L’apoptose ou mort cellulaire programmée joue un rôle prépondérant dans l’homéostasie cellulaire. Ce processus est très finement régulé par les protéines de la famille BCL-2 qui contrôlent la perméabilité de membrane mitochondriale externe et la libération du cytochrome c, deux événements majeurs précédant la mort cellulaire. Les protéines anti-apoptotiques de la famille BCL-2 contribuent à la tumorigenèse et sont impliquées dans la résistance des cancers aux molécules chimiothérapeutiques ; à ce titre, elles représentent des cibles importantes pour le développement de nouvelles thérapies. BCL2A1 est un membre anti-apoptotique de la famille BCL-2 impliqué dans la chimiorésistance de nombreuses tumeurs. La protéine BCL2A1 a pour caractéristique d’avoir une demi-vie courte due à sa dégradation constitutive par le système ubiquitine-protéasome. Ceci régule la stabilité et la fonction anti-apoptotique de BCL2A1 et représente un mécanisme suppresseur de tumeur majeur. Cependant, les enzymes qui contrôlent les modifications post-traductionnelles impliquées dans l’ubiquitination et la dégradation de BCL2A1 demeurent, à ce jour, inconnues. Dans la présente thèse, nous donnons un aperçu des acteurs et des mécanismes impliqués dans la régulation de l’ubiquitination de BCL2A1. Nous présentons des preuves que TRIM28 est une E3 ubiquitine-ligase pour BCL2A1. En effet, les protéines TRIM28 et BCL2A1 endogènes interagissent ensemble au niveau des mitochondries et la déplétion de TRIM28 diminue l’ubiquitination de BCL2A1. Nous montrons aussi que TRIM17 stabilise BCL2A1 en empêchant son interaction avec TRIM28 et son ubiquitination médiée par TRIM28, et que l’activité de GSK3 est impliquée dans l’inhibition de la dégradation de BCL2A1. Ainsi, BCL2A1 et son proche homologue MCL-1 sont régulés par des facteurs communs mais de façon opposé. Finalement, la surexpression de TRIM28 ou l’inactivation de TRIM17 diminue le niveau protéique de BCL2A1 et restaure la sensibilité des cellules de mélanomes aux thérapies utilisant des inhibiteurs de la kinase BRAF. Globalement, nos résultats décrivent un rhéostat moléculaire au sein duquel deux protéines de la famille TRIM régulent de façon antagoniste la stabilité de BCL2A1 et modulent ainsi la mort cellulaire. / Apoptosis or programmed cell death plays a crucial role in tissue homeostasis and is regulated by the Bcl-2 proteins, which control mitochondria membrane permeability and cytochrome c release, two events that precede cell demise. Anti-apoptotic Bcl-2 family members can contribute to tumorigenesis and cause resistance to anti-cancer regimens, therefore representing important targets for novel therapeutics. BCL2A1 is an anti-apoptotic member of the BCL-2 family that contributes to chemoresistance in a subset of tumors. BCL2A1 has a short half-life due to its constitutive processing by the ubiquitin-proteasome system. This constitutes a major tumor-suppressor mechanism regulating BCL2A1 function. However, the enzymes involved in the regulation of BCL2A1 protein stability are currently unknown. Here we provide the first insight into the regulation of BCL2A1 ubiquitination. We present evidence that TRIM28 is an E3 ubiquitin-ligase for BCL2A1. Indeed, endogenous TRIM28 and BCL2A1 bind to each other at the mitochondria and TRIM28 knock-down decreases BCL2A1 ubiquitination. We also show that TRIM17 stabilizes BCL2A1 by blocking TRIM28 from binding and ubiquitinating BCL2A1, and that GSK3 is involved in the phosphorylation-mediated inhibition of BCL2A1 degradation. BCL2A1 and its close relative MCL1 are thus regulated by common factors but with opposite outcome. Finally, overexpression of TRIM28 or knock-out of TRIM17 reduced BCLA1 protein levels and restored sensitivity of melanoma cells to BRAF-targeted therapy. Therefore, our data describe a molecular rheostat in which two proteins of the TRIM family antagonistically regulate BCL2A1 stability and modulate cell death.Sommaire

