Uma questão ambiental preocupante é a contaminação do meio ambiente por substâncias que interagem com os sistemas endócrinos de seres humanos e animais. Tais substâncias são denominadas desreguladores endócrinos e, como outros poluentes ambientais, apresentam uma variedade de fontes e grande potencial agressor à saúde humana. Neste trabalho, buscamos a aplicação de técnicas de espectroscopia vibracional e técnicas eletroquímicas ao estudo de desreguladores endócrinos, em termos de sua detecção, caracterização, estudo de sua reatividade, desenvolvimento de métodos para sua degradação fotoquímica, estudo dos mecanismos envolvidos e características dos produtos de degradação. O enfoque é voltado ao uso de técnicas espectroscópicas, com especial destaque para o desenvolvimento de metodologias de espectroscopia vibracional intensificadas, associadas ou não a sistemas eletroquímicos. Dentre os compostos considerados desreguladores endócrinos, o nosso trabalho é dedicado ao estudo fotoquímico e espectroeletroquímico das seguintes substâncias: o difenilalcano bisfenol A (BPA), os herbicidas triazínicos ametrina e atrazina, e os compostos organoclorados ácido 2,4-diclorofenoxiacético (2,4-D) e 2,4-diclorofenol (2,4-DCF). O estudo de processos de fotólise e degradação fotocatalítica mediada por TiO2 foram realizados para o BPA e para a ametrina. Tais processos mostram-se importantes para a remoção destes compostos do meio ambiente. Os estudos fotocatalíticos foram estendidos ainda para o uso de compósitos TiO2-Pr como catalisadores. Foi observado que a dopagem do TiO2 pelo íon terra rara praseodímio resulta em alterações nas propriedades físico-químicas do material semicondutor, sendo um método valioso para o aumento da atividade fotocatalítica do TiO2. Conclui-se ainda pela necessidade do controle da porcentagem de modificação química para a otimização do processo fotocatalítico, sendo a proporção de 1% praseodímio considerada ótima. O estudo de processos de fotólise e degradação fotocatalítica do BPA mediado por TiO2 mostra diferenças significativas entre o processo de degradação e mineralização, relacionadas à formação de intermediários orgânicos recalcitrantes. Para a ametrina observa-se que a fotólise e a fotocatálise envolvem mecanismos de degradação distintos. Estudos espectroeletroquímicos dos desreguladores endócrinos (DEC) foram conduzidos através de espectroscopia Raman intensificada (efeito SERS) visando a compreensão da natureza da interação química entre os adsorbatos e superfícies metálicas nanoestruturadas, bem como a caracterização vibracional dos produtos de processos faradáicos desses compostos sobre eletrodos. Produtos de reações químicas de oxidação e redução dos DEC foram identificados através de modificações espectrais em função de potenciais eletroquímicos anódicos e catódicos. / An environmental issue of concern is contamination of the environment by substances which interact with the endocrine systems of humans and animals. Such substances are called endocrine disruptors (ED) and, like other environmental pollutants, may produce adverse effects in human health. In this work, we explore the application of vibrational spectroscopy and electrochemical techniques in the study of ED, in terms of their detection, characterization, study of their reactivity, development of methods for their photochemical degradation, study of the involved mechanisms and characteristic of degradation products. The approach employs the use of advanced spectroscopic techniques, with special attention for the development of methodology of surface-enhanced vibrational spectroscopy, associated or not with electrochemical systems. Among the compounds thought to cause endocrine disruption, the present study is dedicated to the photochemical and electrochemical study of the following substances: the plasticizer bisphenol A (BPA), the triazinic pesticides ametrine and atrazine, and the organochlorine compounds 2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenol. The study of photolytic and TiO2-mediated photocatalytic degradation was carried out for BPA and ametrine. Such processes are important for the removal of such substances from the environment. The photocatalytic studies were extended to include the use of TiO2-Pr composite as catalysts. The doping of TiO2 by the rare earth ion praseodymium results in changes in the physicochemical properties of semiconducting material, being a valuable method for enhancing the photocatalytic activity of TiO2. Our results showed the need of percentage control of the chemical modification for the optimization of the photocatalytic process, with the 1% praseodymium/titanium ration the optimal composition. The study of photolysis and TiO2-mediated photocatalytic degradation processes of BPA shows significant differences between the degradation and mineralization, suggesting the production of recalcitrant organic compounds. For ametrine, photolysis and photocatalytic degradation processes were found to occur via different mechanisms. Spectroelectrochemical studies of endocrine disruptors (ED) were carried out through surface-enhanced Raman spectroscopy (SERS effect) aiming at the understanding of the nature of the chemical interaction between these molecules and nanostructured metallic surfaces, and at the vibrational characterization of the products formed by faradaic charge transfer processes. Products of oxidation and reduction chemical reactions were identified through spectral changes observed in the SERS spectra as a function of cathodic and anodic potentials.

