Die Arbeit umfasst die Synthese von lipophilen Inositolen und Glycerollipiden, welche auf ihre Raftophilie getestet wurden. Des weiteren wurden eine Reihe neuer Ceramide synthetisiert und diese in Bioassays auf ihre Wirksamkeit gegenüber diversen Krankheiten wie Influenza getestet.

T-cell activation is a complex process crucial for a proper function of immune system. It has been extensively studied and its main features are well understood. However, some of the events involved in T-cell signalling are still unclear. After T-cell receptor stimulation, Src-family kinase Lck drives the initiation of signalling by tyrosine phosphorylation. Phosphorylation of several downstream targets is dependent on the redistribution of Lck to the different compartment of the plasma membrane, called lipid rafts. In lipid rafts, active Lck is juxtaposed and activates raft-resident substrates which then trigger downstream signalling. The critical in this process is the mechanism of Lck translocation to lipid rafts which has not been studied so far and represents the topic of great academic and clinical interests. Previously, we identified the adaptor protein RACK1 as a candidate protein mediating the redistribution of Lck to lipid rafts by linking it to the microtubular network. In this thesis, we analysed the structural features and functional role of RACK1 in its interaction with Lck. We show here, using the SYF cell lines expressing the wild type and various mutated forms of Lck, that intact SH3 or SH2 domains of Lck are required for an effective RACK1-Lck complex formation. We also documented...

La maladie d’Alzheimer (MA) est une démence progressive caractérisée dans ses stades précoces par une perte synaptique et une dégénérescence neuronale. Aucun traitement efficace n’est disponible à ce jour et les mécanismes moléculaires impliqués sont encore mal connus. Des études récentes, menées notamment par notre équipe, indiquent que les oligomères de peptide amyloïde-ß (Aß) interagissent avec la membrane plasmique et y induisent un stress cellulaire impliquant des cascades apoptotiques. Ce travail a été consacré à l’étude de l’influence du statut lipidique sur la sensibilité des neurones au peptide Aß soluble et sur la signalisation pro-apoptotique. Nous avons montré que ce peptide induit un remodelage membranaire conduisant à la mort neuronale en activant des voies pro-apoptotiques et en inhibant des voies de survie. Par contre, la supplémentation du milieu en acide docosahexaénoïque (DHA), acide gras polyinsaturé de type ?3, protège les neurones de la mort induite par le peptide Aß. Cet effet préventif semble dépendre d’une incorporation du DHA pouvant localement induire un remodelage membranaire discret capable de préserver des voies de croissance et de survie cellulaire. Le DHA n’agit toutefois pas en inhibant l’activation de la phospholipase A2 cytosolique (cPLA2) et le stress oxydant induits par le peptide Aß, mécanismes que nous avons observés être impliqués dans la voie pro-apoptotique menant à la production de céramides suite à l’activation des sphingomyélinases. Ces enzymes occupent une position essentielle dans la toxicité du peptide Aß, puisqu’un prétraitement par le DHA ou par la sphingosine-1-phosphate, connue pour son potentiel antiapoptotique, protège les neurones en inhibant la sphingomyélinase acide. Les travaux de cette thèse ont donc montré que les lipides, molécules structurales mais aussi médiateurs de signalisation, sont des acteurs essentiels à considérer pour le développement de stratégies préventives ou thérapeutiques contre la MA / Alzheimer’s disease (AD) is a progressive dementia characterised in the early stages by synaptic loss and neurodegeneration. Currently, no effective treatments are available and the molecular mechanisms implicated are still unknown. Recent studies, including ours, indicate that the direct interaction between soluble amyloid-ß (Aß) oligomers and the plasma membrane initiates a cellular stress involving apoptotic cascades. The present work focuses on the impact of lipid status on neuronal susceptibility to the toxicity of soluble Aß peptide and on the pro-apoptotic signalling pathways. We showed that this peptide induces membrane remodelling, thereby activating pro-apoptotic pathways and inhibiting survival signalling, which leads to neuronal cell death. This was however prevented in neurons cultured in a medium supplemented with docosahexaenoic acid (DHA), an ?3 polyunsaturated fatty acid. This protective effect seems to rely on subtle local membrane remodelling due to DHA incorporation, which maintains active survival and growth pathways. DHA did not prevent either cytosolic phospholipase A2 (cPLA2) or oxidative stress upon soluble Aß peptide exposure. These two events have already been reported by our team to be implicated in a pro-apoptotic cascade leading to ceramide production due to the activation of sphingomyelinases. These enzymes very likely play a central role in Aß peptide toxicity, as pretreatments with DHA or sphingosine-1-phosphate, a well-known anti-apoptotic compound, lead to neuronal protection through the inhibition of acid sphingomyelinase. This PhD thesis demonstrates that lipids, as structural molecules as well as signalling mediators, are essential players that could be used to develop strategies for the prevention or treatment of AD