A produção comercial de bovinos produzidos por transferência nuclear de célula somática permitiu o desenvolvimento de novo modelo no estudo da placenta. A necessidade da cesariana para o nascimento do bezerro favorece a coleta das membranas fetais. Alterações como número total de placentônios, tamanho, formato, árvores vilosas, cordão umbilical, assim como alterações no recém nascido já foram mostradas anteriormente. Gestações produzidas por transferência de núcleo freqüentemente apresentam retenção de placenta. Durante a palpação retal após dois dias da cesariana notou-se placentônios sem diminuição no tamanho e sem alteração na consistência. A observação da ausência de regressão levaram à formulação da hipótese de placentônios provenientes de gestações produzidas por transferência de núcleo apresentam menor freqüência de apoptose quando comparados com placentônios provenientes de gestações produzidas por monta natural. Para testar essa hipótese 15 placentônios provenientes de clones e 3 placentônios provenientes de monta natural foram coletados e congelados em nitrogênio líquido. A extração de RNA foi realizada mediante protocolo Trizol (Invitrogen, Brasil) e a reação de trascriptase reversa feita com Kit Impron II (Promega, Brasil), usando 1µg de RNA total. A quantificação relativa foi desenvolvida no "7500 Real Time PCR System" (Applied Biosystems, EUA), em reação de 25µL contendo 1X "Power SYBR Green PCR Master Mix" (Applied Biosystems, EUA), 1µg de cDNA e 0,6µM de cada primer (BAX, BCL2, e GAPDH). Para a quantificação dos genes ITM2B e PI3K, também se utilizou o gene GAPDH como gene endógeno e reação contendo 1x "Taqman Universal Master Mix" (Applied Biosystems, EUA), 40ng de cDNA, 0,72µM de cada primers (ITM2B, PI3K e GAPDH) e 0,2µM de cada sonda. O programa "LinRegPCR" (RAMAKERS et al., 2003) foi usado para o cálculo da eficiência individual de cada reação e para o cálculo estatístico usou-se o programa "REST2005" (PFAFFL et al., 2002). Os resultados mostraram que o gene BAX possui redução da expressão relativa no grupo transferência de núcleo (P=0,04), enquanto os genes BCL2, ITM2B e PI3K foram iguais entre grupo transferência de núcleo e monta natural. A relação BAX/BCL2 foi maior que 1 em 12 dos indivíduos analisados 12/15 (80%). Nenhuma diferença significativa foi encontrada quando comparadas variáveis como sexo, sobrevivência e peso ao nascimento para todos os genes estudados. A redução da expressão do BAX no grupo transferência de núcleo sugere que a apoptose é menor neste grupo e a relação BAX/BCL2 indica que a redução na taxa de morte celular programada pode ser responsável pela redução na taxa de regressão dos placentônios. / The study of placenta from somatic cell nuclear transfer pregnancy originated a new model of study after the commercial production of bovine nuclear transfer clones, this technique requires caesarean section, that allows collection of fetal membranes. Alterations as differences in the placentomes total number, size, villous trees, umbilical cord and new born alterations were already described in NT placenta. These nuclear transfer gestations frequently have placental retention and during rectal palpation two days after caesarean section, we observed placentomes with normal characteristics as before section, suggesting an absence of recending. This information about the receding absence lead us to hypothesize that placentomes produced from nuclear transfer pregnancies show lower frequency of apoptosis thus produced from natural. For testing this hypothesis we collected 15 fragments of placentomes from nuclear transfer fetus and 3 of natural mating. The RNA extraction was performed with Trizol (Invitrogen, Brazil) protocol and the reverse transcriptase by Impon II (Promega, Brazil), using 1µg of total RNA. We performed the real time relative quantification technique in the "7500 SDS System" (Applied Biosystems, USA) to investigate the mRNA expression of BAX, BCL2 as apoptosis genes and GAPDH as endogenous gene, with concentrations of 1X Power SYBR Green PCR Master Mix (Applied Biosystems, USA), 1µg of cDNA and 0.6µM of each primer (BAX, BCL2, and GAPDH) in 25µL of total reaction. The ITM2b and PI3K as apoptosis genes and GAPDH as endogenous gene with concentrations of 1x Taqman Universal Master Mix (Applied Biosystems, USA), 40ng of cDNA, 0.72µM of primers (ITM2B, PI3K and GAPDH) and 0.2µM of each probe. The "LinRegPCR" software (RAMAKERS et al., 2003) was used for individual efficiency calculation and the "REST2005" (PFAFFL et all, 2002) software for statistical analysis, using the standard efficiency. The results show that BAX gene is down regulated in the nuclear transfer group (P=0.04), while the BCL2, ITM2B and PI3K are equal in nuclear transfer and natural mating groups. The relation BAX/BCL2 was greater than 1 in 12 nuclear transfer samples 12/15 (80%). No significant difference was found when evaluated variables such as survival, calv sex, birth weight for all studied genes. The lower expression of BAX gene in nuclear transfer groups suggests that apoptosis is lower in this group and the relation BAX/BCL2 is indicative that a lower rate of apoptosis may be responsible for the lower rate of placentome receding.