Há evidências crescentes de que os desreguladores endócrinos (DE) podem afetar negativamente os seres humanos. Águas superficiais e subterrâneas são as principais fontes de obter a sua potabilidade, no entanto, podem ser contaminadas por DE, que não são totalmente removidos pelos processos convencionais de tratamento de água e esgoto. Alguns problemas de saúde estão relacionados à exposição dos seres humanos aos DE, sendo a obesidade um deles. Atualmente vive-se um aumento da prevalência da obesidade em todo o mundo, fato considerado preocupante, considerando-se seu potencial impacto no sistema de saúde, pois, as principais doenças crônicas como diabetes e doenças cardiovasculares, tem a obesidade como grande fator de risco. Por meio de revisão de literatura, este artigo buscou reunir publicações científicas relacionando a exposição aos DE com a obesidade, com o objetivo de verificar a importância de sua remoção dos corpos hídricos, preservando a saúde da população e da biota aquática. A maioria dos estudos selecionados sugere associação entre DE e obesidade em humanos. / There is growing evidence that endocrine disrupters (ED) may adversely affect humans. Surface and ground water are the main sources to get their potability however, can be contaminated by ED, which are not completely removed by conventional processes of water and sewage treatment. Some health problems are related to the exposure of humans to ED, obesity being one of them. Currently lives an increase in obesity prevalence worldwide, a fact considered worrying, considering its potential impact on the health care system, for major chronic diseases like diabetes and cardiovascular diseases have obesity as a major risk factor. Through literature review, this paper aims to gather scientific publications relating to exposure to ED with obesity, in order to verify the importance of their removal from water bodies, preserving the population\'s health and aquatic biota. Most of the selected studies suggest an association between ED and obesity in humans.

Orientador: Sebastião Roberto Taboga / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T04:58:37Z (GMT). No. of bitstreams: 1 Biancardi_ManoelFrancisco_D.pdf: 15374332 bytes, checksum: b70012fe11a604131b1622adb07d2654 (MD5) Previous issue date: 2014 / Resumo: Pesquisas recentes tem mostrado que a predisposição às desordens prostáticas tem origem nos momentos iniciais da vida. Portanto, o entendimento detalhado da organogênese prostática e dos mecanismos envolvidos na predisposição à estas desordens são de fundamental importância para se compreender a etiologia das patologias prostáticas. Assim, o objetivo deste trabalho foi avaliar a organogênese prostática normal e a influência da exposição à testosterona durante a vida intrauterina e puberal sobre a próstata de fêmeas e machos senis do gerbilo da Mongólia. Para isto, o presente trabalho foi dividido em duas etapas. Na primeira, grupos de fêmeas grávidas de gerbilo receberam injeções subcutâneas de testosterona (500 µg/dose) 4 dias antes do parto (grupos TG e TG + T) e/ou durante a vida pós-natal (grupos C + T e TG + T). Os filhotes destas mães envelheceram até serem mortos com 1 ano de idade. Na segunda etapa, foi realizado um estudo normativo da organogênese do lobo ventral da próstata de machos. Para isto, fetos machos de 20 a 24 dias de gestação (E20 - E24), além de recém nascidos de 1 dia de idade (P1), foram empregados no estudo. Todos os fragmentos teciduais foram dissecados e fixados em metacarn ou paraformaldeído 4%, sendo processados para inclusão em parafina. Posteriormente, as seções teciduais foram submetidas à análises biométricas, morfológicas, estereológicas, imuno-histoquímicas, de imunofluorescência e de reconstrução tridimensional. Os resultados mostraram que somente os machos velhos do grupo TG + T foram acometidos por hiperplasia adenomatosa associada à inflamação, embora nos animais dos outros grupos experimentais também foram observados focos hiperplásicos na próstata. Já as fêmeas expostas à testosterona na fase pré-natal (grupos TG e TG + T) apresentaram uma maior heterogeneidade de alterações em decorrência do tratamento, que envolveram desde malformações do sistema reprodutor até a formação de tecido ectópico ao redor da vagina, além da presença de lesões prostáticas também caracterizadas por focos hiperplásicos adenomatosos. Estes resultados mostram que a exposição anormal à testosterona afeta a organogênese prostática