À l’heure actuelle, le paludisme reste un problème de santé majeur et demeure une des maladies parasitaires les plus menaçantes. Parmi les cinq espèces de malaria infectant l’homme, Plasmodium falciparum est la forme la plus mortelle. Lors de la phase érythrocytaire de son cycle de vie, causant tous les symptômes du paludisme, P.falciparum utilise les phospholipides pour créer les membranes nécessaires au développement de cellules filles. Chez P. falciparum, la phosphatidylcholine est principalement obtenue grâce à la voie de synthèse de novo, dite voie de Kennedy. Dans cette voie de biosynthèse, la seconde étape catalysée par la CTP:phosphocholine cytidylyltransferase [EC 2.7.7.15] est limitante et apparait essentielle pour la survie du parasite murin P. berghei lors de la phase sanguine. Les objectifs de mon travail de thèse ont été de caractériser structuralement cette enzyme et d’identifier des effecteurs, principalement grâce à des approches de « fragment-based drug design » (FBDD). Ainsi, la première structure cristalline du domaine catalytique de l’enzyme (PfCCT) a été déterminée avec une résolution de 2.2 Å. De plus, les structures de trois complexes enzyme-substrat (en présence de CMP, de phosphocholine ou de choline) et d’un complexe enzyme-produit (CDP-Choline) ont été déterminées. Ces structures cristallographiques apportent des informations détaillées sur la poche de liaison de l’enzyme et elles ont révélé des informations sur le mécanisme de la réaction catalytique à l’échelle atomique. La seconde partie de ma thèse présente les méthodes développées pour identifier des inhibiteurs potentiels de la PfCCT. Une approche de FBDD a été utilisée pour identifier et sélectionner de petites molécules (fragments, PM<300 Da) se liant à la PfCCT. Diverses techniques biophysiques (fluorescence-based thermal shift assay, différence de transfert de saturation par RMN, dénaturation chimique isotherme) ont permis la sélection de 23 fragments à partir du criblage d’une bibliothèque (~ 300 molécules). En parallèle, un criblage in silico de plus grandes bibliothèques de fragments (environ 15 000 composés) a permis d’identifier 100 fragments “hits”. Enfin, 5 composés déjà connus pour inhiber la croissance parasitaire (Malaria Box fournit par Medecines for Malaria Venture) ont été sélectionnés pour leur inhibition de l’activité de la PfCCT recombinante. L’ensemble de ces données ouvre la voie pour l’élaboration de futurs composés ciblant la PfCCT et inhibant la biosynthèse de phosphatidylcholine chez P. falciparum. / Malaria remains a major global health problem and the most threatening parasitic disease. Among the 5 malaria species that affect humans, Plasmodium falciparum is the most deadly form. During its life cycle, in erythrocytic stage, which causes all the malaria symptoms, P. falciparum relies on phospholipids to build the membranes necessary for daughter cell development. Approximately 85% of parasite phospholipids consist of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) synthesized by the parasite through the de novo Kennedy pathways. In the pathway of phosphatidylcholine biosynthesis, the second step catalyzed by CTP:phosphocholine cytidylyltransferase [EC 2.7.7.15] is rate limiting and appears essential for the parasite survival at its blood stage. In this PhD thesis I focus on the structural characterization of this enzyme and the identification of effectors mainly by fragment-based drug design approach (FBDD). The first reported crystal structure of the catalytic domain of the enzyme target (PfCCT) has been solved at resolution 2.2 Å. Four other crystal structures of PfCCT in complex with substrates (CMP, phosphocholine and choline) or product (CDP-choline) have been determined. These structural data give detailed images of the binding pocket and reveal the enzyme structures at all catalytic steps that provide crucial information on the catalytic mechanism at atomic level. The second part of the project present the methods developed to identify potential PfCCT inhibitors. A FBDD approach was used in order to identify and select small molecules (fragments, MW< 300 Da) binding to the PfCCT. A combination of biophysical techniques (fluorescence-based thermal shift assay, saturation transfer difference NMR and isothermal chemical denaturation) allowed the selection of 23 fragment hits from the screenings of fragment library (~ 300 molecules). In parallel in silico screening of larger fragment libraries (~15,000 compounds) resulted in 100 selected hits. Finally, 5 compounds already known to inhibit parasite growth (Malaria Box from Medicines for Malaria Venture) were selected for their inhibition of the recombinant PfCCT activity. The results obtained within this thesis brought important knowledge and structural insights on the catalytic mechanism of PfCCT. Taken together, these results pave the way for future structure-based drug design to target PfCCT and to inhibit the essential phosphatidylcholine biosynthesis in P. falciparum.

