No description available.

Bisphenol A (BPA) is an alkylphenol compound used in plastic manufacturing, which enters the aquatic environment through wastewater treatment plants and landfill leachates. Exposure of fish to BPA results in developmental defects, decreased hatching, and increased occurrence of the egg yolk precursor protein vitellogenin in male fish. BPA is an estrogenic like compound (xenoestrogen) that can bind and activate the nuclear estrogen receptors, ERα and ERβ, causing changes in gene transcription (genomic mechanism). However, the ability of BPA to activate rapid estrogen signaling and to disrupt nongenomic physiological responses to estrogens is not known. One important nongenomic estrogen action in fish is to maintain meiotic arrest of oocytes and to prevent precocious oocyte maturation through activation of the transmembrane G-protein coupled estrogen receptor (Gper). Binding of estrogens to Gper on zebrafish oocytes results in rapid activation of epidermal growth factor receptor (Egfr) signaling and subsequent Mapk3/1 activation. We show here that BPA and three structurally-related chemicals, tetrachlorobisphenol A, tetrabromobisphenol A and nonylphenol, can mimic estrogen by activating this mechanism of meiotic arrest through Gper in zebrafish (Danio rerio). BPA bound to zebrafish Gper and inhibited spontaneous oocyte maturation (OM) of denuded oocytes. Treatment of oocytes with Actinomycin D did not block the effects of BPA, suggesting that this inhibition of OM is through a nongenomic mechanism. Incubation of oocytes with a selective GPER antibody and the specific GPER antagonist G-15 blocked the effects of BPA on OM, further suggesting that BPA inhibition of OM is through its interaction with the receptor. Various inhibitors of the EGFR pathway were utilized to determine if the inhibition of OM by BPA is mediated through this mechanism. BPA activation of the Egfr pathway resulted in Mapk3/1 (also known as Erk 1/2) phosphorylation. The results show that BPA disrupts oocyte maturation through a novel mechanism involving activation of a Gper/Egfr/Mapk3/1 pathway with potential adverse impacts on reproductive success. / text

O estrógeno é importante para o desenvolvimento de redes neuronais. Assim, investigamos mecanismos celulares relacionados à neuroproteção, através da sinalização rápida mediada pelo GPER-1 em cultura mistas e enriquecida de neurônios submetidas ou não à privação de glicose/oxigênio (PGO). Mostramos que as células corticais em cultura expressam o receptor GPER-1 e esta marcação encontra-se dispersa tanto no citosol como no núcleo. Nossos resultados mostraram que a proteção, via sinalização estrogênica, foi dependente da composição celular. A ausência da sinalização via GPER-1 previamente à PGO aumentou a morte celular induzida pela PGO, sugerindo que o bloqueio desta sinalização via GPER-1 pode estar relacionado ao pior prognóstico de lesões isquêmicas, e a suplementação com G1 no meio de cultura durante a privação e reperfusão atenuaram estes efeitos. Além disso, nossos resultados apontam para a influência das células da glia como mediadores do papel neuroprotetor, via sinalização estrogênica não-nuclear, neste contexto de privação de glicose/oxigênio. / Estrogen is important to the development of neural networks. Thus, we investigated the cellular mechanisms related to neuroprotection through the rapid signaling mediated by GPER-1 in mixed culture and enriched neurons submitted or not to glucose/oxygen deprivation (OGD). We showed that cortical cell cultures express GPER-1 receptor and this are dispersed both in the cytosol and the nucleus. Our results showed that protection via estrogen signaling was dependent on the cellular composition. The lack of a signaling pathway GPER-1 before OGD increased cell death induced by OGD, suggesting that blocking of GPER-1 signaling pathway could be related to poor prognosis of ischemic lesions and G1 supplementation of culture media during deprivation and reperfusion attenuated these effects. In addition, our results point to the influence of glial cells as mediators of the neuroprotective role via non-nuclear estrogen signaling in this context of glucose/oxygen deprivation.

