Insulin and insulin-like growth factor 1 (IGF-1) and 2 (IGF-2) are related protein hormones with different but overlapping biological functions. All the hormones interact with a receptor within the insulin-IGF system (insulin receptor A and B, IGF-1 receptor), however with different affinity. The different interaction with individual receptors is just one of the main tools for regulation of the system that is essential for the proper functioning of the organism. Although the residues directly interacting with receptors are mainly located in A and B domains, the C and D domains probably play a role in receptor specificity. Here, we firstly focused on the impact of D domains of IGF-1 and 2 (D1 and D2 domains) and C domain of IGF- 2 (C2 domain). To probe the impact of C and D domains, we prepared insulin analogues containing a part of or an entire domain following a pattern seen in IGFs. The receptor-binding affinities of these analogues and their receptor autophosphorylation potentials were characterised. Our results revealed that the initial part of D1 domain has a detrimental effect on IR affinity that is only slightly enhanced by the rest of the D1 domain. D2 domain has rather neutral effect on IR affinity. We further showed that the addition of amino acids derived from the C2 domain to the...

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Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O objetivo deste estudo foi avaliar o efeito da administração de leptina no lobo ventral da próstata de ratos adultos. Vinte ratos Wistar machos e adultos foram divididos em 2 grupos: L - animais foram injetados com 50 μL diária de leptina (8 μg / 100 g PC, subcutânea) durante quatro dias e C - animais receberam o mesmo volume de solução salina. Perfil lipídico e níveis séricos de testosterona foram avaliados. O lobo ventral da próstata foi processado para análise histomorfométrica. Expressão dos genes da aromatase, receptor de andrógeno, receptores de estrógeno (α e β) e as isoformas dos receptores de leptina longa (Ob-Rb) e curta (Ob-Ra) foram avaliados por PCR em tempo real. Proliferação celular foi avaliada por imuno-histoquímica com PCNA. Os dados foram expressos como média  erro padrão e analisados pelo teste t de Student. Níveis séricos de colesterol aumentaram (C = 39,7 4,2; L = 55,2 4,2, mg / dL, P ≤ 0,02) e de testosterona (C = 1,6 0,43; L = 0,6 0,15, ng / dL, P ≤ 0,03) diminuíram no grupo L. A análise histomorfométrica mostrou uma redução na densidade de células (C = 8868 242; L = 8.211 210, mm2; P ≤ 0,04), na área total (C = 0,24 0,026; L = 0,10 0,009, mm2; P ≤ 0,001) e na área interna dos ácinos (C = 0,16 0,009; L = 0,08 0,006, mm2; P ≤ 0,0002). Por outro lado, houve um aumento na altura do epitélio (C = 17,3 0,3; L = 22,8 0,2 m, P ≤ 0,0001) e no número de ácinos (C = 7,0 0,2; L = 8,7 0,1, mm2; P ≤ 0,0002). As análises histomorfométrica juntamente com os resultados imuno-histoquímicos para PCNA sugerem que a leptina aumenta a proliferação celular. Em relação à expressão gênica, o tratamento de leptina aumentou a expressão de todos os genes, mas ER-α, em mais de 200 vezes em comparação com a expressão no grupo C. Em conclusão, neste trabalho mostramos que a leptina tem um efeito direto sobre a próstata de ratos adultos levando a um aumento na proliferação celular e na expressão gênica da aromatase, receptor de androgênio, nas isoformas dos receptores de leptina e receptores de estrogênios alfa e beta que são importantes para a fisiologia normal do tecido prostático / The aim of this study was to evaluate the effect of leptin administration on the ventral prostate lobe of adult rat. Twenty adult male rats were divided into 2 groups: L - animals were daily injected with 50 μL of leptin (8 g / 100 g BW, subcutaneous) for four days and C -animals received the same volume of saline solution. Lipid profile and testosterone serum