les études de biologie structurale (RMN, Rayons X, microscopie électronique, spectroscopies optiques et vibrationnelles, spectrométrie de masse), la conception chimique ou physicochimique d’édifices biomimétiques vecteurs sont aussi importantes que la maîtrise de la biologie cellulaire et moléculaire, la production et le test de molécules actives par microbiologie. Plan de formation 2007 et suivants Le laboratoire a été créé en janvier 2007 et Bernard Desbat a accepté d’être correspondant de formation fin février. Il s’est d’abord formé pendant 3 jours sur le rôle du correspondant de formation. Il a lancé en juillet 2007 une enquête générale par courrier électronique sur les besoins en formation. Les réponses positives à ce sondage regroupent environ 20% des effectifs du laboratoire. On peut penser que c’est assez faible et y trouver deux origines l’éclatement géographique du laboratoire et le fait qu’au sein du laboratoire la pluridisciplinarité est de mise ce qui donne déjà de fait une bonne formation dans les domaines de la chimie, biologie et physique. Les demandes de formation regroupent les aspect suivants : • Langages informatiques spécifiques: Python, Bash, C, site web en PHP et MySQL • Langues: anglais espagnol • Techniques particulières : préparation de cryosection o microscopie à champ proche o purification des protéines o spectroscopies UV-Vis, Fluorescence o gestion d'un autoclave • Formation hygiène sécurité: Sur le bilan des formations réellement suivies depuis 2007 on peut voir dans le tableau, cidessous, qu’elles concordent bien avec les souhaits exprimés lors de l’enquête. Formations/années Informatiques/logiciels : Python,Unicorn, Photoshop Hygiène /sécurité : Secourisme/risques biologiques Langues : Anglais/Espagnol Techniques : Cryomicroscopie

 

2008 1 0 4 0

2009 3 5 1 2

167

Le nombre général de formations par années est assez faible, mais l’on peut remarquer que le nombre a nettement augmenté en 2009. L'analyse des demandes montre que c'est surtout les ingénieurs, techniciens et étudiants qui suivent des formations. Par ailleurs, les demandes concernent assez peu la formation à de nouvelles techniques. Pour l'année qui vient ce sont les mêmes types de formations qui sont désirées. Il y a cependant une demande particulière pour une formation à des techniques spécifiques de spectrométrie de masse, organisée lors d'un congrès international aux Etats-Unis. Le CBMN a également vocation à dispenser des formations car il dispose en son sein de techniques et de méthodes tout à fait originales. • école thématique sur les protéines • école thématique nano-objets aux interfaces • spectroscopie Raman des systèmes biologiques • spectroscopie IR des surfaces  

 

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