In recent times, endocrine disrupting chemicals (EDCs) have been associated with the rise in neurodevelopmental disorders such as autism, attention deficit hyperactivity disorder (ADHD) and decreased intelligence quotient (IQ) in children. This effect is suspected to be induced at pre-/peri-natal development, via an alteration in hormonal signaling, thus interfering with neuronal differentiation, with subsequent effect on normal brain development and function in exposed children. This issue increases the need for chemical screening for potential developmental neurotoxicity (DNT) effect. The current available EDC induced DNT test guideline is based on in-vivo testing that requires animal use. Here, a multipotent neural progenitor cell line, the C17.2 cell-line, generated from neural stem cells of the external germinal layer of mouse cerebellum, with potential to differentiate to neurons or astrocytes, is introduced for in-vitro EDC induced DNT testing. This project focused on optimizing the C17.2 cell-line for the detection of EDC-induced DNT with emphasis on the disruption of the oestrogen, androgen, and thyroid hormone systems. It aimed at validating the involvement of oestrogen, androgen, and thyroid hormone on molecular and cellular endpoints relevant for the differentiation of the C17.2 cells.  Herein, the cells were exposed to the hormonal agonist and antagonist at a range of concentrations for a 10-day differentiation period. After exposure, LDH, viability assay and morphological changes (percentage of neurons in culture and neurite outgrowth) were evaluated. The results showed no morphological changes induced by androgen receptor (AR) agonist/antagonist at relevant physiological concentrations. The thyroid receptor (TR) agonist and antagonist on the other hand showed a response in the form of increased neurite outgrowth in relation to the negative control at a concentration range of 40-200 nM and 40 nM respectively. The oestrogen receptor (ER) antagonist at 100 nM also increased percentage neuron in culture. Additionally, in-silico analysis of microarray and RNA sequencing data were used to map out target genes regulated by ER, AR and TR and involved in neurodevelopment. With this approach, 29 marker genes were identified. Validation of the marker genes by means of gene expression (qPCR) was carried out, ER and TR agonist/antagonist were observed to modulate the expression of examined genes. In summary, the model could not be established for detecting EDC induced DNT via androgenic and oestrogenic pathway, while it is a promising model for identifying DNT induced by thyroid hormone signalling disruption.

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Le développement et l’homéostasie cellulaire de la prostate impliquent le contrôle strict des voies de signalisation induites par les androgènes et les facteurs de croissance. Ces diverses voies sont profondément altérées dans le cancer de la prostate, notamment lors des stades les plus avancés. Dans ce travail, une lignée immortalisée à partir de l’épithélium de prostate humaine, la lignée RWPE-1, a été utilisée pour étudier certains signaux régulant la prolifération cellulaire, ainsi que les connexions entre les voies de signalisation correspondantes. La prolifération des cellules RWPE-1 est sous la dépendance de l’EGF (Epidermal Growth Factor) qui intervient physiologiquement dans le développement épithélial. Les récepteurs apparentés à l’EGF-R sont également impliqués dans la prolifération au cours de la progression tumorale. La prolifération des cellules RWPE-1 en réponse à l’EGF est strictement dépendante de la voie ERK1/2, qui est donc considérée comme un point d’intégration des signaux. L’utilisation d’inhibiteurs du récepteur aux androgènes a permis de montrer le rôle essentiel qu’il joue dans l’activation d’ERK1/2 en réponse à l’EGF. Le récepteur aux androgènes s’associe avec plusieurs molécules de signalisation dans les cellules RWPE-1. Je démontre ici pour la première fois une association entre le récepteur aux androgènes et la kinase Raf-1, activatrice de la voie ERK1/2. Ainsi, le récepteur aux androgènes contrôlerait directement un processus essentiel à la prolifération épithéliale selon un mode d’action non-génomique. Par ailleurs, j’ai montré que la réponse proliférative des cellules RWPE-1 à