La polarité apico-basolatérale, qui est essentielle pour le maintien de l'architecture tissulaire et pour des fonctions de l'épithelium sain, est fréquemment fragilisée, voire perdue, dans des lésions prénéoplasiques et au cours de la tumorigénèse. Les patients atteints de l'hépatite C chronique ont un risque accru de développer des carcinomes hépatocellulaires. Le contexte inflammatoire et cirrhotique, caractéristique de l'infection chronique par le virus de l'hépatite C (VHC), constitue un facteur de risque important de la tumorigenèse. De plus, le virus exerce des effets directs sur la physiopathologie de la cellule hôte, qui semblent favoriser la transformation oncogénique. Mon travail de thèse a permit de développer et de caractériser un modèle de culture tridimensionnelle qui reproduit la morphologie complexe des hépatocytes pour ensuite étudier l'effet d'une proteine virale, NS5A sur la physiopathologie de cellules hépatiques dans un contexte tridimensionnelle. Des cellules hépatiques, immortalisées ou primaires, s'organisent en organoides polarisés et acquièrent l'expression de marqueurs hépatocytaires matures. L'organisation tridimensionnelle et la polarité des organoides influent sur des voies de signalisation intracellulaires, dont la voie PI3K/Akt, un acteur crucial de la physiologie de la cellule et de sa transformation. Dans notre modèle, l'activation constitutive de la kinase Akt perturbe l'organisation tridimensionnelle des hépatocytes. NS5A, une des protéines du VHC, active la voie PI3K/Akt et interfère avec l'intégrité des organoides. Dans des précurseurs hépatiques primaires, les BMELs l'expression de NS5A conduit à la perte de marqueurs épithéliaux et à l'acquisition de marqueurs mésenchymateux et au phénotype migratoire et invasif. Ces effets de NS5A, qui suggèrent une transition épithelio-mésenchymateuse (TEM), sont additifs à l'action du TGF-β, un inducteur connu de la TEM. / Apico-basolateral polarity is essential to maintain tissue architecture and function of healthy epithelium. It is weakened or lost in preneoplastic lesions and in the course of carcinogenesis. Patients suffering from chronic hepatitis C are at risk for hepatocellular carcinoma. In addition to the necroinflammatory liver microenvironment, which favours tumorigenesis, direct effects of the virus on its host cell physiopathology also participate in the initiation of oncogenic transformation.To study the effects of viral proteins on cellular polarity and function, we developped an in vitro threedimensional (3D) culture model that reproduces complex hepatocyte morphology. Both immortalized cells and primary hepatocyte precursors organise themselves into intricate 3D organoids and acquire markers of mature hepatocytes. Their organisation and polarity impacts on several intracellular signal transduction pathways, including the PI3K/Akt axis, a major actor of physiology and of oncogenic transformation. Interestingly, constitutive Akt signalling perturbs hepatocyte 3D organization.NS5A, an HCV viral protein with pleiotropic activities, is an upstream activator of Akt. NS5A expression interferes with organoid formation and integrity. In primary hepatocyte precursors it leads to downregulation of epithelial and to acquisition of mesenchymal markers, suggesting an induction of epithelial-to-mesenchymal transition (EMT). Moreover, the effects of NS5A are additive to that of TGF-beta, a bona fide EMT inducer relevant to HCV-related pathologies. NS5A is also inducing pro-migratory and invasive phenotypes in BMEL cells as well as the hepatic cell line AML12. The signaling pahways underlying this results might involve Twist factors, well known actors of EMT, as NS5A is capable of their transcriptional regulation.The molecular mechanisms linking viral proteins to alterations of hepatocyte morphology are under investigation. Alterations of cell shape and function are of major interest in the context of virus-induced phenotype, relevant to tumour initiation and progression.

Made available in DSpace on 2016-04-28T20:18:28Z (GMT). No. of bitstreams: 1 2006-Juliana Solozabal Martins da Rocha.pdf: 3772089 bytes, checksum: 41414ee2cbb97d4f355ca0c9f47ec737 (MD5) Previous issue date: 2006-02-09 / The microfilariaes when found should be identified correctly, for obtaining a correct diagnosis, since Dipetalonema is apathogenic and the infection doesn't request treatment, while Dirofilariosis, known as Heartworm Disease, it can take the dog to the death. This work had as objective to contribute to the diagnosis using histochemical differentiation in twelve sanguine samples of microfilaremics dogs and aid in the recognition of the identified species with that staining, based on the individual acid fosfatase activity and to confirm the use of a commercial kit for this procedure. They were identified Dirofilaria immitis and Dipetalonema reconditum, making possible this technique in the taxonomy of those species. / As microfil?rias quando encontradas devem ser identificadas corretamente, para realiza??o de um diagn?stico preciso, j? que o Dipetalonema ? apatog?nico e a infec??o n?o requer tratamento, enquanto a Dirofilariose, conhecida como Doen?a do Verme do Cora??o, pode levar o c?o ? morte. Este trabalho teve como objetivo contribuir ao diagn?stico pela diferencia??o histoqu?mica em doze amostras sangu?neas de c?es microfilar?micos e auxiliar no reconhecimento das esp?cies identificadas por essa marca??o, baseada na atividade da fosfatase ?cida individual, confirmando a utiliza??o de um kit comercial para este procedimento. Foram identificadas histoquimicamente, Dirofilaria immitis e Dipetalonema reconditum, possibilitando esta t?cnica na taxonomia dessas esp?cies.