Orientador: Ronilson Agnaldo Moreno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T08:52:50Z (GMT). No. of bitstreams: 1 Mazuqueli_AnaCristina_M.pdf: 7140095 bytes, checksum: c193ad443d7fe35cf3c6d7b25f3175e8 (MD5) Previous issue date: 2008 / Resumo: O objetivo deste estudo foi avaliar a bioequivalência de duas formulações de acetato de ciproterona e etinilestradiol em drágea vs comprimido (Selene® comprimidos, Laboratório Eurofarma Ltda., formulação Teste e Diane® 35, Schering do Brasil, como Referência) após administração oral em 48 voluntárias adultas sadias. O estudo foi do tipo aberto, randomizado com duas fases, em que as voluntárias receberam uma dose única de acetato de ciproterona 2mg e etinilestradiol 0,035mg . As amostras de plasma foram obtidas em um período total de 240h. As concentrações plasmáticas de acetato de ciproterona e etinilestradiol foram analisadas por um método baseado na cromatografia líquida acoplada ao espectrômetro de massa usando como fonte de ionização photospray ( LC-MS/MS ) utilizando finasterida como padrão interno do acetato de ciproterona e 17-a-etinilestradiol-D4 como padrão interno do etinilestradiol. A concentração plasmática do acetato de ciproterona não teve diferença significante após a administração de ambas as formulações (formulação teste e referência do Diane®35). A média geométrica da razão entre o medicamento teste e referência com 90% IC, foi 90,66% (84,39% - 97,40% ) para Cmax, 96,20% (90,45% - 102,33%) para ASC0-240 e 95,86% (89,81% - 102,31%) para ASC0-inf. Já para o Etinilestradiol a média geométrica da razão entre o medicamento teste e referência com 90% IC, foi de 109,92% (IC 90% = 102,67% - 117,69% ) para Cmax, 90,63% (83,75% - 98,08%) para ASC0-120 e 83,85% (69,98% - 100,47%) para ASC0-inf. Diante dos resultados encontrados de Cmax e ASC0-t e estando dentro do intervalo de confiança entre 80% e 125% proposto pela Agência Nacional de Vigilância Sanitária (ANVISA) e pelo Food and Drug Administration (FDA), conclui-se que o Acetato de Ciproterona 2mg e o Etinilestradiol 0.035mg da Eurofarma Laboratórios Ltda. em comprimido é bioequivalente ao Diane®35 da Schering do Brasil, de acordo com sua taxa e extensão de absorção. / Abstract: The aim of this study was to assess the bioequivalence of two cyproterone acetate + ethinylestradiol tablet formulations (Selene® tablet formulation elaborated by Eurofarma Laboratórios Ltda., Brazil, as test formulation, and Diane ® 35 tablet formulation by Schering of Brazil, as reference formulation) after their oral administration to 48 healthy adult females. The study was conducted using an open, randomized two-period crossover design, in which twenty-four healthy volunteers received a single oral dose of cyproterone acetate + ethinylestradiol (2mg + 0.035mg) tablet. Plasma samples were obtained over a 240-hour period. Plasma cyproterone acetate + ethinylestradiol concentrations were analyzed by a method based on liquid chromatography by positive-ion photospray ionization (LC-MS/MS), using finasteride as internal standard of cyproterone acetate and 17-a-ethinyl estradiol-D4 as internal ethinylestradiol standard. The plasma concentration of CYP acetate did not differ significantly after the administration of both formulations (test formulation and the reference Diane®), according to the geometric mean ratio between the test and reference formulations, with 90% CI: 90.66% (84.39% - 97.40% ) for Cmax, 96.20% (90.45% - 102.33%) for ASC0-240 and 95.86% (89.81% - 102.31%) for ASC0-inf. Conversely, the geometric mean ratio between the test and reference formulations for ethinylestradiol, with 90% CI, was 109.92% (CI 90% = 102.67% - 117.69% ) for Cmax, 90.63% (83.75% - 98.08%) for ASC0-120 and 83.85% (69.98% - 100.47%) for ASC0-inf. Considering the results of Cmax and ASC0-t within the confidence interval between 80% and 125% proposed by the Brazilian National Agency for Sanitary Surveillance (ANVISA) and for the US Food and Drug Administration (FDA), it was concluded that the cyproterone acetate 2mg and ethinylestradiol 0.035mg by Eurofarma Laboratórios Ltda. in tablet is bioequivalent to the Diane®35 by Schering of Brazil, according to its absorption and extension rate. / Mestrado / Mestre em Farmacologia