l’IL-6 requiert l’activation de la voie ERK1/2, et l’activité kinase de l’EGF-R, suggérant la transactivation de ce récepteur par l’IL-6. L’utilisation de divers inhibiteurs chimiques a permis de démontrer que les métalloprotéases de la famille ADAM (a disintegrin and metalloprotease), notamment ADAM17, sont impliquées dans ce processus. Ainsi, l’activation de protéines ADAM par l’IL-6 conduirait au clivage d’un ligand membranaire de l’EGF-R, aboutissant à l’activation de la voie ERK1/2. Ce nouveau mécanisme pourrait être impliqué dans les situations inflammatoires conduisant à une prolifération excessive de l’épithélium prostatique, prélude à la transformation tumorale. En conclusion, les voies de signalisation étudiées sont fortement connectées dans les cellules épithéliales normales. Les deux nouveaux mécanismes décrits ici aboutissent à l’activation des kinases ERK1/2, point d’intégration et de convergence des voies de signalisation dans les cellules épithéliales de prostate normale. / Prostate development and cell homeostasis involve strict control of androgen and growth factors induced signaling pathways. These signaling pathways are deeply altered in prostate cancer, especially during late stages. In this work, the RWPE-1 immortalized cell line derived from human prostate epithelium has been used to study the signaling pathways regulating cell proliferation and their crosstalk. Optimal RWPE-1 proliferation is dependent on EGF (Epidermal Growth Factor), that also controls normal epithelial development. EGF-R family is also involved in cancer cell proliferation. EGF-dependent RWPE- 1 cell proliferation relies strictly on the ERK1/2 pathway which is then seen as a signal integrating node. Specific inhibitors showed essential role of androgen receptor in EGF mediated ERK1/2 activation. Androgen receptor is associated with several signaling molecules in RWPE-1 cells. I show here for the first time the physical interaction between the androgen receptor and the ERK1/2 activating kinase Raf1. Then, the androgen receptor could directly regulate an essential pathway for epithelial cells proliferation through a non-genomic mechanism. In addition, I showed that IL-6 dependent RWPE-1 cells proliferation requires both ERK1/2 and EGF-R kinase activities, suggesting an IL-6 mediated transactivation of EGF-R. By using several inhibitors, I showed that ADAM (a disintegrin and metalloprotease) family metaloproteases, especialy ADAM17, are involved in this process. IL-6-mediated ADAM proteins activation could lead to the cleavage of a membrane bound EGF-R ligand, leading to ERK1/2 pathway activation. This new mechanism could be involved in the inflammatory situations inducing hyperproliferation of the prostate epithelium, the first step of the transformation process. To conclude, the signaling pathways I studied are strongly connected in normal epithelial cells. The two new mechanisms described in this study lead to ERK1/2 kinases activation, an integrating node of signaling pathways in normal prostate epithelial cells.

Introdução: O desenvolvimento psicossexual humano inicia no período pré-natal e é composto pelo papel de gênero (PG), pela identidade de gênero (IG) e pela orientação sexual (OS). Em indivíduos com DDS 46,XY, vários fatores podem comprometer esse desenvolvimento, levando a incongruência de identidade de gênero e à mudança de gênero. Nesses pacientes, a exposição androgênica pré-natal e o grau de virilização da genitália externa tem sido avaliados como possíveis influenciadores destes desfechos, mas seu papel ainda não foi esclarecido. Objetivos: Avaliar os desfechos psicossexuais - IG, PG e OS - e aspectos da vida sexual em uma coorte de indivíduos com DDS 46,XY na idade adulta com diagnostico etiológico caracterizado do ponto de vista clínico e molecular e investigar a influência da exposição androgênica pré-natal e do grau de virilização da genitália externa nesses desfechos e na prevalência de disforia de gênero (DG). Pacientes: 144 pacientes com diagnóstico etiológico confirmado de DDS 46,XY acompanhados do HCFMUSP com idade entre 16 e 60 anos foram incluídos neste estudo. Métodos: Os componentes do desenvolvimento psicossexual (IG, PG, OS) foram avaliados usando questionários e por teste psicológico projetivo (HTP - House-Tree-Person). O escore de Sinnecker