O diabetes é uma doença crônica que afeta mais de 8% da população mundial. As alterações vasculares estão relacionadas às principais complicações do diabetes. A aldosterona contribui para a disfunção endotelial após a ativação do receptor mineralocorticóide (MR). Recentemente, foi demonstrado que a aldosterona ativa o receptor de estrógeno acoplado à proteína G (GPER). A ativação de GPER está relacionada a efeitos benéficos na vasculatura. Entretanto, ainda não existem estudos em artérias de resistência relacionando aldosterona e GPER e essa interação no diabetes. Dessa forma, o presente estudo testou a hipótese de que a aldosterona ativando receptores GPER promove efeitos benéficos na vasculatura, mas esses efeitos estão diminuídos no diabetes. Os objetivos foram investigar os níveis de expressão de GPER nos animais controle e db/db [camundongos com mutação no receptor de leptina que desenvolvem obesidade e diabetes tipo 2], quais efeitos da aldosterona são mediados por ativação de GPER e quais mecanismos envolvidos nessa ativação e verificar se estão alterados no diabetes. O grupo diabético apresentou maior expressão de GPER, mas não de MR. A aldosterona promoveu aumento da resposta máxima contrátil à fenilefrina (PE) somente no grupo controle, que foi revertido pelo uso do antagonista de GPER, G15. No grupo diabético, a resposta à PE já é aumentada, o uso dos antagonistas de MR e GPER reduziram a resposta da PE. A aldosterona ainda reduziu a potência de relaxamento da acetilcolina (ACh) em ambos os grupos, por ativação de MR. O antagonismo de GPER por G15 promoveu um redução adicional na potência do relaxamento no grupo controle, mas não afetou a resposta do grupo diabético. Esses achados confirmam a hipótese de que GPER exerce um papel benéfico na vasculatura e esse efeito é perdido no diabetes. Nossos resultados contribuem para a compreensão dos mecanismos que a aldosterona influencia os danos vasculares no diabetes através da ativação de receptores MR e GPER. / Diabetes is a chronic disease that affects more than 8 % of the world population. Vascular changes are related to the major complications of diabetes. Aldosterone contributes to endothelial dysfunction after activation of the mineralocorticoid receptor (MR). It was recently shown that aldosterone also activates the G protein-coupled estrogen receptor (GPER) and induces non genomic effects. The activation of GPER by estrogen or G1 is related to beneficial effects on the vasculature. However, there are no studies demonstrating the relationship between aldosterone and GPER in diabetes mellitus. Therefore, we hypothesized that the beneficial effects of aldosterone mediated by the activation of GPER receptors on vascular reactivity are decreased in diabetes mellitus. Female control and diabetic (db/db) mice [leptin receptor knockout mice that develop obesity and type 2 diabetes] were used. We determined the expression of GPER and the effect of aldosterone in the presence of MR and GPER antagonists in arteries from control and db/db mice and the major signaling pathways involved. The diabetic group showed increased GPER expression, but not MR. In the presence of aldosterone the control increased the maximal contractile response to phenylephrine (PE), and this increase was reversed by the use of GPER antagonist, G15. The response to PE is already increased in the diabetic group. Although aldosterone did not cause further increase the use of MR and GPER antagonists reduced the maximum response to PE at the same level of control. Aldosterone also reduced the potency of acetylcholine (ACh)-induced relaxation in both groups by the activation of MR. GPER antagonism caused further decrease in the potency of ACh-induced relaxation in the control group, while not affecting the response of the diabetic group. These findings confirm the hypothesis that the beneficial effects of aldosterone mediated by the activation of GPER receptors are decreased in diabetes mellitus. Our results contribute to understanding the mechanisms that aldosterone influences the vascular damage in diabetes through activation of MR and GPER receptors.