The 14-3-3 family of proteins are important signalling proteins involved in a number of cellular processes. These include cell cycle regulation, apoptosis, signal transduction and cell signalling. There is also considerable evidence in the literature that 14-3-3 proteins play a vital role in the pathology of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, Huntington’s and Prion disease. The neurodegenerative disease of focus in this research is Spinocerebellar Ataxia Type 1 (SCA1). SCA1 is a polyglutamine-repeat disease and the interaction of the disease protein ataxin-1 with 14-3-3 proteins leads to the toxic accumulation and subsequent protein aggregation which is characteristic of this disease. This study focused on attempting to elucidate the structure of various domains of the disease protein and also in identifying potential inhibitors of this deleterious interaction. Unfortunately, structural studies were not successful due to a number of caveats encountered in the expression and purification of the ataxin-1 protein domains. By utilising computational methods and small molecule inhibitors, a number of potential lead compounds which possess the ability to at least partly disrupt the interaction of 14- 3-3ζ have been identified. As 14-3-3 proteins play roles in other neurodegenerative diseases, successful identification of potential drug lead treatments can have far reaching benefits in a number of neurodegenerative diseases including SCA1. Lipid rafts are also involved in neurodegenerative disease pathology. Lipid rafts are cholesterol and sphingolipid rich domains which organise the plasma membrane into discrete microdomains and act as signalling platforms and processing centres which attach specific proteins and lipids. A number of disease proteins are processed at these membrane regions, including those involved in Alzheimer’s, Parkinson’s and Prion disease. This processing is a step which is critical in the pathology of disease and abnormal processing leads to the formation of toxic protein aggregates. Previous research in the lab identified the association of low levels of the five main brain isoforms of 14-3-3 proteins with rafts. This study expanded on this to positively identify the presence of the two phospho-forms of 14-3-3, α and δ. The mechanism by which 14-3-3 proteins associate with rafts was also investigated, indicating that 14-3-3 associates with rafts via an unidentified raftbound protein(s). In addition, the phosphorylation status and quaternary structure of 14-3-3 in the presence of sphingolipids has been explored.

A theoretical explanation of the existence of lipid rafts in cell membranes remains a topic of lively debate. Large, micrometer sized rafts are readily observed in artificial membranes and can be explained using thermodynamic models for phase separation and coarsening. In live cells such domains are not observed and various models are proposed to describe why the systems do not coarsen. We review these attempts critically and show within a phase field approach that membrane bound proteins have the potential to explain the different behaviour observed in vitro and in vivo. Large scale simulations are performed to compute scaling laws and size distribution functions under the influence of membrane bound proteins and to observe a significant slow down of the domain coarsening at longer times and a breakdown of the self-similarity of the size-distribution function. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The conformational conversion of the cellular prion protein (PrPc) to the misfolded isoform (PrPsc) is the central pathogenic event in the transmissible neurodegenerative prion diseases. The recently shown transmissibility of variant Creutzfeldt-Jakob disease by blood transfusion emphasizes the need for better understanding of the PrPc in blood. In the current thesis, we focused on blood platelet PrPc, which has not been very well described so far. In the first part of the thesis, platelet PrPc was characterized as glycosylphosphatidylinositol- anchored glycoprotein with dominant diglycosylated form. Platelet PrPc was shown to be sensitive to cleavage with proteinase K, which is a feature discriminating between cellular and pathological prion protein. We have confirmed that platelet PrPc binds copper ions by its N- terminal octapeptide repeat region. Regarding quantity of PrPc molecules expressed on blood elements we have proved that both platelets and red blood cells express considerable amount of PrPc and thus can not be neglected in the problematic of prions transmission by blood transfusion. The detailed study regarding PrPc localization in blood platelets is presented in the second part of the thesis. PrPc was shown to be expressed in -granules as well as on the cytoplasmic membrane of...

