京都大学 / 新制・課程博士 / 博士(医学) / 甲第23409号 / 医博第4754号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 武藤 学, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

As células-satélite musculares vêm sendo muito estudadas em diferentes pesquisas, especialmente com o objetivo de aumentar a compreensão do mecanismo de sua ação na regeneração muscular e as respectivas implicações nas diferentes miopatias, visando identificar possíveis alvos terapêuticos. Dois modelos para distrofias, os camundongos Largemy d e Lama2dy2j/J possuem um padrão de degeneração intenso e bastante semelhante, mas com diferenças na expressão de genes envolvidos na cascata de regeneração. Por isso, estes constituem interessantes modelos para o estudo de possíveis diferenças no mecanismo de ativação e atuação das células-satélite no músculo distrófico. Assim, os objetivos específicos deste projeto consistiram em: 1) isolar e caracterizar por citometria de fluxo as populações de células-satélite dos modelos Largemy d e Lama2dy2j/J em comparação com o normal C57Black6, quanto a expressão de marcadores de miogênese e de células-tronco pluripotentes; e 2) estudar e comparar o perfil de expressão gênica destas populações de células satélites através de microarray de expressão. Na caracterização fenotípica das células isoladas do músculo normal, aquelas que aderem mais precocemente (PP1) e mais tardiamente (PP2) mostram um padrão fenotípico semelhante entre si e com características mais próximas às miogênicas. Já a população de células de adesão bem mais tardia (PP6) apresentou um padrão de marcação misto, mantendo as características miogênicas, mas apresentando tambem padrão de células-tronco mesenquimais, sugerindo o fenótipo de células mais imaturas. Nos músculos distróficos, identificamos diferenças na constituição do pool de células presentes inicialmente no músculo, onde na linhagem Lama2dy2j/J há indícios de uma população em estágio proliferativo, enquanto que na linhagem Largemy d há presença de células com maior imaturidade. Os resultados da analise de expressão gênica nas populações caracterizadas se mostraram concordantes com os fenótipos celulares avaliados por citometria. Identificamos a hipo-expressão de genes ligados à regeneração e remodelamento muscular em ambos modelos distróficos. Considerando os genes diferentemente expressos somente em cada um dos modelos, os resultados sugerem ativação de proliferação celular e inibição da diferenciação na linhagem Lama2dy2j/J e distúrbios na miogênese na linhagem Largemy d. Assim, pudemos identificar vias importantes alteradas em cada um dos modelos que explicam parte das diferenças encontradas nos trabalhos anteriores / Muscle satellite cells have been widely studied, especially to understand their mechanism of action in muscle regeneration and correspondent implications in the different dystrophic processes, aiming the identification of potential therapeutic targets. Two mice models for muscular dystrophies, Largemyd and Lama2dy2j/J, have a pattern of an intense and very similar degeneration, but with differences in the expression of genes involved in the regeneration cascade. Therefore, they are interesting models to study possible differences in the mechanism of activation and action of satellite cells in the dystrophic muscle. The main objectives of this project are: 1) to isolate and characterize by flow cytometry, populations of satellite cells from Largemyd and Lama2dy2j/J models, as compared to normal C57Black6, evaluating the presence of myogenic and pluripotent stem cells markers; and 2) to study and compare gene expression profiles of these populations of satellite cells using microarray technique. In the phenotypic characterization of cells harvested from normal muscle, both faster (PP1) and slower (PP2) populations to adhere in culture flasks show similar phenotypic characteristics, which were closer to myogenic phenotype. On the other hand, the population of cells with very delayed adhesion ability (PP6) presented a mixed pattern, maintaining the myogenic characteristics, but associated to positive mesenchymal stem cell´s markers, suggesting a phenotype of more immature cells. In dystrophic muscles, we could identify differences in the constitution of the first pool of cells present in the Lama2dy2j/J muscle where there is evidence of a population in proliferative stage, while in the Largemyd strain, we found more immature cells. Gene expression profile in the characterized populations showed consistent concordance with the cellular phenotypes assessed by flow citometry. In both dystrophic models, we identified down-regulated genes related to regeneration and remodeling of the muscle. Considering only the genes differently expressed in each dystrophic model, data suggest the activation of cell proliferation and inhibition of differentiation pathways in Lama2dy2j/J strain and altered myogenesis in the Largemyd model. Thus, we identified important altered pathways in each of the dystrophic models that could explain most of the differences in gene expression profile in the muscle, described in our previous work