Orientadores: Sebastião Roberto Taboga, Fernanda Cristina Alcântara dos Santos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T18:00:07Z (GMT). No. of bitstreams: 1 Perez_AnaPauladaSilva_D.pdf: 9263858 bytes, checksum: 6ed11b7c4d0102ec555e0f333c5a35e7 (MD5) Previous issue date: 2014 / Resumo: Os disruptores endócrinos (DE) são agentes exógenos que interferem no funcionamento do sistema endócrino. O 17?-etinilestradiol (EE), um importante componente dos contraceptivos orais é um exemplo de DE. Alguns estudos com roedores machos e fêmeas relataram que a exposição aos DE durante o período pré-natal foi capaz de eliciar proliferações patológicas no sistema reprodutor, incluindo a próstata do animal adulto. Entretanto, pouco se sabe sobre a real ação do estrógeno nas próstatas de machos e fêmeas, principalmente quando se leva em consideração o comportamento de ambas às próstatas durante o desequilíbrio dos níveis de hormônios esteroides que ocorre na puberdade e durante o envelhecimento dessas glândulas. Assim o presente estudo, teve como objetivos avaliar por análises morfológicas, sorológicas e imunohistoquímica quais foram os efeitos da exposição à baixa dose de EE durante os períodos pré-natal e puberal sobre a próstata ventral masculina e na próstata feminina de gerbilo senil. Deste modo, nós dividimos os grupos experimentais de acordo com o período de exposição ao EE (15µg/kg/dia). EE/PRÉ durante o período pré-natal, EE/PUB durante a puberdade e o EE/PRÉ-PUB durante o período pré-natal e puberdade. A exposição à estrógenos sintéticos durante o desenvolvimento afeta o eixo hipotálamo-pituitária gonadal, alterando a produção de hormônios esteroides. Os resultados revelaram que a exposição ao EE durante o desenvolvimento prostático alterou os níveis de hormônios esteroides, diminuindo os níveis de testosterona nos machos senis dos grupos EE/PRÉ e EE-PRÉ/PUB e aumento, nas fêmeas senis do grupo EE/PRÉ. Os níveis de estradiol aumentaram nas fêmeas do grupo EE/PRÉ-PUB. A interação epitélio-estroma também foi afetada pela exposição ao EE durante o desenvolvimento, dado evidenciado pela diminuição da imunorreatividade de ?-actina de músculo liso em regiões com presença de lesões invasivas, principalmente na próstata ventral de macho senil. A frequência de células positivas de p63 diminuiu na próstata ventral masculina do grupo EE/PRÉ, provavelmente a camada basal diminui em locais onde se observa foco com NIP e, aumentou na próstata feminina do grupo EE/PUB. Entretanto, esses dados mostraram que a próstata ventral de machos senil foi mais sensível aos efeitos da exposição ao EE, comparado à próstata de fêmeas senis. A senescência é caracterizada pela queda dos hormônios sexuais¸ e nessa fase aumenta as doenças prostáticas em machos e fêmeas. A exposição ao EE durante fases críticas como o pré-natal e a puberdade acentuaram as alterações na estrutura glandular da próstata e aumentaram o desenvolvimento de lesões prostáticas na senescência. Assim como o período pré-natal, o puberal também foi considerado uma fase crítica na exposição ao EE sobre a próstata de gerbilos machos e fêmeas senis. O consumo de EE durante a gestação ou na puberdade altera significativamente a saúde prostática masculina e feminina durante o envelhecimento, e o gerbilo foi considerado um bom modelo para esse estudo / Abstract: Endocrine disruptor (ED) are exogenous agents that interfere in the endocrine system function. 17?