Submitted by Warne Pedro de Andrade (warne800@hotmail.com) on 2018-06-27T16:37:27Z No. of bitstreams: 1 Mestrado Warne.pdf: 2077163 bytes, checksum: 68e5205b57ffe2680fcdd40ebea34554 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-06-28T16:07:28Z (GMT) No. of bitstreams: 1 andrade_wp_me_bot.pdf: 2077163 bytes, checksum: 68e5205b57ffe2680fcdd40ebea34554 (MD5) / Made available in DSpace on 2018-06-28T16:07:28Z (GMT). No. of bitstreams: 1 andrade_wp_me_bot.pdf: 2077163 bytes, checksum: 68e5205b57ffe2680fcdd40ebea34554 (MD5) Previous issue date: 2018-02-28 / Introdução: As proteínas de choque térmico (“Heat Shock Proteins”) são produzidas em resposta ao estresse patofisiológico nas células animais e não só fazem parte de várias etapas da carcinogênese, atuando principalmente como agentes antiapoptóticos, como também estão implicadas em mecanismos de resistência à quimioterapia em vários tipos de tumores. Objetivo: O presente estudo visa comparar a expressão dos genes TRAP1, HSPB1, HSPD1, HSPA1L e HSPA1A nas amostras de CEO (no tumor primário ou na metástase) com a expressão dos mesmos em amostras de tumores ovarianos benignos e tecido ovariano normal e correlacionar a expressão gênica com o prognóstico das pacientes e com a resistência ao tratamento com platina. Métodos: Foram avaliadas amostras de 51 pacientes operadas no Hospital Vera Cruz, entre os anos de 2008 a 2011, divididas em quatro grupos: CEO primário (n = 14), CEO metastático (n = 11), cistoadenoma seroso ovariano (n = 07) e ovário normal (n = 19). Utilizou-se a técnica de qRT-PCR para determinar o perfil de expressão dos genes. Resultados: As pacientes incluídas neste estudo apresentavam idade média de 56,75 anos. Não houve diferença significativa (valor-P> 0,050) na comparação entre a expressão dos genes e os grupos estudados. Os genes HSPA1A, HSPA1L e TRAP1 foram subexpressos e se diferiram significativamente dos genes em indivíduos com ovário normal. A expressão dos genes analisados não correlacionou se com as variáveis quantitativas, como idade, menarca, e tempo após menopausa bem como em relação as dosagens séricas do CA125, presença de ascite e citorredução. Em relação ao estadiamento, observou que mulheres com CEO no estadio I e II apresentaram a expressão do gene TRAP1 significativamente maior que as pacientes no estadio III e IV (p=0,040). Ao analisar a expressão dos genes em relação a sobrevida global observou se que houve influência significativa do nível de expressão dos gene HSPA1A sobre o risco de morte das mulheres afetadas por CEO. Conclusão: Apesar das limitações em relação ao tamanho da amostra, os resultados obtidos sinalizam para um melhor entendimento da relação entre a expressão destes genes e o câncer de ovário, evidenciando algumas implicações no prognóstico. / Heat Shock Proteins are produced in response to pathophysiological stress and take part in several stages of carcinogenesis, acting primarily as anti-apoptotic agents. They are also implicated in resistance to chemotherapy in several types of tumors. Herein we correlated the expression of genes encoding these proteins and the clinical and pathological aspects of patients with ovarian cancer (OC). METHODS: 51 patients included in the study were divided into four groups: those with primary EOC (n = 14), metastatic EOC (n = 11), ovarian serous cystadenoma (n = 7), with no evidence of ovarian malignancy or control (n = 11). The 57 tumor samples obtained were submitted to RNA extraction and reverse transcription. qRT-PCR was performed to compare the expression of TRAP1, HSPB1, HSPD1, HSPA1A and HSPA1L in primary and HSP60, HSP70, HSPA1L genes did not differ among the groups (p-value> 0.050) .HSPA1A, HSPA1L and TRAP1 we underexpressed in the primary and metastatic EOC groups with HSPA1L showing the lowest expression with compare with normal ovary tissue. TRAP1 expression was higher in tumors at stage I/II than at stages III/IV. Grade II subjects showed higher HSPB1 expression. There was no correlation between HSPs expression and age, menarche, parity, period after menopause initiation and CA-125. HSPA1A gene was negatively correlated with the risk of dying of OC. There was no differences between HSP expression gene evaluated and overall and disease-free survival. In conclusion, we suggest that downregulation of HSPA1A, HSPA1L e TRAP1 could help to clinical evaluated of prognostic in women with EOC.

