INTRODUÇÃO: Carcinomas de mama triplo-negativos (CMTN) constituem um grupo de tumores heterogêneos caracterizados por sobrevida pobre dos pacientes e falta de terapia-alvo. Receptor andrógeno (AR) tem sido descrito no CMTN, mas o impacto prognóstico da expressão nesse subgrupo não está bem claro. OBJETIVO: Investigar a associação do status de expressão AR analisado por imunohistoquímica em casos de CMTN com parâmetros clínicos (idade, sobrevida) e variáveis patológicas (tamanho do tumor, grau do tumor). MÉTODOS: Foram analisados 62 CMTN por imuno-histoquímica automatizada para receptor andrógeno. A imuno-histoquímica foi avaliada por dois diferentes patologistas e a expressão do biomarcador foi avaliada por H-Score (intensidade mais percentagem de coloração). Kaplan-Meyer foi o método usado para avaliar a sobrevida global, tendo as diferenças nas distribuições sido avaliadas com base na expressão do marcador. RESULTADOS: 26% dos CMTNs foram AR positivos (n = 16) e 74% AR negativos (n = 46). Todos os casos de AR positivos ocorreram em mulheres ≥ 40 anos enquanto 13% dos casos de AR negativos foram vistos em mulheres ≤ 40 anos. 93% (52/56) de todos os CMTNs foram carcinoma ductal invasivo do tipo não especial (ICNST) e 93% (14/15) de AR positivos dos casos de ICNST. O tamanho do tumor variou de 2 a 4,9 cm na maioria dos cânceres AR positivos (n = 8/13; 62%). 60% (n = 9/15) dos casos de AR positivos mostraram grau histológico 3 seguidos de 27% de tumores de grau 2. Não se observou diferença entre pacientes AR positivos e AR negativos quando comparados por idade, tamanho do tumor, grau do tumor e tipo do tumor (p > 0,17). A positividade imuno-histoquímica para ARs também não foi associada com melhor sobrevida (p = 0,737; n = 51) ou sobrevida livre da doença (p = 0,552; n = 45) em CMTNs. CONCLUSÃO: Sob o ponto de vista do prognóstico, a imunorreatividade do AR tem sido associada com melhor sobrevida das pacientes. Esse resultado não foi confirmado na presente série. Isso poderia estar relacionado com o pequeno tamanho amostral ou com uma baixa prevalência de casos de AR positivos especificamente nesse subtipo de câncer de mama. Estudos realizados com maiores amostras são necessários para investigar esse biomarcador em CMTNs. / INTRODUCTION: Triple negative breast carcinomas (TNBC) are a heterogeneous group of tumors characterized by poor patient survival and lack of targeted therapies. Androgen receptor (AR) has been described in TNBC but the prognostic impact of the expression in this subgroup of tumors is not clear. OBJECTIVE: To investigate the association of AR expression status by immunohistochemistry in TNBC cases with clinical (age, survival) and pathological variables (tumor size, tumor grade). METHODS: 62 TNBC were analyzed by automated immunohistochemistry for androgen receptor. Immunohistochemistry was scored by two investigators and biomarker expression was assessed by H-Score (intensity plus the percentage of staining). Kaplan-Meier was used to evaluate overall survival, where differences in distributions were evaluated based on marker expression. RESULTS: 26% of TNBC were AR-positive (n = 16) and 74% AR negative (n = 46). All AR-positive cases occurred in women ≥ 40 years, while 13% of AR-negative cases were seen in women ≤ 40 years. 93% (52/56) of all TNBC were infiltrating ductal carcinomas of no special type (ICNST) and 14/15 (93%) of AR-positive cases were ICNST. Tumor size varied from 2-4.9 cm in the majority of AR-positive cancers (n = 8/13; 62%). 60% (n = 9/15) of AR-positive cases showed histological grade 3 tumors, followed by 27% of grade 2 tumors. No differences were observed between AR-positive and AR-negative patients when compared for age, tumor size, tumor grade and tumor type (all p > 0.17). AR immunohistochemical positivity was also not associated with better overall survival (p = 0.737, n = 51) or disease-free survival (p = 0.552; n = 45) in TNBCs. CONCLUSION: From the prognostic point of view, AR immunoreactivity has been associated with better overall patient survival. Unfortunately, this result could not be confirmed in our series. This could be related to the relatively small series of analyzed samples and to the low prevalence of AR-positive cases in this specific breast cancer subtype. Further studies with bigger samples are needed to investigate this biomarker in TNBC.