Les complications vasculaires rétiniennes constituent des évènements qui peuvent être observés au cours de rétinopathies pouvant être à l’origine d’une cécité à tous les stades de la vie. Ces complications concernent particulièrement la rétinopathie du prématuré, la rétinopathie diabétique et la dégénérescence maculaire liée à l’âge. Les lipides offrent de nombreuses possibilités pour prévenir et éventuellement freiner le développement de ces rétinopathies. Parmi eux, la classe des plasmalogènes est particulièrement riche en acides gras poly-insaturés (AGPI), qui sont libérés par une phospholipase indépendante du calcium (iPLA2) et qui sont précurseurs de métabolites biologiquement actifs. Certains de ces métabolites sont connus pour être impliqués dans la modulation de l’angiogenèse rétinienne. L’objectif de ce travail de thèse a été d’évaluer l’implication des plasmalogènes dans le développement vasculaire rétinien par l’intermédiaire de la libération des AGPI par la iPLA2. Pour vérifier cette hypothèse, nous avons caractérisé les évènements cellulaires et moléculaires du développement vasculaire rétinien postnatal chez un modèle animal d’inhibition de la iPLA2 rétinienne que nous avons préalablement développé, ceci de manière comparative avec un modèle de déficience totale en plasmalogènes. Nous avons également tenté de mettre en évidence de potentielles altérations du métabolisme des plasmalogènes chez au cours d’une rétinopathie à composante vasculaire chez l’homme, la rétinopathie diabétique. Nos résultats ont suggéré que les plasmalogènes sont indispensables pour le développement physiologique des vaisseaux rétiniens. Ils seraient impliqués dans le contrôle de la formation de la trame astrocytaire et la mise en place du réseau endothélial par l’intermédiaire des AGPI libérés par la iPLA2. Les mécanismes moléculaires impliqueraient la voie des Angiopoïétines-Tie sans affecter celle du VEGF. Chez l’homme, nous avons noté une réduction des AGPI circulants, en particulier l’acide docosaexanéïque et l’acide arachidonique, sur les phosphatidyl-éthanolamines chez tous les patients diabétiques avec ou sans rétinopathie diabétique, sans implication des formes plasmalogènes. Nos résultats suggèrent une implication du métabolisme des plasmalogènes dans le contrôle du développement vasculaire en période péri-natale mais pas au cours de la rétinopathie diabétique. Ce contrôle serait exercé par l’intermédiaire des AGPI libérés par la iPLA2. / Retinal vascular complications are secondary events of several retinopathies that result in blindness at all ages. Such complications can be observed in retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. Lipids, and particularly polyunsaturated fatty acids (PUFAs), display beneficial properties in the prevention of such retinopathies. Among the different lipid classes, the plasmalogen subclass is particularly interesting since it is known to be rich in PUFAs. These PUFAs are known to be released by a calcium-independent phospholipase (iPLA2) and further converted into biologically active metabolites. Some of these metabolites are known to be involved in the modulation of retinal angiogenesis. The aim of this work was to evaluate the involvement of plasmalogens in retinal vascular development through PUFA release by iPLA2. To check this hypothesis, we have comparatively characterized cellular and molecular mechanisms of postnatal retinal vascular development in an animal model of retinal iPLA2 inhibition as well as in a model of plasmalogens deficiency. On the other hand, we have attempted to identify potential alterations in plasmalogen metabolism in diabetic retinopathy. Our results suggest that plasmalogens are essential for the physiological development of retinal vessels. They are involved in the control of astrocyte template formation and the development of the primary vascular network through PUFA released by iPLA2. Molecular mechanisms by which PUFAs from plasmalogens control retinal vascular development involve Angiopoietin-Tie pathways, without affecting those involving VEGF. In the human study, we have observed a decrease in the bioavailability of circulating PUFAs, and especially docosaexaneic acid and arachidonic acid binded to phosphatidyl-ethanolamine in all diabetic patients with or without diabetic retinopathy. Plasmalogens were not involved in these modifications. Our results suggest that plasmalogen metabolism is involved in the control of primary vascular growth during retinal development but not in diabetic retinopathy. Plasmalogens may control early steps of retinal vascular development through the release of PUFAs by iPLA2.