Les échanges entre les cellules tumorales et le microenvironnement jouent un rôle essentiel dans le développement des tumeurs. Dans ce contexte, la nouvelle famille de métalloprotéases, les protéines ADAM, constituent aujourd’hui des régulateurs majeurs de la progression tumorale en agissant sur la biodisponibilité des médiateurs de la communication cellulaire que sont les cytokines, chimiokines et facteurs de croissance. Au sein de cette famille, ADAM12 est la plus associée au cancer. Elle possède la particularité de jouer un rôle dans la signalisation cellulaire, de façon indépendante de son activité métalloprotéase, notamment dans les voies de signalisation du TGFβ. Notre étude montre pour la première fois un rôle pour la forme membranaire d'ADAM12 dans l'induction de la transition épithélio-mésenchymateuse (EMT), un processus essentiel à l'invasion tumorale dont le TGFβ est un inducteur majeur. Cet effet est médié par l'activation des voies de signalisation du TGFβ, impliquant les protéines SMAD3, AKT et ERK et requiert le domaine cytoplasmique d'ADAM12L mais pas son domaine catalytique. L'activation de ces voies de signalisation pourrait impliquer une relocalisation d'ADAM12L au sein de plates-formes de signalisation dans les radeaux lipidiques. Par ailleurs, nous avons montré qu'ADAM12L interagit avec les protéines ZO-1 et ZO-2, des protéines des jonctions serrées, et pourrait favoriser leur désassemblage au cours de l'EMT. Nos travaux ont permis de mettre en évidence une nouvelle fonction pour ADAM12L dans l'EMT, par un mécanisme impliquant une modulation des signaux régulant ce processus. Une meilleure compréhension de la dynamique de ces mécanismes moléculaires pourrait permettre de développer de nouvelles thérapies ciblées pour lutter contre la progression tumorale. / Communication between tumoral cells and the microenvironnement plays an essential role in the developpement of tumors. In that context, the new family of metalloproteases, the ADAM proteins, are major regulators of the tumoral progression by acting on the bioavaibility of importants mediators of cellular communication as cytokines and growth factors. Among this family, ADAM12 is the most associated with cancer. It has been shown to mediate signaling pathways by a process independant of its metalloproteasis activity, in particular for TGFβ signaling. This study show for the first time a role for the membrane form of ADAM12 in the induction of epithelial to mesenchymal transition (EMT), a essential process involved in tumor invasion, whom TGFβ is a main inducer. This effect is mediated by the activation of TGFβ signaling pathways, SMAD3, AKT and ERK and require the cytoplasmic tail of ADAM12L but not its catalytic activity. Activation of these pathways could involve a relocalisation of ADAM12L in special signaling platform in lipid rafts. Moreover, we have shown that ADAM12L interact with ZO-1 and ZO-2, two proteins of tight junctions, and could facilitate their desassembling during EMT. This work underscore for the first time a new function of ADAM12L in EMT, by a mecanism invovlving a modulation of signals regulating this process. A better understanding of the dynamic of these molecular mecanisms could allow the developpement of new targeted therapies to fight against tumoral progression.

