Mes travaux de thèse ont porté sur l’étude de deux modèles murins d’épilepsies génétiques de l’enfance liées à des mutations des canaux Nav1.1 (impliqués dans l’excitabilité des neurones inhibiteurs) : le Syndrome de Dravet (SD), une épilepsie pharmaco-résistante sévère, et l’Epilepsie Génétique avec Convulsions Fébriles Plus (EGCF+), présentant un phénotype plus modéré.Ils se sont décomposés en 3 axes : - La première partie mettant en évidence un phénomène d’épileptogenèse dans ces modèles murins.- La seconde permettant d’identifier des conditions expérimentales d’induction d'activités épileptiformes spécifiques du modèle murin du SD sur des tranches de cerveau.- La dernière consistant à mettre au point une stratégie pour réduire l’hyperexcitabilité cérébrale / During my thesis, I studied two murine models of childhood genetic epilepsies, caused by mutations of Nav1.1 channels (involved in the excitability of inhibitory neurons): Dravet Syndrome (DS), a severe and drug resistant epilepsy, and Genetic Epilepsy with Febrile Seizures Plus (GEFS+), characterized by a milder phenotype.My work is divided into three parts:- The first one revealed a process of epileptogenesis in these murine models.- In the second, I identified experimental conditions to induce epileptiform activities which are specific of the DS model in brain slices, which could allow pharmacological screens ex-vivo.- The third one was aimed at developing a new strategy to reduce cerebral hyperexcitability

Made available in DSpace on 2016-06-02T19:22:52Z (GMT). No. of bitstreams: 1 2981.pdf: 6989540 bytes, checksum: 48d65263f0c7e6493fadd24c1378e4d0 (MD5) Previous issue date: 2009-10-14 / Financiadora de Estudos e Projetos / ADAMs is a term used to describe the presence of disintegrin and metalloprotease domains(A Disintegrin And Metalloprotease) in a certain class of multi-functional membrane proteins,expressed in several animal species such as mammals and insects. They play important rolesin many physiological processes as in fertilization, myoblast fusion, migration, proliferationand cell survival, as well in diseases including breast, prostate, and pancreas cancer. ADAM9is involved in cellular processes such as adhesion, migration and signaling of tumor cells andit is involved in the metastatic spreading. Aims: To analyze the expression of ADAM9 intumor cell lines (MDA-MB-231 and DU-145), no tumors (FH and C2C12), and to generateknockout clones for ADAM9 expression using silencing RNA techniques for the study ofADAM9 effects on invasion, proliferation and gene expression of MDA-MB-231 cells.Methods: Cells were cultured and plated (2x106 cells/plate of 6cm) for 24 hours in DMEM(10% FBS) and lysed for western blotting and zymography. To check for inhibition ofadhesion to immobilized collagen I, MDA-MB-231 cells and FH (5x106 cells/ml) werelabeled with CMFDA, and then incubated with anti-ADAM9D and anti-RP3ADAM9 indifferent concentrations. For ADAM9 silencing, it was used a kit (Silencer ® siRNA StarterKit - Ambion), 2x105 cells (MDA-MB-231) and the transfection agent (Lipofectamine 2000 -Invitrogen). The cells were plated in 5ml of culture medium DMEM/plate. On the third daycells were treated with the transfection agent and RNA silencing primers. Total RNA wasisolated for RT-PCR, and proliferation and invasion in matrigel assays. Results: All the celllines studied expressed ADAM9 in the tested conditions. MMP-2 (Gelatinase-A) activity