La littérature des dernières décennies regorge de références concernant les bienfaits des acides gras oméga 3, tels que l’EPA (C20:5 n-3) et le DHA (C22:6 n-3) qui jouent un rôle essentiel dans la prévention de nombreuses pathologies comme les maladies neurodégénératives (type Alzheimer). Les sources majeures d’EPA et DHA sont celles d’origine marine. C’est dans ce contexte que cette étude a choisi de s’intéresser aux phospholipides provenant de têtes de saumon. L’objectif était de les extraire, de purifier la phosphatidylcholine (PC) issue du mélange de lipides et d’en déterminer ses propriétés d’auto-assemblage en bicouches. Une approche expérimentale par la caractérisation physico-chimique de ce PC a été complétée par une étude théorique du même composé en utilisant les techniques de simulation par dynamique moléculaire qui a permis une caractérisation à l’échelle moléculaire des bicouches lipidiques. La caractérisation a permis d’aboutir au profil détaillé de la composition du mélange PC saumon, d’élaborer le diagramme de phase PC –eau, de déterminer les propriétés d’empaquetage d’hydratation de ce lipide. Les paramètres choisis pour l’étude en dynamique moléculaire ont permis de reproduire de manière fidèle les résultats expérimentaux, validant ainsi le modèle et les conditions de simulations déterminés au préalable. La caractérisation des propriétés structurales de la PC de saumon sous forme de multicouches a permis d’approfondir la compréhension des mécanismes d’interactions à l’échelle moléculaire existant entre les lipides insaturés eux-mêmes. / The literature of recent decades is replete with references regarding the benefits of omega 3 fatty acids such as EPA (C20:5 n-3) and DHA (C22:6 n-3) which play an essential role in preventing many diseases such as neurodegenerative diseases (Alzheimer's type). The major sources of EPA and DHA are those of marine origin. It is within this context that this study chose to deal with phospholipids from salmon heads. The objective was to extract, purify phosphatidylcholine (PC) derived from the mixture of lipids and determine its properties of self-assembly into bilayers. An experimental approach by the physicochemical characterization of this PC was supplemented by a theoretical study of the same compound using the techniques of molecular dynamics simulation that allowed a molecular-scale characterization of lipid bilayers. The characterization resulted in detailed profile of the mixture composition of salmon PC, to draw up the phase diagram of PC-water, to determine the packing and hydration properties of this lipid. The parameters chosen for the study of molecular dynamics have faithfully reproduced the experimental results, thus validating the model and simulation conditions determined in advance. The characterization of structural properties of the PC as a multilayer salmon has deepened the understanding of interaction mechanisms at the molecular level between unsaturated lipids themselves.

Advanced fluorescence techniques were used to explore tree distinct topics concerning biological membrane and their interactions. Following thesis is according to the topic divided into three parts: 1) Ionic effects were studied employing time dependent fluorescence shift experiments and molecular dynamic simulations. Combination of these two approaches are suitable to reveal characteristic like mobility and hydration of particular bilayer segment, lipid packing or ion binding sites. Halide anions were reported to adsorb to the cationic lipid bilayer specifically, altering membrane mobility and organization. Changes in observed parameters follows Hofmeister order. Their effect is mediated either by direct ionic interaction (soft, polarizable ions) as well as via alteration of water structure (hard, non-polarizable ions) in proximity of ion molecule. Further, divalent calcium was shown to bind strongly to neutral and negatively charged lipid bilayers. Several types of binding sites depending on calcium concentration were identified. 2) Two complementary lipopeptides, CPK and CPE, incorporated into distinct lipid bilayers serve as a minimal model inducing membrane fusion. Effectiveness of fusion event might be influenced by lipopeptide-membrane and lipopeptide-lipopeptide interaction. To reveal...