foi utilizado para a mensuração do grau de virilização da genitália externa. A exposição androgênica pré-natal foi estimada de acordo com a etiologia do DDS 46,XY. Aspectos da vida sexual foram avaliados através de questionário específico.Todas as variáveis categóricas foram analisadas usando teste X². A força de associação foi avaliada pelo cálculo do V de Cramer. O índice kappa foi usado para avaliar concordância entre resultados dos testes. Resultados: Houve uma associação positiva entre exposição androgênica pré-natal e a maior incidência de desfechos psicossexuais masculinos em indivíduos com maior exposição. O grau de virilização da genitália externa não interferiu nos desfechos psicossexuais. Houve uma prevalência de 19% (27/144) de disforia de gênero em toda a coorte. Em 93% (25/27), a DG foi do sexo feminino para o masculino e ocorreu em 50% (16/32) de casos de deficiência de 5alfa-RD2, seguido de 33% (5/15) dos casos de deficiência da 17beta-HSD3 e se associou com exposição androgênica pré-natal (p < 001; V=0,461), mas não com a virilização da genitália externa. A mediana de idade do desejo de mudar de sexo foi de 8 anos (5 - 9) enquanto que a da idade da mudança de sexo foi 15 anos (10.5 - 20). Os desfechos psicossexuais mostraram maior concordância com o sexo social final (PG - k=0.81; IG - k=0.65 e OS - k=0.85) do que com o sexo de registro (PG - k=0.1; IG - k=0.25 e OS - k=0.15). Quanto a sexualidade, alguns parâmetros (fantasias sexuais, masturbação e parceiro sexual fixo) foram melhores no sexo masculino comparado ao feminino. No entanto, não houveram diferenças em relação aos parâmetros da vida sexual comparando indivíduos do sexo feminino com e sem atipia genital e indivíduos do sexo masculino que mantiveram o sexo social com os que mudaram para este sexo. Conclusões: A exposição androgênica pré-natal influenciou o desenvolvimento psicossexual em indivíduos com DDS 46,XY, de uma forma exposição-dependente, favorecendo desfechos masculinos, enquanto que o grau de virilização da genitália externa não influenciou estes desfechos. A DG do feminino para o masculino foi comum entre esses indivíduos e também foi influenciada pela exposição androgênica pré-natal. Os parâmetros psicossexuais nesses pacientes concorda muito mais com o sexo social final do que com o sexo de registro. A sexualidade dos indivíduos do sexo masculino tem aspectos mais satisfatórios que o feminino. Atipia genital no sexo feminino não afetou a sexualidade destas pacientes assim a sexualidade dos indivíduos que mudaram para o sexo masculino são semelhantes aos que foram registrados no sexo masculino Behavioral and Psychosexual Aspects of 46,XY DSD Individuals At Adulthood / Introduction: The human psychosexual development begins at prenatal period and is composed by gender role, gender identity and sexual orientation. In 46,XY DSD individuals a variety of factors may jeopardize an adequate psychosexual development and sometimes results in desire to change the gender. The effects of prenatal androgen exposure and the impact of atypical genitalia in the psychosexual outcomes have been suggested as influencing factors in the human psychosexual development but there is not conclusive evidence, especially in DDS 46, XY. Methods: We evaluated the psychosexual compounds - gender role (GR) at childhood gender identity (GI) and sexual orientation (SO) in individuals a large cohort of 144 46,XY DSD individuals, 86% of them raised in the female social sex, from a single tertiary medical center. The same psychologist, specialized in DSD, performed the psychosexual evaluation. We used a questionnaire and a projective psychological test (HTP test) to measure the psychosexual compounds. Prenatal androgen exposure was estimated considering the 46,XY etiology. Sinnecker\'s score was used to measure the external genitalia virilization. All ordinal variables were analyzed using Wilcoxon test. Categorical variables were analyzed using X2 test with posterior Cramer\'s V to measure the association strength. The kappa index was calculated as a concordance measure. Results: We found an association between prenatal androgen exposure and major prevalence of male psychosexual outcomes and a higher incidence of female to male gender dysphoria. There was not difference in the psychosexual outcomes according by