-ethinylestradiol, an important component of oral contraceptives is an example of ED. Studies with male and female rodents have reported that exposure to ED during the prenatal period is able to elicit aberrant pathological proliferations in the reproductive system, inclusive in the prostate of the adult animal. However, the amount known about the actual action of estrogen on male and female prostate, mainly as is the behavior of both prostates during imbalance between androgen and estrogen that occurs in pubertal period and during senescence of these glands. Thus the present study aimed to evaluate by morphological, serological, histopathological and immunohistochemical methods, as the exposure to low dose of ethinylestradiol during the prenatal and pubertal periods acts on ventral male prostate and female prostate of senile gerbil. Thus, we divided the experimental groups according to the period of exposure to EE (15?g/kg/day). EE/PRE during the prenatal period, EE/PUB during puberty and EE/PRE-PUB during the prenatal period and puberty. Exposure to synthetic estrogens during development affects the hypothalamic-pituitary-gonadal axis this alters the production of steroid hormones. The results showed that exposure to EE during developing prostate changed steroid hormones levels, decreasing testosterone levels in senile male of EE/PRE and EE-PRE/PUB groups and increased in senile female of EE/PRE group. The estradiol levels enhanced in females of EE /PRE-PUB group. In addition, EE exposure during the prenatal and pubertal periods altered the immunoreactivity of AR, ER? and ER?, as the function of these receptors are critical for prostate development, changes these signaling pathways contributed to increase of development of lesion and inflammation prostate in males and females during senescence. The stromal-epithelium interaction was also affected by exposure to EE during developing, was observed by decreased immunoreactivity of smooth muscle ?-actin in regions where noted invasive lesions, mainly in the ventral male prostate of senile gerbil. Frequencies positive cells of p63 decreased in the ventral male prostate of EE/PRE group, the basal layer decreases in locals with NIP focus, and these frequencies increased the female prostate EE/PUB group. However, these data showed that the ventral male prostate of senile gerbil was more sensible to effects of exposure to EE compared to female prostate. Senescence is characterized by reducing of sexual hormones, in this phase increases the prostatic diseases in males and females. Exposure to EE during critical periods as prenatal and puberty accentuated the changes in prostate glandular structure and increased the developing prostatic lesions in senescence. As prenatal period, pubertal was also considered a critical phase during the exposure to EE on male and female prostate of senile gerbil. The use of EE during gestational or puberty phases significantly alters male and female prostate health during aging, and the gerbil was considered a good model for this study / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Organic and inorganic compounds are present as contaminants in varying concentrations throughout our water cycle. Examples of these contaminants include the endocrine disrupting compounds (EDCs) bisphenol-A (BPA) and 17β-estradiol (E2) from plastics and pharmaceutical use. It can be necessary to obtain the concentration of these compounds within the water cycle for analysis by interested parties such as research groups, regulatory agencies, and private organizations. These concentrations, however, can be too dilute within the initial sample for analysis. Therefore it is necessary to concentrate the compound of interest (analyte) prior to analysis. One such way to do this is by way of Solid Phase Extraction (SPE). SPE uses a small cartridge which contains chromatographic packing material to chemically extract analytes from a water sample onto a solid phase. To increase concentration, these analytes are then transferred (eluted) to a substantially smaller volume of organic solvent for eventual analyses. These commercially available cartridges are relatively inexpensive, approximately $5 each. However, these cartridges are labeled as single use. In large-scale analyses, this can quickly add up to a sizable percentage of the analysis budget. Additionally, sizable waste volumes can be generated from these analyses