Androgens are well documented as important regulators of male health, primarily in the maintenance and development of male sexual characteristics. However, a decline in circulating androgens has also been associated with co-morbidities such as obesity, cardiac disease and metabolic syndrome. Previous research has focussed upon the body wide impact of adrenal androgens, however whilst androgen receptor (AR) is abundantly expressed in the adrenal cortex of both rodents and humans, surprisingly little is known about androgen action on the adrenal cortex itself. This gap in our understanding is at least in part due to the perceived lack of suitable animal models. Rodents have largely been overlooked as a model system as their adrenals are unable to produce androgens due to lack of 17α Hydroxylase and 17, 20 lyse activity and they therefore do not have a zona reticularis. However, historical studies using castrated mice showed that removal of androgens leads to the redevelopment of an additional cortex zone known as the transient X-zone. The foetal adrenal is thought to give rise the adult adrenal cortex in human and rodents. These foetal cells are maintained for a period postnatally and regress differently depending on species and sex. In the human this zone is known as the ‘foetal zone’, and the rodent homologue termed the ‘X-zone’. The mechanisms underpinning the regression of the X-zone and its purpose and maintenance postnatally still aren’t clearly understood. To provide a comprehensive overview of androgen signalling in the adrenal cortex, multiple mouse models were utilised. First, Cre/loxP technology was used to ablate AR specifically from the adrenal cortex. Further androgen manipulation was achieved through castration (removal of androgens) and human chorionic gonadotropin (hCG) treatment (increased androgens). The initial study investigates the impacts on the male mouse adrenal. Histology analysis revealed the presence of an X-zone in all experimental cohorts following loss of AR or circulating androgens, confirmed by 20- α-hydroxysteroid dehydrogenase (20 alpha-HSD) expression. These data demonstrate that androgens signalling via AR is required for X-zone regression during puberty. However, interrogation of morphology of hCG treated cohorts revealed no phenotypic changes compared to controls, this demonstrates that hyper stimulation with androgens does not negatively impact the adrenal cortex or influence X-zone morphology. Differences in X-zone morphology and 20 alpha-HSD localization prompted cortex measurements which revealed significant differences in X-zone depth and cell density depending on ablation of AR, circulating androgens or both. This suggests that androgens and androgen receptor are working together and also independently to regulate the adrenal cortex. This result was strengthened through analysis of steroid enzyme genes and cortex markers, which revealed that normal AKR1B7 expression was absent following loss of androgens but not androgen receptor. A final part of this study examined the impacts long term androgen receptor ablation and long term castration in ageing animals. A final part of this study examined the impacts long term androgen receptor ablation and long term castration in ageing animals. These results demonstrate that following prolonged loss of androgens that there is no major disruption to the adrenal cortex. Morphology analysis and X-zone measurements revealed that X-zone regression was occurring in mice with long term castration, characterized by a reduction in size and pockets of vacuolization throughout the X-zone. This phenotype is also observed in ageing females with X-zone regression via vacuolization. These data suggest that following prolonged loss of androgens, the male adrenal is feminized and behaves as such. In contrast, AR ablation only, results in an enlarged adrenal with large spindle cell lesions and X-zone expansion confirmed by X-zone measurements. Initial experiments have demonstrated that androgens can work independently of AR to regulate the adrenal cortex. Together these data suggests that AR is required to control the appropriate action of circulating androgens in the adrenal cortex, with loss of AR resulting in off target signalling from circulating androgens in the adrenal leading to spindle cell hyperplasia, X-zone expansion and X-zone mislocation. A second set of studies were carried out to determine the role of androgen signalling in the female adrenal, specifically, if loss of AR leads to the absence of normal X-zone regression during pregnancy. To answer this question the same selective AR ablation model was used. Analysis of litters comparing observed and expected genetic distribution revealed significantly fewer females being born carrying complete ablation of adrenal AR. Morphology analysis of these mice revealed severe cortex disruption and spindle cell hyperplasia similar to that observed in mutant males. Investigation of adrenals following pregnancy revealed that X-zone regression still occurred despite loss of AR. This result shows that X-zone regression in the female is under different regulation compared to male adrenal and occurs via an androgen-independent signalling mechanism. However, loss of AR still leads to anatomical dysregulation of the adrenal cortex. AR ablation revealed changes in glucocorticoid receptor (GR) expression in the adrenal cortex. To dissect this relationship further a final study was conducted, attempting to ablate GR from the adrenal cortex also using the Cyp11a1 Cre. Initial observations of these mice revealed excessive hair loss through barbering, curved spines and stressed behaviour when monitored in the cage under normal conditions. Immunohistochemistry was used to confirm GR ablation in the adrenal cortex, however, to our surprise, GR expressing cells were not steroidogenic and thus were not targeted by the Cre recombinase. Despite no GR ablation in the adrenal, morphology analysis revealed severe disruption to the adrenal cortex. The Cyp11a1 Cre not only targets the adrenal but is expressed in the hindbrain. To determine if GR ablation in the hindbrain explains the phenotype, we next used PCR analysis interrogating hindbrain genomic DNA to determine if there was recombination of GR. Results confirmed GR recombination in the hindbrain. Due to the observation of stressed behaviour and adrenal cortex disruption, we wanted to determine if this was a result of hyperactivity of the adrenal cortex. Serum corticosterone was analysed and was elevated in these animals. These data revealed that GR ablation in the hindbrain results in adrenal cortex disruption and an elevated stress response, potentially highlighting a new model to investigate stress disorders and their impact on the hypothalamic-pituitary-adrenal axis. Together this data defines new roles for AR signalling in the adrenal cortex and the role of the hindbrain GR signalling in regulating adrenal morphology and function.