external genitalia virilization in male and in female individuals. There was an incidence of 19% of gender dysphoria (27 out from 144). In 93% (n=25), the gender change was from female to male (F to M). The ethological diagnosis related with F to M GD were 5alpha-RD2 deficiency (5ARD2) in 16/32 (50%), followed by 5/15 (33%) in 17beta-HSD3 deficiency (17betaHSD3). Others diagnosis related with F to M GD were: partial gonadal dysgenesis (n=3/24; 12%) and 3betaHSD2 (n=1/3; 33%). Both cases of male to female (M to F) GD occurred in partial gonadal dysgenesis (8%; n=2/24). The median of GD age (desire to belong to another gender) was 8 years old (5-9), and the median of gender change itself was 15 years old (10.5 - 20). In F to M GD, gender change was associated with prenatal androgen exposure (p < 001; V=0,461). The psychosexual components showed higher concordance index with final gender (GI - k=0.81; GI - k=0.65 and SO - k=0.85) then with the assigned sex (GI - k=0.1; GI - k=0.25 and SO - k=0.15). Conclusion: Prenatal androgen exposure affects the psychosexual development, favoring more male outcomes. This influence was observed in GI, GR and SO. The degree of external genitalia virilization did not influence the psychosexual development. Female to Male GD is common in 46,XY DSD raised in female social sex, especially in 5ARD2 and 17?HSD3 deficiencies. There is a strong relationship between prenatal androgen exposure and F to M GD. On the other hand, M to F gender change was rare in 46,XY DSD and occurred only in partial gonadal dysgenesis patients

INTRODUÇÃO: Os carcinomas HER-2 positivos representam cerca de 20- 30% de todos os tumores da mama e se caracterizam por curso clínico mais agressivo, com alta proliferação celular e resistência a apoptose, determinados por cascatas de sinalizações intracelulares, tais como a via PI3K/AKT. O trastuzumabe, um anticorpo monoclonal humanizado que se liga à molécula de HER-2, é o tratamento padrão destas pacientes. A resposta a monoterapia com trastuzumabe varia de 12-30% e a persistência da ativação da via PI3K/AKT é um dos mecanismos de resistência. A ativação do AKT começa com a fosforilação do PIP2 a PIP3 pela PI3K. A desfosforilação do PIP3 é mediada pela PTEN e sua deficiência é um dos fatores possivelmente implicados na resistência ao trastuzumabe. Além da resistência à terapêutica, os tumores HER-2 positivos são heterogêneos quanto ao seu comportamento biológico. A busca de diferentes padrões morfológicos e moleculares neste grupo de carcinomas pretende identificar subgrupos prognósticos e preditivos, permitindo a individualização terapêutica. OBJETIVOS: Estudar a expressão imunoistoquímica de duas moléculas da via de sinalização PI3K/AKT (PTEN e AKT fosforilada) e explorar a via de sinalização androgênica através da expressão do receptor de androgênio e dos perfis morfológico e molecular apócrinos. METODOLOGIA: O estudo foi retrospectivo com revisão dos preparados histológicos e construção de blocos de microarranjos com amostras dos tumores para estudo imunoistoquímico. Na revisão foram avaliados: tipo histológico, características morfológicas apócrinas, presença de componente in situ, graus histológico e nuclear, receptores de estrogênio e progesterona, e atividade proliferativa através da expressão imunoistoquímica do Ki-67. Os preparados histológicos foram submetidos à pesquisa de PTEN, AKT fosforilada e receptor de androgênio. Pacientes, familiares e médicos foram contatados para recuperação do seguimento e evolução. RESULTADOS: Foram estudadas 104 pacientes portadoras de carcinoma primário da mama. A expressão de PTEN esteve reduzida em 20/104 (19,2%) dos casos e foi mais freqüente nos tumores com AKT positivo (p= 0,06). O grupo de tumores sem perda de expressão de PTEN apresentou maior atividade proliferativa. A AKT foi positiva em 71/104 (68,3%) casos e se associou a maior grau de diferenciação e à expressão de receptor de androgênio. O receptor de androgênio foi positivo em 89/104 (85,6%) dos casos e esteve associado ao menor grau histológico (p=0,018), receptor de estrogênio (p=0,008) e menor atividade proliferativa (p=0,001). A ausência da expressão do receptor de estrogênio (perfil molecular apócrino) foi identificada em 41/104 casos (39,4%) e se associou a tumores com grau histológico mais alto. O perfil morfológico