O estrógeno é importante para o desenvolvimento de redes neuronais. Assim, investigamos mecanismos celulares relacionados à neuroproteção, através da sinalização rápida mediada pelo GPER-1 em cultura mistas e enriquecida de neurônios submetidas ou não à privação de glicose/oxigênio (PGO). Mostramos que as células corticais em cultura expressam o receptor GPER-1 e esta marcação encontra-se dispersa tanto no citosol como no núcleo. Nossos resultados mostraram que a proteção, via sinalização estrogênica, foi dependente da composição celular. A ausência da sinalização via GPER-1 previamente à PGO aumentou a morte celular induzida pela PGO, sugerindo que o bloqueio desta sinalização via GPER-1 pode estar relacionado ao pior prognóstico de lesões isquêmicas, e a suplementação com G1 no meio de cultura durante a privação e reperfusão atenuaram estes efeitos. Além disso, nossos resultados apontam para a influência das células da glia como mediadores do papel neuroprotetor, via sinalização estrogênica não-nuclear, neste contexto de privação de glicose/oxigênio. / Estrogen is important to the development of neural networks. Thus, we investigated the cellular mechanisms related to neuroprotection through the rapid signaling mediated by GPER-1 in mixed culture and enriched neurons submitted or not to glucose/oxygen deprivation (OGD). We showed that cortical cell cultures express GPER-1 receptor and this are dispersed both in the cytosol and the nucleus. Our results showed that protection via estrogen signaling was dependent on the cellular composition. The lack of a signaling pathway GPER-1 before OGD increased cell death induced by OGD, suggesting that blocking of GPER-1 signaling pathway could be related to poor prognosis of ischemic lesions and G1 supplementation of culture media during deprivation and reperfusion attenuated these effects. In addition, our results point to the influence of glial cells as mediators of the neuroprotective role via non-nuclear estrogen signaling in this context of glucose/oxygen deprivation.

O diabetes é uma doença crônica que afeta mais de 8% da população mundial. As alterações vasculares estão relacionadas às principais complicações do diabetes. A aldosterona contribui para a disfunção endotelial após a ativação do receptor mineralocorticóide (MR). Recentemente, foi demonstrado que a aldosterona ativa o receptor de estrógeno acoplado à proteína G (GPER). A ativação de GPER está relacionada a efeitos benéficos na vasculatura. Entretanto, ainda não existem estudos em artérias de resistência relacionando aldosterona e GPER e essa interação no diabetes. Dessa forma, o presente estudo testou a hipótese de que a aldosterona ativando receptores GPER promove efeitos benéficos na vasculatura, mas esses efeitos estão diminuídos no diabetes. Os objetivos foram investigar os níveis de expressão de GPER nos animais controle e db/db [camundongos com mutação no receptor de leptina que desenvolvem obesidade e diabetes tipo 2], quais efeitos da aldosterona são mediados por ativação de GPER e quais mecanismos envolvidos nessa ativação e verificar se estão alterados no diabetes. O grupo diabético apresentou maior expressão de GPER, mas não de MR. A aldosterona promoveu aumento da resposta máxima contrátil à fenilefrina (PE) somente no grupo controle, que foi revertido pelo uso do antagonista de GPER, G15. No grupo diabético, a resposta à PE já é aumentada, o uso dos antagonistas de MR e GPER reduziram a resposta da PE. A aldosterona ainda reduziu a potência de relaxamento da acetilcolina (ACh) em ambos os grupos, por ativação de MR. O antagonismo de GPER por G15 promoveu um redução adicional na potência do relaxamento no grupo controle, mas não afetou a resposta do grupo diabético. Esses achados confirmam a hipótese de que GPER exerce um papel benéfico na vasculatura e esse efeito é perdido no diabetes. Nossos resultados contribuem para a compreensão dos mecanismos que a aldosterona influencia os danos vasculares no diabetes através da ativação de receptores MR e GPER. / Diabetes is a chronic disease that affects more than 8 % of the world population. Vascular changes are related