levels were evaluated. The prostate ventral lobe was processed for histomorphometric analysis. Gene expression of aromatase, androgen receptor, leptin and estrogen receptors isoforms was evaluated by real-time PCR. Cell proliferation was evaluated by PCNA immunohistochemistry. Data were expressed as mean  standard error and analyzed by students t-test. Serum levels of cholesterol (C = 39.7 4.2; L = 55.2 4.2, mg / dL; P ≤ 0.02) increased and testosterone (C = 1.6 0.43; L = 0.6 0.15, ng / dL; P ≤ 0.03) decreased in L group. The histomorphometric analysis showed a reduction in cell density (C = 8868 242; L = 8211 210, mm2; P ≤ 0.04), in total (C = 0.24 0.026; L = 0.10 0.009, mm2; P ≤ 0.001) and in the internal acini areas (C = 0.16 0.009; L = 0.08 0.006, mm2; P ≤ 0.0002). On the other hand, there was an increase in the epithelial height (C = 17.3 0.3; L = 22.8 0.2, m; P ≤ 0.0001) and in the number of acini (C = 7.0 0.2; L = 8.7 0.1, mm2; P ≤ 0.0002). The histomorphometric analyses together with PCNA immunohistochemistry results suggest that leptin increases cell proliferation. In relation to the gene expression, leptin treatment increased the expression of all genes, but ER-α, in more than 200 times compared to the expression in C group. In conclusion, in this paper we showed that leptin has a direct effect on the prostate gland of adult rats leading to an increase in proliferation and in the gene expression of aromatase, androgen, leptin and estrogen receptors isoforms that are important for the physiology of the prostate gland.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O objetivo deste estudo foi avaliar o efeito da administração de leptina no lobo ventral da próstata de ratos adultos. Vinte ratos Wistar machos e adultos foram divididos em 2 grupos: L - animais foram injetados com 50 μL diária de leptina (8 μg / 100 g PC, subcutânea) durante quatro dias e C - animais receberam o mesmo volume de solução salina. Perfil lipídico e níveis séricos de testosterona foram avaliados. O lobo ventral da próstata foi processado para análise histomorfométrica. Expressão dos genes da aromatase, receptor de andrógeno, receptores de estrógeno (α e β) e as isoformas dos receptores de leptina longa (Ob-Rb) e curta (Ob-Ra) foram avaliados por PCR em tempo real. Proliferação celular foi avaliada por imuno-histoquímica com PCNA. Os dados foram expressos como média  erro padrão e analisados pelo teste t de Student. Níveis séricos de colesterol aumentaram (C = 39,7 4,2; L = 55,2 4,2, mg / dL, P ≤ 0,02) e de testosterona (C = 1,6 0,43; L = 0,6 0,15, ng / dL, P ≤ 0,03) diminuíram no grupo L. A análise histomorfométrica mostrou uma redução na densidade de células (C = 8868 242; L = 8.211 210, mm2; P ≤ 0,04), na área total (C = 0,24 0,026; L = 0,10 0,009, mm2; P ≤ 0,001) e na área interna dos ácinos (C = 0,16 0,009; L = 0,08 0,006, mm2; P ≤ 0,0002). Por outro lado, houve um aumento na altura do epitélio (C = 17,3 0,3; L = 22,8 0,2 m, P ≤ 0,0001) e no número de ácinos (C = 7,0 0,2; L = 8,7 0,1, mm2; P ≤ 0,0002). As análises histomorfométrica juntamente com os resultados imuno-histoquímicos para PCNA sugerem que a leptina aumenta a proliferação celular. Em relação à expressão gênica, o tratamento de leptina aumentou a expressão de todos os genes, mas ER-α, em mais de 200 vezes em comparação com a expressão no grupo C. Em conclusão, neste trabalho mostramos que a leptina tem um efeito direto sobre a próstata de ratos adultos levando a um aumento na proliferação celular e na expressão gênica da aromatase, receptor de androgênio, nas isoformas dos receptores de leptina e receptores de estrogênios alfa e beta que são importantes para a fisiologia normal do tecido prostático / The aim of this study was to evaluate the effect of leptin administration on the ventral prostate lobe of adult rat. Twenty adult male rats were divided into 2 groups: L - animals were