Com a utilização dos atuais protocolos de tratamento, 70-80% dos casos de leucemia linfóide aguda (LLA) na infância têm obtido sobrevida livre de eventos em cinco anos. Entretanto, os 20% restantes, que se mostram resistentes ao tratamento, apresentam recidivas e as causas desse insucesso no tratamento ainda permanecem desconhecidas. Dessa forma, com o intuito de melhor compreender os mecanismos moleculares que participam desse processo, o presente trabalho teve por objetivo avaliar o perfil de expressão de um painel de genes que foram previamente associados à resistência e/ou sensibilidade aos quimioterápicos: prednisona (F8A, CDK2AP1, BLVRB, CD69), vincristina (RPLP2, CD44, TCFL5, KCNN1, TRIM24), daunorrubicina (MAP3K12, SHOC2, PDCH9, EGR1, KCNN4) e asparaginase (GPR56, MAN1A1, CLEC11A, IGFBP7, GATA3). Para a realização do estudo, foram utilizadas inicialmente amostras de medula óssea de pacientes portadores de LLA pertencentes a quatro grandes centros de oncologia pediátrica do Estado de São Paulo e que foram submetidos ao protocolo de tratamento do GBTLI-99. A análise da expressão gênica foi realizada pela técnica de PCR quantitativa em tempo real, utilizando-se o reagente SYBR Green, o gene GUS? como controle endógeno e amostras de medula óssea normais como referência. A análise dos dados de expressão gênica em relação aos diversos parâmetros clínicos e laboratoriais avaliados na LLA, demonstrou associações importantes entre os diversos genes estudados e variáveis clínicas importantes como contagem de glóbulos brancos ao diagnóstico, presença do antígeno CD10 (CALLA), translocação TEL/AML1, presença de doença residual mínima entre outras. Dentre os genes avaliados destacaram -se como possíveis marcadores de bom prognóstico os genes SHOC2 e GPR56. Posteriormente, reavaliou-se o perfil de expressão desses genes em pacientes submetidos ao protocolo de tratamento europeu do grupo BFM com o intuito de verificar o padrão de expressão em pacientes com um background genético distinto e submetidos a um protocolo terapêutico distinto. Os resultados confirmaram os dados encontrados anteriormente e demonstraram a hiperexpressão do gene SHOC2 (que foi previamente associado à sensibilidade à daunorrubicina) associada ao grupo de pacientes bons respondedores, sugerindo a correlação desse gene com critérios favoráveis de prognóstico. Para verificar o nível de interação desse gene avaliou-se ainda a expressão protéica do mesmo, que confirmou os padrões de expressão gênica obtidos por RQ-PCR. A função do gene SHOC2, que embora não esteja completamente elucidada, já foi anteriormente descrita pela literatura, que demonstra a participação do gene no processo de ativação da proteína Erk pela via Ras. Finalmente para melhor compreender os possíveis mecanismos que envolvem o gene SHOC2 no processo de melhor resposta à quimioterapia, utilizou-se a técnica de RNAi para silenciá- lo na linhagem celular leucêmica Jurkat. Os resultados demosntraram a associação da expressão do gene SHOC2 com proliferação celular e também com a indução de apoptose. Esses dados sugerem que a hiperexpressão desse gene pode ser importante para o processo de sensibilidade das células leucêmicas ao tratamento. Como conclusão, este trabalho demonstrou a associação de diversos genes com importantes parâmetros clínicos da LLA e destaca principalmente o papel do gene SHOC2 como possível alvo terapêutico para o tratamento da leucemia linfóide aguda. / Major improvements have been made in the ALL treatment, which achieved successful rates of approximately 80% of long-terms survival. Despite the significant percentage of success, the remaining 20 % still presents treatment failure and the molecular mechanisms involved in the resistance process remains unclear. The present study was undertaken to analyze and validate the gene expression pattern of the previously described genes related to prednisolone (F8A, CDK2AP1, BLVRB, CD69), vincristine (RPLP2, CD44, TCFL5, KCNN1, TRIM24), daunorubicin (MAP3K12, SHOC2, PDCH9, EGR1, KCNN4) and Lasparaginase (GPR56, MAN1A1, CLEC11A, IGFBP7, GATA3) in order to better inderstand these mechanisms. Bone marrow samples of ALL patients, obtained at diagnosis, in four oncology centers and treated according to the Brazilian protocol (GBTLI-99). The relative mRNA expression levels were quantified using real-time PCR analysis. Amplification of the specific sequences was performed with SYBR® Green reagent; GUSB was used as the reference gene and normal bone marrow samples used as calibrator. The expression profile analisis showed important associations among the studied genes and clinical features as WBC count at diagnosis, CALLA, TEL/AML1 translocation and minimal residual disease. Among the analyzed genes, possible therapy targets were found at SHOC2 and GPR56. Further we addressed the expression profile of these genes in ALL patients, treated according to the BFM protocol, which chacarterize a group of distinct genetic\'s background. The results confirmed the data previously obtained. The overexpression of the gene SHOC2, that was primaraly associated to sensibility to dauborubicin, was related to patients who presented good prednisone response, suggesting the correlation of SHOC2 with good prognostic factors. In order to acess the interaction level of this gene, the protein expression was analyzed and confirmed the mRNA expression data. Despite its lack of information, the data on SHOC2 shows its role as na important element in the Erk activation by Ras induced pathway. Finally, to better understand the possible mechanisms which involve SHOC2 gene to the chemotherapy response process, Jurkat cells was transfect with siRNA to silence the gene SHOC2. Further, functional assays were done to characterize the mechanisms involved. The results showed the association of SHOC2 gene expression with processes of cell proliferation and apoptosis induction, thus suggesting that the overexpression of SHOC2 could play an important role in leukemic cell\'s sensibility to chemotherapy agents, and consequently in patients\' treatment outcome. In conclusion, this work demonstrated the association of the expression profile of many genes with important clinical and laboratorial features. Furthermore, this data present the gene SHOC2 as a possible therapy target to acute lymphoblastic leukemia \'s treatment.