to the major complications of diabetes. Aldosterone contributes to endothelial dysfunction after activation of the mineralocorticoid receptor (MR). It was recently shown that aldosterone also activates the G protein-coupled estrogen receptor (GPER) and induces non genomic effects. The activation of GPER by estrogen or G1 is related to beneficial effects on the vasculature. However, there are no studies demonstrating the relationship between aldosterone and GPER in diabetes mellitus. Therefore, we hypothesized that the beneficial effects of aldosterone mediated by the activation of GPER receptors on vascular reactivity are decreased in diabetes mellitus. Female control and diabetic (db/db) mice [leptin receptor knockout mice that develop obesity and type 2 diabetes] were used. We determined the expression of GPER and the effect of aldosterone in the presence of MR and GPER antagonists in arteries from control and db/db mice and the major signaling pathways involved. The diabetic group showed increased GPER expression, but not MR. In the presence of aldosterone the control increased the maximal contractile response to phenylephrine (PE), and this increase was reversed by the use of GPER antagonist, G15. The response to PE is already increased in the diabetic group. Although aldosterone did not cause further increase the use of MR and GPER antagonists reduced the maximum response to PE at the same level of control. Aldosterone also reduced the potency of acetylcholine (ACh)-induced relaxation in both groups by the activation of MR. GPER antagonism caused further decrease in the potency of ACh-induced relaxation in the control group, while not affecting the response of the diabetic group. These findings confirm the hypothesis that the beneficial effects of aldosterone mediated by the activation of GPER receptors are decreased in diabetes mellitus. Our results contribute to understanding the mechanisms that aldosterone influences the vascular damage in diabetes through activation of MR and GPER receptors.

Encefalomielite Autoimune Experimental (EAE) é um modelo animal para o estudo da Esclerose Múltipla, doença autoimune na qual células do sistema imune atacam a bainha de mielina e os oligodendrócitos, levando a desmielinização, perda axonal e morte neuronal. Astrócitos são importantes na doença e na modulação da neuroinflamação. O estrógeno apresenta ação protetora, porém a ação via receptor acoplado a proteína G (GPER) é pouco conhecido. Como GPER está presente nos astrócitos, o objetivo deste projeto foi verificar se o tratamento com G1 (agonista seletivo de GPER) seria capaz de modular o processo inflamatório presente no SNC. O tratamento com o G1 (3mg/Kg, via subcutânea, durante três dias, iniciando no 5º dia após a indução da EAE) atenuou o escore clínico no pico da doença e alterou a morfologia dos astrócitos da medula espinhal, tanto na substância branca como na cinzenta, sugerindo um efeito anti-inflamatório do G1. Estudos subsequentes in vitro foram feitos para tentar elucidar possíveis vias de ativação relacionadas ao GPER. / Experimental autoimmune encephalomyelitis (EAE) is an animal model for the study of Multiple Sclerosis, an autoimmune disease in which cells of the immune system attack the myelin sheath and oligodendrocytes, leading to demyelination, axonal loss, and neuronal death. Astrocytes are important in disease and modulation of neuroinflammation. Estrogen has protective action, but the action by G protein-coupled estrogen receptor (GPER) is little known. GPER is present in astrocytes, the objective of this project was to verify if treatment with G1 (selective agonist of GPER) would be able to modulate the inflammatory process present in the CNS. Treatment with G1 (3 mg / kg, subcutaneously for three days, beginning on the 5th day after EAE induction) attenuated the clinical score at the peak of the disease and altered the morphology of the spinal cord astrocytes both in white matter and Suggesting an anti-inflammatory effect of G1. Subsequent in vitro studies have been done to try to elucidate possible pathways of activation related to GPER.

No description available.

