Mon travail de thèse concerne l’étude des leucémies aiguës lymphoblastiques T (LAL-T). Il se décline en deux projets. Le premier, Le Saracatinib affecte les LAL-T humaines en ciblant la kinase LCK dans les cellules riches en radeaux lipidiques, nous a permis d’identifier une nouvelle voie métabolique importante pour la prolifération des cellules leucémiques. Nous avons montré que la kinase LCK est intégrée dans les radeaux lipidiques et impliquée dans la croissance des cellules leucémiques. L’inhibiteur de LCK Saracatinib affecte les cellules de LAL-T in vitro et in vivo en ciblant particulièrement les cellules les plus agressives ayant beaucoup de radeaux lipidiques à leur surface. Ces résultats permettent d’envisager une nouvelle stratégie thérapeutique pour traiter les LAL-T et ont fait l’objet d’une publication parue dans Leukemia en janvier 2018. Le second projet, Les leucémies aiguës lymphoblastiques T produisent l’interleukine 7 de manière autocrine, démontre pour la première fois que pour la plupart des LAL-T, les cellules sont capables de sécréter elles-mêmes la cytokine IL-7. Nous avons complété cette étude par une analyse des mécanismes épigénétiques impliqués dans la régulation de cette sécrétion autocrine. Nos résultats montrent qu’elle est activée par la fixation des facteurs de transcription IRF-1 (Interferon Regulatory Factor 1) et IRF-2 au niveau du promoteur du gène IL 7, lorsque celui-ci est peu méthylé. Grâce à l’inactivation du gène IL-7 dans un de nos modèles de LAL-T, nous avons pu démontrer que la sécrétion autocrine favorise le développement de la leucémie chez la souris xénogreffée en impactant la prise de greffe et le nombre de cellules initiatrices de leucémie. Ainsi, la régulation épigénétique de la sécrétion autocrine d’IL-7 pourrait être impliquée dans les premières étapes de la leucémogenèse des LAL-T. / My PhD work concerns T-cells acute lymphoblastic leukemia (T-ALL) and includes two projects. The first one, Saracatinib impairs maintenance of human T-ALL by targeting the LCK tyrosine kinase in cells displaying high level of lipid rafts, allow us to identify a new signaling pathway important for the proliferation of T-ALL cells. We showed that LCK is localized into lipid rafts and is involved in the growth of T-ALL cells. The LCK inhibitor Saracatinib affects T-ALL cells in vitro and in vivo by targeting the most aggressive cells displaying high level of lipid rafts. These results highlight a new therapeutic strategy to treat T-ALL and were published in Leukemia in January 2018. The second project, T cell acute lymphoblastic leukemia produces autocrine interleukin 7, demonstrated for the first time that T-ALL cells are able to produce IL-7 cytokine. We performed an analysis of epigenetic mechanisms involved in the regulation of this autocrine secretion. Our results showed that when the IL-7 gene promoter is low methylated, Interferon Regulatory Factor 1 (IRF-1) and (IRF-2) transcription factors bind IRF-E sequence and upregulate IL-7 gene transcription. Thanks to IL 7 gene inactivation in one of our T ALL models, we demonstrated that autocrine secretion promotes leukemia development on xenografted mice through increasing engraftment cells capacity and leukemia initiating cells number. Thus, epigenetic regulation of IL-7 autocrine secretion may be involved in the leukemogenesis of T-ALL.

In this study we tested whether the prevalence or synaptic distribution of NMDA receptor subunits would be altered in the brain of the MeCP2-null mouse model of Rett syndrome. Detergent resistant membranes (DRMs) and post-synaptic densities (PSDs) were isolated from the synaptic membranes treated with TritonX-100, and resolved by sucrose density gradient centrifugation. Immunoblot analysis of the resulting density gradient fractions revealed that the relative distribution of the different NMDA receptor subunits between the DRM fractions, soluble fractions, and insoluble postsynaptic density fractions was preserved in the MeCP2-null brain. However, analysis of the overall NMDA receptor subunit prevalence within these fractions revealed a significant decrease in the expression of the NR1 and NR2A subunits, but not the NR2B subunit, in the MeCP2-null brain. The preservation of distribution of NMDAR subunits to the synaptic membranes, together with the decrease in NR1 and NR2A prevalence, suggest an imbalance in equilibrium between the mature and the immature synapses in a mouse model of Rett syndrome.