Thesis (Ph. D.)--Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), February 2013. / "February 2013." Cataloged from PDF version of thesis. / Includes bibliographical references. / Intact polar diacylglycerolipids (IP-DAGs) were used to study microbial dynamics in the surface ocean. IP-DAGs from surface ocean seawater were quantified using high performance liquid chromatography-mass spectrometry (HPLC-MS), after first developing a sensitive, high throughput molecular ion independent triple quadrupole MS method for quantification. Using this analytical technique I examined the distribution of the nine most abundant classes of IPDAGs across the Mediterranean, and found that phospholipids as a percent of total IP-DAGs correlated with phosphate concentration. Furthermore, phospholipids were a higher percent of total particulate phosphorus where phosphate was higher, ranging from 1-14%. Thus IP-DAGs can play not only a significant but also a dynamic role in defining planktonic nutrient needs and cellular C:N:P ratios in the environment. Additionally, microcosm incubations were amended with phosphate and ammonium, and in the course of several days this elicited a shift in the ratios of IP-DAGs. This study was the first to demonstrate the dynamic response of membrane lipid composition to changes in nutrients in a natural, mixed planktonic community, and indicated that the change in IP-DAG ratios in response to changing nutrients may be a useful indicator of microbial nutrient stress. In the surface waters of the western North Atlantic I used three experimental approaches to identify the microbial sources of the nine most abundant classes of IP-DAGs. Phytoplankton are the primary source of one class of sulfolipid, sulfoquinovosyldiacylglycerol, and one class of betaine lipid, diacylglyceryl-trimethyl-homoserine, while heterotrophic bacteria are the dominant source of the phospholipids phosphatidylglycerol and phosphatidylethanolamine. In regrowth experiments in the Sargasso Sea and the North Pacific I demonstrated that phospholipid specific production rate is representative of heterotrophic bacterial cell specific growth rate. I measured phospholipid specific production rate and bacterial production rate using uptake of 3H-leucine (³H-Leu) and 3H-thymidine (³H-TdR) across the North Atlantic, across the Mediterranean, and in the North Pacific subtropical gyre. I found that phospholipid specific production rates estimate heterotrophic bacterial cell specific growth rates that are on the order of 1 per day, an order of magnitude faster than cell specific growth rates suggested by uptake of ³H-Leu and ³H-TdR. / by Kimberly J. Popendorf. / Ph.D.

Indiana University-Purdue University Indianapolis (IUPUI) / The details of Polarization Modulation Near-Field Scanning Optical Microscopy (PM-NSOM) are presented. How to properly calibrate and align the system is also introduced. A measurement of Muscovite crystal is used to display the capabilities of the setup. Measurements of supported Lβʹ 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers are presented, emphasizing how it was tooled in exploiting the anisotropic nature of the acyl chains. A discussion of how the effective retardance (ΔS = 2π( n_e-n_o )t/λ) and the direction of the projection of the acyl chains (θ) are measured simultaneously is given, (where t is the thickness of the bilayer and λ is the wavelength of light used). It is shown from ΔS the birefringence (ne-no) of the bilayer is determined, by assuming the acyl chain tilt with respect to the membrane's normal to be ϕ ≈ 32. Time varying experiments show lateral diffusions of ~ 2 x 10-12 cm2/s. Temperature controlled PM-NSOM is shown to be a viable way to determine the main phase transition temperature (Tm) for going from the gel Lβʹ to liquid disorder Lα state of supported DPPC bilayers. A change of ΔS ~ (3.8 +/- 0.3 mrad) at the main phase transition temperature Tm (≈41^o C) is observed. This agrees well with previous values of (ne-no) and translates to an assumed <ϕ> ~ 32^o when T < Tm and 0^o when T > Tm. Evidence of supper heating and supper cooling will be presented, along with a discussion of the fluctuations that occur around Tm. Finally it is shown how physical parameters such as the polarizability are extracted from the data. Values of the transverse (αt) and longitudinal (αl) polarizabilites of the acyl chains are shown to be, αt = 44.2 Å3 and αl = 94.4 Å3, which correspond well with the theoretical values of a single palmitic acid (C16) αt = 25.14 Å3 and αl = 45.8 Å3.