A Leucose Enzoótica Bovina é uma doença crônica de ocorrência mundial que acomete bovinos. Seu agente etiológico é um retrovírus do mesmo grupo dos que causam a leucemia linfocítica crônica e a AIDS, importantes doenças que acometem humanos. Os retrovírus são vírus que se integram ao DNA do hospedeiro e desta forma são potencialmente mutagênicos. Os bovinos infectados podem desenvolver uma linfocitose persistente e/ou linfoma. A gênese das neoplasias está nas mutações de classes de genes como os proto-oncogenes, genes supressores de tumor, genes de reparo do DNA e genes que controlam a apoptose. O gene p53 acumula algumas funções como de parada do ciclo celular, estimulo ao reparo do DNA ou indução da célula a apoptose. O gene bcl-2 é conhecidamente um gene que regula a apoptose. A interferência no processo de apoptose através de mutações provocadas pelos retrovírus nos genes p53 e bcl-2 é um dos mecanismos carcinogênicos associados a LEB. As mutações no gene p53 acabam promovendo o aumento de sua meia vida e em bcl-2 o aumento em sua expressão. Devido a essas alterações o aumento na expressão do gene bcl-2 e o aumento da meia vida da p53 podem ser avaliadas pela técnica de imunoistoquímica. O presente trabalho avaliou as proteínas p53 e bcl-2 em amostras de bovinos infectados pelo VLB através da técnica de imunoistoquímica. Foram pré-definidos escores de imunomarcação em amostras de animais não infectados. Os escores apresentados pelos animais infectados pelo VLB foram significativamente maiores que os animais do grupo controle. Os valores percentuais de imunomarcação apresentados para bcl-2 foram de 50,26 ± 12,25 do grupo afetado contra 10,86 ± 3,30 do grupo controle. Para p53 somente o grupo afetado apresentou imunomarcação, ficando em zero a imunomarcação do grupo controle. O valor percentual médio do grupo afetado foi de 55,95 ± 7,07. A diferença significativa entre os percentuais dos grupos controle e afetado confirma que os genes p53 e bcl-2 podem estar envolvidos no processo de linfomagênese da LEB. / The Enzootic Bovine Leukosis (EBL) is a chronic disease with mundial occurrence in bovines. Its etiologic agent is a retrovirus of the same group that promotes the chronic lymphocytic leukemia and HIV, that wich are important diseases in humans. The retrovirus integrates into the host DNA and thus they are potential mutagenic. The infected bovines can develop a persistent lymphocytosis and/or lymphoma. The neoplasm genesis is associated with genes mutation like the protooncogenes, tumor suppressor, DNA repair and apoptosis control. The p53 gene has some functions how cell cycle arrest, DNA repair stimulus or apoptosis induced. The interference in the apoptosis process through mutations stimulates about the retrovirus in the p53 and bcl-2 genes are the carcinogenic mechanisms associated to EBL. The mutations in the p53 gene promote the increase in its half life and increase of bcl-2 expression. Due this alterations the increase of bcl-2 gene expression and p53 half-life can be evaluate by immunohistochemistry. This work evaluated the bcl-2 and p53 proteins in EBL infected bovines tissue samples by immunohistochemistry. We determined scores of immunomarked in non-infected animal\'s samples. The scores showed by the EBL infected animals were significantly higher than in control group. The infected bovines showed 50.26 ± 12.25% of positive cells to bcl-2 while the control group showed 10.86 ± 3.3%. Only the infected animals presented reactivity against p53, whereas the control group no showed reaction. These results confirm that the p53 and bcl-2 genes can be involved in a process of EBL leukemogenesis.

A Leucose Enzoótica Bovina é uma doença crônica de ocorrência mundial que acomete bovinos. Seu agente etiológico é um retrovírus do mesmo grupo dos que causam a leucemia linfocítica crônica e a AIDS, importantes doenças que acometem humanos. Os retrovírus são vírus que se integram ao DNA do hospedeiro e desta forma são potencialmente mutagênicos. Os bovinos infectados podem desenvolver uma linfocitose persistente e/ou linfoma. A gênese das neoplasias está nas mutações de classes de genes como os proto-oncogenes, genes supressores de tumor, genes de reparo do DNA e genes que controlam a apoptose. O gene p53 acumula algumas funções como de parada do ciclo celular, estimulo ao reparo do DNA ou indução da célula a apoptose. O gene bcl-2 é conhecidamente um gene que regula a apoptose. A interferência no processo de apoptose através de mutações provocadas pelos retrovírus nos genes p53 e bcl-2 é um dos mecanismos carcinogênicos associados a LEB. As mutações no gene p53 acabam promovendo o aumento de sua meia vida e em bcl-2 o aumento em sua expressão. Devido a essas alterações o aumento na expressão do gene bcl-2 e o aumento da meia vida da p53 podem ser avaliadas pela técnica de imunoistoquímica. O presente trabalho avaliou as proteínas p53 e bcl-2 em amostras de bovinos infectados pelo VLB através da técnica de imunoistoquímica. Foram pré-definidos escores de imunomarcação em amostras de animais não infectados. Os escores apresentados pelos animais infectados pelo VLB foram significativamente maiores que os animais do grupo controle. Os valores percentuais de imunomarcação apresentados para bcl-2 foram de 50,26 ± 12,25 do grupo afetado contra 10,86 ± 3,30 do grupo controle. Para p53 somente o grupo afetado apresentou imunomarcação, ficando em zero a imunomarcação do grupo controle. O valor percentual médio do grupo afetado foi de 55,95 ± 7,07. A diferença significativa entre os percentuais dos grupos controle e afetado confirma que os genes p53 e bcl-2 podem estar envolvidos no processo de linfomagênese da LEB. / The Enzootic Bovine Leukosis (EBL) is a chronic disease with mundial occurrence in bovines. Its etiologic agent is a retrovirus of the same group that promotes the chronic lymphocytic leukemia and HIV, that wich are important diseases in humans. The retrovirus integrates into the host DNA and thus they are potential mutagenic. The infected bovines can develop a persistent lymphocytosis and/or lymphoma. The neoplasm genesis is associated with genes mutation like the protooncogenes, tumor suppressor, DNA repair and apoptosis control. The p53 gene has some functions how cell cycle arrest, DNA repair stimulus or apoptosis induced. The interference in the apoptosis process through mutations stimulates about the retrovirus in the p53 and bcl-2 genes are the carcinogenic mechanisms associated to EBL. The mutations in the p53 gene promote the increase in its half life and increase of bcl-2 expression. Due this alterations the increase of bcl-2 gene expression and p53 half-life can be evaluate by immunohistochemistry. This work evaluated the bcl-2 and p53 proteins in EBL infected bovines tissue samples by immunohistochemistry. We determined scores of immunomarked in non-infected animal\'s samples. The scores showed by the EBL infected animals were significantly higher than in control group. The infected bovines showed 50.26 ± 12.25% of positive cells to bcl-2 while the control group showed 10.86 ± 3.3%. Only the infected animals presented reactivity against p53, whereas the control group no showed reaction. These results confirm that the p53 and bcl-2 genes can be involved in a process of EBL leukemogenesis.

Les cancers du sein sont une pathologie complexe et très hétérogène qui peut-être divisée en plusieurs sous-types selon leurs caractéristiques biologiques. Parmi ces sous-types, les cancers dits “triple négatifs” (TN) sont caractérisés par un marquage immunohistochimique négatif pour les récepteurs à l’oestrogène et à la progestérone et ne présentent pas de surexpression de HER2. Ces tumeurs très agressives représentent 10 à 20% des cancers du sein et sont actuellement traitées par chimiothérapie classique contrairement aux tumeurs non-TN qui bénéficient de thérapies ciblées (traitements hormono-dépendants, anticorps neutralisants). Un taux élevé de rechute et de métastases dans les cinq ans après diagnostic est observé chez les patientes TN, en lien avec le développement de résistances à la chimiothérapie. De ce fait, la compréhension des mécanismes moléculaires impliqués dans ce processus est importante pour le développement de meilleures thérapies. Les études précédemment menées par notre équipe sur le récepteur de mort CD95 