In this study we tested whether the prevalence or synaptic distribution of NMDA receptor subunits would be altered in the brain of the MeCP2-null mouse model of Rett syndrome. Detergent resistant membranes (DRMs) and post-synaptic densities (PSDs) were isolated from the synaptic membranes treated with TritonX-100, and resolved by sucrose density gradient centrifugation. Immunoblot analysis of the resulting density gradient fractions revealed that the relative distribution of the different NMDA receptor subunits between the DRM fractions, soluble fractions, and insoluble postsynaptic density fractions was preserved in the MeCP2-null brain. However, analysis of the overall NMDA receptor subunit prevalence within these fractions revealed a significant decrease in the expression of the NR1 and NR2A subunits, but not the NR2B subunit, in the MeCP2-null brain. The preservation of distribution of NMDAR subunits to the synaptic membranes, together with the decrease in NR1 and NR2A prevalence, suggest an imbalance in equilibrium between the mature and the immature synapses in a mouse model of Rett syndrome.

Dietary n-3 polyunsaturated fatty acids (PUFA) have been shown to potently attenuate T cell-mediated inflammation, in part, by suppressing T cell activation and proliferation. Apoptosis is an important mechanism for preventing chronic inflammation by maintaining T cell homeostasis through the contraction of populations of activated T cells. We hypothesized that dietary n-3 PUFA would promote T cell apoptosis, thus, providing an additional mechanism to explain the anti-inflammatory effects. We specifically examined activation-induced cell death (AICD) since it is the form of apoptosis associated with peripheral T cell deletion involved in immunological tolerance and T cell homeostasis. Female C57BL/6 mice were fed diets containing either n-6 PUFA (control) or n-3 PUFA for 14 d. Splenic T cells were stimulated with CD3/CD28, CD3/PMA, or PMA/Ionomycin for 48 h followed by reactivation with the same stimuli for 5 h. Apoptosis was measured using Annexin V/propidium iodide and flow cytometry. Cytokine analyses revealed that n-3 PUFA enhanced AICD only in T cells expressing a Th1-like cytokine profile (high IFN, low IL-4) compared to mice fed the n-6 PUFA control diet. Dietary n-3 PUFA significantly altered the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine in T cell membranes. To examine the apparently selective effect of dietary n-3 PUFA on AICD in Th1 cells, CD4+ T cells were polarized in vitro to a Th1 phenotype by culture with IL-4, IL-2, and IL-12 for 2 d, followed by culture with IL-2 and IL-12 for 3 d in the presence of diet-matched homologous mouse serum (MS) to prevent loss of cell membrane fatty acids. Following polarization and reactivation, we observed that n-3 PUFA enhanced Th1 polarization and AICD only in cells cultured in the presence of MS, but not in fetal bovine serum. The n-3 PUFA enhancement of Th1 polarization and AICD was associated with the maintenance of diet-induced changes in EPA (20:5n-3) and DHA (22:6n-3) in plasma T cell membrane lipid rafts. Overall, these results suggest that dietary n-3 PUFA enhance both the polarization and deletion of pro-inflammatory Th1 cells, possibly as a result of alterations in lipid raft fatty acid composition.