daily injected with 50 μL of leptin (8 g / 100 g BW, subcutaneous) for four days and C -animals received the same volume of saline solution. Lipid profile and testosterone serum levels were evaluated. The prostate ventral lobe was processed for histomorphometric analysis. Gene expression of aromatase, androgen receptor, leptin and estrogen receptors isoforms was evaluated by real-time PCR. Cell proliferation was evaluated by PCNA immunohistochemistry. Data were expressed as mean  standard error and analyzed by students t-test. Serum levels of cholesterol (C = 39.7 4.2; L = 55.2 4.2, mg / dL; P ≤ 0.02) increased and testosterone (C = 1.6 0.43; L = 0.6 0.15, ng / dL; P ≤ 0.03) decreased in L group. The histomorphometric analysis showed a reduction in cell density (C = 8868 242; L = 8211 210, mm2; P ≤ 0.04), in total (C = 0.24 0.026; L = 0.10 0.009, mm2; P ≤ 0.001) and in the internal acini areas (C = 0.16 0.009; L = 0.08 0.006, mm2; P ≤ 0.0002). On the other hand, there was an increase in the epithelial height (C = 17.3 0.3; L = 22.8 0.2, m; P ≤ 0.0001) and in the number of acini (C = 7.0 0.2; L = 8.7 0.1, mm2; P ≤ 0.0002). The histomorphometric analyses together with PCNA immunohistochemistry results suggest that leptin increases cell proliferation. In relation to the gene expression, leptin treatment increased the expression of all genes, but ER-α, in more than 200 times compared to the expression in C group. In conclusion, in this paper we showed that leptin has a direct effect on the prostate gland of adult rats leading to an increase in proliferation and in the gene expression of aromatase, androgen, leptin and estrogen receptors isoforms that are important for the physiology of the prostate gland.

The role of NMDAR subtypes in synaptic plasticity is very controversial, partially caused by the lack of specific GluN2A containing NMDA receptor (GluN2AR) antagonists. Here we took a novel approach to selectively modulate NMDAR subtype activity and investigated its role in the induction of plasticity. Whole cell recording in both acutely isolated CA1 cells and hippocampal slices demonstrated that pituitary adenylate cyclase activating peptide 1 receptors (PAC1 receptors), which are Gαq coupled receptors, selectively recruited Src kinase and enhanced currents mediated by GluN2ARs. In addition, biochemical experiments showed that the activation of PAC1 receptors phosphorylated GluN2ARs specifically. In contrast, vasoactive intestinal peptide receptors (VPAC receptors), which are Gαs coupled receptors, selectively stimulated Fyn kinase, potentiated currents mediated by GluN2B containing NMDARs (GluN2BRs). Furthermore, dopamine D1 receptor activation (another Gαs coupled receptor) specifically phosphorylated GluN2BRs. Interestingly, field recording experiments showed that PAC1 receptor activation lowered the threshold for LTP whilst LTD was enhanced by dopamine D1 receptor activation. In conclusion, the activity of GPCRs can signal through different pathways to selectively modulate absolute contribution of GluN2ARs versus GluN2BRs in CA1 neurons via Src family kinases. Furthurmore, Epac, activated by some Gαs coupled receptors, also modulated NMDAR currents via a PKC/Src dependent pathway, but whether it selectively modulates NMDAR subtypes, and has capacity to change the induction of plasticity, requires further study. By this means, we can investigate the role of NMDAR subtypes in the direction of synaptic plasticity by selectively modulating the activity of GluN2ARs or GluN2BRs. In addition, based on my work, some interfering peptides and drugs can be designed and used to selectively inhibit the activity of GluN2BRs and GluN2ARs by interrupting Fyn- and Src - mediated signaling cascade respectively. It will provide new candidate drugs for the treatment of some neurological diseases such as Alzheimer disease (AD) and schizophrenia.