The conformational conversion of the cellular prion protein (PrPc) to the misfolded isoform (PrPsc) is the central pathogenic event in the transmissible neurodegenerative prion diseases. The recently shown transmissibility of variant Creutzfeldt-Jakob disease by blood transfusion emphasizes the need for better understanding of the PrPc in blood. In the current thesis, we focused on blood platelet PrPc, which has not been very well described so far. In the first part of the thesis, platelet PrPc was characterized as glycosylphosphatidylinositol- anchored glycoprotein with dominant diglycosylated form. Platelet PrPc was shown to be sensitive to cleavage with proteinase K, which is a feature discriminating between cellular and pathological prion protein. We have confirmed that platelet PrPc binds copper ions by its N- terminal octapeptide repeat region. Regarding quantity of PrPc molecules expressed on blood elements we have proved that both platelets and red blood cells express considerable amount of PrPc and thus can not be neglected in the problematic of prions transmission by blood transfusion. The detailed study regarding PrPc localization in blood platelets is presented in the second part of the thesis. PrPc was shown to be expressed in -granules as well as on the cytoplasmic membrane of...

Indiana University-Purdue University Indianapolis (IUPUI) / Omega-3 polyunsaturated fatty acids (n-3 PUFAs) relieve the symptoms of a wide variety of chronic inflammatory disorders. Typically, they must be obtained in the diet from sources such as fish oils. Docosahexaenoic acid (DHA) is one of these n-3 PUFAs. As yet the structural mechanism responsible for the health benefits within the body is not completely understood. One model that has emerged from biochemical and imaging studies of cells suggests that n-3 PUFAs are taken up into phospholipids in the plasma membrane. Thus the focus here is on the plasma membrane as a site of potential structural modification by DHA. Within cellular membranes, the huge variety of molecules (called lipids) which constitute the membrane suggest inhomogeneous mixing, thus domain formation. One potential domain of interest is called the lipid raft, which is primarily composed of sphingomyelin (SM) and cholesterol (chol). Here the molecular organization of [2H31]-N-palmitoylsphingomyelin (PSM-d31) mixed with 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as a monounsaturated control, and cholesterol (chol) (1:1:1 mol) in a model membrane was examined by solid state 2H NMR spectroscopy. Solid state 2H NMR spectroscopy extracts details of molecular orientation and anisotropy of molecular reorientation by analysis of the lineshape. This essentially non-invasive technique allows for a direct measurement of dynamics in bulk materials which has been extensively applied to biological materials. It is a niche area of NMR for which standard software often lack necessary features. Two software programs, “EchoNMR processor” and “EchoNMR simulator”, collectively known as “EchoNMR tools”, that were developed to quickly process and analyze one-dimensional solid-state NMR data, will be described along with some theoretical background of the techniques used. EchoNMR tools has been designed with a focus on usability and the open-source mindset. This is achieved in the in the MATLAB® programming environment which allows for the development of the graphical user interfaces and runs as an interpreter which allows the code to be open-source. The research described here on model membranes demonstrates the utility of the software. The NMR spectra for PSM-d31 in mixtures with PDPC or POPC with cholesterol were interpreted in terms of the presence of nano-sized SM-rich/chol-rich (raft-like) and PC-rich/chol-poor (non-raft) domains that become larger when POPC was replaced by PDPC. An increase in the differential in order and/or thickness between the two types of domains is responsible. The observation of separate signals from PSM-d31, and correspondingly from [3α-2H1]cholesterol (chol-d1) and 1-[2H31]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d31), attributed to the raft-like and non-raft domains enabled the determination of the composition of the domains. Most of the SM (84%) and cholesterol (88%) was found in the raft-like domain. There was also a substantial amount of PDPC (70%) in the raft-like domain that appears to have minimal effect on the order of SM. PDPC molecules sequestering into small groups to minimize the contact of DHA chains with cholesterol is one possible explanation that would also have implications on raft continuity. These results refine the understanding of how DHA may modulate the structure of raft domains in membranes.