in the form of non-degradable polypropylene plastic. If these cartridges can be re-used, material costs as well as waste volumes can be substantially reduced. However, little is known regarding how the quality of analysis degrades with cartridge re-use. The objective of this project is to evaluate the number of times SPE cartridges can be reused without compromising the results of the subsequent analyses. Based on a review of prior literature, I identified and developed protocols for extracting analytes (BPA and E2) from water via SPE, then analyzing them with gas chromatography and mass spectrometry (GC-MS). These protocols have been developed to mimic those employed by research labs, industry, and other entities for which the results of this study would be most applicable. The only deviation is the re-use of the cartridge rather than disposal and replacement. One type of commercially available SPE cartridge (Oasis HLB, Waters Inc., Milford, MA) was used and two water types were tested. The water was spiked with fixed concentrations of BPA and E2, and then analyzed by way of SPE/GC-MS. For both water types, I performed multiple SPE runs on 10 cartridges each. I tracked the history of GC-MS peak areas, which indicate apparent analyte concentration. Peak area data were analyzed as a function of the number of analyses performed (run number), and evaluated for statistically significant changes as well as overall trends. Statistically significant change and/or trends would indicate that the cartridge had exceeded the maximum allowable number of re-uses and would thereby identify the number of times the “single-use” cartridge can reliably be re-used. Peak area history for 20 SPE runs per cartridge for pure water samples and 10 SPE runs for wastewater effluent showed no statistically significant changes or trends on peak area. This indicates that cartridges can be re-used at least 10 times without compromising the integrity of water sample analysis for the EDCs considered in this study.

Following the BP Deepwater Horizon oil spill event, there was a critical need to assess the effects of the oil and dispersant chemicals on the coastal pelagic fish complex in the Gulf of Mexico and the Florida Straits. The objective of this study was to determine if spilled crude oil and dispersant chemicals have posed an ecological risk to the coastal pelagic fish complex through the detection of vitellogenesis. Crude oil containing polycyclic aromatic hydrocarbons (PAHs), dispersant chemicals and other estrogen-mimicking compounds are suspected to induce vitellogenin production in male and immature female fish, normally only produced by sexually mature females. Blood plasma and surface mucus were collected from wild-caught adult and juvenile males and females from as many representative coastal pelagic species as possible (including yellowfin tuna Thunnus albacares and swordfish Xiphias gladius). To create a control for this experiment, crevalle jacks (Caranx hippos) and lookdowns (Selene vomer) were injected with estradiol-17β (10 μg/g body weight) into the peritoneal cavity to induce vitellogenesis, regardless of sex or reproductive stage. The mucus and blood plasma of each injected fish was collected 7 to 11 days post-injection. Mucus and blood plasma samples of wild-caught and experimentally-injected fishes were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, stained with a phosphoprotein specific fluorescent dye (Pro-Q Diamond®), and visualized through ultraviolet transillumination. Vitellogenin was visibly detectable in the mucus collected from the control-injected fish, suggesting a disruption in the endocrine system as a result of estrogen exposure (estradiol-17β). However, there were no elevated levels of vitellogenin detected in any wild-caught fish mucus or blood plasma samples, indicating no vitellogenesis. From this, we infer that there has been no detectable endocrine disruption to the sampled coastal pelagic fish complex in the Gulf of Mexico two years after the start of the spill.