Questiona-se um possível papel deletério da endometriose sobre a qualidade oocitária (QO), que é, em grande parte, condicionada pelo papel das células do cumulus. A função dessas células envolve a expressão de diversas moléculas codificadas por genes específicos, regulada a nível de transcrição, pós-transcrição e tradução. Os microRNAs atuam como reguladores póstranscricionais da expressão gênica, de modo que qualquer alteração nesse mecanismo pode levar a anormalidades no desenvolvimento oocitário. Desta forma, propusemos um estudo inédito da análise de microRNAs em células do cumulus (CC) de mulheres inférteis com e sem endometriose com o objetivo de evidenciar processos biológicos e vias de atuação correlacionados com o papel da endometriose na infertilidade e seu possível impacto na aquisição da competência oocitária. Foram incluídas no estudo 15 pacientes inférteis (5 controles com fator tubário e/ou masculino, 5 com endometriose estágios I/II e 5 com endometriose estágios III/IV) submetidas à estimulação ovariana controlada (EOC) para injeção intracitoplasmática de espermatozoide (ICSI). Imediatamente após a captação oocitária, as CCs foram isoladas e armazenadas para extração dos miRNAs. O perfil de 754 miRNAs foi analisado por meio da técnica de TaqMan®Array Human MicroRNA Cards. Considerou-se significativo p<0,05. Os miRNAs hsa-let-7f-1#, hsa-miR-1291, hsa-miR-140-5p, hsa-miR-218, hsa-miR-30b e hsa-miR-629-5p foram identificados menos expressos nas pacientes com endometriose I/II comparadas às controles. Os miRNAs hsa-miR-1291, hsa-miR-187-3p, hsamiR-30b, hsa-miR-532-3p e hsa-miR-629-5p foram identificados menos expressos nas pacientes com endometriose III/IV em relação às controles. Ao comparar-se os grupos endometriose I/II e endometriose III/IV entre si, os miRNAs hsa-miR-187-3p e hsa-miR-532- 3p foram menos expressos e os miRNAs hsa-let-7f-1# e hsa-miR-362-3p mais expressos nas pacientes com endometriose III/IV. A análise de enriquecimento identificou os genes regulados pelos miRNAs e as respectivas vias metabólicas em que estão envolvidos, sugerindo aumento de apoptose, diminuição de proliferação celular, alterações no controle do ciclo celular e no metabolismo energético das CCs de mulheres com a doença inicial e avançada. Os dados apontam para alterações na regulação pós-transcricional em CCs de mulheres inférteis com endometriose inicial e avançada, o que pode afetar processos e vias essenciais à aquisição de competência oocitária e estar envolvido na infertilidade associada à doença. Este estudo contribui para o entendimento da etiopatogênese da infertilidade relacionada à endometriose identificando mecanismos pós-transcricionais relacionados à piora da qualidade gamética nestas mulheres. / It is questioned a possible deleterious role of endometriosis on oocyte quality (OQ), which is largely conditioned by the function of cumulus cells. The role of these cells involve the expression of several molecules encoded by specific genes, regulated at transcription level, post-transcription and translation. The microRNAs act as transcriptional regulators of gene expression, thus any alteration in this mechanism can lead to abnormalities in oocyte development. In this way, we proposed an unpublished study of the microRNAs analysis in cumulus cells (CC) of infertile women with and without endometriosis, aiming to evidence the biological processes and pathways of action correlated with the presence of endometriosis in infertility and its possible impact on the acquisition of oocyte competence. Fifteen infertile patients (5 controls with tubal factor and/or male, 5 with stage I/II of endometriosis and 5 with stages III/IV of endometriosis) submitted to the controlled ovarian stimulation (EOC) for intracytoplasmic sperm injection (ICSI) were included in the study. Immediately after oocyte uptake, CCs were isolated and stored for miRNA extraction. The 754 miRNAs profile was analyzed using the TaqMan®Array Human MicroRNA Cards technique. Significant values were considered when p <0.05. The hsa-miR-218, hsa-miR-301 and hsa-miR-629-5p miRNAs were identified as less expressed in the patients with endometriosis I/II compared to controls. The miRNAs hsa-miR-1291, hsa-miR-187-3p, hsa-miR-30b, hsa-miR-532-3p and hsa-miR- 629-5p were identified as less expressed in patients with endometriosis III/IV compared to controls. Comparing the groups with endometriosis I/II and endometriosis III/IV, hsa-miR-187- 3p and hsa-miR-532-3p miRNAs were less expressed and hsa-let-7f-1# and hsa-miR-362-3p more expressed in patients with endometriosis III/IV. The enrichment analysis identified the genes regulated by the miRNAs and the respective metabolic pathways in which they are involved, suggesting apoptosis increase, decreased cell proliferation, alterations in the cell cycle control and energetic metabolism of the CCs of women with initial and advanced disease. The data point to changes in post-transcriptional regulation in CCs of infertile women with early and advanced endometriosis, which may affect processes and pathways essential for acquiring oocyte competence and resulting in infertility associated with the disease. This study contributes to the understanding of the etiopathogenesis of infertility related to endometriosis by identifying post-transcriptional mechanisms related to the deterioration of quality of gametes in these women.