Les recherches que nous présentons dans ce mémoire s'inscrivent dans le cadre du projet européen IBIOLAB[1] qui vise à la création de biolubrifiants écolabellisables. Tout d'abord, une approche d'ingénierie inverse a été réalisée pour déterminer le rôle des tensioactifs natifs des graines oléoprotéagineuses dans la stabilisation des oléosomes dans l'eau. Ceci a permis par la suite une approche générique avec la mise en œuvre d'un procédé intégré qui permet la libération des lipides sous forme émulsionnée. Une étape optionnelle consiste en l'hydrolyse enzymatique in situ des triglycérides. L'activité de la lipase utilisée a été évaluée lors d'une étude d'estérification des acides gras avec différents alcools par une méthode de séparation triphasique (TPP three-phase partitioning). / The present work was performed within the framework of the European project IBIOLAB* which aims to create environmentally friendly biolubricants (able to be eco-labelled). First of all, a reverse engineering methodology was developed to understand the role of oilseed native surfactants in the stability of oil-bodies in water. Subsequently, using a general approach, it was possible to develop a process that allows releasing the lipid fraction into an emulsion. If an in-situ enzymatic hydrolysis of triglycerides is added to this process, an emulsion containing the free fatty acids will be obtained. The activity of the remaining lipase in the emulsion was used to study the esterification of free fatty acids with different alcohols using a three-phase partitioning method.

O objetivo deste trabalho foi desenvolver biosensores de membranas baseados na técnica de Ressonância Plasmônica de Superfície (SPR) e aplicá-los no estudo da interação do citocromo c (cit c) com modelos de membranas. SPR é uma técnica ótica, que através de medidas de variações de índice de refração (n) próximas a uma interface mensura com alta sensibilidade a adsorção ou ligação de moléculas. Inicialmente desenvolvemos um sistema de gradiente de fluxo acoplado ao SPR, denominado FIG-SPR, e demonstramos a determinação automatizada da variação de n em função da concentração (dn/dC) de diferentes compostos e biopolímeros. O desenvolvimento dos biosensores de membranas iniciou-se com o estudo dos fatores que afetam a formação de uma membrana de bicamada híbrida (HBM). HBMs são compostas de uma monocamada de alcanotiol adsorvida sobre o ouro, e sobre esta uma camada fosfolipídica. A formação da HBM depende da fusão de vesículas em superfícies hidrofóbicas e que não é bem compreendido no nível molecular. Nossos estudos mostraram que na presença de cálcio e espermina a formação da HBM é favorecida, de tal forma que a monocamada de fosfolipídio alcança valores de espessura próximos àqueles previstos, cerca de 20 &#197;\'. Além disso, mostramos que em soluções de baixa força iônica a camada lipídica não é homogênea. Demonstramos também que a presença de cálcio na concentração 150 mM diminui o tempo de formação da monocamada lipídica cerca de 14 vezes quando comparado ao tempo indicado na literatura. A homogeneidade da HBM e a carga superficial da mesma foram verificadas com a adsorção e a dissociação de cit c e de albumina bovina (BSA). Utilizando HBMs de composição lipídica variada demonstramos a adsorção e a dissociação de cit c induzida por cálcio em HBMs mistas, incluindo um modelo mimético da membrana mitocondrial interna (IMM) constituído de fosfatidilcolina, fosfatidiletanolamina e cardiolipina (PC/PE/CL) na proporção (4,5:3,5:2,0). Demonstramos que a adsorção de cit c nativo segue um perfil cooperativo e padrões esperados de variação de afinidade e cooperatividade em pHs 6,8, 7,4 e 8,0. Um modelo matemático foi desenvolvido para tratar as curvas de ligação de cit c, que é uma adaptação do modelo de Hill para adsorção de proteínas em superfícies. Os resultados de SPR juntamente com dados obtidos por Microscopia de Força Atômica (AFM) sugerem que a ligação cooperativa de cit c com HBM ocorre devido à reorganização das moléculas de CL e formação de domínios fosfolipídicos. O tratamento dos resultados cinéticos da dissociação de cit c por cálcio indica a existência de duas constantes de velocidade de dissociação (kd), sendo a primeira constante (kd1) relacionada à perda das interações eletrostáticas entre a proteína e a HBM, e a segunda (kd2) à perda