Rapid increases in plasma androgens are generally associated with short-term aggressive challenges in many breeding vertebrates. However, some animals such as song sparrows (Melospiza melodia) are aggressive year-round, even during the non-breeding season, when gonads are regressed and systemic testosterone (T) levels are non-detectable. In contrast, levels of the prohormone dehydroepiandrosterone (DHEA) are elevated year-round in the plasma and brain. The local conversion of brain DHEA to potent androgens may be critical in regulating non-breeding aggression. 3β-hydroxysteroid dehydrogenase/Δ4-Δ5 isomerase (3β-HSD) catalyzes DHEA conversion to androstenedione (AE) and the cofactor NAD⁺ assists in this transformation. In this thesis, I asked whether brain 3β-HSD activity is regulated by social encounters in seasonally breeding male songbirds. In Experiment 1, I looked at the long-term seasonal regulation of brain 3β-HSD activity. 3β-HSD activity was highest in the non-breeding season compared to the breeding season and molt. In Experiment 2, I hypothesized that brain 3β-HSD activity is rapidly regulated by short-term social encounters during the non-breeding season. A 30 min social challenge increased aggressive behavior. Without exogenous NAD⁺, there was ~355% increase in 3β-HSD activity in the caudal telencephalon and ~615% increase in the medial central telencephalon compared to controls (p<0.05). With exogenous NAD⁺, there was no effect of social challenge on 3β-HSD activity. These data suggest that endogenous cofactors play a critical role in the neuroendocrine response to social challenges. The increase in brain DHEA conversion to AE during social challenges may be a mechanism to rapidly increase local androgens in the non-breeding season, when there are many costs of systemic T. / Science, Faculty of / Zoology, Department of / Graduate

La différenciation sexuelle du système nerveux central est régie par les hormones stéroïdes sexuelles. Chez la femelle, les ovaires sont silencieux pendant la période fœtale et le cerveau est protégé de l’action masculinisante de l’œstradiol périnatal par l’action de l’α-fœtoprotéine. Des études révèlent un rôle actif de l’œstradiol ovarien postnatal dans la mise en place de la puberté et la féminisation des circuits neuraux gouvernant l’expression du comportement sexuel. A l’âge adulte, l’œstradiol a un rôle activationnel dans la régulation de l’axe hypothalamo-hypophysaire-gonadotrope (HPG) et les comportements reproducteurs. Il agit principalement via les récepteurs des œstrogènes nucléaires ERα et ERβ. L’ERα neural est indispensable à la maturation et la régulation de l’axe HPG et à l’expression du comportement sexuel. Le rôle de l’ERβ neural restait à clarifier. Au cours de ma thèse, nous avons généré un modèle de souris invalidées de manière sélective pour l’ER neural. Les femelles mutantes sont fertiles et ne présentent pas d’altération de l’axe HPG, ni de l’expression du comportement sexuel. Elles présentent un retard de puberté, corrélé à une diminution de l’expression prépubertaire de la kisspeptine dans le noyau rostral périventriculaire du 3e ventricule. Nous avons aussi étudié les effets d’une exposition maternelle au bisphénol A (BPA) sur les fonctions de reproduction femelle. Les résultats obtenus révèlent un rôle de l’ER dans la mise en place de la puberté et la régulation de l’expression de la kisspeptine dans le RP3V, et un effet du BPA probablement pendant la période postnatale/prépubertaire sur les fonctions neuroendocrines et comportementales femelles. / Sexual differentiation of the central nervous system is governed by the sex steroid hormones. In females, the ovaries are inactive during the fetal period, and the brain is protected from the masculinizing action of perinatal estradiol by the action of α-fetoprotein. Recent studies suggest an active role of postnatal ovarian estradiol in the establishment of puberty and feminization of neural circuits involved in the expression of female sexual behavior. In adulthood, estradiol has an activational role in the regulation of the hypothalamic-pituitary-gonadal (HPG) axis and reproductive behaviors. Estradiol acts mainly via nuclear estrogen receptors ERα and ERβ. Neural ERα is essential for the maturation and the regulation of the HPG axis, as well as the expression of sexual behavior. The role of neural ERβ remained to be clarified. During my thesis, we generated a mouse model selectively lacking ERβ in the nervous system. The mutant females show normal fertility, HPG axis regulation, and expression of sexual behavior. However, these mice have delayed puberty, correlated with a significant decrease in prepubertal expression of kisspeptin in the rostral periventricular area of the third ventricle (RP3V). Furthermore, we studied the effects of maternal exposure to bisphenol A (BPA) on the regulation of female reproductive functions. The overall results highlight the importance of ERβ in the postnatal/prepubertal regulation of kisspeptin expression and pubertal onset, as well as the potential effects of BPA during this period on neuroendocrine and behavioral responses.