et son ligand, le CD95L, ont mis en évidence leur fonction pro-oncongénique dans les cancers TNs. En effet, en comparaison aux patientes non-TNs, les patientes TNs présentent un taux plus élevé de CD95L (cl-CD95L) dans leur sérum, ce qui est associé à un mauvais pronostic. De plus, in vivo, le cl-CD95L promeut la dissémination métastatique des cellules TNs à travers la formation d’un complexe appelé MISC (Motility-Inducing Signalling Complex) et l’induction de la voie non-apoptotique PI3K/Akt/mTOR. Bien que ces nouvelles données enrichissent notre compréhension du processus oncogénique des cancers TNs, beaucoup reste à faire pour le développement de nouvelles thérapies ciblées. Deux axes de recherche ont été abordés durant mes travaux de thèse. Le premier axe s’inscrit dans la continuité des résultats précédemment obtenus par l’équipe et a consisté à développer de nouveaux inhibiteurs pour bloquer le processus de migration induit par le récepteur de mort. Le second axe propose de définir comment les acteurs majeurs de la machinerie apoptotique, et plus particulièrement les protéines anti-apoptotiques Bcl-2 et BclxL, contribuent à promouvoir la dissémination métastatique des cellules cancéreuses mammaires. Nos travaux ont mis en évidence que l’inhibition de ces membres de la famille Bcl-2, par l’utilisation de BH3 mimétiques, pourrait s’avérer être une stratégie thérapeutique originale pour prévenir la dissémination de métastases chez les patientes TNs. / Breast cancer represents a complex and heterogeneous pathology that can be divided in many subtypes according to biological characteristics. Among them, triple negative breast cancers (TNBCs) are characterized by a negative immunohistochemical staining for estrogen (ER) and progesterone (PR) receptors, and without overexpression of the human epidermal growth factor-2 (HER2). TNBCs represent 10 to 20% of breast cancers and are currently treated by chemotherapy contrary to non-TNBCs, which receive targeted therapies such as hormone therapy or neutralizing antibodies. Compared to non-TNBC, higher rates of relapse and metastasis are observed within five years after diagnosis due to the development of chemotherapy resistance. Therefore, identification of molecular mechanisms implicated in this process is crucial to develop improved therapies. Recent studies carried out by our group on the death receptor CD95 and its ligand CD95L highlighted their pro-oncogenic function in TNBCs. Indeed, in comparison to non-TNBC patients, TNBC patients present higher levels of the naturally cleaved CD95L (cl-CD95L), which is correlated with poor prognosis. Furthermore, cl-CD95L promotes in vivo metastatic dissemination of TNBC cells through the formation of the Motility-Inducing Signalling Complex (MISC) and the induction of the non-apoptotic signaling pathway PI3K/Akt/mTOR. Unless, these new findings increase our understanding of the oncogenic process in TNBC tumours, many things remain to be done to develop new targeted therapies. During my thesis, two lines of research were investigated. The first one, in agreement with previous results obtained by our team, consisted in the development of new inhibitors able to block the migration process induced by the death receptor. The second one was to define how the main actors of the apoptotic machinery, especially anti-apoptotic Bcl-2 and BclxL proteins, promote metastatic dissemination of breast cancer cells. Our work revealed that inhibiting these Bcl-2 family members using BH3-mimetics may turn out to be an original therapeutic strategy to prevent metastatic dissemination in TNBC patients.

Made available in DSpace on 2015-07-22T20:50:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Em casos de carcinoma gástrico, para contribuir ao conhecimento do processo de carcinogênese: Objetivo: Estudar a expressão das proteínas da família Bcl-2 (BcI-2, Bcl-xl, Bak, Bad, Bax). Correlacionar a expressão destas proteínas com 0 índice apoptótico mediante a expressão da proteína Caspase 3 clivada, com 0 índice mit6tico mediante a expressão da proteína Ki-67 e com a expressão da proteína p53. Método: Técnica de arranjo em matriz de amostras teciduais (TMA): em 87 amostras de adenocarcinomas gástricos (grupo teste) e de mucosa gástrica não tumoral (grupo controle) foi avaliada a imuno-expressão das proteínas da família BcI-2 (BeI-2, Bcl-xl, Bak, Bad, Bax), da proteína p53, da proteína caspase 3 e da proteína Ki-67. Resultados: Todas as proteínas examinadas foram observadas nos adenocarcinomas e mucosa não tumoral, porem com diferenças de expressão em relação à porcentagem de positividade e intensidade. Observamos: i) Houve associação entre 0 tamanho do tumor e a proteína p53. ii) Houve associação da proteína Bad no adenocarcinoma com a idade dos pacientes. iii) Associação das proteínas Bax, Bad e Ki-67 com 0 adenocarcinoma de tipo intestinal. iv) As proteínas Bcl-xl, Bak, Bad, p53 e Ki-67 apresentaram diferenças estatisticamente significantes entre a imuno-expressão no tumor e na mucosa não tumoral. v) Associação das proteínas Bax, Bak e Bad na mucosa não tumoral. vi) Não houve correlação da imunoexpressão das proteínas com a sobrevida dos pacientes. Conclusão: A expressão aumentada da proteína Bcl-xl nos adenocarcinomas, com evidente diferença de expressão entre 0 grupo teste e 0 grupo controle, esta relacionada com 0 efeito anti-apoptótico da proteína. A expressão reduzida das proteínas Bak e Bad e a expressão aumentada das proteínas p53 e Ki-67 nos adenocarcinomas demonstram 0 desequilíbrio entre morte e proliferação celular, permitindo 0 crescimento descontrolado das células neoplásicas. / Purpose: To study the immunoexpression of Bcl-2 family proteins (Bcl-2, Bcl-xl, Bax, Bak, Bad) and to evaluate the correlation between the immunoexpression of these proteins with the cleaved caspases 3, Ki-67 and p53 immuno-expression. Methods: A TMA paraffin block was constructed with gastric carcinoma tissue (test group) and normal gastric adjoining mucosa (control group) of 87 patients. The TMA block was submitted to immunohistochemistry for Bcl-2, Bcl-xl, Bax, Bak, Bad, p53 and-cleaved Caspase 3. Results: All studied proteins were present in tumor and normal gastric adjoining mucosa, but with different intensity and amount of positive cells. i) There was an association between tumor size and p53 expression. ii) association between Bad expression in the tumor and patient’s age. iii) Intestinal type adenocarcinoma was positively correlated with the expression of Bax, Bad and Ki-67. iv) The protein Bcl-xl, Bak, Bad, p53 and Ki-67 showed statistically significant differences between the immuno-expression in tumor and normal gastric adjoining mucosa. v) There was an association between the proteins Bax, Bak and Bad expression in the normal gastric adjoining mucosa. vi) No correlation between patient’s survival rates and the expression of the proteins was observed. Conclusions: The higher expression of Bcl-xl protein in adenocarcinoma, the difference of Bcl-xl expression between test group and control group, might be related with the anti-apoptotic effect of this protein. The lower expression of Bak and Bad and the increased expression of p53 protein and Ki-67 protein in adenocarcinomas demonstrate the imbalance between death and cellular proliferation, which allows the uncontrolled tumor cell proliferation. / FAPESP: 04/09932-4 / FAPESP: 06/54187-0 / TEDE

A produção comercial de bovinos produzidos por transferência nuclear de célula somática permitiu o desenvolvimento de novo modelo no estudo da placenta. A necessidade da cesariana para o nascimento do bezerro favorece a coleta das membranas fetais. Alterações como número total de placentônios, tamanho, formato, árvores vilosas, cordão umbilical, assim como alterações no recém nascido já foram mostradas anteriormente. Gestações produzidas por transferência de núcleo freqüentemente apresentam retenção de placenta. Durante a palpação retal após dois dias da cesariana notou-se placentônios sem diminuição no tamanho e sem alteração na consistência. A observação da ausência de regressão levaram à formulação da hipótese de placentônios provenientes de gestações produzidas por transferência de núcleo apresentam menor freqüência de apoptose quando comparados com placentônios provenientes de gestações produzidas por monta natural. Para testar essa hipótese 15 placentônios provenientes de clones e 3 placentônios provenientes de monta natural foram coletados e congelados em nitrogênio líquido. A extração de RNA foi realizada mediante protocolo Trizol (Invitrogen, Brasil) e a reação de trascriptase reversa feita com Kit Impron II (Promega, Brasil), usando 1µg de RNA total. A quantificação relativa foi desenvolvida no "7500 Real Time PCR System" (Applied Biosystems, EUA), em reação de 25µL contendo 1X "Power SYBR Green PCR Master Mix" (Applied Biosystems, EUA), 1µg de cDNA e 0,6µM de cada primer (BAX, BCL2, e GAPDH). Para a quantificação dos genes ITM2B e PI3K, também se utilizou o gene GAPDH como gene endógeno e reação contendo 1x "Taqman Universal Master Mix" (Applied Biosystems, EUA), 40ng de cDNA, 0,72µM de cada primers (ITM2B, PI3K e GAPDH) e 0,2µM de cada sonda. O programa "LinRegPCR" (RAMAKERS et al., 2003) foi usado para o cálculo da eficiência individual de cada reação e para o cálculo estatístico usou-se o programa "REST2005" (PFAFFL et al., 2002). Os resultados mostraram que o gene BAX possui redução da expressão relativa no grupo transferência de núcleo (P=0,04), enquanto os genes BCL2, ITM2B e PI3K foram iguais entre grupo transferência de núcleo e monta natural. A relação BAX/BCL2 foi maior que 1 em 12 dos indivíduos analisados 12/15 (80%). Nenhuma diferença significativa foi encontrada quando comparadas variáveis como sexo, sobrevivência e peso ao nascimento para todos os genes estudados. A redução da expressão do BAX no grupo transferência de núcleo sugere que a apoptose é menor neste grupo e a relação BAX/BCL2 indica que a redução na taxa de morte celular programada pode ser responsável pela redução na taxa de regressão dos placentônios. / The study of placenta from somatic cell nuclear transfer pregnancy originated a new model of study after the commercial production of bovine nuclear transfer clones, this technique requires caesarean section, that allows collection of fetal membranes. Alterations as differences in the placentomes total number, size, villous trees, umbilical cord and new born alterations were already described in NT placenta. These nuclear transfer gestations frequently have placental retention and during rectal palpation two days after caesarean section, we observed placentomes with normal characteristics as before section, suggesting an absence of recending. This information about the receding absence lead us to hypothesize that placentomes produced from nuclear transfer pregnancies show lower frequency of apoptosis thus produced from natural. For testing this hypothesis we collected 15 fragments of placentomes from nuclear transfer fetus and 3 of natural mating. The RNA extraction was performed with Trizol (Invitrogen, Brazil) protocol and the reverse transcriptase by Impon II (Promega, Brazil), using 1µg of total RNA. We performed the real time relative quantification technique in the "7500 SDS System" (Applied Biosystems, USA) to investigate the mRNA expression of BAX, BCL2 as apoptosis genes and GAPDH as endogenous gene, with concentrations of 1X Power SYBR Green PCR Master Mix (Applied Biosystems, USA), 1µg of cDNA and 0.6µM of each primer (BAX, BCL2, and GAPDH) in 25µL of total reaction. The ITM2b and PI3K as apoptosis genes and GAPDH as endogenous gene with concentrations of 1x Taqman Universal Master Mix (Applied Biosystems, USA), 40ng of cDNA, 0.72µM of primers (ITM2B, PI3K and GAPDH) and 0.2µM of each probe. The "LinRegPCR" software (RAMAKERS et al., 2003) was used for individual efficiency calculation and the "REST2005" (PFAFFL et all, 2002) software for statistical analysis, using the standard efficiency. The results show that BAX gene is down regulated in the nuclear transfer group (P=0.04), while the BCL2, ITM2B and PI3K are equal in nuclear transfer and natural mating groups. The relation BAX/BCL2 was greater than 1 in 12 nuclear transfer samples 12/15 (80%). No significant difference was found when evaluated variables such as survival, calv sex, birth weight for all studied genes. The lower expression of BAX gene in nuclear transfer groups suggests that apoptosis is lower in this group and the relation BAX/BCL2 is indicative that a lower rate of apoptosis may be responsible for the lower rate of placentome receding.

Le cancer est un problème majeur de santé public, tuant chaque année plusieurs millions de personnes. L’inhibition de la mort cellulaire programmée, l’apoptose, est considérée comme l’un des paramètres principaux impliqués dans son initiation et son développement. La régulation de la voie intrinsèque (mitochondriale) de l’apoptose est régulée par la famille Bcl-2. Jusqu’à maintenant, on pensait que la protéine PUMA, une protéine pro-apoptotique, était principalement exprimée au niveau mitochondrial. Nous avons montré qu’à l’état basal, PUMA était exprimé au niveau du cytosol des lymphocytes B humains. Cependant, suite à un signal apoptotique, PUMA est capable de transloquer du cytosol à la mitochondrie, de façon indépendante des caspases mais dépendante de l’activation de la MAPKinase p38, permettant ainsi son interaction avec les protéines anti-apoptotiques Bcl-2 et Mcl-1 dont l’inhibition conduit à l’apoptose. Les protéines anti-apoptotiques, Mcl-1 notamment, sont souvent surexprimées dans les tumeurs. Mcl-1 est une protéine à courte demi-vie, rapidement dégradée par le protéasome. Cette dégradation dépend de son ubiquitination réalisée par des E3 ligases (E3). Quelques E3 et une déubiquitinase (DUB), hydrolysant les chaînes d’ubiquitine, régulant l’expression de Mcl-1 ont été décrites. Cependant, ces protéines sont soit très peu exprimées, soit leur inhibition n’a pas d’impact sur l’expression de Mcl-1 dans notre modèle. Nous avons donc entrepris de caractériser de nouvelles E3 et DUB régulant l’ubiquitination de Mcl-1. Après une immunoprécipitation de Mcl-1 dans nos cellules, suivie d’une analyse par spectrométrie de masse, nous avons identifié la DUB USP14. Lorsque son expression est diminuée, l’expression et la stabilité de Mcl-1 augmentent de façon sélective. Nos résultats pourraient contribuer à une approche à double-tranchant dans le traitement du cancer, en retirant les freins à l’apoptose via une diminution de l’expression de Mcl-1 d’une part et en l’activant via PUMA de l’autre. / Cancer is a major public health issue, killing millions of people worldwide each year. The inhibition of apoptosis, a programmed cell death, in its onset and development has been well documented, making it one of the hallmarks of cancer. The regulation of the intrinsic (mitochondrial) pathway of apoptosis is regulated by the Bcl-2 (B cell lymphoma-2) family. Up until now, PUMA, a pro-apoptotic protein, was thought to be mainly expressed at the mitochondria, based on experiments where it had been overexpressed. We showed that endogenous PUMA is mainly expressed in the cytosol of activated or resting B cells. However, upon apoptotic stress, PUMA was able to translocate from the cytosol to the mitochondria, in a caspase-independent but p38-dependent manner, allowing PUMA to bind and inhibit the anti-apoptotic proteins Bcl-2 and Mcl-1, and thereby leading to cell death. The anti-apoptotic proteins, especially Mcl-1, are often overexpressed in tumors. Mcl-1 is a protein with a short half-life, degraded rapidly by the proteasome. This degradation is ubiquitin-dependent, requiring E3 ligases (E3). A handful of E3s and one deubiquitinase (DUB), that hydrolyses the ubiquitin chains, have been reported to regulate Mcl-1 expression. However, they were either very poorly expressed or their inhibition had no impact on Mcl-1 expression in our model. We thus undertook to characterize new E3s and DUBs mediating Mcl-1 ubiquitination. After an immunoprecipitation of Mcl-1 in our cells, followed by a mass spectrometry analysis, we identified the DUB USP14. When knockdown, Mcl-1 expression was selectively increased and its stability enhanced. Our results could help build “double-edge” therapies, removing the breaks on apoptosis on one hand via Mcl-1 downregulation while activating it on the other via PUMA translocation.

Les homologues Bcl-2 constituent une famille de protéines structurellement apparentées qui jouent un rôle central dans la régulation de l'apoptose. Les mécanismes de régulation de l'apoptose entérocytaire sont peu connus. Afin de mieux comprendre cette régulation, le but de ce travail était d'analyser l'établissement des mécanismes de contrôles de l'apoptose entérocytaire au cours du développement de l'intestin humain. Pour ce faire nous avons étudié l'expression épithéliale des homologues Bcl-2 proapototiques (Bax, Bak et Bad) et anti-apoptotiques (Bcl-2, Mcl-1 et BCI-X L ), ainsi que la molécule associée anti-apoptotique Bag-1, le long de l'axe cryptevillosité et pour les différents segments (jéjunum, iléon et côlon) de spécimens foetaux humains âgés de 9 à 20 semaines de gestation. Dans un premier temps, afin d'analyser l'expression épithéliale des homologues Bcl-2 le long de l'axe crypte-villosité, nous avons effectué des immunofluorescences indirectes en utilisant des anticorps spécifiques pour les homologues étudiés. Dans un deuxième temps, afin d'analyser l'expression épithéliale des homologues Bcl-2, nous avons effectué des Western blots."--Résumé abrégé par UMI.

Plusieurs évidences suggèrent que des membres impliqués dans le contrôle de l'activation de la cascade apoptotique et particulièrement les membres de la famille de protéines Bcl-2 pourraient jouer un rôle dans le phénomène de résistance observé dans les tumeurs ovariennes. Le but de cette étude est de déterminer l'importance relative de la protéine Bcl-2 dans le phénomène de résistance clinique à la chimiothérapie dans le cancer ovarien, plus précisément au cisplatin. L'analyse de l'expression de la protéine Bcl-2 dans des cellules d'ovaire normales et cancéreuses a démontré une surexpression de la protéine dans les cellules cancéreuses par rapport aux cellules d'ovaire normales, mais aucune corrélation entre l'expression de la protéine Bcl-2 et la sensibilité au cisplatin des cellules d'ovaire cancéreuses n'a pu être établie. Pour pouvoir mieux évaluer le rôle de la protéine Bcl-2 dans le phénomène de résistance, nous avons utilisé un anticorps monovalent modifié (scFv) dirigé contre cette dernière. Ce scFv agit essentiellement comme un inhibiteur spécifique de la protéine Bcl-2 ans les cellules.