tanto em machos quanto em fêmeas, alterando os processos normais do desenvolvimento e aumentando a suscetibilidade ao desenvolvimento de doenças ao longo do envelhecimento. Em relação ao estudo da organogênese prostática, foi observado que os brotos ventrais emergem do epitélio uretral entre o 20º e o 21º dia de vida pré-natal, atingindo o pé mesenquimal ventral (VMP) e iniciando o processo de morfogênese de ramificação no primeiro dia de vida pós-natal. Os achados também demonstraram que a camada de musculatura lisa parece ter um papel central neste processo, a qual pode atuar como uma barreira física à expansão dos brotos em direção ao VMP. Em relação aos receptores nucleares, o receptor de andrógeno apresentou uma expressiva imunomarcação no mesênquima periuretral, enquanto que células com marcação para receptor alfa de estrógeno foram identificadas tanto nos brotos como no mesênquima periuretral. Por fim, os resultados demonstram que a organogênese prostática, além de envolver uma série de eventos finamente regulados, é um processo extremamente sensível e determinante para a saúde da glândula ao longo da vida / Abstract: Recent researches have been shown that the predisposition to prostatic disorders has its origin during moments of early life. Therefore, the detailed understanding of prostate organogenesis and the mechanisms underlying in predisposing to these disorders are extremely important in order to highlight the etiology of prostatic pathologies. Thus, the aim of this work was to evaluate the normal prostate organogenesis and the influence of testosterone exposure during prenatal and pubertal life on the prostate of male and female Mongolian gerbil. To this, the present work was divided into two phases. First, groups of pregnant female received subcutaneous injections of testosterone (500 µg/dosage) four days before the parturition (groups TG and TG + T) and/or during postnatal life (groups C + T and TG + T). The offspring of these mothers aged, being killed with one-year-old. In a second moment, we realized a normative study of the organogenesis of male ventral prostate lobe. To this, male fetus with 20 and 24 (E20 - E24) days of gestation, besides newborn with one-day-old (P1), were employed in this study. All tissue fragments were dissected and fixed in methacarn or 4% paraformaldehyde, being processed for paraffin embedding. Following, tissue sections were submitted to biometrical, morphological, stereological, immunohistochemical, immunofluorescence and three dimensional reconstruction analyses. The results showed that only the old males of TG + T group were affected by adenomatous hyperplasia associated with inflammation, although in other male experimental groups we also observed hyperplastic foci in the prostate. The female exposed to testosterone during prenatal phase (TG and TG + groups) have demonstrated a higher heterogeneity of alterations due treatment, being characterized by reproductive system malformations, formation of ectopic prostatic tissue surrounding vaginal wall, besides the presence of prostatic lesions characterized by adenomatous hyperplasic foci. These findings show that abnormal prenatal exposure to testosterone affect the prostate morphogenesis in both male and female, disrupting normal developmental processes and increasing the susceptibility to the development of prostatic diseases during aging. Regarding the prostate organogenesis study, it was observed that the first ventral buds emerge from the ventral urethral epithelium between the 20th and 21th day of prenatal life, reaching the ventral mesenchymal pad (VMP) and initiating the branching process at the first day of postnatal life. We also noted that the smooth muscle layer seems to have a central play during this process, which may act as physical barrier to the buds heading to VMP. Regarding the nuclear receptors, the androgen presented a high immunomarking at the periurethral mesenchyme, whereas cells with positive marking for estrogen receptor alpha were identified either in the periurethral mesenchyme or in the buds. Finally, the results demonstrate that the prostate organogenesis, besides involving a series of events finely regulated, is an extremely sensitive and crucial to the health of the gland throughout the life / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Orientador: Wilson de Figueiredo Jardim / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-19T06:01:40Z (GMT). No. of bitstreams: 1 