Recently we have begun to investigate a novel role of EphB receptors in opiate-dependant analgesia. EphB2-β-galactosidase knockins demonstrate that EphB2 is persistently expressed within a number of neural pathways involved in MOR-mediated nociception in vivo and that EphB2 colocalizes with markers of the MOR at the cellular level in the spinal cord and dorsal root ganglia. Despite demonstrating wild-type levels of sensory and motor activity, EphB2 null mice exhibit a significantly altered analgesic response to repeated (but not naive) opiate exposure compared to controls. Investigation of EphB2 null mice and wild type animals revealed no differences in MOR protein levels or affinity. Analysis of this opiate-mediated tolerance suggests that associative phenomena play a substantial role in mediating the analgesic effects observed, possibly due to defeciencies in CA1-mediated learning. Therefore, loss of EphB2 may diminish context-dependent learning and that such learning plays a substantial role in regulating morphine-dependent tolerance.

In 1999, the discovery of the Steroid Receptor RNA Activator (SRA) was unprecedented in the field of steroid receptor co-regulator research. It was the first time that an RNA molecule was demonstrated to function similarly to its protein counterpart and modulate the activity of steroid receptors. This peculiar steroid receptor co-activator thus attracted the attention of numerous research groups. Over the years, studies were reported deciphering SRA mechanisms of action, its role in co-regulating nuclear receptors and its possible implication in human diseases. While SRA was originally thought to exist solely as a non-coding RNA, our laboratory has identified longer SRA RNA isoforms with the theoretical capacity to encode for a protein. This discovery impelled us to investigate the existence of a Steroid Receptor RNA Activator Protein or SRAP. In this thesis, we first demonstrated the existence and function of endogenous evolutionary conserved SRA proteins. Based on these results we further explored SRAP expression in breast tumors. Interestingly, Western blot analysis of a small cohort of estrogen positive breast tumors suggested that SRAP expression correlates with a better overall survival in patients treated with tamoxifen. This observation prompted us to explore the biological role of SRAP. We found that MCF-7 cells stably expressing coding SRA isoforms had lower ligand dependent estrogen receptor alpha transcriptional activity. In order to dissect the function of the protein independently of its RNA counterpart, we separated the functions of the protein by introducing extensive silent mutations into the RNA sequence. Using this model, we established that SRAP, independent of its RNA counterpart, enhances estrogen receptor alpha activity in a ligand and response-element dependent manner. Furthermore, we showed for the first time that SRAP physically interacts with multiple transcription factors and is recruited to specific promoter regions. Moreover, by artificially recruiting SRAP to the promoter of a luciferase reporter gene under the control of the strong transcriptional activator VP16, we observed a decrease in transcription. These latter results suggest that SRA could function as a repressor through direct association with promoters. Overall, we believe that SRA is a very peculiar example of a bi-faceted system consisting of a functional RNA and its corresponding protein. Altogether our data suggest that SRAP, similarly to its RNA counterpart, is involved in many critical pathways that directly participate in gene expression regulation.

Recently we have begun to investigate a novel role of EphB receptors in opiate-dependant analgesia. EphB2-β-galactosidase knockins demonstrate that EphB2 is persistently expressed within a number of neural pathways involved in MOR-mediated nociception in vivo and that EphB2 colocalizes with markers of the MOR at the cellular level in the spinal cord and dorsal root ganglia. Despite demonstrating wild-type levels of sensory and motor activity, EphB2 null mice exhibit a significantly altered analgesic response to repeated (but not naive) opiate exposure compared to controls. Investigation of EphB2 null mice and wild type animals revealed no differences in MOR protein levels or affinity. Analysis of this opiate-mediated tolerance suggests that associative phenomena play a substantial role in mediating the analgesic effects observed, possibly due to defeciencies in CA1-mediated learning. Therefore, loss of EphB2 may diminish context-dependent learning and that such learning plays a substantial role in regulating morphine-dependent tolerance.