The medial amygdala (MeA) is a central node in the interwoven circuits that regulate social behavior based on pheromones. Aromatase-expressing (arom+) neurons in the MeA are key for the establishment and maintenance of sex differences. Here, we characterized the intrinsic electrophysiological properties of arom+ neurons and non-aromatase (arom-) neurons in the MeA of male and female mice. Most electrophysiological properties were similar for arom+ neurons in the MeA between sexes, but the relative refractory period was twice as large in female mice. We also show that the firing pattern and firing frequency is markedly different between arom+ and arom- neurons. The activity of MeA neurons could be modulated by estradiol, which reduced activity in arom+ neurons in males. The differences between arom+ and arom- neurons were observed in both sexes suggesting that aromatase expression delineates a neural population in the MeA with similar and unique electrophysiological properties.

Submitted master's thesis is dealing with the problem of occurrence of residues of specific anthropogenic pollution of drinking water sources, with accent on possibilities of elimination of these compounds during drinking water treatment. There was pay attention to two estrogenic hormones – estradiol and ethinylestradiol. In laboratory scale there was tested efficiency of coagulation, activated carbon adsorption and ozonation in elimination of studied compounds from artificially contaminated water.

La 1α,25(OH)2 vitamine D3 (1,25-D3) est synthétisée à partir du cholestérol par l'exposition solaire de la peau. Les effets de cette hormone sont médiées par le récepteur de la vitamine D (VDR) dans le noyau et à la membrane plasmique, et avec le récepteur PDIA3 ils médient des effets génomiques et non-génomique. La vitamine D joue un rôle important dans la reproduction, puisque la réduction de la fertilité a été observée chez les rats déficients en vitamine D. L'estradiol (E2) est synthétisé à partir de la testostérone par l'enzyme aromatase (CYP19). L’E2 a des effets génomiques et non génomiques médiée par les récepteurs ESR1, ESR2 et GPER. L’objectif de ce travail a été d’étudier l’effet de l’E2 sur le métabolisme et les voies de signalisation de la vitamine D dans des testicules des rats à différents âges ainsi qu’une perturbation éventuelle initiée par un perturbateur endocrinien à activité estrogénique, le Bisphénol A (BPA). Dans le première axe de travail, trois protocoles expérimental (PE) ont été réalisés, où l’E2 et le BPA ont été administrés: traitement de J15pp à J30pp et euthanasie immédiate à J30 (PE1), traitement de J15 à J30 et euthanasie différée à J75 (PE2) et traitement à l’âge adulte de J60 à J75 et euthanasie immédiate à J75 (PE3). Dans le PE1, le traitement avec l’E2 a diminué l'expression du CYP27A1. L’E2 et le BPA ont diminué l'expression du VDR. Cet effet n'a pas été vérifié dans l'expression de la protéine VDR. Dans le PE2, l’E2 a augmenté l'expression des gènes VDR, PDIA3 et CYP27A1, et l'expression de la protéine VDR et CYP27A1. Les traitements n’ont eu aucun effet dans le PE3, ce qui indique qu’un traitement en période prépubère entraîne à la fois un effet immédiat et différé alors que le traitement à l’âge adulte semble sans effet. Dans le deuxième axe de travail, des effets non-génomiques du BPA ont été étudiés par la technique d’afflux de 45Ca2+ dans les testicules de rat prépubères. Le BPA a stimulé l’afflux de 45Ca2+ de manière un peu pareille avec les effets de l’E2. Cet effet semble ne pas impliquer les récepteurs classiques des estrogènes, mais semble se produire de manière compatible avec l'activation d'un récepteur couplé à la protéine G, comme le GPER. Cet effet se produit par la modulation de la fonction des canaux ioniques, comme des canaux potassiques, TRPV1 et des canaux chlorés. Aussi le BPA module le calcium du stock intracellulaire par l’inhibition de la SERCA et l’activation du récepteur IP3. Également des protéines kinases PKA, PKC, MEK et p38MAPK participent de l’effet du BPA, qui pourrait déclencher un cross talk avec les voies de signalisation nucléaires résultant la médiation des réponses génomiques. Dans le troisième axe de travail, l'expression de certains gènes impliqués dans le métabolisme et la signalisation de 1,25-D3 et E2 a été analysée dans des cellules de Leydig. La 1,25-D3 a diminué l'expression du CYP27A1, un effet qui a également été observé lorsque les cellules étaient co-incubées avec l'E2. L’E2 a diminué l'expression des gènes ESR1 et CYP19. Les deux hormones ont démontré un mécanisme de retours négatifs sur leur métabolisme