Orientador: Agnaldo Lopes da Silva Filho / Resumo: Introdução: As proteínas de choque térmico (“Heat Shock Proteins”) são produzidas em resposta ao estresse patofisiológico nas células animais e não só fazem parte de várias etapas da carcinogênese, atuando principalmente como agentes antiapoptóticos, como também estão implicadas em mecanismos de resistência à quimioterapia em vários tipos de tumores. Objetivo: O presente estudo visa comparar a expressão dos genes TRAP1, HSPB1, HSPD1, HSPA1L e HSPA1A nas amostras de CEO (no tumor primário ou na metástase) com a expressão dos mesmos em amostras de tumores ovarianos benignos e tecido ovariano normal e correlacionar a expressão gênica com o prognóstico das pacientes e com a resistência ao tratamento com platina. Métodos: Foram avaliadas amostras de 51 pacientes operadas no Hospital Vera Cruz, entre os anos de 2008 a 2011, divididas em quatro grupos: CEO primário (n = 14), CEO metastático (n = 11), cistoadenoma seroso ovariano (n = 07) e ovário normal (n = 19). Utilizou-se a técnica de qRT-PCR para determinar o perfil de expressão dos genes. Resultados: As pacientes incluídas neste estudo apresentavam idade média de 56,75 anos. Não houve diferença significativa (valor-P> 0,050) na comparação entre a expressão dos genes e os grupos estudados. Os genes HSPA1A, HSPA1L e TRAP1 foram subexpressos e se diferiram significativamente dos genes em indivíduos com ovário normal. A expressão dos genes analisados não correlacionou se com as variáveis quantitativas, como idade, menarca, e tempo ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Heat Shock Proteins are produced in response to pathophysiological stress and take part in several stages of carcinogenesis, acting primarily as anti-apoptotic agents. They are also implicated in resistance to chemotherapy in several types of tumors. Herein we correlated the expression of genes encoding these proteins and the clinical and pathological aspects of patients with ovarian cancer (OC). METHODS: 51 patients included in the study were divided into four groups: those with primary EOC (n = 14), metastatic EOC (n = 11), ovarian serous cystadenoma (n = 7), with no evidence of ovarian malignancy or control (n = 11). The 57 tumor samples obtained were submitted to RNA extraction and reverse transcription. qRT-PCR was performed to compare the expression of TRAP1, HSPB1, HSPD1, HSPA1A and HSPA1L in primary and HSP60, HSP70, HSPA1L genes did not differ among the groups (p-value> 0.050) .HSPA1A, HSPA1L and TRAP1 we underexpressed in the primary and metastatic EOC groups with HSPA1L showing the lowest expression with compare with normal ovary tissue. TRAP1 expression was higher in tumors at stage I/II than at stages III/IV. Grade II subjects showed higher HSPB1 expression. There was no correlation between HSPs expression and age, menarche, parity, period after menopause initiation and CA-125. HSPA1A gene was negatively correlated with the risk of dying of OC. There was no differences between HSP expression gene evaluated and overall and disease-free survival. In conclusion, we ... (Complete abstract click electronic access below) / Mestre

La résistance à la chimiothérapie constitue un frein majeur à son efficacité. Notre équipe a récemment pu montrer que le masitinib, un nouvel inhibiteur de protéines tyrosine kinases, possède une activité de resensibilisation des cellules tumorales résistantes à la chimiothérapie lorsqu'il est combiné à certaines chimio-drogues. L'objectif des travaux de cette thèse est de déterminer les voies de signalisation, modulées par l'action du masitinib, qui sont impliquées dans la resensibilisation aux chimiothérapies et amélioration de l'activité anti-tumorale.Dans la première partie de la thèse, nous avons pu identifier la nucléoside kinase dCK (désoxycytidine kinase), protéine activatrice d'un grand nombre de chimiothérapies, comme nouvelle cible du masitinib. Cette première étude nous a permis de mettre en évidence un nouveau concept thérapeutique: le masitinib, un composé chimique de type inhibiteur de protéines tyrosine kinases, peut jouer en même temps le rôle d'activateur de nucléoside kinase.Nous avons pu mettre en évidence dans la deuxième partie de la thèse que le traitement combiné entre l'épi-drogue décitabine et le masitinib peut être plus efficace pour la réexpression de certains gènes non ou peu induits par la décitabine seule.En conclusion, ces travaux nous ont permis de mettre en évidence l'interaction entre un inhibiteur de protéine tyrosine kinases et une nucléoside kinase, dans un concept d'activation enzymatique qui pourra certainement servir de base pour l'élaboration de nouvelles petites molécules chimiques spécifiques de l'activation de dCK ou d'autres nucléosides kinases nécessaires à l'activation des drogues de chimiothérapie. / Resistance to chemotherapy is considered as one of the major blockers of its efficacy. Recently, our team demonstrated that masitinib, a new tyrosine kinases inhibitor, possesse a resensitization activity of cell lines resistant to chemotherapy when associated with chemodrugs.The aim of this work is to determine signaling pathways, modulated by masitinib action, that could explain the resensitization to chemotherapy and improvement of anti-tumoral activity.In the first part of this work, we identified the nucleoside kinase dCK (deoxycytidine kinase), a chemotherapy activating protein, as a new target of masitinib. In summary, this first part of the work allowed us to describe a new and never described concept: masitinib, a small molecule belonging to tyrosine kinases group, can also play a role as nucleoside kinase activator.We were able to demonstrate through the second part of the work that the combined treatment of the epidrug decitabine and masitinib can be more effective than decitabine treatment for the re-expression of some genes non or weakly induced by decitabine when used alone.In conclusion, These data allowed us to introduce an interaction between a tyrosine kinases inhibitor and a nucleoside kinase, as an enzymatic activation new concept. This could be used as a base for the design of new small chemical molecules specific for dCK or other nucleoside kinases essential for the activation of chemodrugs. This concept will obviously help to imagine and evaluate more potential therapeutic combinations of chemodrugs and small chemical molecules to overcome the resistance to chemotherapy dependent on nucleoside kinases.