apócrino foi identificado em 71 (68,3%) dos casos e se associou a alto grau histológico e nuclear. O seguimento foi possível em 55 casos e observamos tendência a menor sobrevida livre de doença nos tumores AKTpositivos e RA-negativos. CONCLUSÕES: Nossos resultados comprovam a heterogeneidade dos carcinomas mamários HER-2 positivos e indicam diferenças em pelos menos duas vias de sinalização celulares como possíveis explicações para as mesmas: a via PI3K/AKT e a androgênica / BACKGROUND: HER-2 positive carcinomas represent about 20-30% of all breast tumors and are characterized by a more aggressive clinical course with high cell proliferation and apoptosis resistance, determined by cascades of intracellular signals, such as the PI3K/AKT pathway. Trastuzumab, a humanized monoclonal antibody that binds to HER-2 molecule, is the standard treatment for these patients. The response to monotherapy with trastuzumab ranges from 12-30% and the persistence of activation of the PI3K/AKT pathway is one of mechanisms of resistance. Activation of AKT begins with the phosphorylation of PIP2 to PIP3 by PI3K. The dephosphorylation of PIP3 is mediated by PTEN and its deficiency is one of the factors possibly involved in resistance to trastuzumab. In addition to resistance to therapy, HER-2 positive tumors are heterogeneous in their biologic behavior. The search for different morphological and molecular patterns of carcinomas in this group aims to identify prognostic and predictive subgroups, allowing for customized therapy. OBJECTIVES: To study the immunohistochemical expression of two molecules of the signaling pathway PI3K/AKT (phosphorylated AKT and PTEN) and to explore the androgen signaling pathway through the expression of androgen receptor and apocrine morphological and molecular profiles. METHODS: This study retrospectively reviewed the histological preparations and built tissue microarray with tumor samples for immunohistochemical study. We assessed histologic type, apocrine morphology, presence of in situ component, histologic and nuclear grade, estrogen and progesterone receptors and proliferative activity through the immunohistochemical expression of Ki-67. The tissue preparations were examined for PTEN, phosphorylated AKT and androgen receptor. Patients, relatives and physicians were contacted for retrieval of follow-up data. RESULTS: We studied 104 primary breast cancer patients. The expression of PTEN was reduced in 20/104 (19.2%) cases and was more frequent in tumors with positive AKT (p = 0.06). The group of tumors without loss of PTEN expression showed higher proliferative activity. AKT was positive in 71/104 (68.3%) cases and was associated with a higher degree of differentiation and with expression of androgen receptor. The androgen receptor was positive in 89/104 (85.6%) cases and was associated with lower histological grade (p = 0.018), estrogen receptor (p = 0.008) and lower proliferative activity (p = 0.001). The absence of expression of estrogen receptor (apocrine molecular profile) was identified in 41/104 cases (39.4%) and was associated with tumors of higher histologic grade. The apocrine morphological profile was identified in 71 (68.3%) cases and was associated with high histological grade and nuclear. Follow-up was possible in 55 cases and a trend for shorter disease-free survival was observed in AKT-positive and AR-negative tumors. CONCLUSIONS: Our results confirmed that HER-2-positive breast cancers are heterogeneous and indicate that differences in at least two cellular signaling pathways PI3K/AKT and androgen pathway might underliy such a heterogeneity

La castration représente le traitement de référence du cancer de la prostate à un stade avancé. Cependant, la plupart des patients rechute du fait notamment de l’émergence de variants tronqués constitutivement actifs du récepteur des androgènes (RA). Le microenvironnement tumoral, en particulier les fibroblastes associés au cancer (CAFs), favorisent largement la progression tumorale. Ils sont très hétérogènes et dérivent de plusieurs types cellulaires dont les cellules souches mésenchymateuses (MSCs). Afin de mettre à jour l’impact des variants du RA sur le microenvironnement tumoral, mon projet de thèse a porté sur l’étude des effets de ces variants du RA sur la différenciation des MSCs en CAFs. Les