das interações hidrofóbicas. Além disso, a dissociação do cit c do modelo estudado requer uma concentração mínima de cálcio de 30 &#181;M para se tornar significativa. O estudo da interação entre moléculas de cit c foto-oxidadas (citc405) e a HBM de PC/PE/CL sugerem que ela ocorre com menor afinidade, nos três pHs estudados, se comparados aos resultados com cit c nativo. Além disso, nossos resultados sugerem que o citc405 não é facilmente dissociado por cálcio devido à perda da cooperatividade na interação. Possíveis implicações em eventos celulares destas descobertas, como a liberação do cit c da IMM e a iniciação da apoptose, são discutidas / The aim of this work was to develop membrane biosensors based on Surface Plasmon Resonance (SPR) and to apply them to study the interactions between cytochrome c (cyt c) and model membranes. SPR is an optical technique that provides high-sensitivity measurements of refractive index (n), allowing the characterization of the adsorption and desorption of molecules near interfaces. Initially we developed a flow gradient system connected to SPR, which was called FIG-SPR, and demonstrated the automated determination of the concentration gradient of refractive index (dn/dC) of different materials and biopolymers. The development of the membrane biosensors was initiated by studying the factors that affect the formation of a hybrid bilayer membrane (HBM). HBMs are composed of two monolayers: an alcanethiol monolayer adsorbed on gold over which is adsorbed a layer of phospholipids. The formation of an HBM depends on the fusion of phospholipid vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed that in the presence of calcium and spermine the complete formation of an HBM is facilitated, i.e., the phospholipid monolayer reaches the expected thickness of about 20&#197;\'. However, in low ionic strength solutions the lipid layer that is formed is not homogeneous. We have also demonstrated that in the presence of 150 mM of calcium the time necessary for the formation of the lipid monolayer is reduced 14 times when compared to the times suggested in the literature. The homogeneity of the HBM and its superficial charge were verified with the adsorption and desorption of cyt c and bovine serum albumine (BSA). The adsorption and desorption of cyt c in different HBMs were studied including a model of the internal mitochondrial membrane (IMM), which is made of phosphatidylcholine, phosphatidylethanolamine and cardiolipin (PC/PE/CL) in the ratio (4,5: 3,5: 2,0). We demonstrated that the adsorption of native cyt c follows a cooperative profile showing expected changes in affinity and cooperativity in different solution pHs of 6,8, 7,4 and 8,0. A mathematical model, which is an adaptation of the Hill model for adsorption of proteins in surfaces, was developed to treat the binding curves of cyt c. The results of SPR together with those obtained by Atomic Force Microscopy (AFM) suggested that the cooperative binding of cyt c in HBMs occurs due to the reorganization of CL molecules and formation of phospholipid domains. The kinetic results of the dissociation of cyt c induced by calcium indicates the existence of two velocity constants (kd), being the larger (kd1) related to the dissociation of cyt c interacting electrostatically with the HBM, and the smaller (kd2) related to the dissociation of cyt c interacting hydrophobically with the HBM. Moreover, the dissociation of cyt c from the HBM requires a minimum calcium concentration of 30 &#181;M. The study of the interaction between photo-oxidized cyt c molecules (cytc405) and the PC/PE/CL HBM suggests that it occurs with smaller affinity when compared with the results obtained with the native cyt c. Moreover, cytc405 is not easily dissociated by calcium due to the loss of the interaction cooperativity with the HBM. Possible implications of these discoveries in cellular events, such as the release of cyt c from the IMM and the initiation of apoptosis, are discussed