Raimundo_CassianaCarolinaMontagner_D.pdf: 6486329 bytes, checksum: eb2082b97701f19b66fc4e6dc033da3f (MD5) Previous issue date: 2011 / Resumo: O objetivo deste trabalho foi avaliar, através de um perfil anual, a qualidade dos mananciais e da água tratada de Campinas (SP) em relação à presença de cafeína, estrona, 17b-estradiol, estriol, progesterona, testosterona, 17a-etinilestradiol, mestranol, levonorgestrel, dietilestilbestrol, triclosan, 4-n-octilfenol, 4-n-nonilfenol, bisfenol A, fenolftaleína e atrazina empregando extração em fase sólida (SPE) e cromatografia líquida acoplada ao espectrômetro de massas (LC-MS/MS), e verificar o potencial estrogênico das amostras usando o bioensaio Bioluminesce Yeast Estrogen Screening (BLYES). Os resultados mostraram a presença de seis dos 16 compostos investigados em pelo menos uma amostra de água tratada. A estrona foi detectada apenas uma vez. Concentrações de atrazina de até 687 ng L foram determinadas nas amostras provenientes do rio Capivari, e nas amostras de água tratada provenientes do rio Atibaia as concentrações não passaram de 42 ng L. A fenolftaleína foi determinada em apenas uma campanha em todas as amostras provenientes do rio Atibaia em concentrações de até 20 ng L. As concentrações médias de bisfenol A e triclosan foram 7 e 17 ng L, respectivamente. Nos mananciais foram determinados 13 dos 16 compostos investigados. Todas as amostras de água bruta e tratada apresentaram concentrações de cafeína que variaram significativamente entre os períodos de seca e cheia dos rios. As estações de tratamento de água de Campinas, operando no sistema convencional foram capazes de remover entre 30 e 99% dos contaminantes investigados. No mais, 40 % das amostras de água tratada apresentaram atividade estrogênica positiva (entre 0,01 e 1,33 ngE2equiv L) e todas as amostras de água bruta apresentaram estrogenicidade entre 0,06 e 6,54 ngE2equiv L. Sendo que estas foram maiores nos períodos de estiagem / Abstract: The purpose of this work was to evaluate an annual profile of both source of supply and drinking water quality in the city of Campinas (São Paulo State) in relation to the presence of caffeine, estrone, 17b-estradiol, estriol, progesterone, testosterone, 17a-ethynylestradiol, mestranol, levonorgestrel, diethylstilbestrol, triclosan, 4-n-octylphenol, 4-n-nonylphenol, bisphenol A, phenolphthalein and atrazine using Solid Phase Extraction (SPE) and Liquid Chromatography with Tandem Mass Spectrometry Detection (LC-MS/MS), and make sure the estrogenic potential of samples using Bioluminesce Yeast Estrogen Screening bioassay (BLYES). Results showed the presence of six of the 16 compounds investigated in least one sample of drinking water (estrone, triclosan, bisphenol A, phenolphthalein, atrazine and caffeine). Atrazine concentrations up to 687 ng L were found in drinking water samples from the Capivari River. In drinking water samples from the Atibaia River, the concentrations did not exceed 42 ng L. Phenolphthalein was determined in only one campaign in both raw and treated water from the Atibaia River at concentrations around 20 ng L. Average concentrations of bisphenol A and triclosan in drinking water samples were 7 and 17 ng L, respectively. Thirteen of the 16 investigated compounds were determined in surface waters. All samples of raw and treated water showed concentrations of caffeine that varied significantly between dry and wet seasons. The conventional water treatment plants in Campinas were able to remove between 30 and 99 % of the investigated contaminants. The estrogenic activity was evaluated using BLYES bioassay, where 40% of drinking water samples showed positive estrogenic activity (between 0.01 and 1.33 ngE2equiv L) and all samples of surface water presented estrogenic activity between 0.06 and 6.54 ngE2equiv L. These values were higher in dry winter periods / Doutorado / Quimica Analitica / Doutor em Ciências