La phagocytose est un processus par lequel des cellules spécialisées du système immunitaire comme les macrophages ingèrent des microorganismes envahisseurs afin de les détruire. Les microbes phagocytés se retrouvent dans un compartiment intracellulaire nommé le phagosome, qui acquiert graduellement de nombreuses molécules lui permettant de se transformer en phagolysosome possédant la capacité de tuer et dégrader son contenu. L’utilisation de la protéomique a permis de mettre en évidence la présence de microdomaines (aussi nommés radeaux lipidiques ou radeaux membranaires) sur les phagosomes des macrophages. Notre équipe a démontré que ces radeaux exercent des fonctions cruciales au niveau de la membrane du phagosome. D’abord nous avons observé que la survie du parasite intracellulaire L. donovani est possible dans un phagosome dépourvu de radeaux lipidiques. Parallèlement nous avons constaté qu’un mutant de L. donovani n’exprimant pas de LPG à sa surface(LPG-) est rapidement tué dans un phagosome arborant des radeaux membranaires. Pour comprendre le mécanisme de perturbation des microdomaines du phagosome par la molécule LPG, nous avons provoqué la phagocytose de mutants LPG- du parasite et comparé par microscopie les différences avec le parasite de type sauvage. Nous avons ainsi démontré que le LPG de L. donovani est nécessaire et suffisant au parasite pour empêcher la maturation normale du phagosome. Nous avons également découvert que la molécule LPG permet d’empêcher la formation des radeaux lipidiques sur le phagosome et peut aussi désorganiser les radeaux lipidiques préexistants. Enfin, nous avons montré que l’action de LPG est proportionnelle au nombre d’unités répétitives de sucres (Gal(β1,4)-Manα1-PO4) qui composent cette molécule. Nos travaux ont démontré pour la première fois le rôle important de ces sous-domaines membranaires dans la maturation du phagosome. De plus, nos conclusions seront des pistes à suivre au cours des études cliniques ayant pour but d’enrayer la leishmaniose. Le second objectif de ce travail consistait à effectuer la caractérisation des radeaux lipidiques par une analyse protéomique et lipidomique à l’aide de la spectrométrie de masse. Nous avons ainsi entrepris l’identification systématique des protéines présentes dans les radeaux membranaires des phagosomes et ce, à trois moments clés de leurmaturation. Le traitement des phagosomes purifiés avec un détergent nous a permis d’isoler les «Detergent Resistent Membranes» (DRMs) des phagosomes, qui sont l’équivalent biochimique des radeaux membranaires. Nous avons ainsi établi une liste de 921 protéines associées au phagosome, dont 352 sont présentes dans les DRMs. Les protéines du phagosome sont partagées presque également entre trois tendances cinétiques (augmentation, diminution et présence transitoire). Cependant, une analyse plus spécifique des protéines des DRMs démontre qu’une majorité d’entre elles augmentent en fonction de la maturation. Cette observation ainsi que certains de nos résultats montrent que les radeaux lipidiques des phagosomes précoces sont soit très peu nombreux, soit pauvres en protéines, et qu’ils sont recrutés au cours de la maturation du phagosome. Nous avons aussi analysé les phospholipides du phagosome et constaté que la proportion entre chaque classe varie lors de la maturation. De plus, en regardant spécifiquement les différentes espèces de phospholipides nous avons constaté que ce ne sont pas uniquement les espèces majoritaires de la cellule qui dominent la composition de la membrane du phagosome. L’ensemble de nos résultats a permis de mettre en évidence plusieurs fonctions potentielles des radeaux lipidiques, lesquelles sont essentielles à la biogenèse des phagolysosomes (signalisation, fusion membranaire, action microbicide, transport transmembranaire, remodelage de l’actine). De plus, la cinétique d’acquisition des protéines de radeaux lipidiques indique que ceux-ci exerceraient leurs fonctions principalement au niveau des phagosomes ayant atteint un certain niveau de maturation. L’augmentation du nombre de protéines des radeaux membranaires qui s’effectue durant la maturation du phagosome s’accompagne d’une modulation des phospholipides, ce qui laisse penser que les radeaux membranaires se forment graduellement sur le phagosome et que ce ne sont pas seulement les protéines qui sont importées. / Macrophages are specialized cells of the immune system which mediate destruction and killing of invading micro-organisms. They do so by engulfing them by a process called phagocytosis. Microbes are then captured in an intracellular compartment, the phagosome, which gradually acquire molecules able to attack and degrade its cargo. Use of proteomics let us demonstrate the presence of flotillin-1 enriched microdomains (also called lipid rafts or membrane rafts) on the phagosomes. Our team demonstrated the crucial importance of these rafts in the phagocytosis process. Indeed, survival of L. donovani correlates with its presence in a ‘raftless’ phagosome while a mutated L. donovani without LPG is rapidly killed in a phagosome containing lipid rafts. To understand the membrane raft destabilisation mechanism mediated the LPG molecule, we induced phagocytosis of parasites devoid of LPG (LPG-) and compared it to the wild type parasite by microscopy. We first demonstrated that LPG alone is necessary to prevent normal maturation of the phagosome. Additionally, we discovered that the LPG molecule not only inhibits lipid rafts formation on the phagosome but also disorganise pre-existing lipid rafts. This effect of LPG is proportional to the number of repetitive sugar units (Gal( 1,4)-Man 1-PO4) which compose this molecule. Our work demonstrated for the first time an important role of the membrane rafts in the phagosome maturation. Moreover, our conclusions will give new interesting leads for clinical studies on leishmaniosis. The second goal of this work was to characterise them with proteomics and lipidomics tools. To do this, we undertook the systematic identification of proteins present on both subdomains of the phagosome (lipid rafts versus the rest of the phagosomal membrane). To achieve this, we purified phagosomes, from which we isolated lipid rafts by floating Triton X-100 insoluble membranes (DRMs for Detergent Insoluble Membranes). After that, we identified proteins by mass spectrometry.

