Le myélome multiple (MM) est un cancer hématologique qui se caractérise par une prolifération et une accumulation de cellules plasmocytaires malignes au niveau de la moelle osseuse (MO). Il représente 10% des hémopathies malignes et 2% de la mortalité par cancer dans le monde occidental. La principale conséquence de l’expansion plasmocytaire clonale médullaire est la sécrétion excessive d’une immunoglobuline (Ig) unique qui va être à l’origine du caractère multi-symptomatique de cette pathologie. Ainsi, les manifestations du MM se caractérisent par des lésions osseuses, une atteinte rénale, une anémie, une hypercalcémie et une immunodéficience humorale conduisant à des infections récidivantes. Son pronostic est mauvais avec une médiane de survie qui se situe entre cinq et sept ans sous chimiothérapie qui vise à éliminer les cellules plasmocytaires malignes.A partir de la lignée U266 de myélome, nous avons dérivé des clones résistants au bortézomib (R6). Grâce à une analyse par biopuces à ADN, nous avons identifié 160 gènes significativement régulés dans les cellules R6 par rapport à la lignée parentale U266. Nous avons établi, par une approche fonctionnelle, que la surexpression de la protéine HspB8 conduit, via l’activation de la dégradation autophagique, à l’élimination des agrégats protéiques et à compenser l’effet de l’inhibition du protéasome conférant ainsi la résistance des cellules de myélome aux inhibiteurs du protéasome.Dans un second temps, nous nous sommes intéressés à l’implication de la protéine Bcl-B (BCL2L10) dans la pathogenèse du MM. Nous avons confirmé que Bcl-B est impliqué dans la pathogénèse du MM (patients et modèle murin). / Velcade is one of the inescapable drug to treat patient suffering from multiple myeloma (MM) and resistance to this drug represents a major drawback for patients. However, the mechanisms underlying velcade resistance remain incompletely understood. We derived several U266 MM cell clones that resist to velcade. We derived several U266 MM cell clones that resist to velcade. U266- resistant cells were resistant to velcade-induced cell death but exhibited a similar sensitivity to various proapoptotic stimuli. Careful analysis of proteosomal subunits and proteasome enzymatic activities showed that neither the composition nor the activity of the proteasome was affected in velcade-resistant cells.In addition, pangenomic profiling of velcade-sensitive and resistant cells showed that the small heat shock protein HSPB8 was overexpressed in resistant cells. Finally, gain and loss of function experiment demonstrated that HSPB8 is a key factor for velcade resistance. In conclusion, HSPB8 plays an important role for the elimination of aggregates in velcade-resistant cells that contributes to their enhanced survival. Multiple myeloma (MM) evolves from a premalignant condition known as monoclonal gammopathy of undetermined significance (MGUS). However, the factors underlying the malignant transformation of plasmocytes in MM are not fully characterized. We report an MM phenotype in transgenic mice with Eμ-directed expression of the Bcl-B protein. With age, Eμ-bcl-b transgenic mice develop the characteristic features of human MM. In addition, this MM-like disease is serially transplantable, underlying the tumoral origin of plasmocytes.

Submitted by ANA CLAUDIA SILVA BRAGA null (anabragga@gmail.com) on 2017-08-31T16:15:53Z No. of bitstreams: 1 Tese Doutorado Ana Claudia Silva Braga.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-09-01T14:16:37Z (GMT) No. of bitstreams: 1 braga_acs_dr_sjrp.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) / Made available in DSpace on 2017-09-01T14:16:37Z (GMT). No. of bitstreams: 1 braga_acs_dr_sjrp.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) Previous issue date: 2017-08-04 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O vírus da hepatite C (HCV) causa a doença da Hepatite C e estima-se que cerca de 3% da população mundial esteja infectada com o vírus. A infecção por HCV promove a alteração na expressão de várias proteínas celulares. Estudos têm demonstrado que muitas proteínas de choque térmico (HSPs) possuem um perfil de expressão alterado na presença do vírus e algumas HSPs interagem diretamente com proteínas do HCV. Assim, o presente estudo teve como objetivo avaliar in vitro os níveis de expressão de proteínas de choque térmico na presença e ausência de HCV. Com este propósito, células de hepatoma humano Huh7.5 e células Huh7.5 infectadas com o vírus (HCV JFH-1) foram submetidas à extração de RNA e síntese de cDNA. A expressão diferencial de 84 HSPs e chaperonas foi avaliada por qPCR Array. Os resultados demonstram que cinco genes apresentaram expressão aumentada (em Log2 2), enquanto outros cinco apresentaram expressão reduzida. Para validar estes resultados os 10 genes diferencialmente expressos foram testados por qPCR em três modelos celulares para o HCV: células contendo replicon subgenômico do HCV (SGR-JFH-1), células infectadas com JFH-1 (ambos do genótipo 2a) e células contendo o replicon subgenômico S52 (genótipo 3). O gene HSPB8 mostrou expressão aumentada nos três modelos testados, condizente com os resultados obtidos por qPCR Array. Em seguida, promovemos o silenciamento de HSPB8 e foi observado um aumento na replicação viral. Em contraste, quando aumentamos a expressão de HSPB8, o HCV teve uma diminuição na taxa de replicação. O mesmo procedimento foi adotado para o gene DNAJC5B, validado no modelo viral genótipo 3, e o HCV mostrou padrão de replicação semelhante ao observado para o gene anterior. Esses resultados sugerem que HSPB8 pode atuar como um fator intracelular contra a replicação do vírus da hepatite C e DNAJC5B apresenta a mesma função, mas específico para o genótipo 3. Também avaliamos interações diretas com proteínas do HCV e os resultados demonstraram uma interação física entre a proteína NS4B de HCV e HSPB8. Esses resultados podem contribuir para uma melhor compreensão dos mecanismos envolvidos na replicação do HCV. / Hepatitis C virus (HCV) causes Hepatitis C disease and it is estimated that about 3% of world population are infected with the virus. HCV infection promotes alteration in the expression of several cellular proteins. Studies have shown that many heat shock proteins (HSPs) have an altered expression profile in the presence of the virus and some HSPs interact directly with HCV proteins. Thus, the present study aimed to evaluate in vitro the expression levels of heat shock proteins in the presence and absence of HCV. With this purpose, human hepatoma Huh7.5 cells and Huh7.5 cells infected with the virus (HCV JFH-1) were subjected to RNA extraction and cDNA synthesis. The differential expression of 84 HSPs and chaperones was assessed by qPCR Array. The results demonstrate that five genes showed increased expression (over Log2 2), while five other presented reduced expression. To validate these results, the 10 differentially expressed genes were tested by real-time PCR in three different HCV cell culture models: subgenomic HCV replicon cells (SGR-JFH-1), JFH-1 infected cells (both genotype 2a) and subgenomic S52 cells (genotype 3). The HSPB8 gene showed increased expression in all of three tested models, consistent with qPCR Array results. Then we promoted the silencing of HSPB8 and observed an increase in viral replication. In contrast, when we increased an expression of HSPB8, HCV had a decrease in replication rate. The same procedure was adopted for the DNAJC5B, validated in the viral model genotype 3, and HCV showed replication pattern similar to that observed for the previous gene. These results suggest that HSPB8 may act as an intracellular factor against hepatitis C virus replication and DNAJC5B have the same function, but genotype 3 specific. We also evaluated direct interactions with HCV proteins and the results demonstrated a physical interaction between the HCV NS4B protein with HSPB8. These results can contribute for a better understanding of the mechanisms involved in HCV replication. / FAPESP: 2013/17253-9