Les inhibiteurs de la protéase virale (IPs) sont utilisés avec succès dans le cadre d'une thérapie anti-VIH-1. L'efficacité de ce traitement est incontestable notamment en terme de réduction de la charge virale et de maintient du taux de lymphocytes CD4 circulants. Depuis l'incorporation des IPs dans la prise en charge thérapeutique, on note une réduction significative de la morbidité et de la mortalité ainsi qu'un allongement de la durée de vie des patients. Cependant, la prise de ces molécules anti-rétrovirales s'accompagne de nombreux effets secondaires. Les plus préoccupants d'entre eux sont : une lipodystrophie partielle, une hyperlipidémie, une insulino-résistance, une athérosclérose prématurée, ainsi que des infarctus du myocarde. Les patients sont également confrontés à l'apparition de maladies généralement associées à l'âge telles que la neurodégenérescence, l'ostéopénie et le développement de tumeurs malignes. Les mécanismes cellulaires et moléculaires impliqués dans ces altérations métaboliques n'ont pas encore été élucidés. L'objectif de cette étude était d'étudier les effets cellulaires de quatre IPs (Atazanavir, Lopinavir, Ritonavir et Saquinavir) sur la cellule musculaire striée squelettique humaine. Ces travaux ont permis d'identifier une augmentation de la production d'espèces oxygénées réactives (ERO), une altération morphologique du réticulum sarco/endoplasmique (RS/RE) ainsi qu'une augmentation de l'expression de CHOP, un marqueur du stress de ce compartiment et une diminution de l'expression et de la localisation dans les microdomaines membranaires (lipid rafts) de la cavéoline 3 et de la flotilline 1. Enfin, l'utilisation d'un antioxydant, le Resvératrol protége le myotube primaire humain de ces différentes altérations engendrées par les IPs. Ces données suggèrent un rôle central de la surproduction d'ERO dans le développement du stress du RE/RS et de la perte de localisation des protéines résidantes des microdomaines membranaires. De plus , en l'absence de persceptive vaccinale concrète, le Resvératrol, au travers de ces effets protecteurs, pourrait se révéler un atout de choix dans l'atténuation des effets secondaires des IPs en contribuant ainsi à l'amélioration de la prise en charge du patient séropositif. / HIV protease inhibitors (PI) have been successfully used in highly active antiretroviral therapy (HAART) of HIV-1 infection, the most effective treatment currently available. Incorporation of protease inhibitors in HAART has significantly reduced the morbidity and mortality and prolonged the lifespan of patients with HIV infection. Protease inhibitor benefits are unfortunately compromised by a number of clinically important adverse side-effects. Most patients on HAART develop a metabolic syndrome associated with partial lipodystrophy, hyperlipidemia, insulin resistance, premature atherosclerosis and myocardial infarction. Moreover, these patients face a growing number of other age-related comorbidities, such as neurodegeneration, osteopenia and malignancies. The cellular and molecular mechanisms underlying protease inhibitor-associated metabolic abnormalities remain elusive, but they seem to be related to overproduction of reactive oxygen speci es (ROS), induction of endoplasmic reticulum stress and activation of the unfolded protein response (UPR). The objective of this thesis was to determine the cell effects of four PIs (Atazanavir, Lopinavir, Ritonavir and Saquinavir) in cultures of primary human skeletal myotubes. This study showed that PIs increased ROS production, altered sarco/endoplasmic reticulum (SR/ER) morphology, increased expression of C/EBP homologous protein, a SR/ER stress marker, and decreased expression and localization at lipid rafts of Caveolin 3 and Flotillin 1. In addition, we showed that the antioxidant Resveratrol protected the human primary myotube of these iatrogenic effects. These data suggest a central role of the overproduction of ERO in the development of SR/ER stress and mislocalization of lipid raft proteins induced by PIs. Besides, in absence of vaccine, Resveratrol may be used by a potentiel therapeutic agent to attenuate PI-induced side effects