La spectroscopie de fluorescence de molécule individuelle a révolutionné le domaine des sciences biophysiques, en permettant la visualisation des interactions moléculaires dynamiques et des caractéristiques nanoscopiques avec une haute résolution spatio-temporelle. Le contrôle des réactions enzymatiques et l'étude de la dynamique de diffusion de molécules individuelles permet de comprendre l'influence et le contrôle de ces entités nanoscopiques sur plusieurs processus biophysiques. La nanophotonique basée sur la plasmonique offre des nouvelles opportunités de suivi d'évènements à molécule unique, puisque il est possible de confiner des champs électromagnétiques dans les hotspots à nano-échelle, à dimensions spatiales comparables à une molécule unique. Dans ce projet de thèse, nous explorons plusieurs plateformes de nanoantennas photoniques avec des hotspots, et nous avons démontré les applications dans l'amélioration de la spectroscopie de fluorescence de molécule individuelle. En utilisant la fluorescence burst analysis, l'analyse de fluctuations temporelle de fluorescence,TCSPC, nous quantifions les facteurs d'amélioration de fluorescence, les volumes de détection de nanoantennas; ainsi, nous discutons l'accélération de fluorescence photo dynamique. En alternative aux structures plasmoniques, des antennes diélectriques basées sur les dimères en silicone ont aussi démontré d'améliorer la détection de fluorescence à molécule unique, pour des concentrations micro molaires physiologiquement pertinentes. En outre, nous explorons des systèmes planaires antennas in box pour l'investigation de la dynamique de diffusion de la PE et de la SM dans les membranes des cellules vivantes. / Single-molecule fluorescence spectroscopy has revolutionized the field of biophysical sciences by enabling visualization of dynamic molecular interactions and nanoscopic features with high spatiotemporal resolution. Monitoring enzymatic reactions and studying diffusion dynamics of individual molecules help us understand how these nanoscopic entities influence and control various biochemical processes. Nanophotonic antennas can efficiently localize electromagnetic radiation into nanoscale spatial dimensions comparable to single bio-molecules. These confined illumination hotspots there by offer the opportunity to follow single-molecule events at physiological expression levels. In this thesis, we explore various photonic nanoantenna platforms and demonstrate their application in enhanced single-molecule fluorescence detection. Using fluorescence burst analysis, fluorescence correlation spectroscopy (FCS), time-correlated TCSPC measurements, and near field simulations, we quantify nanoantenna detection volumes, fluorescence enhancement factors and discuss the fluorescence photodynamic accelerations mediated by optical antennas. Further, using resonant planar antenna-in-box devices we investigate the diffusion dynamics of phosphoethanolamine and sphingomyelin on the plasma membrane of living cells and discuss the results in the context of lipid rafts. Together with cholesterol depletion experiments, we provide evidence of cholesterol-induced nanodomain partitioning within less than 10~nm diameters and characteristic times being ~100 microseconds.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Cell membranes have remarkable properties both at the microscopic level and the molecular level. The current research describes the use of physisorbed polymer-grafted lipids in model membranes to investigate some of these properties on both of these length scales. On the microscopic scale, plasma membranes can be thought of as heterogenous thin films. Cell membranes adhered to elastic substrates are capable of sensing substrate/film mismatches and modulating their membrane stiffness to more closely match the substrate. Membrane/substrate mismatch can be modeled by constructing lipopolymer-enriched lipid monolayers with different bending stiffnesses and physisorbing them to rigid substrates which causes buckling. This report describes the use of atomic force microscopy and epimicroscopy to characterize these buckled structures and to illustrate the use of the buckled structures as diffusion barriers in lipid bilayers. In addition, a series of monolayers with varying bending stiffnesses and thicknesses are