L’un des plus importants défis de la toxicologie est de pouvoir extrapoler les résultats issus des différentes phases de l’analyse du risque sanitaire à partir de systèmes expérimentaux vers les populations humaines. Dans ce contexte, les techniques globales dites "omiques" sont de plus en plus utilisées pour caractériser les différents états des systèmes biologiques. Ainsi, la toxicogénomique permet non seulement d’étudier les mécanismes d’action des composés, d’identifier des marqueurs d’exposition, mais aussi de générer des signatures moléculaires à potentiel prédictif. En effet, des composés ayant des signatures moléculaires semblables ont également de forts risques de présenter les mêmes effets toxicologiques. L’objectif de cette thèse est d’appliquer ce concept de manière systématique, en explorant les données publiées et disponibles dans les banques dédiées à la toxicogénomique via des modèles statistiques multivariés. Ces analyses ont pour objectif de permettre le regroupement et donc la classification des composés sur la base des gènes dont ils affectent l’expression. L’appartenance de produits toxiques bien caractérisés aux classes ainsi constituées permet alors d’émettre des hypothèses quant à la toxicité des autres composés. Un jeu de données quantitatives intégrant 18 études réalisées avec la même technologie de puce à ADN et chez une seule espèce a été assemblé. De ce jeu de données, 3022 signatures toxicogénomiques correspondant à 452 composés différents ont été obtenues. Des approches de classification non supervisées afin de définir des classes de traitements induisant des altérations transcriptionnelles proches ont été mises en place. 95 et 104 classes ont été obtenues en fonction des méthodes utilisées. Finalement, une attention toute particulière a été portée sur les potentiels nouveaux perturbateurs endocriniens et xénobiotiques reprotoxiques sur-représentés dans trois classes spécifiquement. 22 composés sont en cours de test sur une lignée cellulaire humaine exprimant les enzymes de la stéroïdogenèse (NCI-H295R) pour évaluer leur potentiel effet perturbateur endocrinien. L’ensemble de ce travail a ainsi permis de démontrer la pertinence de nos approches de toxicogénomique pour la prédiction des effets toxiques de composés testés dans d’autres organes et/ou chez d’autres espèces. Il se poursuit à l’heure actuelle par la mise en place d’une base de données, TOXsIgN, permettant l’hébergement et l’accès à l’ensemble de signatures de toxicogénomique générées dans ce projet. De même, mon travail a également permis la mise en place de plusieurs outils dédiés à la toxicologie prédictive et à la visualisation de données, tels que le navigateur de génomes comme le ReproGenomics Viewer (RGV). / The core aim of my thesis project is to develop predictive toxicology approaches based on the integration of massive toxicogenomics datasets using bioinformatics and biostatistics methodologies. Specific objectives include: (1) classification of chemicals based on toxicogenomics signatures, i.e. the set of genes whose expression is known to be positively or negatively altered after exposure to these compounds; (2) the association of the resulting classes with human disorders or deleterious phenotypes based on the well-known toxicants present in those classes; (3) the prediction of novel reprotoxicants and/or endocrine disruptors based on toxicogenomics signature similarities with known chemicals affecting testis development and function. The assembled toxicogenomics dataset contains 23,657 samples covering 7092 experimental conditions (one chemical, one dose, one exposure time, one tissue) for 541 chemicals in seven distinct tissues in the rat from 18 different studies. From this dataset, 3,022 experimental conditions corresponding to 452 distinct compounds are associated to a toxicogenomics signature containing more than ten genes showing an altered expression pattern after exposure. Using unsupervised classification methods, 95 chemical clusters were defined showing close toxicogenomics signatures. The phenotype association analysis using data extracted from de Comparative Toxicogenomics Database (CTD) allowed us to identify three clusters significantly enriched in known endocrine-disrupting chemicals. Currently, 22 compounds are being tested on a human cell line expressing the enzymes of steroidogenesis (NCI-H295R) to evaluate their potential endocrine disrupting effects. These researches allowed us to demonstrate the relevance of integrating massive toxicogenomics datasets to predict adverse effects of compounds tested in different organs. It is currently being pursued through the development of a novel repository, TOXsIgN. This resource provides a flexible environment to facilitate online submission, storage and retrieval of toxicogenomics signatures by the scientific community. Similarly, the current PhD project also yielded to the implementation of several tools dedicated to predictive toxicology and data visualization including the ReproGenomics Viewer (RGV).