In recent years, with the microarray technique widely adopted, a large amount of biomedical literatures are published to provide a lot of useful information. However, some relationships among disease, genes and drug are still to be explored, since the authors only focus on part of the significant genes to the disease or the significant genes to the drug but not connect them to obtain new relationships. There are several methods proposed for finding out the hidden relationships, however many of them requires manual involvements. The main objective of this dissertation is to discover the hidden connections between human diseases and genes and the connections between drugs and the same genes. In order achieve this goal, the intermediate nodes (signification genes) must be found first. When a gene has more significant difference in observed group (abnormal patients) than in control group (normal persons), this gene is called significant genes to the disease. These signification genes often play a crucial role in cancer diagnosis and treatment. Via classifying the microarray gene expression data to find these significant genes, doctors can obtain the feasible and appropriate information for treatments that can give to the patients according to their cancer symptoms. A variety of existing classifiers have been proposed for this problem. However, most of them often work inefficiently when attributes grow up over thousands. To further improve the accuracy and the speed of the existing classifiers, a novel microarray attribute reduction scheme (MARS) is proposed for selecting significant genes to the disease. Experimental results demonstrate that combining the proposed scheme with multiclass support vector machine (MCSVM) obtains better performance than other different gene selection methods with the same MCSVM. In addition, the proposed scheme with MCSVM performs better than the results listed in the existing literature.. Furthermore, 19 of 22 genes selected by the proposed scheme in acute lymphoblastic leukemia and acute myeloid leukemia (AML-ALL) dataset are related to the AML and ALL diseases that have been reported in the literatures. Thus the proposed scheme not only can significantly reduce large amount of attributes (genes) for gene expression classification problem, but also increase the classification accuracy. MARS finds related gene set according to a threshold determined by using receiver operating characteristic (ROC) curve. However, it requires repeating the experiment many times to determine the best threshold. Hence, we propose a novel disease-oriented feature selection algorithm (DOFA) to improve MARS. DOFA uses the Genetic Algorithm (GA) in the selection method for automatic picking up the related genes and Support Vector Machine (SVM) and K-nearest-neighborhood (KNN) as the classifier. DOFA is tested on picking up related genes for AML-ALL and Colon datasets. For AML-ALL and Colon datasets, it selects 21 genes and 25 genes, respectively. Based on the literatures, it shows that 20 of 21 genes are related to the disease or cancers related for AML-ALL dataset and one of these genes is still uncertain. And 20 of 25 genes are directly related to the disease colon cancer or cancers related and 5 of these genes are still uncertain. Three more experiments are conducted to verify the discriminability of the genes selected by DOFA. Experimental results all indicate that DOFA obtains better performance than other competing methods. Thus DOFA not only can select the genes related to the diseases, but also increase the classification accuracy. After obtaining the significant gene group, we can further use these genes to obtain the hidden connections. We propose a high specificity literature mining method based on microarray expression profile for discovering hidden connections among disease, drug, and genes. The proposed method can automatically select related genes from the disease or drug microarray expression profiles, and use the disease names or the drug names and gene names or aliases of the selected genes to obtain the related abstract collections. An alias expansion scheme and a weight function are used to eliminate the unrelated literatures. We perform three scenarios to verify the proposed method. Experimental results show that using the proposed method can obtain the hidden connections among diseases, genes and drugs. The (ROC) curve shows that the proposed method can not only find the hidden connections between diseases and drugs but also have high specificity. Concluding this dissertation, our goal is to discover the hidden connections between the diseases and the drugs. In order to achieve this goal, we first proposed MARS to select the significant genes to the diseases. And then, we proposed DOFA to improve the ability of MARS. We proposed a high specificity literature mining method based on microarray expression profile for discovering the hidden connections among diseases, genes, and drugs. The proposed method combines the power of searching significant genes to the disease of DOFA to further obtain the hidden connections. Experimental results show that the proposed method not only can obtain the hidden connections among diseases, genes, and drugs, but also has high specificity.

