Thèse doctorat : Aspects Moléculaires et Cellulaire de la Biologie : Strasbourg 1 : 2006. / Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 19 p.

Etude du rôle des lysosomes et du cholestérol au cours de la différenciation des kératinocytes épidermiques (par Ralph Jans) La majorité des rôles protecteurs de l’épiderme, couche superficielle de la peau, sont garantis par les kératinocytes qui se différencient de manière progressive et terminale dans les couches suprabasales de ce tissu, dont le renouvellement est assuré par la prolifération de cellules-souches dans la couche basale. Toute perturbation des mécanismes qui contrôlent la prolifération et/ou la différenciation des kératinocytes conduit à des dysfonctionnements. Comprendre ces mécanismes reste donc un défi majeur pour la biologie cutanée. Plusieurs types cellulaires subissent l’exocytose de lysosomes quand il y a entrée d’ions calcium dans les cellules. Puisque les kératinocytes subissent une entrée de calcium au cours de leur différenciation, ces cellules pourraient exocyter des lysosomes dans ces circonstances. Un traitement de kératinocytes avec un ionophore induit en effet une sécrétion de la forme mature lysosomale de la cathepsine D, une libération de l’activité des enzymes lysosomales solubles cathepsine C et β-galactosidase, ainsi que l’apparition des protéines lysosomales membranaires Lamp-1 et Lamp-2 au niveau de la membrane plasmique. L’exocytose de lysosomes peut donc faire partie de la différenciation des kératinocytes, mais permet aussi à ces cellules de réparer des ruptures de la membrane plasmique. Puisque le cholestérol pourrait contrôler certaines voies de signalisation au cours de la différenciation des kératinocytes, nous avons analysé les effets d’une déplétion en cholestérol induite par un traitement avec la méthyl-β-cyclodextrine sur le phénotype et la signalisation du kératinocyte. Cette déplétion induit une augmentation de l’expression de l’involucrine, marqueur de différenciation tardive, et une répression de l’expression de la kératine 10, marqueur de la différenciation précoce, et de la kératine 14, marqueur des kératinocytes non-différenciés. Ce traitement active le récepteur de l’EGF et HER2, ainsi que la MAP kinase p38 dont l’activation (en particulier l’activation de p38α) est responsable de l’augmentation de l’expression de l’involucrine. En résumé, nos observations suggèrent un nouveau rôle pour les lysosomes au cours de la différenciation des kératinocytes épidermiques, mais suggèrent surtout un rôle critique du cholestérol dans la régulation de ce processus. Role of lysosomes and cholesterol during the differentiation process of epidermal keratinocytes (by Ralph Jans) A major part of the protective role of the epidermis, superficial layer of the skin, is guaranteed by the keratinocytes that differentiate progressively and terminally in the suprabasal layers of this tissue. The renewal of the epidermis is performed by proliferating stem cells in the basal layer. Perturbation of the mechanisms that regulate the proliferation and/or the differentiation of keratinocytes leads to an invalid barrier function. Therefore, elucidating these mechanisms is a major challenge for skin researchers. In several cell types, lysosomes undergo exocytosis upon entry of calcium into the cells. Since keratinocytes are subjected to an entry of calcium during their differentiation in vivo, these cells could exhibit an exocytosis of lysosomes under these circumstances. The results presented in this work show that an incubation of keratinocytes with the calcium ionophore ionomycin triggers the secretion of the enzymatic activities of the lysosomal enzymes cathepsin C and β-galactosidase as well as the release of the lysosomal form of cathepsin D. This treatment also induces the appearance of the lysosomal membrane proteins Lamp-1 and Lamp-2 at the plasma membrane of keratinocytes. Exocytosis of lysosomes could be part of the keratinocyte differentiation process, but could also allow these cells to repair their plasma membrane upon disruption due to mechanical stresses. Since cholesterol could be involved in the regulation of several signal transduction pathways during keratinocyte differentiation, we have investigated the effects of a depletion of cholesterol on the keratinocyte phenotype and on selected signaling pathways. Cholesterol depletion was induced by incubating the cells with methyl-β-cyclodextrin. This treatment, followed by an inhibition of cholesterol neosynthesis using lovastatin, triggers an upregulation of the expression of involucrin, a late differentiation marker, and a downregulation of keratin 10 and keratin 14, which, respectively, are markers of early-differentiating and undifferentiated keratinocytes. Cholesterol depletion activates the membrane receptors EGFR and HER2 and the MAP kinase p38. Using the specific inhibitor PD169316, we demonstrate that the p38α isoform is responsible for the upregulation of involucrin during cholesterol depletion. In summary, our observations suggest a novel role for lysosomes during keratinocyte differentiation and indicate a critical role for cholesterol in the regulation of this process.