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No. of bitstreams: 1 GUSTAVO_DALTO_BARROSO_MACHADO.pdf: 989422 bytes, checksum: 22ea376b88186b8cd072559da6413b3b (MD5) Previous issue date: 2017-12-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The present study investigated if the model of memory dysfunction induced by iron overload, so far only tested in male rats, would be able to induce cognitive decline in female rats, aiming to validate it as an experimental model to study gender differences in neurodegenerative diseases. We also evaluated the effects of the activation of the G protein coupled estrogen receptor (GPER) on iron- and ovariectomy-induced memory deficits and the role of the PKA/CREB pathway as a mediator of its physiologic effects. Four experiments were performed: I ? female rats received iron (30mg/kg, per oral, p.o.) or vehicle (sorbitol, p.o.) in the neonatal period and after, in adulthood, they were submitted to the surgical protocol: ovariectomy (OVX) or false-surgery (SHAM), after 3 weeks, behavioral tasks were performed using the object recognition (RO); object location (OL) and inhibitory avoidance (IA) tasks; II ? the same two protocols cited before and behavioral tasks 3 weeks from the surgeries, but, immediately after the training sessions the animals received the selective agonist of the GPER, G1 (10mg/kg, subcutaneously, sc.) or vehicle (veh, oil, sc.), the long-term memory was assessed 24h after the training sessions; III ? dose-response curve in order to determine the the effects of the PKA-inhibitor (H-89) on memory retention of inhibitory avoidance task, the following doses were administered intraperitoneally to na?ve adult female rats: 0.1 mg/kg; 0.25mg/kg or 0.5mg/kg; and finally the experiment IV in which the memory of iron overloaded-ovariectomized female rats was assessed 24h after that they had received H-89 (0.25mg/kg, ip.) or veh 15 minutes previous to the training sessions and G1 (10mg/kg, sc.) or veh immediately after their training in the OL and IA tasks. We observed that the effects of iron overload in female, associated or non-associated to OVX varied according to the behavioral test that the animals were submitted. The lowest recognition index level in OL task was observed in the animals exposed to the iron overload and, subsequently, to the lack of estrogens secondary to ovariectomy. The acute post-training treatment with G-1 increased the recognition index in the OL task and it was also associated with an increasing in the latency to step down ifrom the platform to the grid in the IA task. Finally, all these positive results obtained with the post-training administration of G-1 were abolished when the animals received the pre-training treatment of H-89. Our study opens new avenues in the field of memory and estrogens once it introduces the association of lack of estradiol and iron overload as a pre-clinical phenotypical model of the aging process in women. Besides, from the best of our knowledge, it is the first evidence of the role of the G1 in the consolidation of the emotional memory as well as the first study connecting the GPER directly to the PKA/CREB pathway. / Este estudo investigou se o modelo de disfun??o cognitiva induzido pela sobrecarga de ferro, somente testado em ratos machos at? o momento, induz d?ficits de mem?ria em ratas, objetivando valid?-lo como um m?todo experimental que auxilie na compreens?o das diferen?as fisiopatol?gicas entre os sexos em rela??o ?s doen?as neurodegenerativas. Tamb?m avaliamos, de maneira in?dita, os efeitos da ativa??o receptor de estrog?nio acoplado ? prote?na G (GPER), nesse modelo combinado com o modelo de menopausa experimental. Al?m disso, especulamos o papel da via da PKA/CREB na ativa??o do GPER, atrav?s da inibi??o farmacol?gica da PKA. Foram realizados quatro experimentos para atingir esses objetivos: I ? ratas tratadas com ferro (via oral ? v.o.) ou ve?culo (sorbitol ? v.o.) foram ovariectomizadas ou submetidas ? cirurgia falsa (SHAM) na idade adulta, ap?s tr?s semanas do protocolo cir?rgico foram submetidas ?s tarefas de reconhecimento de objetos (RO), localiza??o de objetos (LO) e esquiva inibit?ria (EI); II ? ratas tratadas com