Le cancer du poumon est la principale cause de décès par cancer chez les hommes et les femmes dans le monde. Malgré les avancées majeures dans les traitements, les thérapies à base de platine restent la thérapie standard pour les patients atteints du cancer du poumon induit par la mutation KRAS. Bien que les composés de platine aient un premier effet sur ces patients, l’apparition d’une rechute constitue le principal défi au niveau clinique.Dans ce contexte, notre projet actuel vise à caractériser l’activation de la voie Notch dans la réponse aux sels de platines d’adénocarcinomes mutés pour K-Ras ainsi que son rôle dans l’acquisition de la résistance à ce traitement. Nous avons mis en évidence que la voie Notch est activée en réponse à divers agents induisant des dommages à l’ADN dans des lignées de cancer du poumon. Nous avons montré que cette activation est dépendante de la voie de checkpoint de Réponse aux Dommages de l’ADN (DDR) via ATM, ATM qui est une des principales kinases de la réponse cellulaire aux lésions de l’ADN telles que les cassures double-brin, causé notamment par la chimiothérapie conventionnelle. Parmi les nombreux substrats d’ATM en réponse à ces dommages, nous montrons un effet direct de MDM2 dans l’activation de la voie Notch en réponse aux dommages à l’ADN causé par le carboplatine. Nos données montrent une nouvelle interaction entre ATM, MDM2 et Notch, lors du traitement au carboplatine, qui pourrait jouer un rôle dans la cascade de signalisation favorisant la survie des cellules tumorales pulmonaires.En utilisant des xénogreffes dérivées de patients atteints d’adénocarcinomes du poumon, ainsi qu’un modèle cellulaire de xénogreffe résistant à la carboplatine, nous avons montré que l’association d’inhibiteurs de Notch et de MDM2 associé au traitement au carboplatine ont un effet accru sur le ralentissement de la croissance de la tumeur et sur la survie, en comparaison avec le traitement actuel de référence que représente le carboplatine.Nos résultats offrent une nouvelle possibilité de thérapie pour les adénocarcinomes de poumons mutés pour KRAS, et pourrait donc répondre à un besoin clinique urgent notamment pour contrecarrer la résistance aux chimiothérapies conventionnelles à base de sels de platine. / Lung cancer is the leading cause of cancer death among men and women worldwide. Despite the major advances in the treatments, platinum-based therapy remains the standard of care for patients affected by KRAS-driven lung cancer. Even though the platinum-compounds display an initial effect on these patients, the onset of the relapse constitutes the main challenge for the clinic. The molecular mechanisms underlying lung adenocarcinoma (LUAD) relapse are not completely elucidated yet, thus it is fundamental to understand them in order to improve survival of patients. Our data show that upon carboplatin treatment, the Notch pathway is activated in vitro. Since this effect was common for several other DNA damage insults, our driving hypothesis connected the DNA Damage Response (DDR) to the activation of the Notch pathway. Our data demonstrates that protein kinase ataxia telangiectasia-mutated (ATM) is a key mediator in the activation of the Notch pathway during DNA damage signalling. ATM is well-known as the chief mobilizer of the cellular response to the most toxic lesions to the DNA, the double-strand breaks which are also the type of damage caused by the conventional chemotherapy. Among the several substrates of ATM in response to the damage, there is the mouse double minute 2 protein (MDM2) that it is known to interact with the Notch pathway. Our data uncovered a new pathway connecting ATM, MDM2 and NICD during carboplatin treatment in LUAD cells.Using LUAD Patient-Derived Xenografts and a new generated carboplatin resistant cellular model, we show in vivo that the combination of Notch and MDM2 inhibitors with carboplatin showed a therapeutic benefit in tumour growth and survival compared to the standard of care, i.e. carboplatin.Our results can offer a new therapeutic window for KRAS-driven LUAD that become resistant to platinum-based therapy, hence tackling an urgent and unmet clinical need.