résultats obtenus m’ont permis de démontrer un impact positif du variant RA Q641X sur l’expression du facteur de différenciation VEGF par les cellules tumorales, ainsi que sur l’expression des marqueurs de différenciation en CAFs FSP-1, CXCL12, PDGFR-β, ainsi que VEGF, au niveau des MSCs. Ces données suggèrent que le variant RA Q641X est capable d’induire la différenciation des MSCs en CAFs, soulignant ainsi l’importance de développer de nouvelles stratégies thérapeutiques ciblant ces variants du RA. / Androgen ablation therapy remains the most common treatment for patients with advanced prostate cancer. However, most patients will relapse due to the emergence of truncated constitutively active androgen receptor (AR) variants. The tumor microenvironment is another necessary feature driving prostate cancer progression. Cancer associated fibroblasts (CAFs) are the major specialized stromal cells that favor tumor progression. These cells are very heterogeneous and derive from several other cell types as mesenchymal stem cells (MSCs). In order to highlight the impact of AR variants on surrounding tumor stroma, the aim of my project was to investigate the effects of these AR variants on MSCs differentiation into CAFs. I noticed that the expression of VEGF, a CAF differentiation factor, was upregulated in tumor cells expressing AR Q641X variant. Similarly, the expression of CAF differentiation markers FSP-1, CXCL12, PDGFR-β, and VEGF was enhanced in MSCs in presence of AR Q641X variant. These data highlight an unknown property of AR Q641X variant in prostate tumor cells that is its ability to induce MSCs differentiation into CAFs, underlining the urgent need to develop novel strategies targeting these AR variants.

BACKGROUND: Prostate cancer is by far the most common cancer among Swedish men. Some patients have an aggressive lethal disease, but the majority of affected men have long expected survival. Unfortunately, the diagnostic tools available are insufficient in predicting disease aggressiveness. Novel prognostic markers are therefore urgently needed. Furthermore, metastatic prostate cancer is generally treated with castration, but the long-term effects are insufficient. Additional studies are therefore needed to explore how the effects of this therapy can be enhanced. Prostate growth and regression is beside testosterone controlled by locally produced regulators. Vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) are two of the major regulators in the normal prostate and in prostate tumours. MATERIALS AND METHODS: VEGF and EGFR were explored in the prostate, by treating rats with either anti-VEGF or anti-EGFR treatment during castration and testosterone-stimulated prostate growth. Rats with implanted androgen-independent prostate tumours were treated with an inhibitor of both VEGF receptor-2 (VEGFR-2) and EGFR. Stereological techniques, immunohistochemistry, western blotting and quantitative real-time PCR were used to evaluate these experiments. Furthermore, prostate tissue from untreated prostate cancer patients was used to retrospectively explore the expression of phosphorylated-EGFR (pEGFR) in relation to outcome. RESULTS: Anti-VEGF treatment during testosterone-stimulated prostate growth, inhibited vascular and prostate growth. Anti-EGFR treatment during castration and testosterone-stimulated prostate growth resulted in enhanced castration effects and inhibited prostate growth. Anti-vascular treatment of androgen-independent prostate cancer with an inhibitor of VEGFR-2 and EGFR, that targets the normal and tumour vasculature, enhanced the effects of castration. Low immunoreactivity for pEGFR in prostate epithelial cells, both in the tumour and also in the surrounding non-malignant tissue, was associated with good prognosis. CONCLUSIONS: Anti-vascular treatment, with an inhibitor of VEGFR-2 and EGFR, in combination with castration could be an effective way to treat androgen-insensitive prostate tumours. VEGF and EGFR signalling are necessary components in testosterone-stimulated prostate growth. Phosphorylation of EGFR could be a useful prognostic marker for prostate cancer patients. Tumours may affect the surrounding non-malignant tissue and pEGFR immunoreactivity in the morphologically normal prostate tissue can be used to retrieve prognostic information.