Les écosystèmes aquatiques constituent la destination finale des contaminants. Les organismes aquatiques sont ainsi impactés par un cocktail de molécules, dont les produits phytosanitaires. Le caractère perturbateur endocrinien (PE) de certains d’entre eux est particulièrement préoccupant. La présente étude explore les effets de pesticides modèles à potentiel PE – la chlordécone (CLD) et le pyriproxyfène (PXF), seuls et en mélange – chez le copépode estuarien Eurytemora affinis par des approches écotoxicogénomiques innovantes. En réponse aux PE, l’accent a été porté sur les effets de ces composés sur la reproduction, la croissance ou le développement. En parallèle, l’affinité des pesticides aux récepteurs aux oestrogènes et aux androgènes humains d’une part, et aux ecdystéroïdes d’autre part, a été étudiée grâce à des outils bio analytiques afin d’évaluer la pertinence de l’utilisation du potentiel PE défini par les tests cellulaires comme critère d’exclusion dans la réglementation européenne des pesticides. Ces travaux de thèse présentent, pour la première fois, les transcriptomes et protéomes d’E. affinis après exposition aux PE. Un effet sexe-dépendant a été observé par les deux approches « omics » révélant un plus fort impact des composés sur les copépodes mâles. Des gènes et protéines, impliqués dans le processus de mue et dans la gamétogénèse ont été identifiés comme candidats pour le développement de biomarqueurs moléculaires de PE chez les crustacés. Les tests cellulaires ont permis de mettre en évidence des interactions entre les contaminants et les récepteurs hormonaux. Toutefois, le screening de molécules par les tests YES/YAS/anti-YES/anti-YAS s’est heurté à quelques limites d’interprétations. Enfin, les résultats du test S2 EcR, souligne la pertinence de l'utilisation couplée de tests spécifiques d’un système endocrinien de vertébrés et d’invertébrés dans l’évaluation du risque des composés PE. / Aquatic ecosystems constitute the chemicals’ final destination. Among the xenobiotics, endocrine disruptors (ED) are compounds of major concern. In this context, ED pesticides effects were investigated in the widespread copepod Eurytemora Affinis using ecotoxicogenomics technics. In response to PE pesticides, focus was made on reproduction, growth and development. In order to evaluate the endocrine activity, in vitro tests (YES/YAS/anti-YES/anti-YAS and S2 EcR) were used to screen the compounds alone and in mixtures. These results were discussed to evaluate the “cut off criterion” used in European assessment. This work presents for the first time, transcriptomes and proteomes of E. affinis after PXF and CLD –alone and in binary mixture- exposure. A sex dependent effect was observed by the two “omics” approaches. Male copepods were more impacted by contaminants than female copepods. Genes and proteins (e.g. chitin deacetylase, kelch protein) were identified as candidates for the development of ED molecular biomarkers. In vitro tests highlighted binding of pesticides with both vertebrate and invertebrate receptors. However, a toxicity for the highest concentrations tested and some limits for the interpretation of mixtures results were limiting in YES/YAS/anti-YES/anti-YAS assays. The last observation represents particularly a major concern for interpretation of the ED mode of action of environmental matrix. S2 EcR highlighted some complementary results about the mode of action of chemicals alone and in binary mixtures. These results accentuate the need to combine vertebrate specific test and invertebrate specific test in ED risk assessment.