INTRODUÇÃO: Carcinomas de mama triplo-negativos (CMTN) constituem um grupo de tumores heterogêneos caracterizados por sobrevida pobre dos pacientes e falta de terapia-alvo. Receptor andrógeno (AR) tem sido descrito no CMTN, mas o impacto prognóstico da expressão nesse subgrupo não está bem claro. OBJETIVO: Investigar a associação do status de expressão AR analisado por imunohistoquímica em casos de CMTN com parâmetros clínicos (idade, sobrevida) e variáveis patológicas (tamanho do tumor, grau do tumor). MÉTODOS: Foram analisados 62 CMTN por imuno-histoquímica automatizada para receptor andrógeno. A imuno-histoquímica foi avaliada por dois diferentes patologistas e a expressão do biomarcador foi avaliada por H-Score (intensidade mais percentagem de coloração). Kaplan-Meyer foi o método usado para avaliar a sobrevida global, tendo as diferenças nas distribuições sido avaliadas com base na expressão do marcador. RESULTADOS: 26% dos CMTNs foram AR positivos (n = 16) e 74% AR negativos (n = 46). Todos os casos de AR positivos ocorreram em mulheres ≥ 40 anos enquanto 13% dos casos de AR negativos foram vistos em mulheres ≤ 40 anos. 93% (52/56) de todos os CMTNs foram carcinoma ductal invasivo do tipo não especial (ICNST) e 93% (14/15) de AR positivos dos casos de ICNST. O tamanho do tumor variou de 2 a 4,9 cm na maioria dos cânceres AR positivos (n = 8/13; 62%). 60% (n = 9/15) dos casos de AR positivos mostraram grau histológico 3 seguidos de 27% de tumores de grau 2. Não se observou diferença entre pacientes AR positivos e AR negativos quando comparados por idade, tamanho do tumor, grau do tumor e tipo do tumor (p > 0,17). A positividade imuno-histoquímica para ARs também não foi associada com melhor sobrevida (p = 0,737; n = 51) ou sobrevida livre da doença (p = 0,552; n = 45) em CMTNs. CONCLUSÃO: Sob o ponto de vista do prognóstico, a imunorreatividade do AR tem sido associada com melhor sobrevida das pacientes. Esse resultado não foi confirmado na presente série. Isso poderia estar relacionado com o pequeno tamanho amostral ou com uma baixa prevalência de casos de AR positivos especificamente nesse subtipo de câncer de mama. Estudos realizados com maiores amostras são necessários para investigar esse biomarcador em CMTNs. / INTRODUCTION: Triple negative breast carcinomas (TNBC) are a heterogeneous group of tumors characterized by poor patient survival and lack of targeted therapies. Androgen receptor (AR) has been described in TNBC but the prognostic impact of the expression in this subgroup of tumors is not clear. OBJECTIVE: To investigate the association of AR expression status by immunohistochemistry in TNBC cases with clinical (age, survival) and pathological variables (tumor size, tumor grade). METHODS: 62 TNBC were analyzed by automated immunohistochemistry for androgen receptor. Immunohistochemistry was scored by two investigators and biomarker expression was assessed by H-Score (intensity plus the percentage of staining). Kaplan-Meier was used to evaluate overall survival, where differences in distributions were evaluated based on marker expression. RESULTS: 26% of TNBC were AR-positive (n = 16) and 74% AR negative (n = 46). All AR-positive cases occurred in women ≥ 40 years, while 13% of AR-negative cases were seen in women ≤ 40 years. 93% (52/56) of all TNBC were infiltrating ductal carcinomas of no special type (ICNST) and 14/15 (93%) of AR-positive cases were ICNST. Tumor size varied from 2-4.9 cm in the majority of AR-positive cancers (n = 8/13; 62%). 60% (n = 9/15) of AR-positive cases showed histological grade 3 tumors, followed by 27% of grade 2 tumors. No differences were observed between AR-positive and AR-negative patients when compared for age, tumor size, tumor grade and tumor type (all p > 0.17). AR immunohistochemical positivity was also not associated with better overall survival (p = 0.737, n = 51) or disease-free survival (p = 0.552; n = 45) in TNBCs. CONCLUSION: From the prognostic point of view, AR immunoreactivity has been associated with better overall patient survival. Unfortunately, this result could not be confirmed in our series. This could be related to the relatively small series of analyzed samples and to the low prevalence of AR-positive cases in this specific breast cancer subtype. Further studies with bigger samples are needed to investigate this biomarker in TNBC.