dans ces cellules. Des effets non génomiques ont été étudiés dans ces cellules, où l’E2 semble avoir un effet inhibiteur tandis que la 1,25-D3 a stimulé l'afflux de 45CA2+. A partir de ces résultats, nous pouvons affirmer que la 1,25-D3, l’E2 et le BPA ont des effets moléculaires importants dans le système reproducteur masculin, par l'expression génique des récepteurs et des enzymes impliqués dans le métabolisme des hormones 1,25-D3 et E2. De plus, les résultats obtenus renforcent la théorie selon laquelle il existe une relation entre les voies de signalisation de la 1,25-D3 et l’E2. Comme la 1,25-D3 et l'E2, le BPA stimule également les effets non-génomiques impliqués dans la signalisation du calcium. / 1α,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D, is synthetized from cholesterol by skin exposure to the sun. This hormone’s actions are mediated by vitamin D receptor (VDR) in the nucleus and in the plasma membrane, resulting in genome actions like gene expression regulation. VDR can also be found in the plasmatic membrane, and together with PDIA3 receptor they mediate 1,25-D3 non-genomic actions. Vitamin D has an important role in reproductive function, since fertility reduction was observed in vitamin D deficient rats, as well as VDR and 1α-hydroxylase deficiency. In these animals, calcium and estrogen supplementation was able to reverse the deleterious effects in reproductive function, indicating that there is a relation between 1,25-D3 and estrogens signalling pathways. Estradiol (E2) is synthetized from testosterone by aromatase enzyme (CYP19). E2 is found in high levels in the male reproductive function, and like 1,25-D3 can induce genomic and non- genomic actions, mediated by ESR1, ESR2 and GPER receptors. Bisphenol A (BPA) is a xenoestrogen utilized in plastic industry, capable of modulating the endocrine system through E2 receptors. The aim of this work was to study metabolism and signaling pathways interactions between 1,25-D3 and E2, as well as BPA influence in testicular cells. In the first line of work, three treatment protocols (TP) were realized, where E2 and BPA were administrated in rats between 15th and 30th days, were a portion of the animals were euthanized at the last day of treatment (TP1) and another portion was kept alive after the treatment until euthanized at adult age with 75 days (TP2). A third animal group also received the same treatments when adults (TP3). In TP1, E2 treatment decreased CYP27A1 gene expression. E2 and BPA decreased VDR gene expression. This effect was not verified in VDR protein expression. In TP2, E2 increased VDR, PDIA3 and CYP27A1 gene expression, and VDR and CYP27A1 protein expression, indicating a compensatory effect over gene expression inhibition in prepubertal age. In TP3, treatments did not change gene expression, indicating that prepubertal age is more susceptible to estrogen exposure. In the second line of work, non-genomic effects of BPA were studied through 45Ca2+ influx in prepubertal rat testis. BPA stimulated 45Ca2+ influx in a similar manner to E2. This effect was independent of classical ERs, consistent with a G protein-coupled receptor mechanism, probably GPER. This effect involves the modulation of ionic channels, such as K+, TRPV1 and Cl- channels. Furthermore, BPA is able to modulate calcium from intracellular storages by inhibiting SERCA and activating IP3 receptor/Ca2+ channels at the endoplasmic reticulum and activate kinase proteins, such as PKA and PKC. The rapid responses of BPA on calcium influx could, in turn, trigger a cross talk by MEK and p38MAPK activation and also mediate genomic responses. In the third line of work, the expression of some genes involved in 1,25-D3 and E2 metabolism and signalling were analysed in Leydig cells. 1,25-D3 decreased CYP27A1 gene expression, an effect that was also observed when cells were coincubated with 1,25-D3 and E2. E2 decreased ESR1 and CYP19 gene expression. Both hormones demonstrated an negative feedback mechanism over their on metabolism in these cells. Non-genomic effects were also studied in these cells, where E2 seems to have an inhibitory effect while 1,25-D3 was able to stimulate calcium influx. From these results we can conclude that 1,25-D3, E2 and BPA have important molecular effects in the male reproductive system, through gene expression control over receptors and enzymes involved in the metabolism of the steroid hormones studied. These results also reinforce the theory that there is a relationship between 1,25-D3 and E2 signalling pathways, as well as 1,25-D3, E2 and BPA also have non-genomic actions in calcium signalling.