wasdetected in the cell extracts of all studied cell lines. Silencing of ADAM9 did not affect therate of MDA-MB-231 proliferation, at 4, 5, 6, 7, 8, 9 and 10 days after silencing (24 or 48hours of incubation in 96-well plates). Silencing of human ADAM9 inhibited tumor cellinvasion in matrigel (71,51±8,02%) when compared to control. Conclusion: The generation ofMDA-MB-231 knockout clones lacking ADAM9 expression using siRNA technique did notaffect the rate of cell proliferation but inhibited tumor cell matrigel invasion, suggesting thatADAM9 is an important molecule in the processes of invasion and metastasis. / ADAMs é um termo usado para descrever a presença de domínios desintegrina emetaloprotease (A Disintegrin And Metalloprotease) em uma determinada classe de proteínasde membrana, multifuncionais, expressas em diferentes espécies animais como mamíferos einsetos. Elas possuem funções importantes em muitos processos fisiológicos como nafertilização, fusão de mioblastos, migração, proliferação e sobrevivência celular, entre outros,bem como em processos patológicos tais como muitos tipos de tumores humanos, incluindomama, próstata, pâncreas, fígado, rins e pele. A ADAM9 está envolvida em diversosprocessos celulares, tais como adesão celular, migração e sinalização de células tumorais,contribuindo para o desenvolvimento de metástases. Objetivos: Analisar a expressão daADAM9 em linhagens de células tumorais (MDA-MB-231 e DU-145) e não tumorais (FH eC2C12), gerar clones com o gene que codifica para a ADAM9 silenciados e verificar o efeitoda ausência desta proteína na invasão, proliferação e expressão gênica das células MDA-MB-231. Métodos: As células foram cultivadas e posteriormente plaqueadas (2x106 células/placade 6cm) por 24 horas e em 5ml de meio de cultura DMEM (10% de FBS) e lisadas para oensaio de western blotting e zimografia. Para o ensaio de inibição de adesão as células MDAMB-231 e FH (5x106 células/ml) foram marcadas com CMFDA (clorometil diacetatofluoresceína) e posteriormente incubadas com os anticorpos anti-ADAM9D e anti-RP3ADAM9 em diferentes concentrações. Para a técnica de RNAi foi utilizado um kit(Silencer® siRNA Starter Kit Ambion), 2,0x105 de células (MDA-MB-231) e agente detransfecção (Lipofectamina 2000 - Invitrogen). As células foram plaqueadas em 5ml de meiode cultura DMEM/placa. No terceiro dia de plaqueamento as células foram tratadas comagente de transfecção e primer de silenciamento do RNA, para serem utilizadas na RT-PCR,ensaio de proliferação e invasão em matrigel. Resultados: Todas as linhagens estudadasexpressaram a ADAM9 nas condições de estudo. A atividade MMP-2 (gelatinase-A)intermediária estava presente em todos os tipos celulares testados. O silenciamento deADAM9 não afetou a taxa de proliferação das células MDA-MB-231 após 4, 5, 6, 7, 8, 9 e 10dias do silenciamento (24 ou 48 horas de incubação). O silenciamento da ADAM9 humanainibiu a invasão celular em células de câncer de mama (MDA-MB-231) em matrigel (71,51 ±8,02%) quando comparados com o controle. Conclusão: A geração de clones knockout sem aexpressão da ADAM9 utilizando a técnica de silenciamento de RNA em células de câncer demama (MDA-MB-231), não afetou a taxa de proliferação celular. No entanto, a invasão dascélulas tumorais em matrigel foi inibida em aproximadamente 70% quando comparada com ocontrole, demonstrando que ADAM9 é uma importante molécula envolvida no processo deinvasão e metástase.

Made available in DSpace on 2016-06-02T19:22:12Z (GMT). No. of bitstreams: 1 6481.pdf: 9627871 bytes, checksum: 5751525bd7b891b47fd19618bcc84a63 (MD5) Previous issue date: 2014-12-12 / Universidade Federal de Minas Gerais / Tumor spreading occurs mainly by two pathways: through blood vessels and by lymphatic vessels, but the last is preferred by breast tumor cells. Some proteins are involved in cell adhesion and proteolysis, causing metastasis, such as ADAMs, a family of multi-domain and multi- functional proteins that contribute in these processes. ADAM9, a member of this family, has been increased in a large number of human carcinomas, including, breast cancer. In this context, the aim of this study was to evaluate the role of ADAM9 in tumor spreading via blood and lymphatic systems, in the search for new targets and focusing the development of new therapeutical tools. Therefore, MDA-MB-231 breast tumor cells were silenced for ADAM9 and tested with respect to their adhesive and invasive activity against blood and lymphatic endothelium. Our results showed that ADAM9 silencing in MDA-MB-231 breast cancer cells inhibited the invasion of this cells in matrigel (71.51 ± 8.02%) when compared to control cells, without affecting cell adhesion, proliferation, migration, and gene expression of the ADAM10, ADAM12, ADAM-17, cMyc, MMP9, VEGF-A, VEGF-C, Osteopontin and Collagen XVII, however, there was a decrease in the expression of the ADAM15 and increased expression of MMP2 when compared to controls. Furthermore, ADAM9 silencing did not affect the adhesion under flow to these vascular endothelial cells (HMEC-1 and HUVEC) and lymphatic (HMVEC-dLyNeo-Der). However, there was a decrease in the rate of trans-endothelial migration through the monolayer endothelial cells (HUVEC, HMEC-1 and HMVEC-dLyNeo-Der) by approximately 50%, 40% and 32%, respectively. In conclusion, ADAM9 showed to be essential in invasion and extravasation of MDA-MB-231 breast cancer cells through the blood and lymphatic vessels in vitro. / A disseminação tumoral ocorre principalmente por duas vias: por vasos sanguíneos e por vasos linfáticos, sendo esta última preferida pelos tumores mamários. Algumas proteínas estão envolvidas na proteólise e na adesão celular, ocasionando metástase, tais como as ADAMs, uma família de proteínas multi-domínios e multi- funcionais que contribuem nesses processos. A ADAM9, um membro desta família, apresenta expressão aumentada em um grande número de carcinomas humanos, entre eles, mama. Nesse contexto, o objetivo desse estudo foi avaliar o papel da ADAM9 na disseminação tumoral via sistema sanguíneo e linfático, visando o desenvolvimento de novas ferramentas terapêuticas. Para tanto, células de tumor de mama MDA-MB-231 foram silenciadas para a ADAM9 e testadas com relação às suas atividades adesivas e invasivas frente ao endotélio sanguíneo e linfático. Nossos resultados mostraram que o siADAM9 inibiu a invasão das células de câncer de mama MDAMB- 231 em matrigel (71,51 ± 8,02%) quando comparado com os controles, sem afetar a adesão celular, proliferação, migração, e expressão gênica da ADAM10, ADAM12, ADAM17, cMYC, MMP9, VEGF-A, VEGF-C, Osteopontina e Colágeno XVII, entretanto houve uma diminuição da expressão da ADAM15 e um aumento da expressão da MMP2 quando comparado com as controles: meio e negativo. O siADAM9 nas células MDA-MB- 231 não afetou sua adesão sob fluxo às endoteliais vasculares (HMEC-1 e HUVEC) e linfáticas (HMVEC-dLyNeo-Der). Entretanto, houve uma diminuição na taxa de transmigração através da monocamada das células endoteliais (HUVEC, HMEC-1 e HMVEC-dLyNeo-Der) em aproximadamente 50%, 40% e 32%, respectivamente. Assim, conclui- se que a ADAM9 mostrou-se essencial no processo de invasão e extravasamento das células de câncer de mama MDA-MB-231 pelos vasos sanguíneos e linfáticos in vitro.

The daytime serial drama, commercially known as the soap opera, has for a long time been diminished as an inferior artform, which previous research has connected to bias towards its historically predominantly female audience as well as its close relation to advertising. This thesis examines Guiding Light (original title The Guiding Light, CBS, 1937-1956, 1952-2009) and its transition from radio to television in the 1950s by using a historiographical approach through analysis of archive artefacts that cover the decade. The archive material, consisting of newspapers and magazines, have two parts to play: the first regarding the influence of the sponsor Procter & Gamble considering the soap opera’s status as a thriving advertisement vehicle of the time, and the second in the relationship between soap opera and its listeners/viewers throughout the era of transition. Throughout the years that it existed as a combination serial; both factors were seen to play a vital role in shaping the serial, both from an industry point-of-view, and as evident in its text. The two perspectives are often contradictory to one another but the co-existence of them is essential to the shaping of the soap opera’s history writing. Gender ties into both angles as the transition added to a growing social panic surrounding the housewife as a pillar for postwar US-American values.