Orientador: Hernandes Faustino de Carvalho / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T10:22:10Z (GMT). No. of bitstreams: 1 Victorio_SheilaCristinadaSilva_M.pdf: 1499738 bytes, checksum: 1208b4b617e591a974a869b7fa20a388 (MD5) Previous issue date: 2007 / Resumo: O crescimento e função prostática dependem da estimulação androgênica e interação epitélio-estroma. Além dos andrógenos, outros fatores interagem com a próstata e são igualmente importantes para sua fisiologia. Sabe-se que os estrógenos exercem um importante papel no desenvolvimento prostático e que, combinados com andrógenos, podem contribuir para o aparecimento de patologias. A insulina é outro hormônio que afeta a atividade destes hormônios sexuais nos tecidos, inclusive na próstata. Os níveis séricos de esteróides sexuais estão intimamente relacionados com a sensibilidade a insulina, embora esta relação ainda seja pouco esclarecida. No estroma prostático, as células musculares lisas são o tipo celular predominante, influenciando a atividade do epitélio por mecanismos parácrinos e modificando a matriz extracelular em situações de remodelação, como no crescimento, na regressão e na invasão tumoral. Sabe-se que estas células apresentam receptores de andrógeno (AR) e que respondem à privação androgênica, alterando sua morfologia. O presente estudo buscou verificar a influência da testosterona, estradiol e insulina sobre a expressão e localização do AR em células musculares lisas da próstata ventral de ratos Wistar cultivadas in vitro. Os resultados mostraram que o estradiol causou alterações nos níveis protéicos, os níveis de RNAm do AR foram pouco afetados e a localização do AR foi predominantemente nuclear independente da dose de estradiol. Os tratamentos feitos com insulina mostraram que a sua presença causou uma queda da expressão da proteína e uma localização predominante nuclear do AR na situação em que os dois hormônios, insulina e testosterona, foram administrados juntamente. Os resultados permitem sugerir que a expressão do AR pode ser modulada por outros fatores como estrógeno e insulina / Abstract: The prostate function and growth depends on the androgenic stimulation and epitheliumstroma interaction. Besides androgens, other factors interact with the prostate and are also important for its physiology. It is known that estrogens exert an important role in prostate development, and combined with androgens they can contribute for the appearance of pathologies. The insulin is another hormone that interacts with these sexual hormones in tissues, and also in the prostate. The serum levels of sexual steroids are closely related with the sensitivity to the insulin, even though this relation is not yet clear. In prostatic stroma, smooth muscle cells are the predominant cell type. They influence the activity of the epithelium through paracrine mechanisms and modify the extracellular matrix in remodeling situations, such as gland growth and regression, and during tumor invasion. It is known that these cells express androgen receptor (AR) and respond to androgen deprivation by modifying its phenotype. The present study was undertaken to verify the influence of testosterone, estradiol and insulin on the expression and localization of the AR in the smooth muscle cells from the Wistar rat ventral prostate cultured in vitro. The results showed that estradiol caused alterations in the AR protein levels, the mRNA level was less affected and AR localization was predominantly nuclear irrespective of the estradiol dose. Insulin treatments caused a decrease in the expression of the protein and a predominant nuclear localization of AR in the presence of testosterone. The results suggest that AR expression and regulation might be modulated by others factors such as estrogen and insulin / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

INTRODUÇÃO: Carcinomas de mama triplo-negativos (CMTN) constituem um grupo de tumores heterogêneos caracterizados por sobrevida pobre dos pacientes e falta de terapia-alvo. Receptor andrógeno (AR) tem sido descrito no CMTN, mas o impacto prognóstico da expressão nesse subgrupo não está bem claro. OBJETIVO: Investigar a associação do status de expressão AR analisado por imunohistoquímica em casos de CMTN com parâmetros clínicos (idade, sobrevida) e variáveis patológicas (tamanho do tumor, grau do tumor). MÉTODOS: Foram analisados 62 CMTN por imuno-histoquímica automatizada para receptor andrógeno. A imuno-histoquímica foi avaliada por dois diferentes patologistas e a expressão do biomarcador foi avaliada por H-Score (intensidade mais percentagem de coloração). Kaplan-Meyer foi o método usado para avaliar a sobrevida global, tendo as diferenças nas distribuições sido avaliadas com base na expressão do marcador. RESULTADOS: 26% dos CMTNs foram AR positivos (n = 16) e 74% AR negativos (n = 46). Todos os casos de AR positivos ocorreram em mulheres ≥ 40 anos enquanto 13% dos casos de AR negativos foram vistos em mulheres ≤ 40 anos. 93% (52/56) de todos os CMTNs foram carcinoma ductal invasivo do tipo não especial (ICNST) e 93% (14/15) de AR positivos dos casos de ICNST. O tamanho do tumor variou de 2 a 4,9 cm na maioria dos cânceres AR positivos (n = 8/13; 62%). 60% (n = 9/15) dos casos de AR positivos mostraram grau histológico 3 seguidos de 27% de tumores de grau 2. Não se observou diferença entre pacientes AR positivos e AR negativos quando comparados por idade, tamanho do tumor, grau do tumor e tipo do tumor (p > 0,17). A positividade imuno-histoquímica para ARs também não foi associada com melhor sobrevida (p = 0,737; n = 51) ou sobrevida livre da doença (p = 0,552; n = 45) em CMTNs. CONCLUSÃO: Sob o ponto de vista do prognóstico, a imunorreatividade do AR tem sido associada com melhor sobrevida das pacientes. Esse resultado não foi confirmado na presente série. Isso poderia estar relacionado com o pequeno tamanho amostral ou com uma baixa prevalência de casos de AR positivos especificamente nesse subtipo de câncer de mama. Estudos realizados com maiores amostras são necessários para investigar esse biomarcador em CMTNs. / INTRODUCTION: Triple negative breast carcinomas (TNBC) are a heterogeneous group of tumors characterized by poor patient survival and lack of targeted therapies. Androgen receptor (AR) has been described in TNBC but the prognostic impact of the expression in this subgroup of tumors is not clear. OBJECTIVE: To investigate the association of AR expression status by immunohistochemistry in TNBC cases with clinical (age, survival) and pathological variables (tumor size, tumor grade). METHODS: 62 TNBC were analyzed by automated immunohistochemistry for androgen receptor. Immunohistochemistry was scored by two investigators and biomarker expression was assessed by H-Score (intensity plus the percentage of staining). Kaplan-Meier was used to evaluate overall survival, where differences in distributions were evaluated based on marker expression. RESULTS: 26% of TNBC were AR-positive (n = 16) and 74% AR negative (n = 46). All AR-positive cases occurred in women ≥ 40 years, while 13% of AR-negative cases were seen in women ≤ 40 years. 93% (52/56) of all TNBC were infiltrating ductal carcinomas of no special type (ICNST) and 14/15 (93%) of AR-positive cases were ICNST. Tumor size varied from 2-4.9 cm in the majority of AR-positive cancers (n = 8/13; 62%). 60% (n = 9/15) of AR-positive cases showed histological grade 3 tumors, followed by 27% of grade 2 tumors. No differences were observed between AR-positive and AR-negative patients when compared for age, tumor size, tumor grade and tumor type (all p > 0.17). AR immunohistochemical positivity was also not associated with better overall survival (p = 0.737, n = 51) or disease-free survival (p = 0.552; n = 45) in TNBCs. CONCLUSION: From the prognostic point of view, AR immunoreactivity has been associated with better overall patient survival. Unfortunately, this result could not be confirmed in our series. This could be related to the relatively small series of analyzed samples and to the low prevalence of AR-positive cases in this specific breast cancer subtype. Further studies with bigger samples are needed to investigate this biomarker in TNBC.