Sarcomas are rare mesenchymally-derived tumors with limited treatment options. Tyrosine kinases may serve as potential targets for sarcoma therapy because many are mutated or overexpressed in sarcomas and cell lines. One potential molecular target for sarcoma treatment is the Src tyrosine kinase. Three independently synthesized Src kinase inhibitors were evaluated in human sarcoma cell lines. Of the three, dasatinib, provided promising results as a potential sarcoma therapy. Until this study, dasatinib activity had not been characterized in sarcoma cells. Based on our previous findings of Src activation in human sarcomas, we evaluated the effects of dasatinib in twelve sarcoma cell lines. Dasatinib inhibited Src activity and downstream signaling at nanomolar concentrations. Inhibition of Src signaling was accompanied by blockade of cell migration and invasion. Moreover, apoptosis was induced in a subset of bone sarcomas at nanomolar concentrations of dasatinib. Inhibition of Src protein expression by siRNA also induced apoptosis, indicating that these bone sarcoma cell lines are dependent on Src activity for survival. These results demonstrate that dasatinib inhibits migration and invasion of diverse sarcoma cell types, and selectively blocks the survival of bone sarcoma cells. Therefore dasatinib may provide therapeutic benefit by preventing the growth and metastasis of sarcomas. Microarray analysis of the sarcoma cell lines lead to the identification of a molecular signature that successfully predicts response to dasatinib by induction of apoptosis. Components of this molecular signature are expressed in primary human sarcomas. Furthermore, expression of the molecular signature in sarcomas can be utilized to cluster tumors based on theoretical response to dasatinib. While the prediction of response in tumors is theoretical, there is encouraging evidence to support further endeavors into validating the potential of this molecular signature to predict response in patients.Together, these studies reveal that, in cell lines, both constitutive Src activation and the presence of a molecular signature that predicts response to dasatinib are important parameters to consider when selecting dasatinib as a treatment for. Furthermore, novel therapeutic approaches that inhibit Src signaling may selectively induce apoptosis in tumor cells and sensitize to chemotherapy those tumors that contain the relevant molecular signature.

As células-satélite musculares vêm sendo muito estudadas em diferentes pesquisas, especialmente com o objetivo de aumentar a compreensão do mecanismo de sua ação na regeneração muscular e as respectivas implicações nas diferentes miopatias, visando identificar possíveis alvos terapêuticos. Dois modelos para distrofias, os camundongos Largemy d e Lama2dy2j/J possuem um padrão de degeneração intenso e bastante semelhante, mas com diferenças na expressão de genes envolvidos na cascata de regeneração. Por isso, estes constituem interessantes modelos para o estudo de possíveis diferenças no mecanismo de ativação e atuação das células-satélite no músculo distrófico. Assim, os objetivos específicos deste projeto consistiram em: 1) isolar e caracterizar por citometria de fluxo as populações de células-satélite dos modelos Largemy d e Lama2dy2j/J em comparação com o normal C57Black6, quanto a expressão de marcadores de miogênese e de células-tronco pluripotentes; e 2) estudar e comparar o perfil de expressão gênica destas populações de células satélites através de microarray de expressão. Na caracterização fenotípica das células isoladas do músculo normal, aquelas que aderem mais precocemente (PP1) e mais tardiamente (PP2) mostram um padrão fenotípico semelhante entre si e com características mais próximas às miogênicas. Já a população de células de adesão bem mais tardia (PP6) apresentou um padrão de marcação misto, mantendo as características miogênicas, mas apresentando tambem padrão de células-tronco mesenquimais, sugerindo o fenótipo de células mais imaturas. Nos músculos distróficos, identificamos diferenças na constituição do pool de células presentes inicialmente no músculo, onde na linhagem Lama2dy2j/J há indícios de uma população em estágio proliferativo, enquanto que na linhagem Largemy d há presença de células com maior imaturidade. Os resultados da analise de expressão gênica nas populações caracterizadas se mostraram concordantes com os fenótipos celulares avaliados por citometria. Identificamos a hipo-expressão de genes ligados à regeneração e remodelamento muscular em ambos modelos distróficos. Considerando os genes diferentemente expressos somente em cada um dos modelos, os resultados sugerem ativação de proliferação celular e inibição da diferenciação na linhagem Lama2dy2j/J e distúrbios na miogênese na linhagem Largemy d. Assim, pudemos identificar vias importantes alteradas em cada um dos modelos que explicam parte das diferenças encontradas nos trabalhos anteriores / Muscle satellite cells have been widely studied, especially to understand their mechanism of action in muscle regeneration and correspondent implications in the different dystrophic processes, aiming the identification of potential therapeutic targets. Two mice models for muscular dystrophies, Largemyd and Lama2dy2j/J, have a pattern of an intense and very similar degeneration, but with differences in the expression of genes involved in the