La voie de signalisation du récepteur des androgènes (RA) est la principale cible thérapeutique des cancers de la prostate métastatiques. Toutefois, l'émergence de variants constitutivement actifs du RA dépourvus de leur partie C-terminale conduit à une résistance au traitement. Pendant ma thèse, j'ai montré que les variants du RA induisent une transition épithélio-mésenchymateuse (EMT) partielle, un phénomène observé lors de la progression tumorale. J'ai ensuite étudié les mécanismes conduisant à cette expression différentielle de marqueurs de l’EMT en me focalisant sur la N-cadhérine (CDH2). Le RA entier (AR-FL) et les variants du RA interagissent tous les deux au niveau des éléments de réponse aux androgènes dans l'intron1 de CDH2. Cependant, une augmentation du niveau d’acétylation des histones est observée uniquement avec les variants du RA. Mes données nous mène à un modèle où l'AR-FL réprimerait l'expression de CDH2 alors que les variants du RA induiraient son expression. / Androgen receptor (AR) pathway is the main therapeutic target for metastatic prostate cancer (Pca).However, the expression of AR variants lacking the carboxy-terminal end lowers therapy efficacy. During myphD, I showed that AR variants induce a partial epithelial-mesenchymal transition (EMT), a phenomenon observed during tumor progression. To understand the mode of action of AR variants, I explored the mechanisms leading to this differential expression of EMT markers focusing my research on N-cadherin(CDH2). While both the full length AR (AR-FL) and AR variants could interact with androgen response elements present in intron 1 of CDH2, I highlighted that they had opposite effects concerning histone modifications. Indeed, increased histone acetylation in this genomic region was observed only in the presence of AR variants. My data lead us to propose a model in which AR-FL represses CDH2 gene, while AR variants favor its expression.

Introdução: O desenvolvimento psicossexual humano inicia no período pré-natal e é composto pelo papel de gênero (PG), pela identidade de gênero (IG) e pela orientação sexual (OS). Em indivíduos com DDS 46,XY, vários fatores podem comprometer esse desenvolvimento, levando a incongruência de identidade de gênero e à mudança de gênero. Nesses pacientes, a exposição androgênica pré-natal e o grau de virilização da genitália externa tem sido avaliados como possíveis influenciadores destes desfechos, mas seu papel ainda não foi esclarecido. Objetivos: Avaliar os desfechos psicossexuais - IG, PG e OS - e aspectos da vida sexual em uma coorte de indivíduos com DDS 46,XY na idade adulta com diagnostico etiológico caracterizado do ponto de vista clínico e molecular e investigar a influência da exposição androgênica pré-natal e do grau de virilização da genitália externa nesses desfechos e na prevalência de disforia de gênero (DG). Pacientes: 144 pacientes com diagnóstico etiológico confirmado de DDS 46,XY acompanhados do HCFMUSP com idade entre 16 e 60 anos foram incluídos neste estudo. Métodos: Os componentes do desenvolvimento psicossexual (IG, PG, OS) foram avaliados usando questionários e por teste psicológico projetivo (HTP - House-Tree-Person). O escore de Sinnecker foi utilizado para a mensuração do grau de virilização da genitália externa. A exposição androgênica pré-natal foi estimada de acordo com a etiologia do DDS 46,XY. Aspectos da vida sexual foram avaliados através de questionário específico.Todas as variáveis categóricas foram analisadas usando teste X². A força de associação foi avaliada pelo cálculo do V de Cramer. O índice kappa foi usado para avaliar concordância entre resultados dos testes. Resultados: Houve uma associação positiva entre exposição androgênica pré-natal e a maior incidência de desfechos psicossexuais masculinos em indivíduos com maior exposição. O grau de virilização da genitália externa não interferiu nos desfechos psicossexuais. Houve uma prevalência de 19% (27/144) de disforia de gênero em toda a coorte. Em 93% (25/27), a DG foi do sexo feminino para o masculino e ocorreu em 50% (16/32) de casos de deficiência de 5alfa-RD2, seguido de 33% (5/15) dos casos de deficiência da 17beta-HSD3 e se associou com exposição androgênica pré-natal (p < 001; V=0,461), mas não com a virilização da genitália externa. A mediana de idade do desejo de mudar de sexo foi de 8 anos (5 - 9) enquanto que a da idade da mudança de sexo foi 15 anos (10.5 - 20). Os desfechos psicossexuais mostraram maior concordância com o sexo social final (PG - k=0.81; IG - k=0.65 e OS - k=0.85) do