La protéine kinase D1, PKD1, est une sérine/thréonine kinase activée par de nombreux facteurs mitogènes. Les études, menées jusqu’à présent sur les fonctions de PKD1, ont montré qu’elle semble jouer un rôle dans la régulation de plusieurs processus biologiques fondamentaux impliqués dans le développement des tumeurs. Cependant, le rôle précis et les cibles de PKD1 restent largement méconnus. Au cours de ce travail, nous avons tout d’abord démontré que l’inhibition de PKD1 dans les cellules de mélanome inhibe la croissance et la motilité cellulaire, induit l’expression de la E-cadhérine et une diminution de la N-cadhérine. D’autre part, nous nous sommes intéressés au rôle des perturbateurs endocriniens dans les cellules tumorales mammaires et avons démontré que PKD1 est une cible des perturbateurs endocriniens (PE). Les PE, tels que le bisphénol A (BPA) et les phtalates, sont des produits chimiques ubiquitaires de notre environnement. Leur rôle dans la croissance tumorale mammaire est bien documenté. Néanmoins, les mécanismes moléculaires précis par lesquels ces molécules agissent demeurent encore inconnus. Au cours de notre travail, nous avons démontré que ces PE induisent de façon dose-dépendante la prolifération des cellules MCF-7 (cellules d’adénocarcinome mammaire) et que ce processus biologique est dépendant de l’expression de PKD1. Ainsi, l’ensemble de ce travail fait apparaître, d’une part, que PKD1 pourrait être une nouvelle cible thérapeutique anti-tumorale potentielle dans le mélanome et que, d'autre part, PKD1est une cible moléculaire de certains PE dans le cancer du sein. / Protein kinase D1, PKD1, is a serine/threonine kinase which can be activated by mitogens and that regulates various functions involved in the development of tumors. However, its precise role and targets are still unclear. Our study demonstrates that PKD1 inhibition in melanoma cells decreased cell growth and motility, and reversed the E- to N-cadherin switch. On the other hand, we examined the role of endocrine disruptors (EDs) in breast cancer cells and identified PKD1 as a target of these compounds. EDs, such as bisphenol A (BPA) and phthalates, have been found molecular mechanisms are still ubiquitously throughout our environment. Their role in breast tumor growth has been well documented. However, their precisemolecular mechanisms are still unknown. Our study demonstrates that EDs induce dose-dependently MCF-7 cell growth and that this biological process is dependent upon PKD1expression. Thus, this work may define PKD1 as a novel potential anti-tumor therapeutic target in melanoma and identifies PKD1 as a new molecular target of some EDs in breast cancer cells.

Orientador: Sebastião Roberto Taboga / Resumo: A próstata é uma glândula componente do sistema genital dos mamíferos e sua principal função prostática é secretar um fluido levemente alcalino (pH 7,29) que contribui para a formação do líquido seminal. As fêmeas em certas espécies, como o roedor gerbilo da Mongólia (Meriones unguiculatus), apresentam próstata cuja morfologia se assemelha à porção ventral do complexo prostático masculino e à próstata feminina em humanos. Nas últimas décadas tem sido relatado um constante aumento da exposição a substâncias capazes de interferir na ação hormonal, conhecidas como disruptores endócrinos. O bisfenol A (BPA) pertence a uma das categorias de disruptores endócrinos presentes no meio ambiente. Estudos demonstram que a exposição a esse composto interfere negativamente na morfofisiologia prostática, favorecendo o estabelecimento de lesões pré-malignas e malignas. Além do BPA, uma das constantes problemáticas dos dias atuais é a exposição ambiental a hormônios exógenos como o estradiol (E2). Desta forma, a administração de fármacos com estrutura semelhante a hormônios esteroides pode exercer efeitos sobre a glândula, uma vez que os mesmos se ligam a seus receptores hormonais afetando a homeostasia prostática. O presente estudo teve como objetivo verificar as alterações morfofuncionais na próstata de fêmeas de gerbilo da Mongólia após tratamento perinatal com dose diária ambiental, ou seja, dose ambiental segura de BPA (50 µg/kg) e doses de 17-β estradiol (35 µg/kg), 3 vezes por semana p... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

17α-ethinylestradiol (EE2) is a synthetic hormone, derivative of the natural hormone estradiol. EE2 is one of the the most prescribed drugs in the world. It belongs to the estrogenic endocrine disrupter chemicals. These compounds are able to alter functions of the endocrine system and cause adverse effects in the organism, offspring and (sub)population. In this thesis, there are observed effects of 17α-ethinylestradiol on enzyme activities of main enzymes involved in phase I of xenobiotic biotransformation, i.e. cytochromes P450 (CYP), in vitro. Isoforms of CYP subfamilies 1A, 2B, 2C, 2E and 3A were studied in rats and humans. Each CYP isoform was incubated with EE2 at two concentrations, 10μM EE2 and the concentration corresponding to the substrate concentration in the specific marker reactions of individual CYP isoforms. The results indicate, that in rat liver microsomes the activity of all studied isoforms except CYP1A2 was decreased in the presence of EE2. When EE2 was added to the incubation mixture at the concentration of the reaction substrate, the greatest decrease in enzyme activity was observed for CYP2C6, with the remaining activity only 36%. In human liver microsomes, the activity of CYP2B6, CYP2C9, CYP2E1 and CYP3A4 was also effected by EE2. As in the case of rat model, CYP2C subfamily...