regeneration cascade. Therefore, they are interesting models to study possible differences in the mechanism of activation and action of satellite cells in the dystrophic muscle. The main objectives of this project are: 1) to isolate and characterize by flow cytometry, populations of satellite cells from Largemyd and Lama2dy2j/J models, as compared to normal C57Black6, evaluating the presence of myogenic and pluripotent stem cells markers; and 2) to study and compare gene expression profiles of these populations of satellite cells using microarray technique. In the phenotypic characterization of cells harvested from normal muscle, both faster (PP1) and slower (PP2) populations to adhere in culture flasks show similar phenotypic characteristics, which were closer to myogenic phenotype. On the other hand, the population of cells with very delayed adhesion ability (PP6) presented a mixed pattern, maintaining the myogenic characteristics, but associated to positive mesenchymal stem cell´s markers, suggesting a phenotype of more immature cells. In dystrophic muscles, we could identify differences in the constitution of the first pool of cells present in the Lama2dy2j/J muscle where there is evidence of a population in proliferative stage, while in the Largemyd strain, we found more immature cells. Gene expression profile in the characterized populations showed consistent concordance with the cellular phenotypes assessed by flow citometry. In both dystrophic models, we identified down-regulated genes related to regeneration and remodeling of the muscle. Considering only the genes differently expressed in each dystrophic model, data suggest the activation of cell proliferation and inhibition of differentiation pathways in Lama2dy2j/J strain and altered myogenesis in the Largemyd model. Thus, we identified important altered pathways in each of the dystrophic models that could explain most of the differences in gene expression profile in the muscle, described in our previous work

Com a utilização dos atuais protocolos de tratamento, 70-80% dos casos de leucemia linfóide aguda (LLA) na infância têm obtido sobrevida livre de eventos em cinco anos. Entretanto, os 20% restantes, que se mostram resistentes ao tratamento, apresentam recidivas e as causas desse insucesso no tratamento ainda permanecem desconhecidas. Dessa forma, com o intuito de melhor compreender os mecanismos moleculares que participam desse processo, o presente trabalho teve por objetivo avaliar o perfil de expressão de um painel de genes que foram previamente associados à resistência e/ou sensibilidade aos quimioterápicos: prednisona (F8A, CDK2AP1, BLVRB, CD69), vincristina (RPLP2, CD44, TCFL5, KCNN1, TRIM24), daunorrubicina (MAP3K12, SHOC2, PDCH9, EGR1, KCNN4) e asparaginase (GPR56, MAN1A1, CLEC11A, IGFBP7, GATA3). Para a realização do estudo, foram utilizadas inicialmente amostras de medula óssea de pacientes portadores de LLA pertencentes a quatro grandes centros de oncologia pediátrica do Estado de São Paulo e que foram submetidos ao protocolo de tratamento do GBTLI-99. A análise da expressão gênica foi realizada pela técnica de PCR quantitativa em tempo real, utilizando-se o reagente SYBR Green, o gene GUS? como controle endógeno e amostras de medula óssea normais como referência. A análise dos dados de expressão gênica em relação aos diversos parâmetros clínicos e laboratoriais avaliados na LLA, demonstrou associações importantes entre os diversos genes estudados e variáveis clínicas importantes como contagem de glóbulos brancos ao diagnóstico, presença do antígeno CD10 (CALLA), translocação TEL/AML1, presença de doença residual mínima entre outras. Dentre os genes avaliados destacaram -se como possíveis marcadores de bom prognóstico os genes SHOC2 e GPR56. Posteriormente, reavaliou-se o perfil de expressão desses genes em pacientes submetidos ao protocolo de tratamento europeu do grupo BFM com o intuito de verificar o padrão de expressão em pacientes com um background genético distinto e submetidos a um protocolo terapêutico distinto. Os resultados confirmaram os dados encontrados anteriormente e demonstraram a hiperexpressão do gene SHOC2 (que foi previamente associado à sensibilidade à daunorrubicina) associada ao grupo de pacientes bons respondedores, sugerindo a correlação desse gene com critérios favoráveis de prognóstico. Para verificar o nível de interação desse gene avaliou-se ainda a expressão protéica do mesmo, que confirmou os padrões de expressão gênica obtidos por RQ-PCR. A função do gene SHOC2, que embora não esteja completamente elucidada, já foi anteriormente descrita pela literatura, que demonstra a participação do gene no processo de ativação da proteína Erk pela via Ras. Finalmente para melhor compreender os possíveis mecanismos que envolvem o gene SHOC2 no processo de melhor resposta à quimioterapia, utilizou-se a técnica de RNAi para silenciá- lo na linhagem celular leucêmica Jurkat. Os resultados demosntraram a associação da expressão do gene SHOC2 com proliferação celular e também com a indução de apoptose. Esses dados sugerem que a hiperexpressão desse gene pode ser importante para o processo de sensibilidade das células leucêmicas ao tratamento. Como conclusão, este trabalho demonstrou a associação de diversos genes com importantes parâmetros clínicos da LLA e destaca principalmente o papel do gene SHOC2 como possível alvo terapêutico para o