In 1999, the discovery of the Steroid Receptor RNA Activator (SRA) was unprecedented in the field of steroid receptor co-regulator research. It was the first time that an RNA molecule was demonstrated to function similarly to its protein counterpart and modulate the activity of steroid receptors. This peculiar steroid receptor co-activator thus attracted the attention of numerous research groups. Over the years, studies were reported deciphering SRA mechanisms of action, its role in co-regulating nuclear receptors and its possible implication in human diseases. While SRA was originally thought to exist solely as a non-coding RNA, our laboratory has identified longer SRA RNA isoforms with the theoretical capacity to encode for a protein. This discovery impelled us to investigate the existence of a Steroid Receptor RNA Activator Protein or SRAP. In this thesis, we first demonstrated the existence and function of endogenous evolutionary conserved SRA proteins. Based on these results we further explored SRAP expression in breast tumors. Interestingly, Western blot analysis of a small cohort of estrogen positive breast tumors suggested that SRAP expression correlates with a better overall survival in patients treated with tamoxifen. This observation prompted us to explore the biological role of SRAP. We found that MCF-7 cells stably expressing coding SRA isoforms had lower ligand dependent estrogen receptor alpha transcriptional activity. In order to dissect the function of the protein independently of its RNA counterpart, we separated the functions of the protein by introducing extensive silent mutations into the RNA sequence. Using this model, we established that SRAP, independent of its RNA counterpart, enhances estrogen receptor alpha activity in a ligand and response-element dependent manner. Furthermore, we showed for the first time that SRAP physically interacts with multiple transcription factors and is recruited to specific promoter regions. Moreover, by artificially recruiting SRAP to the promoter of a luciferase reporter gene under the control of the strong transcriptional activator VP16, we observed a decrease in transcription. These latter results suggest that SRA could function as a repressor through direct association with promoters. Overall, we believe that SRA is a very peculiar example of a bi-faceted system consisting of a functional RNA and its corresponding protein. Altogether our data suggest that SRAP, similarly to its RNA counterpart, is involved in many critical pathways that directly participate in gene expression regulation.

The role of NMDAR subtypes in synaptic plasticity is very controversial, partially caused by the lack of specific GluN2A containing NMDA receptor (GluN2AR) antagonists. Here we took a novel approach to selectively modulate NMDAR subtype activity and investigated its role in the induction of plasticity. Whole cell recording in both acutely isolated CA1 cells and hippocampal slices demonstrated that pituitary adenylate cyclase activating peptide 1 receptors (PAC1 receptors), which are Gαq coupled receptors, selectively recruited Src kinase and enhanced currents mediated by GluN2ARs. In addition, biochemical experiments showed that the activation of PAC1 receptors phosphorylated GluN2ARs specifically. In contrast, vasoactive intestinal peptide receptors (VPAC receptors), which are Gαs coupled receptors, selectively stimulated Fyn kinase, potentiated currents mediated by GluN2B containing NMDARs (GluN2BRs). Furthermore, dopamine D1 receptor activation (another Gαs coupled receptor) specifically phosphorylated GluN2BRs. Interestingly, field recording experiments showed that PAC1 receptor activation lowered the threshold for LTP whilst LTD was enhanced by dopamine D1 receptor activation. In conclusion, the activity of GPCRs can signal through different pathways to selectively modulate absolute contribution of GluN2ARs versus GluN2BRs in CA1 neurons via Src family kinases. Furthurmore, Epac, activated by some Gαs coupled receptors, also modulated NMDAR currents via a PKC/Src dependent pathway, but whether it selectively modulates NMDAR subtypes, and has capacity to change the induction of plasticity, requires further study. By this means, we can investigate the role of NMDAR subtypes in the direction of synaptic plasticity by selectively modulating the activity of GluN2ARs or GluN2BRs. In addition, based on my work, some interfering peptides and drugs can be designed and used to selectively inhibit the activity of GluN2BRs and GluN2ARs by interrupting Fyn- and Src - mediated signaling cascade respectively. It will provide new candidate drugs for the treatment of some neurological diseases such as Alzheimer disease (AD) and schizophrenia.