Orientador: Aparecida Machado de Moraes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T11:25:06Z (GMT). No. of bitstreams: 1 Cordeiro_RaquelCristinaTancsik_D.pdf: 1883576 bytes, checksum: c00bc86b625d6d904784f53f3f6041e6 (MD5) Previous issue date: 2009 / Resumo: A estria atrófica cutânea ou striae distensae (SD) é uma afecção muito comum, sendo causa freqüente de procura por consultas dermatológicas. Ainda que não representem qualquer risco à saúde física, produzem impacto emocional e induzem busca por tratamentos trabalhosos, caros e dolorosos e, com freqüência, inadequados. Além disso, o surgimento de striae distensae pode refletir alteração do tecido conjuntivo e indicar condições patológicas locais e sistêmicas. Alguns autores descrevem as estrias cutâneas como uma condição de estiramento ou distensão da pele, com perda ou ruptura das fibras elásticas na região acometida. Entretanto, vários autores observam que as estrias não surgem com frequência sobre a pele acima de tumores abdominais, ascites, hemorragias extensas ou grandes hérnias. Atualmente admite-se que sua etiopatogenia é multifatorial, englobando aspectos mecânicos, bioquímicos e genéticos. No entanto, considerando-se a multiplicidade de fatores envolvidos, a literatura é divergente e inconclusiva. Portanto, através do estudo de fatores clínicos associados às SD e dos receptores hormonais (estrógeno, andrógeno e glicocorticóide), pretendeu-se entender melhor a participação dos hormônios na fisiopatogênese das estrias. Para o estudo clínico foram selecionados pacientes com queixa de estrias cutâneas e a comparação foi feita com grupo controle de número semelhante, atendido aleatoriamente por outras queixas no Ambulatório Geral de Dermatologia do HC, FCM/UNICAMP. O estudo da expressão dos receptores hormonais foi realizado por Western blot em oito amostras de pele de estrias recentes, com menos de um ano de evolução, comparando com a avaliação de pele sem lesão de região palpebral de oito pacientes que se submenteram a blefaroplastia. Observou-se que fatores como adolescência, gestação e obesidade estão significativamente relacionados ao surgimento das SD. Constatou-se ainda que a idade materna jovem e o ganho ponderal durante a gestação são importantes fatores associados ao desenvolvimento das lesões. Além disso, a localização das lesões correlaciona-se ao fator causador das estrias. Em relação ao estudo dos receptores hormonais, observou-se que na SD recentes há duas vezes mais expressão de receptores de estrógeno e 1,7 vezes mais expressão receptores de andrógeno e glicocorticóide. Alguns autores interpretam SD como cicatrizes. Após influência hormonal, haveria uma reação inflamatória inicial que determinaria a destruição de fibras elásticas e colágenas. O processo seria seguido de regeneração das fibras, um fenômeno de remodelação dinâmica,ou seja, um balanço entre síntese de colágeno e sua quebra, o qual reestrutura o tecido para acomodar as forças que agem sobre ele, resultando na formação das SD. O balanço entre a expressão de receptores de estrógeno, andrógeno e glicocorticóide poderia induzir as modificações específicas da matriz extracelular, o que levaria a esse fenômeno de remodelação. Em concordância com outros estudos, observamos que as estrias surgem em situação de grande modificação sistêmica, como adolescência e gestação. Através das observações morfológicas e moleculares, nota-se que as SD estão correlacionadas com intensas alterações do tecido conectivo. Os resultados mostram-se relevantes e representam mais um passo na compreensão do mecanismo fisiopatológico da estrias cutâneas e abrem espaço para novas linhas de pesquisa, relacionadas às SD e a outras alterações do tecido conectivo. / Abstract: Stretch marks or striae distensae (SD) are a very common condition which often results in persons searching dermatological treatment. While not presenting any risk to one's physical health, the emotional impact often induces those so affected to demand medical treatments, which are often laborious, expensive and painful, and frequently ineffective. Beyond this, the appearance of SD may indicate other alterations in conjunctive tissues, including both local and systemic pathologies. Some authors described cutaneous strias as a condition of stretching or distension of the skin which results in the loss or rupture of elastic fibers in the affected areas. However, many authors have also observed that SD do not occur frequently associated with abdominal tumors, ascites, extensive hemorrhages or large hernias. Current medical opinion is that its etiopathogenesis is multifactored, englobing mechanical, biochemical and genetic aspects. Nevertheless, in view of the multiplicity of the factors involved, the literature is divergent and inconclusive. This study was proposed to look for better understanding of the role played by hormones in the physiopathology of SD studying clinical factors associated with SD and hormone receptors (estrogen, androgen and glycocorticoid). Patients complaining of SD were selected for inclusion in this clinical study, and comparisons were made with a control group of a similar number of persons who had been treated for other medical conditions at the General Ambulatory Dermatology Care Facility at HC, FCM/UNICAMP. The expression of hormone receptors was undertaken of the Western Blot testing of recent skin striations, in comparison with lesion-free skin taken from the eyelids of patients who had undergone blepharoplasty. The study revealed that factors such as adolescence, pregnancy and obesity are significantly related to the appearance of SD. It was further established that age (the younger the gravid, the greater the possibility of SD) and significant weight gain during pregnancy are important factors associated with the development of SD lesions. In addition, there was a positive correlation with the location of the lesions. In relation to the study of hormone receptors, it was observed that recently-formed SD have two times more expression of estrogen receptors, and 1,7 times more expression of androgen and glycocorticoid receptors. Some authors classify SD as scars. Under hormonal influence, there is an initial inflammatory reaction which results in the destruction of elastic fibers and collagens. This is followed by the regeneration of the destroyed fibers, a phenomenon of dynamic remodeling, a balance between the synthesis and destruction of collagens, which restructures the tissue in order to accommodate the forces acting upon it, resulting in the formation of SD. The balance between the expression of estrogen, androgen and glycocorticoid may elicit the specific modifications of the extracellular matrix which leads to the phenomenon of remodalation. In agreement with other studies, we observed that the strias arise under conditions of significant system modification, such as adolescence and pregnancy. The observation of morphological and molecular changes showed that there is a correlation between SD and with intense alterations in connective tissue. The results of this study are relevant and represent an important step in the understanding of physiopathological mechanisms in stretch marks, opening new horizons for new directions in research of SD and other alterations in connective tissues as well. / Doutorado / Clinica Medica / Doutor em Clínica Médica

Orientador: Hernandes Faustino de Carvalho / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T07:07:28Z (GMT). No. of bitstreams: 1 Oliveira_ElusaCristinade_M.pdf: 10251411 bytes, checksum: b1a5d2634c9831ce84cdf0a70561fdc8 (MD5) Previous issue date: 2008 / Resumo: Em ratos machos, a completa função reprodutiva é criticamente dependente da adequada ação do estrógeno nos diferentes níveis do eixo hipotálamo-hipófise-gônadas. A administração de altas doses de estrógeno durante o período de diferenciação sexual resulta em diversas alterações no sistema reprodutor masculino. De forma geral, os estrógenos têm efeito anti-androgênico atuando no eixo hipotálamo-hipófise-testículos e, assim, reduzindo a produção de testosterona pelos testículos. A administração de estrógeno nos períodos. críticos do desenvolvimento promove um mecanismo conhecido como imprinting estrogênico. Este fenômeno é extremamente importante, pois pode ser acionado pela exposição do recém-nascido a várias substâncias com caráter estrogênico, e não somente ao estrógeno. Um dos efeitos do imprinting estrogênico causado por altas doses de estrógeno é a regulação negativa da expressão de receptores de andrógenos (ARs). na próstata e reduzida resposta a aplicações de andrógenos, o que parece alterar padrões de comportamento em machos. Diante disso, este trabalho investigou os aspectos relacionados aos mecanismos gerais envolvidos no imprinting estrogênico, com o propósito de analisar a participação do estrógeno e a sua influência sobre os ARs no hipotálamo de ratos Wistar. Para tanto, ratos machos foram tratados no período neonatal inicial com três doses de 15 mg/kg de 17p-estradiol (E2) e comparados com ratos controle (tratados apenas com óleo de milho) e com fêmeas que não receberam nenhum tratamento farmacológico, e os efeitos foram observados na idade adulta. Foram verificados os padrões de distribuição do AR por imunoistoquímica, o conteúdo total de AR por Western blotting e do correspondente RNAm por RT-PCR no hipotálamo dos animais submetidos à estrogenização neonatal. A estrogenização promoveu diminuição no conteúdo total de AR e na expressão do RNAm correspondente, e modificações no padrão de distribuição do AR nos núcleos ventromedial, pré-óptico medial anterior, paraventricular e área pré-óptica, aproximando-se do padrão apresentado por fêmeas. Portanto, esses dados indicam que o estrógeno é capaz de promover imprinting neonatal no hipotálamo e alterar a expressão do AR, o que possivelmente pode afetar o comportamento sexual desses animais / Abstract: In male rats, complete reproductive function is critically dependent on adequate estrogen action at different levels of the hypothalamic-pituitary-gonadal axis. The administration of high doses during the period of sexual differentiation results in irreversible alterati ns in the male reproductive system. In brief, the estrogens have anti-androgenic effects t. ey act in the hypothalamus-pituitary-testes axis, reducing the testosterone production by the testes. The administration of estrogen in critical periods of development promotes a mechanism known as estrogen imprinting. This phenomenon is extremely important, because it can be started by the exposure of the neonate to several estrogen-like substances with estrogen characteristics, and not only to estrogen. One of the estrogenic imprinting effects caused by high doses of estrogen is the negative regulation of the expression of androgen receptors (ARs) in the prostate and reduced responses to androgen exposure that seems to alter the male behavior. This study investigated the participation of estrogen and it influence on the ARs in the hypothalamus of Wistar rats afier estrogen imprinting. For this, male rats were treated in the early neonatal period with 17pestradiol (E2) three doses of 15mg/kg and compared with control rats (only treated with com oil) and with not treated females, and the effects were analyzed in the adulthood. The AR distribution pattem was analyzed by immunohistochemistry, the total content of the AR by Westem blotting and it's mRNA by RT-PCR in the hypothalamus oftreated animaIs. The neonatal estrogenization