tratamento da leucemia linfóide aguda. / Major improvements have been made in the ALL treatment, which achieved successful rates of approximately 80% of long-terms survival. Despite the significant percentage of success, the remaining 20 % still presents treatment failure and the molecular mechanisms involved in the resistance process remains unclear. The present study was undertaken to analyze and validate the gene expression pattern of the previously described genes related to prednisolone (F8A, CDK2AP1, BLVRB, CD69), vincristine (RPLP2, CD44, TCFL5, KCNN1, TRIM24), daunorubicin (MAP3K12, SHOC2, PDCH9, EGR1, KCNN4) and Lasparaginase (GPR56, MAN1A1, CLEC11A, IGFBP7, GATA3) in order to better inderstand these mechanisms. Bone marrow samples of ALL patients, obtained at diagnosis, in four oncology centers and treated according to the Brazilian protocol (GBTLI-99). The relative mRNA expression levels were quantified using real-time PCR analysis. Amplification of the specific sequences was performed with SYBR® Green reagent; GUSB was used as the reference gene and normal bone marrow samples used as calibrator. The expression profile analisis showed important associations among the studied genes and clinical features as WBC count at diagnosis, CALLA, TEL/AML1 translocation and minimal residual disease. Among the analyzed genes, possible therapy targets were found at SHOC2 and GPR56. Further we addressed the expression profile of these genes in ALL patients, treated according to the BFM protocol, which chacarterize a group of distinct genetic\'s background. The results confirmed the data previously obtained. The overexpression of the gene SHOC2, that was primaraly associated to sensibility to dauborubicin, was related to patients who presented good prednisone response, suggesting the correlation of SHOC2 with good prognostic factors. In order to acess the interaction level of this gene, the protein expression was analyzed and confirmed the mRNA expression data. Despite its lack of information, the data on SHOC2 shows its role as na important element in the Erk activation by Ras induced pathway. Finally, to better understand the possible mechanisms which involve SHOC2 gene to the chemotherapy response process, Jurkat cells was transfect with siRNA to silence the gene SHOC2. Further, functional assays were done to characterize the mechanisms involved. The results showed the association of SHOC2 gene expression with processes of cell proliferation and apoptosis induction, thus suggesting that the overexpression of SHOC2 could play an important role in leukemic cell\'s sensibility to chemotherapy agents, and consequently in patients\' treatment outcome. In conclusion, this work demonstrated the association of the expression profile of many genes with important clinical and laboratorial features. Furthermore, this data present the gene SHOC2 as a possible therapy target to acute lymphoblastic leukemia \'s treatment.

Questiona-se um possível papel deletério da endometriose sobre a qualidade oocitária (QO), que é, em grande parte, condicionada pelo papel das células do cumulus. A função dessas células envolve a expressão de diversas moléculas codificadas por genes específicos, regulada a nível de transcrição, pós-transcrição e tradução. Os microRNAs atuam como reguladores póstranscricionais da expressão gênica, de modo que qualquer alteração nesse mecanismo pode levar a anormalidades no desenvolvimento oocitário. Desta forma, propusemos um estudo inédito da análise de microRNAs em células do cumulus (CC) de mulheres inférteis com e sem endometriose com o objetivo de evidenciar processos biológicos e vias de atuação correlacionados com o papel da endometriose na infertilidade e seu possível impacto na aquisição da competência oocitária. Foram incluídas no estudo 15 pacientes inférteis (5 controles com fator tubário e/ou masculino, 5 com endometriose estágios I/II e 5 com endometriose estágios III/IV) submetidas à estimulação ovariana controlada (EOC) para injeção intracitoplasmática de espermatozoide (ICSI). Imediatamente após a captação oocitária, as CCs foram isoladas e armazenadas para extração dos miRNAs. O perfil de 754 miRNAs foi analisado por meio da técnica de TaqMan®Array Human MicroRNA Cards. Considerou-se significativo p<0,05. Os miRNAs hsa-let-7f-1#, hsa-miR-1291, hsa-miR-140-5p, hsa-miR-218, hsa-miR-30b e hsa-miR-629-5p foram identificados menos expressos nas pacientes com endometriose I/II comparadas às controles. Os miRNAs hsa-miR-1291, hsa-miR-187-3p, hsamiR-30b, hsa-miR-532-3p e hsa-miR-629-5p foram identificados menos expressos nas pacientes com endometriose III/IV em relação às controles. Ao comparar-se os grupos endometriose I/II e endometriose III/IV entre si, os miRNAs hsa-miR-187-3p e hsa-miR-532- 3p foram menos expressos e os miRNAs hsa-let-7f-1# e hsa-miR-362-3p mais expressos nas pacientes com endometriose III/IV. A análise de enriquecimento identificou os genes regulados pelos miRNAs e as respectivas vias metabólicas em que estão envolvidos, sugerindo aumento de apoptose, diminuição de proliferação celular, alterações no controle do ciclo celular e no metabolismo energético das CCs de mulheres com a doença inicial e avançada. Os dados apontam para alterações na regulação pós-transcricional em CCs de mulheres inférteis com endometriose inicial e avançada, o que pode afetar processos e vias essenciais à aquisição de competência