As estatísticas relacionadas aos cânceres hematológicos indicam que a incidência e mortalidade dessas doenças têm aumentado ao longo dos anos. Embora a maioria dos casos de linfomas e leucemias não possua etiologia definida, sugere-se que fatores genéticos possam estar envolvidos. Nesse contexto, destaca-se a família de proteínas Bcl-2, divididas em anti e pró-apoptóticas. Os genes Bcl-2 e Bcl-XL, membros de uma nova classe de oncogenes, que atuam no mecanismo de morte celular das células cancerígenas, sobretudo apoptose, a qual é controlada por numerosos sinais intra e extracelulares. Uma nova estratégia para o tratamento desta doença inclui a terapia gênica mediada por RNA de interferência, que silencia importantes genes, a exemplo dos genes da família Bcl-2. Visto que o silenciamento isolado de um único gene pode não ter resultados expressivos, o presente trabalho teve por objetivo desenhar um RNA de interferência (RNAi) homólogo a dois tipos distintos de RNA mensageiro (RNAm) e inibir simultaneamente os genes Bcl-2 e Bcl-XL,assim como testar a inibição isolada dos mesmos. Amostras de linhagem tumoral Jurkat e Granta-519 foram avaliadas após transfecção com os seguintes RNA:i Bcl-2,Bcl-XL, Bcl-2/Bcl-XL,Bcl-2+Bcl-XL e scramble. Os nossos achados evidenciam que, na linhagem Granta-519, a sequência do RNAi Bcl-2 inibe, isoladamente ou conjugado ao Bcl-XL, o gene Bcl-2. Deste modo, o RNAi Bcl-2 apresenta-se mais eficiente no mecanismo de silenciamento gênico, uma vez que propicia a morte celular frente a toxicidade do quimioterápico etoposide. / The hematological cancer statistics indicates that its incidence and mortality have increased over the years. Although most cases of lymphomas and leukemias has no definite etiology however is suggested that genetic factors may be involved. In this context there is the Bcl-2 proteins family divided into anti-apoptotic and pro-apoptotic which Bcl-2 and Bcl-XL genes are members of a new class of oncogenes that act in cancer cells death mechanisms, especially apoptosis, that is controlled by numerous intra-and extracellular signals. Among new strategies to treat hematological cancer includes gene therapy mediated by RNA interference, which can decrease expression of genes like Bcl-2 family components. Studies of single gene silencing have not shown significant results so this study aimed to design an RNA interference (iRNA) homologous to two distinct types of messenger RNA (mRNA) and inhibit both genes Bcl-2 and Bcl-XL -XL as well as test the inhibition. Commercial cells Jurkat and Granta-519 were evaluated after transfection with iRNA as follows: Bcl-2, Bcl-XL, Bcl-2/Bcl-XL, Bcl-2+Bcl-XL and scramble. Our findings show that in Granta-519 cell line Bcl-2 RNAi sequence inhibits, alone or conjugated to Bcl-XL, Bcl-2 gene. Thus RNAi Bcl-2 appears more effective in gene silencing mechanism as it promotes cell death due chemotherapeutic agent etoposide toxicity.

As estatísticas relacionadas aos cânceres hematológicos indicam que a incidência e mortalidade dessas doenças têm aumentado ao longo dos anos. Embora a maioria dos casos de linfomas e leucemias não possua etiologia definida, sugere-se que fatores genéticos possam estar envolvidos. Nesse contexto, destaca-se a família de proteínas Bcl-2, divididas em anti e pró-apoptóticas. Os genes Bcl-2 e Bcl-XL, membros de uma nova classe de oncogenes, que atuam no mecanismo de morte celular das células cancerígenas, sobretudo apoptose, a qual é controlada por numerosos sinais intra e extracelulares. Uma nova estratégia para o tratamento desta doença inclui a terapia gênica mediada por RNA de interferência, que silencia importantes genes, a exemplo dos genes da família Bcl-2. Visto que o silenciamento isolado de um único gene pode não ter resultados expressivos, o presente trabalho teve por objetivo desenhar um RNA de interferência (RNAi) homólogo a dois tipos distintos de RNA mensageiro (RNAm) e inibir simultaneamente os genes Bcl-2 e Bcl-XL,assim como testar a inibição isolada dos mesmos. Amostras de linhagem tumoral Jurkat e Granta-519 foram avaliadas após transfecção com os seguintes RNA:i Bcl-2,Bcl-XL, Bcl-2/Bcl-XL,Bcl-2+Bcl-XL e scramble. Os nossos achados evidenciam que, na linhagem Granta-519, a sequência do RNAi Bcl-2 inibe, isoladamente ou conjugado ao Bcl-XL, o gene Bcl-2. Deste modo, o RNAi Bcl-2 apresenta-se mais eficiente no mecanismo de silenciamento gênico, uma vez que propicia a morte celular frente a toxicidade do quimioterápico etoposide. / The hematological cancer statistics indicates that its incidence and mortality have increased over the years. Although most cases of lymphomas and leukemias has no definite etiology however is suggested that genetic factors may be involved. In this context there is the Bcl-2 proteins family divided into anti-apoptotic and pro-apoptotic which Bcl-2 and Bcl-XL genes are members of a new class of oncogenes that act in cancer cells death mechanisms, especially apoptosis, that is controlled by numerous intra-and extracellular signals. Among new strategies to treat hematological cancer includes gene therapy mediated by RNA interference, which can decrease expression of genes like Bcl-2 family components. Studies of single gene silencing have not shown significant results so this study aimed to design an RNA interference (iRNA) homologous to two distinct types of messenger RNA (mRNA) and inhibit both genes Bcl-2 and Bcl-XL -XL as well as test the inhibition. Commercial cells Jurkat and Granta-519 were evaluated after transfection with iRNA as follows: Bcl-2, Bcl-XL, Bcl-2/Bcl-XL, Bcl-2+Bcl-XL and scramble. Our findings show that in Granta-519 cell line Bcl-2 RNAi sequence inhibits, alone or conjugated to Bcl-XL, Bcl-2 gene. Thus RNAi Bcl-2 appears more effective in gene silencing mechanism as it promotes cell death due chemotherapeutic agent etoposide toxicity.

Les facteurs de transcription de la famille MAF sont impliqués d’une part au cours du développement dans des processus de différenciation terminale et d’autre part dans la carcinogenèse. Un découplage fonctionnel est observé. En effet, les gènes cibles mis en jeu au cours des processus cancéreux et de différenciation terminale semblent différents. L'activité oncogénique des protéines MAF dépend du contexte cellulaire. Ainsi, il a été proposé qu'elles exercent leur activité oncogénique dans des tissus où elles ne sont pas exprimées normalement. De plus, leur pouvoir transformant est régulé par phosphorylation. Mon travail de thèse a porté sur le rôle oncogénique de ces protéines en étudiant ces deux aspects. J'ai identifié le rôle oncogénique de NRL, un membre de la famille MAF, dans le Médulloblastome et j'ai étudié la régulation par phosphorylation des MAF dans le Myélome Multiple. L’activité oncogénique du facteur de transcription NRL, connu pour son rôle dans la différenciation terminale d’un type cellulaire de la rétine, n’avait jamais été établie. Alors que NRL n'est pas exprimé dans le cervelet sain, des études de transcriptome ont montré que NRL est surexprimé dans un sous-groupe agressif de médulloblastome, une tumeur pédiatrique du cervelet. J’ai montré pour la première fois que NRL est un oncogène. Il participe directement à la carcinogénèse de médulloblastomes, en protégeant les cellules de l’apoptose, à travers la régulation d’un membre anti-apoptotique de la famille BCL, BCL-XL. L’inhibition des protéines BCL pourrait constituer une stratégie thérapeutique potentielle dans des Médulloblastomes de mauvais pronostic et résistants aux traitements classiques.Au sein du laboratoire, un projet, auquel j’ai participé, a porté sur la régulation de l’activité oncogénique des facteurs de transcription MAF, dans des Myélomes Multiples, de mauvais pronostic. Ce travail établit que l’activité oncogénique de deux membres de la famille MAF, MAFB et c-MAF, est régulée par phosphorylation induite par la Ser/Thr kinase GSK3. La phosphorylation des facteurs de transcription MAF augmente leur activité oncogénique et paradoxalement induit leur dégradation par le protéasome. Ce travail a permis d’identifier un mécanisme de résistance potentiel de ces tumeurs et de proposer une nouvelle approche thérapeutique, basée sur l’inhibition de la phosphorylation des protéines MAF. / MAF (MusculoAponeurotic Fibrosarcoma) transcription factors are involved in terminal differentiation during normal development, and also in oncogenesis. A functional uncoupling is observed between these two functional activities. Indeed, target genes involved in cancer or terminal differentiation look different. The oncogenic activity of MAF proteins is dependent on the cellular context. Thus, it was suggested that they exert their oncogenic activity in tissues where they are not normally expressed. In addition, their transforming ability is regulated by phosphorylation. My work focused on the oncogenic role of these proteins by studying these two aspects. I identified the oncogenic role of NRL, a member of the MAF family in Medulloblastoma and I studied the regulation by phosphorylation of MAF in Multiple Myeloma.The oncogenic activity of the transcription factor NRL, known for its role in the terminal differentiation of a cell type in the retina, has never been established. While NRL is not expressed in the healthy cerebellum, transcriptome studies showed that NRL is overexpressed in an aggressive subgroup of medulloblastoma, a pediatric tumor of the cerebellum. I showed for the first time that NRL is an oncogene. NRL is directly involved in the carcinogenesis of medulloblastoma, protecting cells from apoptosis through regulation of an anti-apoptotic member of the BCL family, BCL-XL. Inhibition of the protein BCL could be a potential therapeutic strategy for medulloblastoma with a poor prognosis and resistant to conventional therapies.In the laboratory, I also participated in a project which focused on the regulation of the oncogenic activity of transcription factors, MAF, in rarely curable multiple myeloma. This work establishes that the oncogenic activity of two MAF members, MAFB and c-MAF, are regulated by phosphorylation-induced Ser/Thr kinase GSK3. Phosphorylation of transcription factors MAF increases their oncogenic activity and paradoxically induces their degradation by the proteasome. This work has identified a potential mechanism of resistance of these tumors providing a new therapeutic approach, based on the inhibition of MAF protein phosphorylation.