que com o sexo de registro (PG - k=0.1; IG - k=0.25 e OS - k=0.15). Quanto a sexualidade, alguns parâmetros (fantasias sexuais, masturbação e parceiro sexual fixo) foram melhores no sexo masculino comparado ao feminino. No entanto, não houveram diferenças em relação aos parâmetros da vida sexual comparando indivíduos do sexo feminino com e sem atipia genital e indivíduos do sexo masculino que mantiveram o sexo social com os que mudaram para este sexo. Conclusões: A exposição androgênica pré-natal influenciou o desenvolvimento psicossexual em indivíduos com DDS 46,XY, de uma forma exposição-dependente, favorecendo desfechos masculinos, enquanto que o grau de virilização da genitália externa não influenciou estes desfechos. A DG do feminino para o masculino foi comum entre esses indivíduos e também foi influenciada pela exposição androgênica pré-natal. Os parâmetros psicossexuais nesses pacientes concorda muito mais com o sexo social final do que com o sexo de registro. A sexualidade dos indivíduos do sexo masculino tem aspectos mais satisfatórios que o feminino. Atipia genital no sexo feminino não afetou a sexualidade destas pacientes assim a sexualidade dos indivíduos que mudaram para o sexo masculino são semelhantes aos que foram registrados no sexo masculino Behavioral and Psychosexual Aspects of 46,XY DSD Individuals At Adulthood / Introduction: The human psychosexual development begins at prenatal period and is composed by gender role, gender identity and sexual orientation. In 46,XY DSD individuals a variety of factors may jeopardize an adequate psychosexual development and sometimes results in desire to change the gender. The effects of prenatal androgen exposure and the impact of atypical genitalia in the psychosexual outcomes have been suggested as influencing factors in the human psychosexual development but there is not conclusive evidence, especially in DDS 46, XY. Methods: We evaluated the psychosexual compounds - gender role (GR) at childhood gender identity (GI) and sexual orientation (SO) in individuals a large cohort of 144 46,XY DSD individuals, 86% of them raised in the female social sex, from a single tertiary medical center. The same psychologist, specialized in DSD, performed the psychosexual evaluation. We used a questionnaire and a projective psychological test (HTP test) to measure the psychosexual compounds. Prenatal androgen exposure was estimated considering the 46,XY etiology. Sinnecker\'s score was used to measure the external genitalia virilization. All ordinal variables were analyzed using Wilcoxon test. Categorical variables were analyzed using X2 test with posterior Cramer\'s V to measure the association strength. The kappa index was calculated as a concordance measure. Results: We found an association between prenatal androgen exposure and major prevalence of male psychosexual outcomes and a higher incidence of female to male gender dysphoria. There was not difference in the psychosexual outcomes according by external genitalia virilization in male and in female individuals. There was an incidence of 19% of gender dysphoria (27 out from 144). In 93% (n=25), the gender change was from female to male (F to M). The ethological diagnosis related with F to M GD were 5alpha-RD2 deficiency (5ARD2) in 16/32 (50%), followed by 5/15 (33%) in 17beta-HSD3 deficiency (17betaHSD3). Others diagnosis related with F to M GD were: partial gonadal dysgenesis (n=3/24; 12%) and 3betaHSD2 (n=1/3; 33%). Both cases of male to female (M to F) GD occurred in partial gonadal dysgenesis (8%; n=2/24). The median of GD age (desire to belong to another gender) was 8 years old (5-9), and the median of gender change itself was 15 years old (10.5 - 20). In F to M GD, gender change was associated with prenatal androgen exposure (p < 001; V=0,461). The psychosexual components showed higher concordance index with final gender (GI - k=0.81; GI - k=0.65 and SO - k=0.85) then with the assigned sex (GI - k=0.1; GI - k=0.25 and SO - k=0.15). Conclusion: Prenatal androgen exposure affects the psychosexual development, favoring more male outcomes. This influence was observed in GI, GR and SO. The degree of external genitalia virilization did not influence the psychosexual development. Female to Male GD is common in 46,XY DSD raised in female social sex, especially in 5ARD2 and 17?HSD3 deficiencies. There is a strong relationship between prenatal androgen exposure and F to M GD. On the other hand, M to F gender change was rare in 46,XY DSD and occurred only in partial gonadal dysgenesis patients