ferro no per?odo neonatal foram ovariectomizadas ou submetidas ? cirurgia SHAM, ap?s recupera??o da cirurgia (3 semanas) foram tratadas anteriormente aos treinos das tarefas de LO e EI com G-1 (agonista seletivo do GPER, 10mg/kg, sc.) ou ve?culo (?leo de girassol, sc.) e testadas 24h ap?s o treino; III ? ratas naive receberam diferentes doses do composto H-89 (inibidor da PKA) para avaliar seu potencial amn?sico (0.1mg/kg, 0.25mg/kg e 0.5mg/kg; ip.) e IV ? ratas submetidas ? sobrecarga neonatal de ferro e OVX, ap?s 3 semanas do protocolo cir?rgico receberam quinze minutos antes dos treinos das tarefas de LO e EI o composto H-89 (0.25mg/kg, ip.) ou ve?culo (salina, ip.) e imediatamente ap?s o treino G-1 (10mg/kg, sc.) ou ve?culo (?leo de girassol, sc.), as ratas foram testadas ap?s 24h dos treinos. Os efeitos do protocolo de sobrecarga neonatal de ferro associado ou n?o a priva??o de estradiol variou em depend?ncia da tarefa comportamental avaliada. Na tarefa de LO os grupos submetidos ? sobrecarga neonatal de ferro e ovariectomia apresentaram os menores ?ndices de reconhecimento. O tratamento com o agonista do GPER ? G1 - foi capaz de melhorar os d?ficits cognitivos induzidos pela sobrecarga neonatal de ferro e/ou pela ovariectomia em ratas no teste de LO e EI, esses efeitos foram inibidos pela administra??o do inibidor da PKA anteriormente ao treino das tarefas. Demonstramos que o modelo de sobrecarga neonatal de ferro associado ? ovariectomia ? um modelo ?til para o estudo dos efeitos delet?rios da diminui??o dos n?veis s?ricos de estradiol na menopausa, auxiliando como modelo fenot?pico do que ocorre na cl?nica, onde as mulheres p?s-menopausa apresentam preval?ncia maior de Doen?a de Alzheimer que homens. Al?m disso, demonstramos de maneira in?dita os efeitos do composto G-1 na tarefa de esquiva inibit?ria. Nossos dados comportamentais tamb?m sugerem que o composto G-1 depende da ativa??o da PKA para exercer seus efeitos sobre a consolida??o da mem?ria.

This thesis examines the rapid, non-genomic effects of estrogens on neural plasticity and learning. Estrogens are classically known to affect gene transcription events, however they have more recently been found to also rapidly activate second messenger systems within 1hr of administration. Therefore, we first examined the rapid effects of 17β-estradiol, and an estrogen receptor (ER) α and ERβ agonist on three different learning paradigms: object placement, object recognition, and social recognition. We found that both systemic injections and intrahippocampal delivery of 17β-estradiol and the ERα agonist improved performance on all 3 learning paradigms within 40min of hormone administration. However, the ERβ agonist administered systemically or intrahippocampally, improved performance only on the object placement learning paradigm, while having no effect on object recognition, and impairing social recognition at high doses. To elucidate how estrogens might rapidly affect learning, we examined how estrogens rapidly affect the neural plasticity of CA1 hippocampal neurons. We found that 17β-estradiol and the ERα agonist increased dendritic spine density in CA1 hippocampal neurons within 40min of administration, suggesting that estrogens rapidly increase the density of synapses within this brain region. Conversely, the ERβ agonist did not affect spine density, or decreased spine density. In addition, by using whole-cell patch clamp recordings of CA1 pyramidal neurons, we were able to determine that 17β-estradiol and the ERα agonist rapidly reduced AMPA receptor (but not NMDA receptor) mediated membrane depolarizations after 15min of hormone application. Similar to above, the ERβ agonist had no effect on AMPA or NMDA receptor mediated membrane depolarizations. These data suggest that estrogens rapidly promote the development of immature synapses (which contain low levels of synaptic AMPA receptors) within the CA1 hippocampus. Immature spines provide synaptic sites at which new memories can be stored and are thought of as “learning spines” (Kasai et al, 2003). Therefore, estrogens (through ERα) may rapidly induce the formation of hippocampal immature spines to promote learning. / Funded by NSERC