promoted decrease of expression oftne AR and its mRNA. Also revealed that the AR distribution, pattem at the ventromedial, anterior medial preoptic and paraventricular nuclei and preoptic area were altered and with a pattem close to not treated females. Therefore, our data indicate that estrogen is able to induce estrogen imprint in the hypothalamus and to alter the AR expression and possibly affecting the sexual behavior / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Orientador: Munir Salomão Skaf / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-21T17:39:27Z (GMT). No. of bitstreams: 1 daSilva_JulioCesarAraujo_D.pdf: 43361733 bytes, checksum: 212cdef85174d1daf286133ff839cfb2 (MD5) Previous issue date: 2012 / Resumo: Receptores de androgênio (AR) são membros da superfamília de receptores nucleares que incluem os receptores de esteroides, entre outros. O AR liga os esteroides sexuais endógenos diidrotestosterona e testosterona. O desenvolvimento normal do fenótipo masculino e do sistema reprodutivo necessita de ações pré- e pósnatais promovidas pela interação do AR com esses hormônios. Mutações no gene do receptor de androgênio podem levar a várias doenças como o câncer de próstata e a síndrome de insensibilidade ao androgênio (AIS). Substituições diferentes no mesmo resíduo de aminoácido podem resultar em impactos variáveis na atividade do receptor levando a diferentes graus de AIS. Um grande número de mutações tem sido reportado para o AR envolvendo AIS e células tumorais de câncer de próstata e sua localização e função podem ajudar a entender como essas doenças devem ser tratadas. Entretanto, pouco se sabe sobre como as mutações mudam a estrutura e a dinâmica do AR, uma vez que apenas poucas estruturas cristalográficas de mutantes foram obtidas. Neste trabalho, apresentamos estudos de simulação de dinâmica molecular de algumas estruturas do AR humano com mutações localizadas no domínio de ligação do ligante (LBD) em comparação com a estrutura nativa complexadas com o ligante sintético metiltrienolona (R1881). Nosso objetivo é investigar as bases moleculares das mudanças sutis no receptor causadas pelas mutações que afetam sua afinidade pelo ligante R1881. Embora nenhum dos resíduos mutados deste estudo interajam diretamente com o ligante, os resultados das simulações indicaram que as mutações causam mudanças estruturais e dinâmicas no AR-LBD na região onde se localiza a mutação e na cavidade de ligação do ligante (LBP). A principal mudança observada foi o deslocamento do resíduo Arg752, facilitando a entrada de moléculas de água no LBP e o reposicionamento de cadeias laterais dos resíduos do domínio F, uma importante região que contribui para a estabilidade da estrutura do AR-LBD e da conformação ativa da hélice 12. Os resultados obtidos mostram que essas mutações, que ocorrem naturalmente, são exemplos de resíduos que não estão em contato direto com o ligante e não pertencem à região de recrutamento do coativador, mas que possuem um importante papel na ligação do ligante e na ativação do receptor por estabilizar a hélice 12 e o domínio F na conformação ativa / Abstract: Androgen receptors (AR) are members of the superfamily of nuclear receptors that includes the steroid receptors, among others. AR binds the male endogenous sex steroids, dihydrotestosterone and testosterone. Normal development of the male phenotype and reproductive system requires pre- and postnatal actions promoted by AR interaction with these hormones. Mutations in the androgen receptor gene may lead to several diseases like prostate cancer (PCa) and the androgen insensitivity syndrome (AIS). Different substitutions at the same amino acid residue may result in variable impact on the activity of the receptor leading to different degrees of AIS. A number of mutations have been reported for the AR in AIS and PCa tumor cells and their location and function may help us to understand how these diseases should be treated. Nevertheless, not much is known about how the mutations change the structure and dynamics of the AR since only a few crystallographic structures of mutants were obtained. In this work we present molecular dynamics simulation (MD) studies of some human AR mutations located in the ligand binding domain (LBD) in comparison with wild type (WT) structure in complex with the synthetic ligand methyltrienolone (R1881). Our goal is to investigate the molecular basis of subtle changes in the receptor caused by mutations in the AR-LBD/R1881 affinity. Although the mutated residues do not interact with the ligand, the simulations results indicated that the mutants cause structural and dynamical changes in the AR-LBD in the region in which the mutation is placed and in the binding pocket (LBP). The principal change observed was the displacement of residue Arg752, facilitating water penetration in LBP, and the repositioning of F-domain side chains, which makes important contributions to the stability of the AR-LBD structure and helix 12 active conformation. The results obtained show that these naturally occurring mutations are examples of residues that are not in contact with the ligand and do not belong to coactivator recruitment region, but which do have an important role in ligand binding and receptor activation by stabilizing the helix H12 and the F-domain in the active conformation / Doutorado / Físico-Química / Doutor em Ciências