Thèse de doctorat : Sciences du vivant. Aspects moléculaires et cellulaires de la biologie : Strasbourg 1 : 2007. / Titre provenant de l'écran-titre. Bibliogr. p. 181-201.

Thèse doctorat : Aspects Moléculaires et Cellulaires de la Biologie : Strasbourg 1 : 2006. / Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 27 p.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Orientador: Lauro Tatsuo Kubota / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-11-01T13:48:03Z (GMT). No. of bitstreams: 1 Kisner_Alexandre_D.pdf: 7108475 bytes, checksum: c8b021ab9cbe477b1fe0b4f1abf6fd48 (MD5) Previous issue date: 2012 / Resumo: Transistores de efeito de campo (FETs) modificados com nanoeletrodos de Au representam uma excelente ferramenta para o estudo eletrofisiológico de células, uma vez que as características da dimensionalidade destes últimos são comparáveis às espécies celulares a serem detectadas e medidas múltiplas podem ser realizadas simultaneamente. Neste trabalho, foram fabricados transistores de efeito de campo com três diferentes tipos de superfície em suas regiões de porta, transistores com somente SiO2, SiO2 e Al2O3 anódica porosa, e ainda SiO2-Al2O3 contendo nanopartículas de Au dentro de seus poros. Os transistores foram caracterizados por microscopia eletrônica de varredura assim como por medidas elétricas convencionais. Estas últimas demonstraram que os processos de anodização e deposição de nanopartículas em sua superfície não comprometem as suas propriedades elétricas. Os transistores com portas nanoporosas foram modificados com torisinase e empregados como biossensores para a detecção de dopamina. Os resultados demonstraram que estes FETs podem detectar dopamina num alcance de concentração normalmente encontrado durante a liberação destas moléculas por células neurais. Transistores com nanopartículas de Au foram empregados na detecção de serotonina utilizando-se uma metodologia de interação eletrostática através de monocamadas auto-organizadas, o que é ainda pouco explorado com FETs. Esta permitiu a detecção de serotonina num alcance linear de 0,1 a 2 µmol L. Experimentos envolvendo a adesão celular e a detecção de prótons liberados por vesículas de células PC12 foram conduzidos, e demonstraram que os processos interfaciais entre as células e os transistores apresentam um dependência das propriedades capacitivas da superfície, e que a presença das nanopartículas pode aumentar a sensibilidade elétrica da porta dos transistores. Estes efeitos interfaciais influenciaram diretamente na razão sinal-ruído da leitura de sinais de exocitose e sugerem que o uso de transistores nanoestruturados representa uma ferramenta promissora para análise deste tipo de célula in vitro / Abstract: Field effect transistors modified with Au nanoparticles represent an excellent tool to electrophysiology analyzes. Because the dimensions of the devices are comparable to the size of the cells, multiple measurements can be performed simultaneously. In this work, field effect transistors were fabricated with three different kinds of surface in their gates, i.e. transistor with only SiO2, SiO2 and porous anodic Al2O3, and SiO2-Al2O3 with Au nanoparticles embedded into the pores of Al2O3. The characterization of the transistors was performed by electron microscopy analysis and conventional electrical characterization. The last one showed that the anodization process and the Au nanoparticles deposition on surface of the transistors did not affect the electrical properties of the devices. The transistors presenting gates with only SiO2-Al2O3 were modified with tyrosinase and employed as biosensors to detect dopamine. The results of these analysis showed that the devices can detect dopamine in a range of concentration usually found when these molecules are released from neuronal cells. Transistors with Au nanoparticles were also applied as biosensors to detect serotonin. In doing so, the surface of the nanoparticles were modified with self-assembled monolayers that were able to interact with serotonin through electrostatic interactions. Although