constructed on rigid substrates to analyze changes in buckling patterns and relate the experimental results to thin film buckling theory. On the molecular scale, plasma membranes can also be thought of as heterogeneous mixtures of lipids where the specific lipid environment is a crucial factor affecting membrane protein function. Unfortunately, heterogeneities involving cholesterol, labeled lipid rafts, are small and transient in live cells. To address this difficulty, the present work describes a model platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains into which transmembrane proteins are incorporated (αvβ3, and α5β1integrins). This flexible platform enables the use of confocal fluorescence fluctuation spectroscopy to quantitatively probe the effect of cholesterol concentrations and the binding of native ligands (vitronectin and fibronectin for αvβ3, and α5β1) on protein oligomerization state and on domain-specific protein sequestration. In particular, the report shows significant ligand-induced integrin sequestration with a low level of dimerization. Cholesterol concentration increases rate of dimerization, but only moderately. Ligand addition does not affect rate of dimerization in either system. The combined results strongly suggest that ligands induce changes to integrin conformation and/or dynamics without inducing changes in integrin oligomerization state, and in fact these ligand-induce conformational changes impact protein-lipid interactions.

No description available.

α-Tocopherol has a well known antioxidant action but is also considered likely to exert significant non-antioxidant effects in cell membranes. Due to its lipophilic nature α-tocopherol inserts into biological membranes where it influences the organisation of the component lipids and may therefore influence biophysical parameters including the membrane dipole potential. The dipole potential has been demonstrated to modulate the function of several membrane associated proteins and perturbation of this physical parameter by α-tocopherol may prove to be a significant non-antioxidant mechanism underlying several of its cellular effects. This study investigates the influence of α-tocopherol, and the non-antioxidant structural analogue α-tocopherol succinate, on the membrane dipole potential employing fluorescence spectroscopy techniques with the dipole potential sensitive probe Di-8-ANEPPS. Similar techniques are utilised with the surface potential sensitive probe FPE to investigate the interaction of the charged α-tocopherol succinate molecule with membranes. α-Tocopherol and α-tocopherol succinate are shown to decrease the dipole potential of egg-phosphatidylcholine vesicles and Jurkat T-lymphocyte cell membranes. This effect is placed in the context of the significant influence of membrane cholesterol oxidation on the dipole potential. 7-ketocholesterol, an oxidised form of cholesterol, significantly influences several cellular processes and is thought to mediate these effects, in part, through its physical effects on the cell membrane. These include altering the composition, and therefore biophysical properties, of rafts; structures which are considered to support the function of a host of membrane proteins. This study attempts to correlate the effect of 7-ketocholesterol on the dipole potential of microdomains with the influence of the oxysterol on the function of two microdomains associated receptors: P-glycoprotein and the insulin receptor, assessed by determining the extent of ligand binding using flow fluorocytometry. α-Tocopherol has been suggested to inhibit the raft-mediated effects of 7-ketocholesterol and the influence of this molecule on the effect of 7-ketocholesterol on the dipole potential are investigated as a potential mechanism for this inhibition. It is hypothesized that α-tocopherols may protect against the deleterious effects of cholesterol oxidation in cell membranes by excluding 7-ketocholesterol from specific microdomains, of which rafts are a subset, acting to preserve their dipole potential and maintain the function of the proteins they support. However, where significant cholesterol oxidation has previously occured the concurrent changes in the microdomain landscape of the membrane is suggested to prevent α-tocopherol succinate from eliciting this protective effect.