Les maladies ostéoarticulaires représentent environ 10% de l’ensemble des pathologiessurvenant en France chaque année. Les difficultés pour identifier les causes de ces maladiesproviennent pour une part d’un manque de compréhension du mécanisme de lubrificationd’une articulation synoviale saine. Dans ce contexte, un premier objectif de ce travail a été d’analyser la structurediscontinue du liquide synovial à partir de prélèvements animaux sains et de la reproduire àpartir de composants biomoléculaires commerciaux afin de comprendre le mécanisme delubrification dans le cas sain. Le deuxième objectif de cette thèse a été d’analyser l’évolution de la structure et despropriétés lubrifiantes du liquide synovial dans le stade précoce de pathologies noninflammatoiresou inflammatoires à partir de prélèvements pathologiques humains. Afind’étudier plus finement l’évolution de la lubrification pathologique cette thèse a visé àdévelopper aussi des modèles de lubrifiants obtenus à partir de cultures cellulaires desynoviocytes humains en combinant l’action de facteurs inflammatoire (cytokines).L’ensemble des résultats montre l’importance de la structure supramoléculaire duliquide synovial dans l’obtention de bonnes propriétés lubrifiantes ; cette relation devraitconstituer d’une part un paramètre clé dans le diagnostic précoce des pathologies articulaireset d’autre part une voie de développement de liquides thérapeutiques à base de lubrifiantsnanostructurés / Osteoarticular diseases account for approximately 10% of all diseases occurring inFrance each year. The difficulties in identifying their causes are also due to a deficiency inunderstanding the lubrication mechanism of healthy synovial joint.In this context, the first objective of the present study was to analyze the discontinuousstructure of synovial fluid samples from healthy animals and to reproduce it using commercialbiomolecular components in order to understand the lubrication mechanism in the healthycase. The second objective of this thesis was to analyze the evolution of the structure andthe lubricating properties of synovial fluid in the early stage of non-inflammatory andinflammatory diseases using pathological human fluid samples. In order to study moreprecisely the evolution of pathological lubrication this work aimed to develop lubricantmodels obtained from cell cultures of human synoviocytes adding the action of pathologicalinflammatory factors (cytokines). All together the results show the importance of the supramolecular structure of thesynovial fluid in obtaining good lubricating properties what may be a key parameter in theearly diagnosis of joint diseases and even more a chance to develop therapeutic fluids basedon nanostructured lubricants

Les vésicules géantes unilamellaires (GUV) permettent d’étudier efficacement les interactions entre les lipides et les peptides. Dans ce manuscrit, il a été montré que les interactions attractives lipides-peptides sont supprimées par l’attachement de polypeptides de type élastine (ELP) sur des peptides riches en arginine et peuvent être modulées par l’auto-assemblage en micelles ainsi que par le nombre de groupements arginine dans la séquence des peptides capables de pénétrer les cellules. De plus, une nouvelle méthode pour former des GUV à partir de systèmes complexes en seulement quelques minutes a été développée. Cette méthode est basée sur le gonflement d’un film de PVA sous une bicouche lipidique. Elle supprime la dégradation des molécules pendant la formation des GUV de lipides synthétiques, tels que des glycolipides et des phospholipides portant des groupements amides, où les méthodes traditionnelles ne réussissent pas à produire des vésicules non endommagées. / Giant Unilamellar Vesicles (GUVs) are a valuable tool to study lipid bilayer-biomolecule interactions in simplified cell-like model systems. In this work, a new method to efficiently form GUVs within minutes from more complex systems was developed. This method is based on swelling of a PVA-film under a lipid bilayer and minimizes damage of involved molecules during GUV formation. It also opens up many interesting perspectives for the formation of GUVs composed from new classes of synthetic lipids, such as glycolipids and amide-bearing phospholipids, where the traditional methods fail to efficiently produce “undamaged” vesicles. GUVs were also used for studying lipid-peptide interactions of a new class of elastin-like polypeptides functionalized with arginine-rich residues. It is shown that attractive interactions between lipids and peptides are suppressed by cargo-attachment and can be tuned by self-assembly into micelles and the arginine-amount of the cell penetrating residue.