Les organes centraux du tractus urogénital sont le testicule et l’ovaire, qui assurent la production de gamètes et d’hormones, et donc la fertilité de l’individu. Ces deux organes, parfaitement distincts et complémentaires, ont pour origine une gonade bipotentielle qui s’engagera vers une trajectoire de différenciation masculine ou féminine au cours de la vie fœtale. Les deux gonades vont par la suite subir plusieurs phases de différenciation et de développement de leurs populations cellulaires, afin d’acquérir leurs fonctions propres qui leur permettront d’assumer leur rôle à l’âge adulte. Depuis plus d’une quinzaine d’années, le concept de syndrome de dysgénésie testiculaire fait état d’un lien entre l’exposition du fœtus à des composés environnementaux et des anomalies du tractus urogénital. Bien que sujette à de vifs débats au sein de la communauté scientifique, cette hypothèse a attiré l’attention de la recherche sur les conséquences de l’exposition des mères aux xénobiotiques sur l’enfant à naître. La différenciation et le développement des gonades fœtales sont gouvernés par des programmes d’expression spécifiques de chaque sexe, dont de nombreuses zones d’ombres subsistent, notamment concernant la fraction non-codante exprimée par le génome humain. La première partie de cette thèse de doctorat présente, pour la première fois, le paysage transcriptionnel contrôlant ces processus complexes entre la 6ième et 17ième semaine de développement chez l’Homme. Grâce à l’avènement des technologies de transcriptomique, il est désormais possible d’identifier et d’observer l’expression des gènes de manière sensible et sans a priori. Le RNA-seq m’a donc permis de décrire de manière exhaustive la dynamique d’expression des gènes, pendant les stades précoces de la différenciation sexuelle, jusqu’aux phénomènes plus tardifs conduisant aux linéages des différentes populations cellulaires spécifiques du testicule et de l’ovaire. Dans une deuxième partie, mon travail de recherche s’est attaché à étudier l’impact de deux perturbateurs endocriniens suspectés, l’ibuprofène et le chlordécone, sur le programme d’expression du testicule fœtal humain. L’utilisation du RNA-seq m’a permis de définir et de comparer la signature toxicogénomique de chaque molécule afin de contribuer à la compréhension de leur mécanisme d’action et d’identifier les populations cellulaires affectées. Enfin, face à l’essor des technologies ultra-haut-débit dans les sciences de la vie, y compris dans les domaines de la reproduction, j’ai activement participé au déploiement d’une nouvelle version du Reprogenomic Viewer dans la dernière partie de ma thèse (http://rgv.genouest.org). Cet outil nternet a pour vocation de centraliser et de rendre accessibles les données de séquençage accumulées au sein de la communauté de la reproduction via des outils de visualisation intuitifs. / Fetal life is a crucial period for sexual reproduction when bipotential gonads differentiate into either a testis or an ovary. Gaining insights into the complex molecular events underlying this process is central to a better understanding of disorders of sexual development. The present work intends to improve the knowledge on molecular pathways at play during gonad development in humans using RNA-sequencing. This project particularly seeks to identify early transcriptional events that may play critical role in the regulatory network driving human sexual differentiation. To address this issue, we defined the transcriptional landscape of fetal human gonads by sequencing total RNA extracted from testes and ovaries between 6 and 17 gestational weeks. The resulting paired-end reads were mapped on the human genome and then assembled into transcripts using the Tuxedo suite. We next defined a high-confidence set of transcripts showing differential expression across samples. Clusters of co-expressed genes were subjected to functional analysis. The analysis of this massive RNA-seq dataset has led to a high-confidence set of 35,194 assembled transcripts; among which 32,391 known and novel isoforms coding genes (mRNAs), 1,209 to long non-coding (lnc) RNAs and 318 to novel unannotated transcripts/genes (NUTs). The dynamic of transcriptional landscape occurring during human fetal gonads development has been described and new genes and interesting candidates, including new genes, have been highlighted as potential key genes governing this biological process. The second interest of this work was the study of the impact of two endocrine disruptors, ibuprofene and chlordecone, on human fetal testis using RNA-seq. The transcriptional alteration induced by these compound in the gonad allowed a deeper understanding of their mechanisms of action of endocrine disruption. The last part of this work was the development of a new version of the ReproGenomics Viewer (http://rgv.genouest.org), a web tool dedicated to the integration and accumulation of sequencing data from studies performed in the field of reproduction.