Orientador: Sebastião Roberto Taboga / Coorientador: Silvana Gisele Pegorin de Campos / Banca: Ana Claudia Polli Lopes / Banca: Ana Paula Girol / Banca: Maria Tercilia Vilela de Azeredo Oliveira / Banca: Claudia Regina Bonini Domingos / Resumo: A incidência de câncer de mama tem aumentado consideravelmente ao longo dos anos. Frente a esta alta taxa epidemiológica a atenção às causas desta doença tem sido significativa em meio a grupos de pesquisa. Fatores genéticos e algumas situações de risco, como a exposição a componentes químicos disruptores endócrinos, podem estimular o seu desenvolvimento. O bisfenol A (BPA), utilizado na manufatura de plásticos e resinas, pode ser liberado destes componentes e exercer efeitos estrogênicos, causando distúrbios na função endócrina e aumentando a predisposição à carcinogênese. Alguns estudos têm demonstrado os efeitos do BPA em longo prazo, de modo que sua exposição durante o período de desenvolvimento embrionário inicial e perinatal pode predispor a glândula mamária (GM) ao desenvolvimento de câncer em indivíduos já adultos. Seu mecanismo de ação, assim como o do estradiol endógeno, baseia-se principalmente na ligação a receptores celulares hormonais, podendo estimular a proliferação, promovendo um desbalanço nos ciclos de renovação celular mamária. Para o estudo de carcinogênese o gerbilo da Mongólia (Meriones unguiculatus) é um modelo experimental interessante, já que possui capacidade de desenvolver tumores espontâneos em órgãos hormônio-responsivos, mimetizando, naturalmente, o ambiente de um indivíduo predisposto à tumorigênese. Assim, os objetivos deste trabalho foram (1) caracterizar morfologicamente a GM de fêmeas de gerbilo da Mongólia em diferentes estágios... / Abstract: The incidence of breast cancer has increased considerably over the years. Facing this high epidemiological rate, attention to the causes of this disease has been significant among research groups. Genetic factors and some risk situations, such as exposure to chemical endocrine disrupting components may stimulate its development. Bisphenol A (BPA), a compound used in the manufacture of plastics and resins, can be released from these components and exert estrogenic effects, causing disturbances of endocrine function and increasing the predisposition to carcinogenesis. Some studies have demonstrated the long-term effects of BPA, in a way that exposure during embryonic development (perinatal phase) may predispose the mammary gland (MG) to development of cancer in adults. Its mechanism of action, as that of endogenous estradiol, is mainly based on cellular hormonal receptors binding, which can stimulate the proliferation, promoting an imbalance of breast cell cycles and renewal. For these carcinogenesis studies, the Mongolian gerbil (Meriones unguiculatus) is an interesting experimental model, since it has the ability to develop spontaneous tumors in hormone-responsive organs, naturally mimicking the environment of an individual predisposed to tumorigenesis. Thus, the aims of this study were to (1) characterize morphologically the mammary gland of Mongolian gerbil females at different stages of functionality, (2) describe morphological changes found in the MG of adult females ... / Doutor