Abstract Gene products that are only expressed in one tissue or cell type are useful models for investigating the biochemical and molecular mechanisms of tissue/cell-specific gene regulation. The regulatory regions of such genes are also practical tools in gene therapy. In this work, prostate-specific and androgen-dependent gene regulation was investigated by using human prostatic acid phosphatase (hPAP) and rat probasin (rPB) as models. In DNase I footprinting, a protected 12 bp region was found in the PB gene between the nucleotides -251 and -240 only with nuclear extracts of prostatic origin. The sequence of this area was GAAAATATGATA. Weak interaction could be detected between the DNA-binding domain of AR and the prostatic transcription factor. The results also suggested that the prostatic regulatory protein makes AR binding to its response element more effective and concomitantly magnifies the effect of androgen. A hPAP construct containing the sequence between the nucleotides -734 and +467 in front of the CAT reporter gene was highly expressed in the prostate of transgenic mice. Five homologues (A-E) for our previously identified prostate-specific GAAAATATGATA DNA-binding site were found in the area where the sites C and E could bind the regulatory protein in EMSA. The prostatic transcription factor complex bound to the GAAAATATGATA site was purified and characterized from a suspension-adapted mass culture of PC-3 prostate cancer cells by using sequence-specific DNA affinity chromatography, mass spectrometry and supershifts. Several potential transcription factors were identified, but only USF2 was confirmed to be part of the transcription factor complex. Two PC-3 cell line variants (anchorage-dependent and suspension-adapted, anchorage-independent variants) were used as a model for advanced, androgen-independent prostate cancer. Genes that were overexpressed in a suspension-adapted PC-3 cell line were further investigated, since they can be considered as putative markers of metastatic activity. The macrophage inhibitory cytokine-1 (MIC-1) gene, which was overexpressed in the suspension-adapted PC-3 cell line, was further investigated in order to clarify the mechanism behind aggressive cell growth and androgen-independent gene regulation. In patient specimens, MIC-1 had no or low expression in benign prostatic hyperplasia and normal prostate but high in prostatic cancer and therefore it could be a useful marker for aggressive prostate cancer. Indomethacin increased the expression of MIC-1 in PC-3 cells, and apoptosis was also induced in this cell line but not in saPC-3 cell line suggesting a block in MIC-1 inducible apoptosis pathway.

Aberrant androgen receptor (AR) activity plays a critical role in the development and progression of both early-staged organ-confined and late-staged metastatic human prostate cancer. Recent large-scale genomic sequencing studies showed that ~50% of organ-confined prostate cancer patients have genetic rearrangements that placed the ETS transcription factors (e.g. ERG, ETV1) under the control of androgen-regulated gene promoters such as TMPRSS2. This results in the upregulation of the ETS transcription factors’ expressions in the presence of androgens. The aberrant overexpression of the ETS transcription factors are shown to induce the expression of genes that promote the cellular motility and invasive potential of prostate-tumor cells. Moreover, the improved therapeutic outcome of the second-generation anti-androgen therapies (e.g. abiraterone and enzalutamide) are encouraging, and prove that aberrant AR activity still drives the progression of metastatic prostate cancer. Although these treatments are initially effective, these cancer cells eventually develop resistance to these AR-targeted therapies termed castration-resistant prostate cancer (CRPC). Since the molecular steps involved in AR activation is still not clearly defined, it is critical to define the interactions required for AR activation prostate cancer cells, which will provide a framework for establishing more effective treatments to inhibit aberrant AR activity in human prostate cancer cells. Here, I developed a cellular system to isolate ligand-dependent interactions of AR in prostate-tumor cells. A siRNA luciferase screen was also developed and identified novel modulators of AR-mediated transcription selected from the proteomic dataset. Further biochemical studies showed that AR is associated with the Golgi membrane in a ligand-sensitive manner. And that the nuclear localization of ARA160, an AR coactivator, is regulated by the COPI retrograde trafficking machinery. Collectively, these results support the use of this cellular system to decipher the known AR-interacting proteins and novel components involved in AR signaling in prostate-tumor cells. I next investigated the androgen-sensitive AR transcriptional complexes and androgen-sensitive microsomes isolated from LNCaP prostate-tumor cells. Both studies yielded results that would further strengthen the diverse AR actions mediated within the cell. These results further support the notion that there is significant crosstalk amongst different cell surface receptor signaling pathways with AR. An extension of the androgen-sensitive microsome findings also led us to study the androgen-sensitive G-protein coupled receptor, CXCR7. I showed that androgens regulate the expressions of CXCR7 and CXCR4 and in turn modulated CXCL12-mediated motility in prostate tumor cells. Lastly, biochemical strategies were developed to detect differences in glycoprotein expression of frozen prostate cancer tissues isolated from human patients. I showed that the workflow successfully solubilized and isolated N- and O-linked glycoproteins from the frozen tissue samples and can be analyzed by quantitative mass spectrometry. This workflow would thus facilitate future biomarker studies. In summary, these data demonstrate the utility of developing methods for the comprehensive mapping of AR-mediated signaling in prostate cancer cells, and thus provide novel target candidates for the therapeutic treatment of metastatic or CRPC.