this approach is scarcely exploited with transistors, it showed promising results. For instance, serotonin could be detect in a linear range of concentration from 0,1 to 2 µmol L. Experiments to analyze the cell adhesion on transistors and detect the release of protons from the extruded matrix of vesicles from PC12 cells were performed and demonstrated that the interfacial processes between cells and transistors were dependent on the capacitive properties of the surface. The presence of nanoparticles can enhance the electrical sensitivity of the gates from the devices. These interfacial effects presented a relationship with the signal to noise ratio of the exocytotic signals measured for vesicles release and suggested that the employment of nanostructurated transistors are promising tools to analyze these events from PC12 cells in vitro / Doutorado / Quimica Analitica / Doutor em Ciências

La dissémination des cellules cancéreuses et la formation de métastases sont des étapes cruciales dans la progression tumorale et constituent une cause majeure des décès dus au cancer. La métalloprotéase transmembranaire MT1-MMP est un acteur clé impliqué dans le franchissement des barrières tissulaires et le remodelage de la matrice extracellulaire (ECM) par les cellules cancéreuses. MT1-MMP est présente dans des vésicules intracellulaires, appelées endosomes, via lesquels elle est adressée à la membrane plasmique (PM) afin d'y dégrader la ECM. Des travaux menés au laboratoire ont identifié le complexe exocyste (CE) comme un acteur important pour la formation d'invadopodes dans la lignée d'adénocarcinome mammaire MDA-MB-231. Ce complexe multiprotéique (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 et Exo84) est impliqué dans l'arrimage des vésicules intracellulaires à la PM. Des cribles double-hybride ont identifiés la protéine WASH comme partenaire potentiel du CE (via les sous-unités Exo84 et Sec3). WASH est capable d'induire la polymérisation de l'actine en activant le complexe Arp2/3. In vitro, nous avons montré que les complexes WASH et exocyste interagissent physiquement et coordonnent le trafic intracellulaire et l'adressage de MT1-MMP à la PM. Ces résultats mettent en évidence une étroite collaboration entre le cytosquelette d'actine et les mécanismes d'exocytose lors des étapes précoces de dégradation de la ECM ainsi que dans l'invasion tumorale. / Cancer cell invasion is a prerequisite to tumor progression and metastasis. In order to disseminate, tumor cells must degrade and remodel the extracellular matrix (ECM) in a process that requires the trans-membrane matrix metalloproteinase MT1-MMP, which is a key component of the ECM remodeling apparatus of cancer cells. MT1-MMP overexpression in cancers is associated with increased invasion and metastasis. Many cellular proteins are involved in the transport and delivery of MT1-MMP-containing vesicles to the PM. Previous work from the laboratory identified the exocyst complex (EC) as a key component required for matrix proteolysis and invasion of cancer cells. This multiprotein complex (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) plays essential roles in docking secretory vesicles at the PM for exocytosis. To better characterize this complex, a yeast two-hybrid screen was performed, identifying the protein WASH as a potential partner of Exo84 and Sec3. WASH is a Nucleation Promoting Factor (NPF) able to activate the actin nucleating Arp2/3 complex. Results of the present study showed that WASH and the exocyst complexes interact and localize on MT1-MMP-positive endosomes in MDA-MB-231 breast cancer cells. This study highlight a direct implication of WASH and exocyst complex in ECM degradation by cancer cells through the docking and exocytosis of MT1-MMP-containing endosomes at the PM through connections between these compartments and the extracellular medium. This WASH- and exocyst-dependent MT1-MMP exocytosis mechanism is required for degradation of adjacent tissue by cancer cells during tumour cell invasion.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.