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

Le co-transporteur Na+-K+-2Cl- spécifique du rein et sensible au bumétanide, NKCC2, joue un rôle essentiel dans l’homéostasie hydro-électrolytique et acido-basique de l’organisme. Les mutations inactivatrices de NKCC2 induisent le syndrome de Bartter anténatal de type 1, une grave maladie rénale caractérisée par une hypotension artérielle associée à des anomalies électrolytiques. À l’opposé, une activité accrue de NKCC2 est associée à une hypertension artérielle sensible au sel. Pourtant, peu est connu sur la régulation moléculaire de NKCC2. Le but de ces travaux de thèse a donc été l’identification des déterminants moléculaires impliqués dans la régulation de l’expression et du trafic intracellulaire de NKCC2, plus spécifiquement dans le contrôle de qualité de ce co-transporteur. Suite au criblage par la technique de double hybride chez la levure d’une banque d’ADNc de rein humain, nous avons identifié OS-9 en tant que partenaire de NKCC2. La léctine OS-9 est un facteur clé de régulation du contrôle de qualité des protéines au niveau du RE. Les analyses de co-immunoprécipitation dans les cellules rénales ont montré qu’OS-9 interagit principalement avec la forme immature de NKCC2. De plus, les expériences d’immunofluorescence ont révélé que cette interaction aurait lieu au niveau du RE. La surexpression d’OS-9 diminue l’abondance totale de NKCC2. Cet effet est aboli suite à l’inhibition de la voie de dégradation protéique par le protéasome par le MG132. De plus, les expériences pulse-chase et cycloheximide-chase ont montré que cette diminution est secondaire à l’augmentation de la dégradation de la forme immature de NKCC2. A l’inverse, le knock-down d’OS-9 endogène augmente l’expression du co-transporteur en augmentant la stabilité de sa forme immature. Enfin, la mutation du domaine MRH (Mannose 6-phosphate Receptor Homology) d’OS-9 n’altère pas son effet sur NKCC2, alors que la mutation des deux sites de N-glycosylation de NKCC2 abolie l’effet d’OS-9. L’ensemble de nos résultats démontre l’implication de la lectine OS-9 dans le système ERAD de NKCC2. Le deuxième volet de ce travail a porté sur l’identification de nouveaux mécanismes Moléculaires impliqués dans le Syndrome de Bartter. Nous avons découvert des mutations dans le gène MAGE-D2, situé sur la chromosome X, responsables d’une nouvelle et très sévère forme du syndrome de Bartter anténatal, caractérisé par un polyhydramnios très précoce avec un risque élevé d’accouchement prématuré et de mortalité. Nous avons montré que les anomalies de MAGE-D2 entraînent un défaut de maturation et d’expression membranaire de NKCC2 ainsi que celle du co-transporteur Na-Cl, NCC, du tubule distal. La comparaison in vitro de l’interactome de MAGED2 sauvage et mutée a révélé que la protéine MAGE-D2 sauvage interagit spécifiquement avec DNAJB1 (HSP40) et/ou GNAS, suggérant l’implication de ces deux partenaires protéiques dans la régulation de NKCC2 et NCC par MAGE-D2 pendant la grossesse. Le troisième volet de ce travail a porté sur l’étude de l’effet de DNAJB1/HSP40, partenaire de MAGE-D2, sur l’expression de NKCC2. HSP40 a été identifiée aussi comme partenaire de NKCC2 par la technique de double hybride réalisée par notre équipe. Nous avons montré que HSP40 et son co-chaperon HSPA1A (HSP70) interagissent avec la forme immature de NKCC2 au niveau du RE. La co-expression de HSP40 et HSP70 augmente l’expression de NKCC2 en augmentant sa stabilité et sa maturation. De plus, ces deux co-chaperons régulent l’expression de NCC de la même manière. Ces observations suggèrent que MAGE-D2 coopère avec DNAJB1/HSP40 et HSPA1A/HSP70 pour protéger NKCC2 et NCC contre la rétention et la dégradation de NKCC2 au niveau du RE durant la grossesse, révélant ainsi une nouvelle voie de régulation du trafic intracellulaire de NKCC2 et NCC. (...) / The kidney-specific Na + -K + -2C1 co-transporter, sensitive to bumetanide, NKCC2, plays an essential role in the body's fluid, electrolyte and acid-base homeostasis. Mutations of NKCC2 cause antenatal type 1 Bartter syndrome, a life-threatening kidney disease characterized by arterial hypotension associated with electrolyte abnormalities. In contrast, an increase in NKCC2 activity is associated with salt-sensitive hypertension. Yet the mechanisms underlying the regulation of NKCC2 trafficking in renal cells are scarcely known. The aim of this work was to identify the protein partners involved in the regulation of the expression and the intracellular trafficking of NKCC2, specifically in the quality control of this co-transporter. Using the yeast tow-hybrid system, we identified OS-9 as a specific binding partner of NKCC2. Lectin OS-9 is a key factor in the regulation of protein quality control at ER. Co-immunoprecipitation assay in renal cells showed that OS-9 interacts mainly with NKCC2 immature forms. Accordingly, immunocytochemistry analysis showed co-localization of the proteins mainly in the ER. Overexpression of OS-9 decreased the total abundance of NKCC2. This effect is abolished following the inhibition of the proteasome protein degradation pathway by MG132. In addition, the pulse-chase and cycloheximide-chase assays demonstrated that the marked reduction in the co-transporter protein levels was essentially due to increased protein degradation of NKCC2 immature forms. Conversely, knock-down endogenous of OS-9 increased the expression of the co-transporter by increasing the stability of its immature form. Finally, inactivation of the Mannose 6-phosphate Receptor Homology domain had no effect on its action on NKCC2, while mutation of the two NKCC2 N-glycosylation sites abolished the effect of OS- 9. In summary, our results demonstrate the involvement of lectin OS-9 in the ERAD of NKCC2. The second part of this work focused on the identification of new molecular mechanisms involved in Bartter Syndrome. We found that MAGE-D2 mutations caused X-linked new and severe form of antenatal Bartter's syndrome, characterized by a very early polyhydramnios with a high risk of premature delivery and mortality. We have shown that MAGE-D2 abnormalities lead to a lack of maturation and membrane expression of NKCC2 as well as that of the Na-Cl co-transporter, NCC, of the distal tubule. In vitro comparison of the wild-type and mutated MAGED2 interactome revealed that wild-type MAGE-D2 interacts specifically with DNAJB1 (HSP40) and / or GNAS, suggesting involvement of these two protein partners in NKCC2 and NCC regulation by MAGE-D2 during pregnancy. The third part of this work focused on the study of the effect of DNAJB1 / HSP40, partner of MAGE-D2, on the expression of NKCC2. HSP40 was also identified as a specific binding partner of NKCC2 by the yeast two-hybrid system realized by our team. We have shown that HSP40 and its co-chaperone HSPA1A (HSP70) interact with the immature form of NKCC2 at the ER. The co-expression of HSP40 and HSP70 increased the expression of NKCC2 by increasing its stability and maturation. In addition, these two co-chaperones regulate the expression of NCC in the same way. These findings suggest that MAGE-D2 cooperates with DNAJB1 / HSP40 and HSPA1A / HSP70 to protect NKCC2 and NCC against retention and degradation of NKCC2 at ER during pregnancy, revealing a new pathway for regulating NKCC2 and NCC intracellular trafficking. A better understanding of NKCC2 and NCC regulatory pathways would help to better understand the pathophysiology of sodium retention and ultimately would provide a new target for a pharmaceutical approach to preventing and / or treating kidney disease related to sodium balance.