oocitária e estar envolvido na infertilidade associada à doença. Este estudo contribui para o entendimento da etiopatogênese da infertilidade relacionada à endometriose identificando mecanismos pós-transcricionais relacionados à piora da qualidade gamética nestas mulheres. / It is questioned a possible deleterious role of endometriosis on oocyte quality (OQ), which is largely conditioned by the function of cumulus cells. The role of these cells involve the expression of several molecules encoded by specific genes, regulated at transcription level, post-transcription and translation. The microRNAs act as transcriptional regulators of gene expression, thus any alteration in this mechanism can lead to abnormalities in oocyte development. In this way, we proposed an unpublished study of the microRNAs analysis in cumulus cells (CC) of infertile women with and without endometriosis, aiming to evidence the biological processes and pathways of action correlated with the presence of endometriosis in infertility and its possible impact on the acquisition of oocyte competence. Fifteen infertile patients (5 controls with tubal factor and/or male, 5 with stage I/II of endometriosis and 5 with stages III/IV of endometriosis) submitted to the controlled ovarian stimulation (EOC) for intracytoplasmic sperm injection (ICSI) were included in the study. Immediately after oocyte uptake, CCs were isolated and stored for miRNA extraction. The 754 miRNAs profile was analyzed using the TaqMan®Array Human MicroRNA Cards technique. Significant values were considered when p <0.05. The hsa-miR-218, hsa-miR-301 and hsa-miR-629-5p miRNAs were identified as less expressed in the patients with endometriosis I/II compared to controls. The miRNAs hsa-miR-1291, hsa-miR-187-3p, hsa-miR-30b, hsa-miR-532-3p and hsa-miR- 629-5p were identified as less expressed in patients with endometriosis III/IV compared to controls. Comparing the groups with endometriosis I/II and endometriosis III/IV, hsa-miR-187- 3p and hsa-miR-532-3p miRNAs were less expressed and hsa-let-7f-1# and hsa-miR-362-3p more expressed in patients with endometriosis III/IV. The enrichment analysis identified the genes regulated by the miRNAs and the respective metabolic pathways in which they are involved, suggesting apoptosis increase, decreased cell proliferation, alterations in the cell cycle control and energetic metabolism of the CCs of women with initial and advanced disease. The data point to changes in post-transcriptional regulation in CCs of infertile women with early and advanced endometriosis, which may affect processes and pathways essential for acquiring oocyte competence and resulting in infertility associated with the disease. This study contributes to the understanding of the etiopathogenesis of infertility related to endometriosis by identifying post-transcriptional mechanisms related to the deterioration of quality of gametes in these women.

Le cancer est un groupe de maladies génétiques opérationnellement défini par uneprolifération cellulaire incontrôlée, impliquant une défaillance del'homeostasie de l'organisme. La recherche sur le cancer vise à fournir desoutils diagnostics précis et des traitements ajustés pour chacune de cesmaladies. La technologie microarray permet la quantification de l'expression detous les produits de transcription du génome humain et constitue donc un outilpour mieux comprendre la nature polygénique du cancer. La technologiemicroarray permet à la fois de découvrir de nouvelles classes de cancers et deprédire l'issue de maladie en fonction de profils d'expression préalables. Enoutre, l'utilisation de signatures d'expression géniques en tant que marqueursreprésentatifs de certains processus physiologiques moléculaires permetl'emploi de données microarray pour tester des hypothèses biologiques.Cette dissertation a deux objectifs: (a) établir la mesure dans laquelledes marqueurs d'expression génique de la différenciation et de la proliférationcellulaire peuvent contribuer à la classification des maladies cancéreuses; et(b) d'évaluer l'étendue des signaux pronostiques dans les transcriptomescancéreux.Nous avons mis au point une méthode objective pour extraire des signatures dedifférentiation organe-spécifiques à partir de données d'expression génique.Nous avons ensuite démontré qu'une signature génique de différentiationtissu-spécifique est capable de distinguer avec précision entre des sous-typeshistologiques de difficile classification dans un modèle thyroïdien. Ceci faitpreuve du potentiel valeur clinique et diagnostique des signatures dedifférentiation dans le domaine oncologique.Nous montrons aussi qu'une fraction non négligeable des transcriptomes cancéreuxest capable de prédire l'issue des respectives maladies, à la suite d'uneanalyse systématique de 114 cohortes de profiles d'expression cancéreuxenglobant 19 types de cancers différents. Cet observation est probablement liéeà une vaste structure de corrélation parmis les profils d'expression cancéreux,partiellement expliquée par des variables techniques et biologiques. Cetteevidence met en cause l'utilisation généralisée d'associations statistiquesentre des marqueurs d'expression géniques et les issues de chaque maladie parmisplusieurs patients afin d'en déduire l'implication de mécanismes biologiquesparticuliers dans la progression du cancer. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished