Dissertationen zum Thema „Leptin receptors“

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第13091号
農博第1596号
新制||農||938(附属図書館)
学位論文||H19||N4217(農学部図書室)
UT51-2007-H364
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 矢野 秀雄, 教授 今井 裕, 教授 久米 新一
学位規則第4条第1項該当

The present study has tested the hypothesis that leptin receptors are localized in caveolae and that caveolae are involved in the leptin-induced stimulation of lipolysis in 3T3-L1 adipocytes. Leptin, a peptide hormone, is secreted primarily by adipocytes and has been postulated to regulate food intake and energy expenditure via hypothalamic-mediated effects. Exposure to leptin increases the lipolytic activity in 3T3-L1 adipocytes. We isolated caveolae from 3T3-L1 adipocytes using a detergent free sucrose gradient centrifugation method. Leptin receptors were localized in the same gradient fraction as caveolin-1. Confocal microscopic studies demonstrated the colocalization of leptin receptors with caveolin-1 in the plasma membrane, indicating distribution of leptin receptors in the caveolae. We disrupted caveolae by treating cells with methyl--cyclodextrin and found that leptin induced lipolytic activity was reduced after caveolae disruption, indicating an important role of caveolae in the signaling mechanism of leptin.

The focus of this dissertation was leptin and the leptin receptor, and the role of these genes (OB and OB-R) in the development of obesity and type 2 diabetes in humans and Psammomys obesus, a polygenic rodent model of obesity and type 2 diabetes. Studies in humans showed that circulating leptin concentrations were positively associated with adiposity, and independently associated with circulating insulin and triglyceride concentrations. Analysis of two leptin receptor sequence polymorphisms in a Caucasian Australian population and a population of Nauruan males, with very high prevalence rates of obesity, showed no associations between sequence variation within the OB-R gene and obesity- or diabetes-related phenotypic measures. In addition, these two OB-R polymorphisms were not associated with longitudinal changes in body mass or composition in either of the populations examined. A unique analysis of the effects of multiple gene defects in the Nauruan population, demonstrated that the presence of sequence alterations in both the OB and OB-R genes were associated with insulin resistance. Psammomys obesus is regarded as an excellent rodent model in which to study the development of obesity and type 2 diabetes in humans. Examination of circulating leptin concentrations in Psammomys revealed that, as in humans, leptin concentrations were associated with adiposity, and independently associated with circulating insulin concentrations. This animal model was utilised to examine expression of OB-R, and the regulation of expression of this gene after dietary manipulation. OB-R is known to have several isoforms, and in particular, OB-RA and OB-RB gene expression were examined. OB-RB is the main signalling isoform of the leptin receptors. It has a long intracellular domain and has previously been shown to play an important role in energy balance and body weight regulation in rodents and humans. OB-RA is a much shorter isoform of OB-R, and although it lacks the long intracellular domain necessary to activate the JAK/STAT pathway, OB-RA is also capable of signalling, although to a lesser degree than OB-RB. OB-RA is found to be expressed almost ubiquitously throughout the body, and this isoform may be involved in transport of leptin into the cell, although its role remains unclear. OB-RA and OB-RB were both found to be expressed in a large number of tissues in Psammomys obesus. Interestingly, obese Psammomys were found to have lower levels of expression of OB-RA and OB-RB in the hypothalamus, compared to lean animals. This finding raises the possibility that decreased leptin signalling in the brain of obese, hyperleptinemic Psammomys obesus may contribute to the leptin resistance previously described in this animal model. However, the primary defect is unclear, as alternatively, increased circulating leptin concentrations may lead to down-regulation of leptin receptors. The effect of fasting on leptin concentrations and gene expression of OB-RA and OB-RB was also examined. A 24-hour fast resulted in no change in body weight, but a reduction in circulating leptin concentrations, and an increase in hypothalamic OB-RB gene expression in lean Psammomys. In obese animals, fasting again did not alter body weight, but resulted in an increase in both circulating leptin concentrations and hypothalamic OB-RB gene expression. In the liver, fasting resulted in a large increase in OB-RA gene expression in both lean and obese animals. These results highlighted the fact that regulation of leptin receptor gene expression in polygenic models of obesity and type 2 diabetes is complex, and not solely under the control of circulating leptin concentrations. Sucrose-feeding is an established method of inducing obesity and type 2 diabetes in rodents, and this experimental paradigm was utilised to examine the effects of longer term perturbations of energy balance on the leptin signalling pathway in Psammomys obesus. Addition of a 5% sucrose solution to the diet of lean and obese Psammomys resulted in increased body weight in both groups of animals, however only obese Psammomys showed increased fat mass and the development of type 2 diabetes. The changes in body mass and composition with sucrose-feeding were accompanied by decreased circulating leptin concentrations in both groups of animals, as well as a range of changes in leptin receptor gene expression. Sucrose-feeding increased hypothalamic OB-RB gene expression in obese Psammomys only, while in the liver there was evidence of a reduction in OB-RA and OB-RB gene expression in both lean and obese animals. The direct effects of sucrose on the leptin signalling pathway are unclear, however it is possible to speculate that the effect of sucrose to decrease leptin concentrations may have been involved in the exacerbation of obesity and the development of type 2 diabetes in obese Psammomys, From these studies, it appears that sequence variation in the OB and OB-R genes is unlikely to be a major factor in the etiology of obesity in human populations. The ability to examine regulation of expression of these genes in Psammomys obesus, however, has demonstrated that the effects of nutritional modifications on leptin receptor gene expression need closer attention. The role of the OB and OB-R genes in metabolism and the development of type 2 diabetes also warrants further examination, with particular attention on the differential effects of dietary modifications on leptin receptor gene expression across a range of tissues.

Thesis (m.s.)--University of Kentucky, 2004.
Title from document title page (viewed Jan. 7, 2005). Document formatted into pages; contains 70 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 58-68).

The highly controlled regulation of pancreatic hormone secretion is vital to keep the body’s glucose concentration at a constant level. Defects in the regulation of glucose levels are involved in several metabolic diseases, including type 2 diabetes and obesity. Leptin is a satiety hormone with important roles in the maintenance of body weight and glucose homeostasis. Mice that lack leptin (ob/ob) or the leptin receptor (db/db) are massively obese and have diabetes symptoms. Leptin has been demonstrated to have an effect on glucose homeostasis that is suggested to be secondary to the obesity these animals are suffering from. Currently, it is unclear how leptin regulates glucose homeostasis. Leptin mediates its effects by interaction with its leptin receptor (LepRb), which is highly expressed in the hypothalamus, and at lower levels in the periphery. Leptin’s effect on glucose homeostasis has been proposed to be mediated via its receptor expressed on pancreatic cells affecting insulin secretion. Previous animal studies have deleted the leptin receptor in pancreatic β- and α-cells using either “leaky” or inefficient Cre-drivers resulting in conflicting results on glucose homeostasis. In this study, we use a β-cell selective Ins1Cre promoter in mice to investigate the role of leptin receptor expressed on pancreatic cells effect on glucose homeostasis. Deletion of LepRb was found to have minor effects on glucose tolerance in female animals an effect that was only detected in 8 weeks old animals. No effect was observed in male animals or in females above the age of 8 weeks. It is well established that the LepRb in hypothalamus plays an important role in regulation of energy balance. However, the LepRb is expressed in several areas outside hypothalamus, such as the nucleus of the solitary tract (NTS). GLP-1-expressing neurons in this area express the LepRb and it is therefore possible that these neurons mediate an effect on energy homeostasis or glucose homeostasis. We have therefore deleted LepRb in GLP-1 expressing neurons with a proglucagon specific promoter iGluCre. In this study, we found no effect on body weight or glucose homeostasis in animals deleted for LepRb in GLP-1 expressing neurons. Hypothalamus is the brain region that plays a key role in the regulation of feeding and energy homeostasis. This area contains anorexigenic and orexigenic neurons and intermingled with these neurons are subpopulation of neurons named RIP2Cre neurons expressing insulin. Due to the neurons location in the feeding area of the brain they are most likely having a role in energy homeostasis. Previous studies have suggested that tumour suppressor LKB1 plays a role on body weight and food intake in these neurons. Therefore, we deleted LKB1 selectively in the RIP2Cre neurons but failed to see a difference in body weight.

Obesity over the last several has become a major health concern in our country as well as the world. Obesity is also one of the risk factors which lead to several inflammatory complications such as diabetes, artherosclerosis, etc. Two leading factors involved in the causes of inflammatory complications include leptin and low dose endotoxin lipopolysaccharide (LPS). However, the mechanism underlying the involvement of these two mediators is not clearly understood. The purpose of this study is to understand the mechanism underlying inflammatory complications caused by leptin and low dose endotoxin most recently coined metabolic endotoxemia. Interleukin-Receptor Associated Kinase 1 (IRAK-1) is an intracellular signaling component shown to activate NFκB which leads to the induction of proinflammatory mediators. Deletion of IRAK-1 in mice has beneficial effects in alleviating inflammatory complications and human variations in IRAK-1 gene are correlated with higher risks for inflammatory diseases. Therefore, we hypothesized that IRAK-1 is critically involved for the induction of proinflammatory mediators induced by leptin and low dose LPS. IL-6 mRNA levels were measured in THP-1 (human monocytic cells) and wild type and IRAK-deficient bone marrow derived macrophages (BMDM) challenged with different combinations of leptin and LPS. Data shows that leptin alone will not induce inflammatory mediators. However, increased induction of IL-6 was observed in a synergistic manner involving both LPS and leptin in an IRAK-1 dependent manner causing a robust inflammatory response. With regard to the effect of low dose LPS, we observed that human monocytic cells treated with low concentrations of LPS showed a mild yet sustained induction of proinflammatory cytokines, which is contrast to the robust and transient induction of cytokines by a high dose LPS. To further determine the molecular mechanisms, we measured several key signaling molecules that include IRAK-1, IKKepsilon, and C/EBPdelta. Our study revealed a novel mechanism that appears to be distinct from the traditional NFï «B pathway responsible for the effect of low dose LPS.
Ph. D.

INTRODUÇÃO: A obesidade na infância e adolescência representa uma epidemia global e figura como um problema de saúde pública proeminente de prevalência crescente. A obesidade frequentemente está associada à compulsão alimentar periódica (CAP) e componentes genéticos participam de sua etiologia multifatorial. Polimorfismos de nucleotídeo único (SNPs) no gene da leptina (LEP) e do receptor da leptina (LEPR) podem modificar a expressão da leptina e de suas vias de sinalização e, consequentemente, alterar a regulação do apetite e da saciedade, contribuindo assim para a etiopatogenia e manutenção da CAP. O objetivo deste trabalho foi investigar a influência dos polimorfismos rs7799039 (G > A) no gene LEP e rs1137100 (A > G), rs1137101 (A > G) e rs8179183 (G > C) no gene LEPR sobre a CAP em crianças e adolescentes obesos, além de caracterizar a população quanto à CAP e verificar a associação dos SNPs com o risco cardiometabólico (RCM) e a obesidade. MÉTODOS: Estudo transversal que incluiu 465 crianças e adolescentes obesos com idade entre 7 e 19 anos avaliados quanto a variáveis antropométricas e metabólicas. Os fatores de RCM consistiram de hipertensão arterial sistêmica, glicemia de jejum alterada, HDL-colesterol baixo e hipertrigliceridemia. A CAP foi avaliada por meio da Escala de Compulsão Alimentar Periódica (ECAP). Para investigar o efeito dos SNPs no risco para a obesidade foi incluído um grupo controle composto por 135 crianças e adolescentes eutróficos. A genotipagem foi realizada por PCR em tempo real e para análise dos SNPs, adotou-se o modelo dominante. Foi calculado o desequilíbrio de ligação entre os SNPs e estimada as frequências dos haplótipos. As comparações entre os grupos foram realizadas estratificadamente por gênero e estádio puberal. Para avaliar a magnitude do risco dos SNPs sobre a CAP e a obesidade foi realizada regressão logística ajustada para variáveis de confusão (idade, Z-IMC e estádio puberal). RESULTADOS: As crianças e adolescentes obesos (12,5 ± 2,9 anos; 52,7% meninas) classificados com CAP apresentaram maior adiposidade e a frequência da CAP foi mais elevada no gênero feminino (OR= 2,146; IC 95% 1,461-3,152; p < 0,001). A frequência do alelo A do rs7799039 foi mais elevada no grupo de obesos (OR= 1,530; IC 95% 1,022-2,292; p= 0,039) e o alelo associou-se ao maior nível de leptina e colesterol total em meninas e à maior glicemia em meninos (p < 0,05). No rs1137100 e o rs1137101, a presença do alelo G em meninas conferiu risco para a hipertrigliceridemia (OR= 1,926; IC 95% 1,010-3,673; p= 0,047 e OR= 2,039; IC 95% 1,057-3,931; p= 0,033, respectivamente). O alelo C do rs8179183 relacionou-se, em meninas, à relação cintura-estatura e glicemia mais elevadas e, em meninos, ao maior percentil de pressão arterial diastólica, glicemia, colesterol total e LDL-colesterol (p <0,05). CONCLUSÃO: Os polimorfismos não foram associados à compulsão alimentar periódica. A CAP foi relacionada ao pior grau de adiposidade e o maior risco foi observado no gênero feminino. O SNP rs7799039 no gene LEP conferiu risco para obesidade, enquanto o rs1137100, rs1137101 e rs8179183 no gene LEPR relacionaram-se ao pior perfil cardiometabólico em crianças e adolescentes obesos
INTRODUCTION: Obesity during childhood and adolescence represents a global epidemic and consists in a prominent public health issue of increasing prevalence. Obesity is frequently associated with binge eating (BE) and genetic factors participate of its multifactorial etiology. Single nucleotide polymorphisms (SNPs) in the leptin (LEP) and leptin receptor (LEPR) genes may modify the leptin expression and its signaling pathways and, consequently, alter appetite and satiety regulation, thus contributing to the etiopathogeny and maintenance of BE. The aim of this study was to investigate the influence of polymorphisms rs7799039 (G > A) in the LEP gene and rs1137100 (A > G), rs1137101 (A > G) and rs8179183 (G > C) in the LEPR gene on BE in obese children and adolescents, besides characterize the population regarding to BE and examine the association of SNPs with cardiometabolic risk (CMR) and obesity. METHODS: Cross-sectional study in which 465 obese children and adolescents aged from 7 to 19 years were enrolled and had anthropometric and metabolic variables assessed. The CMR factors consisted of systemic hypertension, impaired fasting glucose, low HDL-cholesterol levels and hypertriglyceridemia. The BE was evaluated through the Binge Eating Scale (BES). To investigate the effect of SNPs on obesity risk, a control group of 135 eutrophic children and adolescents was enrolled. Genotyping was performed by real-time PCR and for the SNPs analysis, the dominant model was adopted. The linkage disequilibrium between SNPs was calculated and the haplotype frequencies were estimated. Comparisons between groups were performed stratified by gender and pubertal stage. To assess the risk magnitude for the SNPs on BE and obesity, logistic regression adjusted for confounding variables (age, Z-BMI and pubertal stage) was performed. RESULTS: Obese children and adolescents (12.5 ± 2.9 years, 52.7% girls) classified with BE showed greater adiposity and BE frequency was higher among females (OR= 2.146; 95% CI 1.461-3.152; p < 0.001). The observed frequency of A allele of rs7799039 was a higher in the obese group (OR= 1.530; 95% CI 1.022-2.292; p= 0.039) and the allele was associated with higher leptin and total cholesterol levels in girls and higher glucose levels in boys (p < 0.05). For the rs1137100 and rs1137101, the presence of the G allele among girls, conferred risk for hypertriglyceridemia (OR= 1.926; 95% CI 1.010-3.673; p= 0.047 and OR= 2.039; 95% CI 1.057-3.931; p= 0.033, respectively). The C allele of rs8179183 was associated, among girls, with a higher waist-to-height ratio and glucose levels and, among boys, with greater diastolic blood pressure percentile, glucose, total cholesterol and LDL-cholesterol levels (p < 0.05). CONCLUSION: Polymorphisms were not associated with binge eating. BE was related with a more severe adiposity and an increased risk was observed among females. The SNP rs7799039 in the LEP gene contributed to the risk of obesity, whereas the rs1137100, rs1137101 and rs8179183 in LEPR gene were related to a worse cardiometabolic profile in obese children and adolescents

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.

A leptina é um componente importante do complexo sistema fisiológico que regula o armazenamento, o equilíbrio e o uso de energia pelo organismo. É descrito na literatura que a concentração plasmática de leptina e/ou a quantidade de mRNA de leptina nos adipócitos, correlacionam-se positivamente com o gênero, a ingesta calórica, o IMC, e a massa de tecido adiposo. A leptina pode aumentar o gasto energético por meio da estimulação nervosa simpática. A ação tecidual da leptina se da através de sua ligação com seu receptor. O receptor de leptina apresenta heterogeneidade na população humana, com naturais ocorrências de polimorfismos que codificam para o aminoácido das posições 109, 223 e 656 entre outros. Acredita-se que mutações no gene do receptor podem modificar sua função e afetar os níveis séricos de leptina na população em geral, sugerindo que este polimorfismo possa contribuir para a correlação da obesidade com a doença cardiovascular associado à atividade nervosa simpática. Neste estudo, avaliamos a relação entre as variantes alélicas do gene do receptor de leptina e mudanças na composição corporal, nas variáveis metabólicas, neurovasculares e hemodinâmicas de mulheres obesas antes e após um programa de treinamento físico e dieta hipocalórica. Como achado principal do nosso estudo, observamos que as pacientes obesas com polimorfismo Gln223Arg/Arg223Arg apresentaram maior massa gorda em comparação ao grupo de mulheres obesas Gln223Gln no período inicial do estudo. Não foram encontradas diferenças nas demais variáveis antropométricas, metabólicas, neurovasculares e hemodinâmicas do códon 223. As variáveis antropométricas, metabólicas, neurovasculares e hemodinâmicas permaneceram semelhantes entre as formas genéticas dos códons 109, 656. Como esperado, a mudança no hábito de vida impactou em algumas das variáveis antropométricas, metabólicas, neurovasculares e hemodinâmicas, porém as variantes genéticas de cada códon não alteraram essas variáveis. A partir dos dados apresentados, podemos dizer que o polimorfismo Gln223Arg/Arg223Arg influencia no aumento de massa gorda em mulheres obesas. Entretanto, após quatro meses de intervenção nos hábitos de vida dessas pacientes, a associação entre o polimorfismo citado e o aumento da massa gorda não se manteve, o que pode ser atribuído à melhora da resistência a ação da leptina, que sabidamente é favorecida por perda de peso, como ocorreu na nossa população
Leptin is an important component of a complex physiological system that contributes to the regulation of the organic energy balance. Leptin plasmatic concentration or mRNA quantities are positive related to gender, caloric intake, BMI and fat mass. Energy expenditure can be altered by leptin action through the nervous sympathetic stimulus. Leptin acts via its specific receptor. These receptor presents heterogeneity in human population, with natural occurrences of polymorphisms that codify to different proteins. Mutations in gene receptor can modify the leptin level and consequently its action. This suggests that the polymorphism of leptin receptor could contribute to the relation of obesity and cardiovascular disease, and the sympathetic nervous system is involved in this process. In this study we evaluate the relation between allelic variants of leptin receptor gene with changes in corporal composition, metabolic, neurovascular and hemodynamic variables in obese women, before and after a life style change program. The main result of this investigation is that the women that carrier a polymorphism in codon 223, with mutation of glicina to arginina presented higher fat mass than the other polymorphisms. There are no differences in the other antropometric, metabolic, neurovascular and hemodynamic variables in this codon. All the studied variables were similar between the genetic forms 109 and 656. As expected, the life style changes positive impact in some of the anthropometric, metabolic, neurovascular and hemodynamic variables, but the allelic variants did not alter these variables. In conclusion, the data presented permit us to say that the Gln223Arg/Arg223Arg polymorphism influences the fat mass gain in obese women. However, after four months of exercise training and diet, this difference in fat mass gain did not keep, wich can be atributted to the improvement in the leptin resistence and the weigh loss, which occurred in our population

O câncer de próstata é o tumor não cutâneo mais comum entre homens e responsável pela segunda maior mortalidade entre os tumores neste sexo. Diferentes métodos são utilizados com o objetivo de determinar o prognóstico do paciente portador de câncer de próstata, contudo existe grande heterogeneidade quando estes são usados individualmente. A leptina é um hormônio peptídico envolvido na regulação da ingestão alimentar, do metabolismo, do gasto energético, além de função neuroendócrina. Este hormônio parece estar envolvido na patogênese de alguns tipos de tumores, inclusive os adenocarcinomas de próstata. Até o presente não se tem certeza se a leptina e seu receptor possam ser utilizados como fatores prognósticos no cancer de próstata. O objetivo do trabalho foi correlacionar os perfis de imunomarcação da leptina e seu receptor em adenocarcinomas de próstata com diferentes fatores prognósticos e comparar as análises de imunomarcação da leptina e seu receptor em adenocarcinomas de próstata por métodos semiquantitativos e quantitativos (morfometria). Foram analisadas 532 peças cirúrgicas de prostatectomias radicais por câncer prostático. A partir destas amostras, após estudo histopatológico, foi montado um arranjo de matriz tecidual, contendo fragmentos de áreas tumorais e não tumorais (peritumorais) destas amostras. Estas foram imunomarcadas com anticorpos antileptina e antirreceptor de leptina. Análises subjetivas (feitas por dois observadores) e objetivas (através da contagem de pontos) foram realizadas em cada uma das imunomarcações. Estes resultados foram comparados e correlacionados com os seguintes fatores prognósticos: invasão perineural, embolização neoplásica vascular, comprometimento bilateral da próstata, invasão das vesículas seminais, comprometimento de margem vesical, de margem uretral e de margem cirúrgica de ressecção. Houve diferença significativa entre as análises subjetivas dos dois observadores e, portanto, estas não foram utilizadas para as demais comparações. Em relação às análises objetivas, foi verificado que a expressão do receptor de leptina estava diminuída nos tumores com comprometimento de margem cirúrgica, de margem uretral e de vesículas seminais. Ainda foi observado correlação entre a expressão desse receptor e o somátório dos fatores prognósticos analisados. Para as demais análises não foi verificada diferença significativa. Métodos semiquantitativos podem ter grande variação e devem ser preteridos em relação a métodos quantitativos para as análises realizadas neste estudo. Nem a leptina nem o seu receptor apresentaram alterações de sua expressão em amostras neoplásicas de próstata quando comparado àquelas não neoplásicas. O receptor de leptina apresentou uma diminuição da sua expressão em tumores com margem cirúrgica comprometida, margem uretral comprometida e com vesículas seminais comprometidas. Houve correlação negativa entre o percentual de área imunomarcada com o anticorpo antirreceptor de leptina e o somatório dos fatores prognósticos analisados.
Prostate cancer is the most common non-cutaneous tumor among men and accounted for the second highest mortality rate among tumors in this sex. Different methods are used to determine the prognosis of patients with prostate cancer, yet there is great heterogeneity when they are used individually. Leptin is a peptide hormone, involved in the regulation of food intake, metabolism, energy expenditure, and neuroendocrine function. This hormone appears to be involved in the pathogenesis of some types of tumors, including prostate adenocarcinomas. Up to the present, it is uncertain if leptin and its receptor may be used as prognostic factors in prostate cancer. The objective of this study was correlate the profiles of immunostaining of leptin and its receptor in prostate adenocarcinomas with different prognostic factors and compare the analysis of immunostaining of leptin and its receptor in prostate adenocarcinomas by semi quantitative and quantitative methods (morphometry). A total of 532 surgical specimens from radical prostatectomies from prostate cancer had been studied. After pathological examination, these samples were included in a tissue microarray containing fragments of tumoral and non-tumoral areas (peritumoral areas). These were immunostained with antibodies antileptin and antileptin receptor. Subjective analysis (made by two observers) and objective (by counting points) were performed on each sample. These results were compared and correlated with the following prognostic factors: perineural invasion, neoplasic vascular embolization, bilateral involvement of the prostate, seminal vesicle invasion, involvement of vesical margin, involvement of urethral margin and involvement of resection surgical margin. There were significant differences between subjective analyses of the two observers, and, therefore, they were not used for other comparisons. Regarding the objective analysis, it was found that leptin receptor expression was reduced in tumors with involvement of the surgical margin, urethral margin and seminal vesicles invasion. Further, there were correlation between the expression of this receptor and the sum of prognostic factors. For the other analyses, it was not observed any significant difference. Semi quantitative methods can have great variation and should be passed over in relation to quantitative methods for the analysis performed in this study. Neither leptin nor its receptor exhibited changes in their expression in neoplastic prostatic samples compared to those that are not neoplastic. The leptin receptor showed a decreased expression in tumors with positive surgical margin, positive urethral margin and involvement of seminal vesicles. There were a negative correlation between the percentage of immunostaining area with antibody antileptin receptor and the sum of prognostic factors.

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Leptin is a multifunctional peptidic hormone, primarily produced by the adipose tissue, which has receptors (Ob-Rs) in reproductive organs, hypothalamus and hypophysis, acting in the appetite regulation and energetic expenditure, potentially influencing the expression of the reproductive function, at puberty and lactation. Reproductive failure are the main causes for female culling, representing 30% to 40%,of the total female culling. Thus, post-mortem evaluation of the genital organs at slaughter may have an important diagnostic value, helping on the identification of the stage of female`s estrous cycle and aiding the interpretation of reproductive abnormalities. Its long form receptor (OBR-b) performs signal transduction in several cell types via mitogen- activated protein kinases (MAPK) cascade, such as ERK 1/2 and p38. The first stage of this thesis is aimed to evaluate the presence of leptin and MPAK in oocites of pubertal sows and prepubertal gilts. Ovaries from 10 pubertal sows and 10 prepubertal gilts were collected at a slaughterhouse. Slides were analyzed for the presence of oocites included in primordial/primary (OIPF), secondary (OISF) and tertiary follicles (OITF). Immunohistochemistry (IHC) was performed with polyclonal antibodies anti-leptin and anti-phospho-MAPK (ERK 1/2 and p38). The second stage involved 28 pubertal sows and the analyses were performed in hypothalamic neurons, endometrial glands and oocytes. For IHC, the antibodies were polyclonal anti-leptin and anti-leptin receptor. Responses were compared with reproductive data registered for each female. All images in both stages were analyzed by 16-Bit Histogram application from Image J® software. Immunolabeling for leptin and activated ERK 1/2 MAPK were more intense in sows oocytes (p ≤ 0.05), while the immunolabeling for activated p38 MAPK was more intense in gilts oocytes (p = 0.05). In sows, OIPF were more intensely marked for leptin and p38 MAPK (p <0.05), on the other hand, for gilts there was no difference in the marking of follicular oocytes from the analyzed categories. Hypothalamic neurons were more intensely marked in the sows culled by reproductive problems (p <0.05). The hypothalamus, uterus and oocytes of luteal or follicular phase sows showed more intense immunolabeling for leptin and OBR-b than the females with cystic ovaries. The immunolabeling for leptin was more pronounced in the hypothalamus and uterus of sows from parity order 2 group (PO 2-4) (p <0.05). These results demonstrate that leptin may be a marker for oocyte competence. Also, cyclic females having PO 2-4 presented more intense leptin immunolabeling in the hypothalamus and uterus and for OBR-b in the oocyte and uterus. The OBR-b immunolabeling was more pronounced in primiparous and multiparous females than for nulliparous. Therefore, immunolabeling for leptin and OBR-b can be a marker for reproductive performance in swine females.
A leptina é um hormônio peptídico multifuncional, produzido primariamente pelo tecido adiposo, com receptores (Ob-Rs) presentes em órgãos reprodutivos, hipotálamo e hipófise, que atua na regulação do apetite e no gasto energético, com potencial influência sobre a expressão da função reprodutiva, na puberdade e após a lactação. Desordens reprodutivas são as causas mais comuns, atribuídas ao total dos descartes, entre 30 e 40%. Considerando a alta proporção de descartes por estes problemas, a avaliação dos órgãos genitais no abate tem importante valor diagnóstico, auxiliando na identificação do estágio do ciclo estral da fêmea e na interpretação de possíveis anormalidades reprodutivas. O seu receptor de forma longa (OBR-b) realiza transdução de sinal em diversos tipos celulares via cascata das proteínas quinases ativadas por mitógenos (MAPK), tais como ERK 1/2 e p38. A primeira etapa do trabalho teve por objetivo avaliar a presença da leptina, e das MAPK nos oócitos de fêmeas suínas púberes e pré-púberes. Envolveu ovários de 10 fêmeas pré-púberes e 10 púberes coletados no abate e lâminas foram analisadas de oócitos inclusos em folículos primordiais/primários (OIFP), secundários (OIFS) e terciários (OIFT). Para a técnica de imuno-histoquímica (IHQ) os anticorpos policlonais instilados foram anti-leptina, anti-phospho- MAPK (ERK 1/2 e p38). A segunda etapa envolveu 28 porcas púberes descartadas, das quais foram analisados os neurônios do hipotálamo, glândulas endometriais e oócitos. Para a IHQ foram utilizados anticorpos policlonais anti-leptina e anti-receptor da leptina e objetivou comparar as respostas com os dados produtivos e reprodutivos a partir do registro de cada fêmea. Em ambas as etapas de trabalho foram utilizadas as modas obtidas pelo aplicativo 16 Bit-Histograma do software Image J®. A imunomarcação da leptina e a presença de ERK 1/2 ativada MAPK foram mais intensas em oócitos de porcas (p≤0,05), enquanto que a marcação para p38 MAPK ativada foi mais intensa em oócitos de leitoas (p=0,05). Em porcas, os OIFP estavam mais intensamente marcados para a leptina e p38 MAPK (p<0,05), mas para leitoas não foi observada diferença na marcação dos oócitos independente do estádio folicular. No hipotálamo a imunomarcação para a leptina foi mais intensa nas porcas descartadas por problemas reprodutivos (p<0,05). O hipotálamo, útero e oócitos das porcas que estavam na fase lútea ou fase folicular apresentaram imunomarcação para leptina e OBR-b mais acentuada que naquelas que possuíam cistos ovarianos. A imunomarcação para a leptina foi mais acentuada no hipotálamo e útero daquelas porcas do grupo ordem de parto 2 (OP 2-4) (p<0,05). Estes resultados demonstram que a leptina pode ser um dos marcadores para competência oocitária. Além disso, pode também ser observado que fêmeas cíclicas com OP 2-4 partos apresentam imunomarcação mais intensa para leptina no hipotálamo e útero, e OBR-b nos oócitos e útero. O OBR-b obteve marcação mais acentuada em fêmeas primíparas e multíparas, que nulíparas. Portanto, a imunomarcação para leptina e OBR-b podem ser um marcador de sinalização reprodutivo em fêmeas suínas.

Leptin, a 16kDa protein, is secreted primarily by adipose tissue. Its effects are pleiotropic, with known roles in body mass regulation, haematopoiesis, immune function and reproduction. Studies of the leptin system in females were undertaken to complement existing animal and human studies. Serum leptin levels were found to increase in the luteal phase of the menstrual cycle raising the possibility of a role for leptin at the time of implantation. RT-PCR studies showed that leptin receptors were expressed in the human endometrium throughout the menstrual cycle. In these preliminary studies, these receptors appeared to be functional in response to leptin; increasing proliferation and decreasing TNF-a production in cultured endometrial cells. This implies that leptin has a local effect at the level of the endometrium. Many cytokines are bound to proteins in serum; a proportion of this binding can be attributed to a soluble cytokine receptor. Soluble receptors and binding proteins have a variety of roles, acting as scavengers, carriers, agonists and antagonists. In order to investigate this phenomenon in the leptin system, a radio-receptor assay was developed to measure leptin-binding activity (LBA). The leptin receptor has an extracellular domain that is common to all isoforms and therefore, LBA may also reflect the binding parameters of cell surface receptors. Serum leptin levels of LBA were found to be low at birth, high in early childhood, to fall steadily through puberty and remain at the post-pubertal levels throughout adult life. This suggests that leptin and LBA has an important role in the initiation of puberty. There was no significant variation in LBA during the menstrual cycle. Several single nucleotide polymorphisms (SNP) exist in the leptin receptor gene. Studies of two of these SNPs, GLN223ARG and LYS656ASN, present in the extracellular domain of the receptor were undertaken to assess if genetic changes are associated with differences in phenotype or effect ligand binding. Homozygosity of the G allele of GLN223ARG is associated with lower fat mass, BMI and leptin levels in postmenopausal Caucasian females. This polymorphism changes the binding characteristics of the receptor, with a higher LBA being associated with homozygosity of the G allele. This suggests that the actual binding of leptin to its receptor may be an important factor in the regulation of body weight and adiposity.

Par sa fréquence et sa gravité, le CCR représente un réel problème de santé publique. Avec 37000 nouveaux cas par an et 15% des décès, il constitue la 2ème cause de mortalité par cancer en France. Malgré d'importants progrès thérapeutiques durant cette dernière décennie, il rest un cancer de mauvais pronostic. Des facteurs génétiques et environnementaux ont été impliqués dans la genèse de ce cancer. La caractérisation moléculaire du CCR a permis d'identifier les tumeurs par instabiblité génique, appelées MSI (Microsatelite Instability) possédant des anomalies de réparation d'appariement d'ADN (MMR mismatch repair). Celles-ci sont fréquemment retrouvées (80%) dans les CCRs familiaux et rarement (15%) dans les cancers sporadiques. Les tumeurs avec phénotype MSI sont de bon pronostic. Le rôle possible de l'alimentation et particulièrement celui du fuel énergétique sur la survenue d'anomalies d'appariement d'ADN a été suggéré. De nombreuses hormones et en particulier la leptine ont été rapportées comme facteur de promotion tumorale. De plus, la leptine possède de nombreuses propriétés immunrégulatrices. Son effet sur l'immunité colique tiendrait autant à sa capacité à initier la production de cytokines à partir des cellules épithéliales digestives qu'à sa capacité à contrôler la prolifération des lymphocytes. Nous avons formulé l'hypothèse que la leptine pouvait réguler des fonctions immunes dans le microenvironnement tumoral. L'ensemble de ces données souligne l'importance de l'étude chez l'homme. L'analyse des données prospectives de 171 patients avec CCR permet de noter une surexpression du récepteur de la leptine dans un sous groupe de tumeurs. Les relations entre le récpteur de la leptine et la réponse immunitaire ont été analysées dans le microenvironnement tumoral humain, par des modèles cellulaires in vitro et animaux in vivo. Nous avons découvert que l'effet pro immunitaire de leptine dépendait du niveau d'expression de sonrécepteur et du degré d'instabilité microsatellitaire dans la cellule tumorale. L'expression du récepteur de la leptine pourrait être considérée comme un marqueur pronostique dans le CCR humain
Cancer of the colon and rectum (CRC) is a real challenge in Western countries because of the prevalence, cost and bad prognosis. With they 37,000 new cases each year and 15% of mortality it is currently the 2nd cause of cancer death in France. Despite significant advances in diagnosis and treatment over the past decade, it remains with bad prognosis. Genetic and environmental factors were involved in the genesis of this cancer. Molecular characterization of CRC leaded to the identification of gene instability (MSI) in tumors with mismatch repair (MMR) abnormalities. This is found frequently (80%) the CRC hereditary no polyposis colon cancer family (HNPCC) and rarely (15%) in sporadic cancers. Those tumors with MSI phenotype are considered to be of good prognosis. The possible role of food and particulary energy balance on the occurrence of MMR abnormalities has been suggested. Several hormones including leptin have been reported to promote tumour growth. In addition, leptin may regulate immune response tin GIT. Its pro immunogenic effect results from cytokines production by gastrointestinal epithelial cells as well as its ability to control the proliferation of lymphocytes. We hypothesised that leptin might regulate anti tumour immune response. The analysis of prospective data from 171 patients with CRC showed that overexpression of leptin receptor in subset of tumours. Relationships between leptin recptor and tumour immune response have been studied in the tumour microenvironment in human tissues, and in culture cells in vitro as well as in animal models in vivo. Results showed intensity of immune response was depended on the level of leptin receptor expression and MSI in colon tumour cells. Thus leptin receptor expression may be considered as a prognostic marker in colon and rectal cancer in human

Abstract Leptin is a hormone secreted by adipose tissue. It is involved in the regulation of appetite and energy expenditure. Leptin binds to its receptor (LEPR) that is expressed in the central nervous system as well as in other tissues including adipocytes and endothelial cells. Plasma leptin level reflects the amount of adipose tissue and previously, it has been shown to be associated with the risk for coronary artery disease. Two LEPR polymorphisms, Lys109Arg and Gln223Arg, have been extensively studied and they have been associated with several risk factors of atherosclerosis. Earlier studies have also shown that the risk for developing atherosclerosis and various other diseases might already be determined during the fetal period or immediately after birth. It seems that intrauterine undernourishment might cause changes on epigenetic level and result in alterations in gene expression. It has been suggested that the impaired fetal growth could affect plasma leptin level and leptin messenger RNA expression from adipose tissue. Long-term fructose consumption has also been shown to result in leptin resistance. Recently, leptin has been observed to be associated with autophagy. Autophagy has been demonstrated to act in several interesting processes such as fat storage in adipocytes and liver. Autophagy and the leptin system might also regulate each another. The aim of this thesis was to investigate the association of LEPR polymorphisms with thickness of the wall of carotid artery as well as with fatal and nonfatal cardiovascular disease events. In addition, we aimed to clarify the effects of fetal undernourishment and fructose consumption on the leptin system and autophagy. We were also interested in studying the role of the leptin system and autophagy in elevated triglycerides and liver fat accumulation seen as a result of high-fructose diet. In our studies, we observed that LEPR polymorphisms, Lys109Arg and Gln223Arg, are associated with intima-media thickness of carotid artery. Moreover, 19-year follow-up study showed that 109Arg homozygotes display lower incidence of cardiovascular events and lower total mortality. In our animal experiments, we were able to detect that fructose diet affects both LEPR isoform and autophagy gene expression. It seems that these changes might partly explain the mechanism behind the rise in blood triglyceride levels and liver fat accumulation caused by fructose diet. In conclusion, the results of this study clarify the role of leptin receptor in cardiovascular diseases. In addition, they offer new information especially about the effects of fructose diet on the leptin system, the dysfunction of which might predispose to the development of diseases
Tiivistelmä Leptiini on rasvakudoksen tuottama hormoni. Se osallistuu ruokahalun ja energiankulutuksen säätelyyn. Leptiini sitoutuu reseptoriinsa (LEPR), joita on sekä keskushermostossa että muissakin kudoksissa, myös adiposyyteissä ja endoteelisoluissa. Plasman leptiinitaso heijastaa rasvakudoksen määrää ja sen on aiemmin osoitettu olevan yhteydessä sepelvaltimotaudin riskiin. Erityisesti kahta LEPR:n polymorfiaa, Lys109Arg ja Gln223Arg, on tutkittu aiemmin ja niiden on osoitettu olevan yhteydessä useisiin ateroskleroosin riskitekijöihin. Aiemmat tutkimukset ovat myös osoittaneet, että ateroskleroosiin ja useisiin muihin sairauksiin sairastumisen riski voi osittain määräytyä jo sikiöaikana tai varhain syntymänjälkeisen kehityksen aikana. Vaikuttaa siltä, että sikiöaikainen aliravitsemus voi aikaansaada muutoksia epigeneettisellä tasolla ja aiheuttaa näin muutoksia geeniekspressiossa. On ehdotettu, että sikiön heikentynyt kasvu vaikuttaisi plasman leptiinitasoon ja rasvakudoksen leptiinin lähetti-RNA:n ilmentymiseen. Pitkäaikaisen fruktoosinkulutuksen on myös osoitettu aiheuttavan leptiiniresistenssiä. Hiljattain leptiinin on havaittu olevan yhteydessä myös autofagiaan. Autofagian on osoitettu vaikuttavan useisiin kiinnostaviin prosesseihin, kuten rasvan varastoitumiseen adiposyytteihin sekä maksaan. Autofagia ja leptiinijärjestelmä mahdollisesti myös säätelevät toisiaan. Tämän väitöskirjan tavoitteena oli tutkia LEPR-polymorfioiden yhteyttä kaulavaltimon seinämän paksuuteen sekä sydän- ja verisuonitautitapahtumiin ja kuolleisuuteen. Pyrimme lisäksi selvittämään sikiöaikaisen aliravitsemuksen ja fruktoosin käytön vaikutusta leptiinijärjestelmään sekä autofagiaan ja olimme kiinnostuneita tutkimaan näiden osuutta fruktoosin kulutuksen seurauksena nähtävien metabolisten muutosten, kuten kohonneiden triglyseridien sekä maksan rasvoittumisen, synnyssä. Tutkimuksessamme havaittiin yhteys LEPR polymorfioiden Lys109Arg ja Gln223Arg sekä kaulavaltimon paksuuden välillä. Lisäksi 19-vuoden seurantatutkimus osoitti 109Arg-homotsygotian liittyvän pienentyneeseen sydän- ja verisuonitapahtumien ilmaantuvuuteen sekä matalampaan kokonaiskuolleisuuteen. Eläinmallissamme havaitsimme sekä LEPR-muotojen että autofagiageenien ilmentymisen muuttuneen fruktoosidieetin vaikutuksesta. Vaikuttaa siltä, että nämä muutokset voisivat osaltaan selittää esimerkiksi fruktoosiruokavalion aiheuttaman veren triglyseriditasojen nousun sekä maksan rasvoittumisen rotilla. Tutkimuksen tulokset selventävät leptiinireseptorin roolia sydän- ja verisuonitautien taustalla. Lisäksi ne tarjoavat uutta tietoa erityisesti fruktoosinkulutuksen vaikutuksesta leptiinijärjestelmään, jonka häiriöt altistavat sairauksien kehittymiselle

Extreme deviations from what is considered normal body weight, from anorexia to obesity, have been linked to reduced reproductive function in females. Discovered in 1994, leptin is a signaling hormone released from adipose tissue to mediate satiety effects in the hypothalamus. Leptin secretion is directly proportional to amount of body fat and evidence has accumulated that leptin and its receptor (Lepr) are found in a variety of tissues including granulosa cells (GCs) of the ovary. Thus, leptin through its receptor may play a role in reproductive function in females. Many studies have examined the effects of Lepr in GCs and the ovary, however the results are contradictory and all have been in vitro. Here we present the first in vivo study to examine the role of Lepr in GCs during follicular development and ovulation. Immature superovulated mice were used in all studies and GCs collected by follicle puncture. We first determined the expression profiles of Lepr isoforms (LeprA, LeprB) during follicular and luteal development along with leptin-related signaling molecules and targets. We also analyzed transcription factors potentially regulating Lepr expression in GCs. To examine the response of GCs to leptin in vivo, leptin hormone was administered at various times of follicular and luteal development. Lastly, we blocked Lepr action using a Lepr antagonist (SMLA) and determined its effects on ovulation. LeprA and LeprB were upregulated at 4h post- human chorionic gonadotropin (hCG) with LeprA being the most abundant isoform showing a 23-fold increase from 0 to 4h post-hCG. Leptin was upregulated at the same time and Lepr signaling molecules: signal transducer and activator of transcription 3 (Stat3), and suppressor of cytokine signaling 3 (Socs3), were upregulated just after Lepr induction at 7 and 12h post-hCG, respectively. CCAAT/enhancer-binding protein beta (Cebpb), which was induced at 1h post-hCG, was shown to associate with the Lepr promoter and thus regulate Lepr expression. Early growth response protein 1 (Egr1) protein and mRNA data revealed it to be another potential regulatory transcription factor with upregulation at 1h post-hCG, just prior to Lepr upregulation. Thus, the mRNA profiles of genes examined provide evidence of a role for Lepr during the periovulatory period. This was further confirmed as the in vivo response of GCs to a physiological dose of leptin was enhanced at 6h post-hCG evidenced by phosphorylation of mitogen-activated protein kinase (Mapk) and Stat3 proteins; however showed no change during the early follicular or luteal periods. Leptin treatment also increased expression of ovulation genes: a disintegrin and metalloproteinase with thrombospondin motifs 1 (Adamts1), programmed cell death 1 (Pdcd1), and Egr1. Antagonizing Lepr action reduced ovulation rate by 60% in SMLA-treated animals. This reduction appeared, at least in part, to be due to deregulated gene expression of Adamts10, Adamts19, Hyaluronan synthase 2 (Has2), amphiregulin (Areg), Pentraxin-related protein (Ptx3), and Forkhead box protein O1 (Foxo1). Overall, the results of this study provide molecular mechanisms for Lepr induction and signaling in GCs. In addition, it provides evidence that leptin and Lepr play a positive role during ovulation and are thus essential for optimal female fertility.
Les écarts extrêmes de poids par rapport à ce qui est considéré comme un poids normal, telles que l'anorexie et l'obésité, sont liées à des problèmes de la fonction reproductrice chez la femme. Découverte en 1994, la leptine est une hormone sécrétée par le tissu adipeux dans le but d'informer l'hypothalamus sur l'état de satiété de l'organisme. La libération de leptine est directement proportionnelle à la quantité de tissu adipeux et la présence de l'hormone et de son récepteur (Lepr) a été montrée dans différents tissus incluant les cellules de la granulosa des ovaires. Par conséquent, la leptine, via son récepteur, joue un rôle dans la fonction reproductrice de la femme. De nombreuses études ont étudié les effets de Lepr dans les cellules de la granulosa et dans l'ovaire, mais elles ont toutes été réalisées in vitro et les résultats sont contradictoires. Nous présentons ici la première étude in vivo dans le but d'examiner le rôle de Lepr dans les cellules de la granulosa pendant le développement folliculaire et l'ovulation. Des souris immature produisant un grand nombre d'ovocytes ont été utilisées dans toutes nos expériences et les cellules de la granulosa ont été collectées par ponction folliculaire. Les profils d'expression des isoformes de Lepr (LeprA et LeprB) durant les développements folliculaire et lutéal ont été d'abord déterminés, ainsi que ceux des molécules de la voie de signalisation de la leptine et leurs cibles. Les facteurs de transcription régulant potentiellement l'expression de Lepr dans les cellules de la granulosa ont aussi été analysés. Pour évaluer la réponse des cellules de la granulosa à la leptine in vivo, l'hormone leptine a été administrée à différents moments des développements folliculaire et lutéal. Enfin, le récepteur Lepr a été bloqué grâce à l'utilisation d'un antagoniste de Lepr (SMLA) et les effets de ce blocage sur l'ovulation ont été analysés. L'expression de LeprA et LeprB ont augmenté 4h après administration d'hCG, LeprA étant l'isoforme la plus abondante et présentant une expression 23 fois plus importante de 0 à 4h post-hCG. L'expression de la leptine a augmenté durant le même temps ainsi que celle des molécules de la voie de signalisation de Lepr, Stat3 et Socs3, juste après l'induction de Lepr, respectivement 7h et 12h post-hCG. Cebpb, qui a été induit 1h post-hCG, a été identifié comme étant associé au promoteur de Lepr et donc comme étant un régulateur de l'expression de Lepr. Les données concernant la protéine Egr1 et ses ARNm suggèrent qu'il peut s'agir d'un autre potentiel facteur de transcription régulant l'expression de Lepr, notamment en raison d'une augmentation de son expression 1h post-hCG, juste avant l'augmentation de l'expression de Lepr. Les profils d'ARNm des gènes examinés ont donc fourni la preuve du rôle de Lepr durant la période périovulatoire. Ceci a été ensuite confirmé par l'augmentation de la réponse des cellules de la granulosa in vivo suite à une dose physiologique de leptine 6h post-hCG , mise en évidence par la phosphorylation des protéines Mapk et Stat3. Le traitement avec la leptine a aussi accru l'expression des gènes impliqués dans l'ovulation Adamts1, Pdcd1 et Egr1. Le blocage de l'action de Lepr a réduit le taux d'ovulation de 60% chez les animaux traités avec SMLA. Cette réduction apparaît être due, au moins en partie, à la dérégulation de l'expression des gènes de Adamts10, Adamts19, Has2, Areg, Ptx3, et Foxo1. En conclusion, les résultats de cette étude éclairent les mécanismes moléculaires de l'induction du récepteur Lepr ainsi que de la voie de signalisation qui lui est associée dans les cellules de la granulosa. Pour finir, cette étude fournit des preuves concernant le rôle positif joué par la leptine et Lepr durant l'ovulation, ce qui est essentiel pour optimiser la fertilité de la femme.

A leptina é um hormônio peptídico multifuncional, produzido principalmente pelo tecido adiposo, possuindo receptores (Ob-Rs) nos órgãos reprodutivos, hipotálamo e hipófise. Os glicocorticóides, como o cortisol, também influenciam diretamente a produção de leptina. A importância desses dois hormônios na atividade reprodutiva tem sido descrita por diversos autores, que observaramo envolvimento de ambos, na melhoria dos índices reprodutivos, seja por sua influência direta no eixo hipotalâmico-hipofisário-gonadal, na maturação dos oócitos ou na preparação uterina para receber o concepto. No entanto, quando em excesso na circulação, podem influênciar negativamente o sistema reprodutivo. Marcadores como a leptina e seu receptor funcional de cadeia longa, estão sendo estudados no endométrio de espécies como humanos, bovinos e suínos, apresentando resultados que correlacionam a diminuição destes, com a infertilidade ou perda embrionária, porém até o momento não foi possível encontrar estudos que abordassem esse tema em equinos. Neste estudo foram avaliados: 1) a presença de leptina (Ob) e seu receptor (Ob-Rb) no endométrio de éguas, observando a influência da obesidade e do ciclo estral nesses marcadores e 2) em uma condição de manejo adverso e estressante, a influência do cortisol intrafolicular, nos níveis de leptina no fluido folicular (FF) e nos marcadores de Ob e Ob-Rb no endométrio durante as fases do ciclo estral. Os resultados demonstraram que existe a presença da leptina e seu receptor (Ob-Rb) no endométrio de equinos, com imunomarcação no epitélio luminal e glandular em todas as fases do ciclo estral avaliadas, apresentando, no entanto, uma marcação imunológica mais intensa nos Ob-Rb (142.68±4.97, P< 0,0001) no epitélio glandular durante o diestro em éguas de escore corporal moderado. Não foi possível observar a influência do aumento do cortisol intrafolicular (FF) nas variáveis avaliadas, pois o cortisol se manteve dentro dos valores fisiológicos para a espécie, no entanto pode-se verificar uma correlação positiva entre os níveis intrafoliculares de cortisol e leptina, estando o cortisol aumentado 8 (30.1±0.07ng/ml, P< 0,05) nos folículos mais próximos a ovulação. Pode-se perceber também, que a marcação imunológica do receptor de leptina no epitélio glandular foi mais intensa (144.52±3.17, P< 0,0001) nos animais que apresentavam folículos até 22 mm, estando a imunomarcação de ambos Ob e Ob-Rb correlacionado de forma negativa (r: -0.7836; P < 0.0001, r:- 0.7343; P < 0.0001), com os níveis de cortisol no FF.
Leptin is a multifunctional peptidic hormone, mainly produced by adipose tissue, having receptors (Ob-Rs) in the reproductive organs, hypothalamus and pituitary. Glucocorticoids, such as cortisol, also directly influence leptin production. The importance of these two hormones in reproductive activity has been described by several authors, who observed the involvement of both, in the improvement of reproductive indices, either by their direct influence on the hypothalamic-pituitary-gonadal axis, oocyte maturation or the uterine preparation to receive the concept. However, when in excess in circulation, they can negatively influence the reproductive system. Markers such as leptin and its long-chain functional receptor are being studied in the endometrium of species such as humans, cattle and pigs, presenting results that correlate the decrease of these with infertility or embryonic loss, but to date it has not been possible to find studies to address this issue in horses. In this study we evaluated: 1) the presence of leptin (Ob) and its receptor (Ob-Rb) in the endometrium of mares, observing the influence of obesity and estrous cycle on these markers and 2) in an adverse and stressful management condition, the influence of intrafollicular cortisol, leptin levels in the follicular fluid (FF), and the Ob and Ob-Rb markers in the endometrium during the estrous cycle phases. The results showed that leptin and its receptor (Ob-Rb) are present in the equine endometrium, with immunostaining in the luminal and glandular epithelium in all stages of the estrous cycle evaluated, however, showing a more intense immunological labeling in Ob -Rb (142.68 ± 4.97, P <0.0001) in the glandular epithelium during the diestrous in mares of moderate body score. It was not possible to observe the influence of intrafollicular cortisol (FF) on the variables evaluated, because cortisol remained within the physiological values for the species, however a positive correlation can be observed between intrafollicular cortisol and leptin levels, being the 10 cortisol increased (30.1 ± 0.07ng/ml, P <0.05) in the follicles closest to ovulation. It can also be noticed that the immunological labeling of the leptin receptor in the glandular epithelium was more intense (144.52 ± 3.17, P <0.0001) in the animals that presented follicles up to 22 mm, and the immunostaining of both Ob and Ob-Rb correlated negatively (r: -0.7836; P <0.0001, r: - 0.7343; P <0.0001), with cortisol levels in FF.

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Introdução: Fatores genéticos e ambientais estão envolvidos na patogênese da obesidade. Vários estudos demonstraram associações de variantes nos genes da leptina (LEP) e do receptor de leptina (LEPR) com a obesidade. Objetivo: O objetivo deste estudo foi investigar a influência dos polimorfismos G2548A, no gene LEP, e Gln223Arg, no gene LEPR, em parâmetros antropométricos, perfil lipídico, glicemia e no consumo alimentar de carboidratos. Metodologia: As participantes foram avaliadas por uma anamnese no Ambulatório de Nutrição da Univates, onde os dados do consumo alimentar foram obtidos pelo recordatório de 24 horas e calculados pelo programa Dietwin Profissional (versão 2008). As medidas antropométricas avaliadas foram: peso, altura, circunferência da cintura, circunferência do quadril, e a composição corporal por bioimpedância. Foi calculado o IMC e RCQ. Amostras de sangue foram coletadas para extração de DNA e para análise de parâmetros laboratoriais para avaliação do perfil lipídico (CT, HDL, TG) e glicose jejum. Os polimorfismos dos genes LEP (rs7799039) e LEPR (rs1177101) foram genotipados pela técnica de discriminação alélica TaqMan (Applied Biosystems). Resultados: A amostra foi composta por 378 mulheres saudáveis, com idade média de 25,1 anos (±6,2). Não foram detectadas associações significativas dos polimorfismos investigados com o perfil lipídico, a glicose jejum e o consumo de carboidratos. O genótipo AA do polimorfismo G2548A foi associado com o aumento de peso, IMC, RCQ, CC, e % de gordura corporal. O genótipo GG do polimorfismo Gln223Arg foi associado com o aumento de IMC. Conclusão: Os alelos de risco dos dois polimorfismos foram associados com o aumento das medidas antropométricas, em indivíduos saudáveis, demonstrando uma possível associação com aumento de risco para obesidade.
Introduction: Genetic and environmental factors are involved in the pathogenesis of obesity. Several studies have demonstrated associations between variants in the leptin (LEP) and in the leptin receptor (LEPR) genes with obesity. Objective: The aim of this study was to investigate the influence of the polymorphisms G2548A, in the LEP gene, and Gln223Arg, in the LEPR gene, on anthropometric parameters, lipid profile, fasting glucose and carbohydrates intake. Methods: Participants were assessed by an interview at the outpatient Nutrition Program at Universidade do Vale do Taquari Univates. Food consumption profile was obtained by the 24-hour recall method and calculated by the Dietwin Professional Program (2008 version). The anthropometric measurements were: weight, height, waist circumference, hip circumference, and fat body composition by bioelectrical impedance. BMI and WHR were calculated. Blood samples were collected for DNA extraction and for the analysis of biochemical parameters (TC, HDL, TG and fasting glucose). The polymorphisms (LEP-rs7799039 and LEPR-rs1177101) were genotyped by allelic discrimination TaqMan (Applied Biosystems). Results: The sample was composed by 378 healthy women with an average age of 25.1 years (± 6.2). No significant associations between the polymorphisms investigated and lipid profile, fasting glucose and carbohydrate intake were detected. The AA genotype of the G2548A polymorphism was associated with increased weight, BMI, WHR, WC, and% body fat. The GG genotype of the Gln223Arg polymorphism was associated with increased BMI. Conclusion: The risk alleles of the two polymorphisms were associated with increased anthropometric measurements in healthy subjects, suggesting a possible association with increased risk for the development of obesity.

Truncal adiposity impairs ventilation in obese adults by altering normal ventilatory mechanics. Leptin, an inflammatory adipocytokine, is elevated in obesity and has been shown to alter ventilatory responses to exercise. Leptin’s bioavailability appears to be regulated by its soluble receptor (LRe), which is reduced in obesity. Roux-en-Y gastric bypass surgery (RYGBS) is a weight loss intervention that reduces total fat mass and improves several obesity related co-morbidities including pulmonary dysfunction. The purpose of this study was to first evaluate the differences between ventilatory responses to carbon dioxide (VE/VCO2 slope) during progressive treadmill walking in morbidly obese and normal weight females. Second, we will analyze the relationships between the VE/VCO2 slope, truncal adiposity, serum leptin, and LRe. Lastly, we want to evaluate the changes in the ventilatory responses to exercise (VE/VCO2 slope), truncal adiposity, serum leptin, and LRe 3 months following Roux-en Y gastric bypass surgery. Thirteen obese (OB 37.7 ±11.4 years, 42.0 ± 4.8 kg/m2) and 12 normal weight females (NW 36.1 ±8.0 years, 22.8 ± 1.2 kg/m2) participated in this study. Blood samples for measure of fasting serum leptin and soluble leptin receptor were obtained prior to exercise. Cardiopulmonary variables were measured throughout exercise. Regional adiposity was determined through dual energy x-ray absorptiometry. Truncal adiposity was significantly greater in the obese group than the normal weight group. Serum leptin was greater in the obese group while LRe was lower than the normal weight group. The VE/VCO2 slopes were lower in obese group when compared to the normal weight group. There were no significant group differences in maximal ventilation, tidal volume or respiratory rate. Stepwise regression determined that truncal adiposity accounted for 31.5% of variance in VE/VCO2 slope (R= 0.561, R2 =0.315, p = 0.004). At 3 months post-surgery we observed significant reductions in the obese group in total percentages of fat, truncal adiposity, serum leptin. The soluble leptin receptor was not changed at any measured time point following RYGBS. There were no changes in 3 months post-surgery VE/VCO2 slopes in the obese group. Truncal adiposity, serum leptin and LRe were associated with reduced ventilatory responses to weight bearing exercise (VE/VCO2 slope) in obese females when compared to normal weight females. There were no differences between obese and normal weight females in maximal minute ventilation, tidal volume or respiratory rate. This result suggests that differences in VE/VCO2 slopes may not be entirely from maximal pulmonary capacity. Rather, the differences in VE/VCO2 slope may be attributed to truncal adiposity and its positive relationship with leptin. Elevated leptin in the obese group may indicate a state of central leptin resistance which has been shown to reduce the ventilatory responses to exercise. At 3 months post RYGBS significant reductions in total percent fat, serum leptin, truncal adiposity and BMI were observed. However, despite improvement in fat mass and serum leptin there were no changes in the VE/VCO2 slope and LRe at 3 months post RYGBS. Therefore, it is possible that the improvements in body composition and leptin following RYGBS were not sufficient to increase ventilation responses to weight bearing exercise in obese females.

Neste estudo, foi avaliada a relação entre polimorfismos dos genes da leptina (LEP) e receptores da leptina (LEPR) e parâmetros antropométricos, leptinemia glicemia e lipídeos séricos, em indivíduos da população brasileira. Foram incluídos 238 indivíduos com idade entre 30 e 80 anos. Foram medidos o índice de massa corporal (IMC), a cintura abdominal (CA) e a razão cintura quadril (RCQ). Amostras de sangue periférico foram obtidas para análise do perfil bioquímico e extração de DNA. Os polimorfismos de nuleotideo único (SNPs) LEP G-2548A e LEPR Lys109Arg, Gln223Arg e Lys656Asn foram detectados por PCR-RFLP. Os SNPs LEPR Lys109Arg e Gln223Arg foram associados com obesidade e com IMC e CA aumentados (p<0.05). Estes polimorfismos também foram associados com leptina e glicose elevada (p<0,05). O perfil lipídico sérico foi influenciado pelo polimorfismo LEPR Lys109Arg (p<0.05). A relação entre os SNPs LEPR Lys109Arg e Gln223Arg e o perfil lipídico foi modificada pelo gênero. Os haplótipos LEP G-2548/ LEPR Lys109Arg foram relacionados com diferenças no IMC de obesos. Os haplotipos LEPR Lys109Arg/Gln223Arg foram associados com diferenças na CA e glicemia e lipídeos séricos. Em conclusão, os polimorfismos LEPR Lys109Arg e Gln223Arg estão associados com obesidade e alterações de leptina, glicose e lipídeos circulantes de forma dependente do gênero.
We have assessed the relationship between polymorphisms of the leptin (LEP) and the leptin receptor (LEPR) genes and anthropometric parameters, plasma leptin and glucose and serum lipids in individuals of the Brazilian population. We included 238 individuais with 30 to 80 years. Body mass index (BMI), abdominal circumference (AC) and waist-to-hip ratio (WHR) were measured. Peripheral blood samples were collected for analysis of the biochemical profile and DNA extraction. The single nucleotide polymorphisms (SNP) LEP G-2548A and LEPR Lys109Arg, Gln223Arg and Lys656Asn were detected by PCR-RFLP. The SNPs LEPR Lys109Arg and Gln223Arg were associated with obesity and with increased BMI and AC (p <0.05). These polymorphisms were also associated with increase leptin and glucose (p<0,05). The serum lipid profile was influenced by the LEPR Lys 1 09Arg (p<0.05). The relationship between the SNPs LEPR Lys 1 09Arg and Gln223Arg and the lipid profile was modified by gender. The haplotypes LEP G-2548A1 LEPR Lys109Arg were related with differences on BMI in obese group. The haplotypes LEPR Lys109Arg/Gln223Arg were associated with differences on AC, glucose and serum lipids. In conclusion, the LEPR Lys109Arg and Gln223Arg polymorphisms are associated with obesity and alterations in blood leptin, glucose and lipids in a gender-dependent manner.

The work presented in this thesis deals with the analysis of protein crystal structures with an emphasis on the stereochemical aspects of the folded conformation of proteins. The various analyses described have been performed on a data-set of 250 high resolution and non-homologous protein structures derived from the Protein Data Bank. The overall objective of the work has been to analyse conformational features of the non-secondary structural regions in proteins and identify structural motifs present therein. The results can be useful in the three-dimensional modelling of proteins, altering the stability of proteins, design of peptide mimics and in understanding the structural rules that guide protein folding. The contents of this thesis can be broadly classified into three parts, (a) Conformational preferences of amino acid residues to occur in the partially allowed regions of the Ramachandran map, (b) conformational features of structural motifs formed by side-chain/main-chain hydrogen bonds by polar residues and (c) analysis and characteristic features of isolated β-strands. Chapter 1 of the thesis gives an introduction, briefly discussing the conformation of polypeptide chains, structural features of globular proteins and applications of protein structural analysis etc. Chapter 2 describes the occurrence of left-handed α-helical conformation in protein structures. A data-set of 250 high resolution (< 2.0A) non-homologous protein crystal structures derived from the Protein Data Bank (PDB) has been analysed for occurrences of left-handed α-helical (αL) conformations. A total of 2,573 αL residues were identified from the data-set. About 59% of the observed examples of at conformations were found to be glycyl residues and about 41% non-glycyl. Continuous long stretches of αL residues are seldom found in protein structures. They are most commonly found as singlets represented by 78% of the observed αL examples. The doublets, triplets and quadruplets account for a very minor fraction of the observed examples. There is only a single example of a stretch of four contiguous αL residues, from the protein thermolysin, which forms a single turn of a left-handed α-helix. A majority of the αL residues are nevertheless part of well-defined substructures in proteins. They play singular roles as part of β-turns and helix termination sites in maintaining the characteristic main-chain hydrogen bonds needed for the stability of these structures. They are also found to be effective in the termination of β-strands. The stereo-chemistry and sequence environment around such structures are discussed. The analysis of the side-chain torsion angles of αL residues indicate that the g+ rotamer is highly unfavourable due to stereo-chemical violations posed by the atoms of the side-chain with those of the backbone. The αL residues are highly conserved by residue type as well as conformation among related proteins indicating their vital importance in protein structures Chapter 3 provides an explanation for the unusual preference of glycyl residues to occur in the bridge regions of the Ramachandran map. The Ramachandran steric map and energy diagrams for the glycyl residue are fully symmetric. Though a plot of the (Φ,Ψ) angles of glycyl residues derived from a data-set of 250 non-homologous and high-resolution protein structures is also largely symmetric, there is a clear aberration in the symmetry. While there is a cluster of points corresponding to the right-handed a-helical region, the "equivalent" cluster is shifted to centre around the (Φ,Ψ)values of (90°, 0°) instead of being centred at the left-handed a-helical region of (60°, 40°). An analysis of glycyl conformations in small peptide structures and in "coil" proteins, which are largely devoid of helical and sheet regions, shows that glycyl residues prefer to adopt conformations around (±90°, 0°) instead of right and left handed a-helical regions. Using theoretical calculations, such conformations are shown to have highest solvent accessibility in a system of two-linked peptide units with glycyl residue at the central Cα atom. This is found to be consistent with the observations from 250 non-homologous protein structures where glycyl residues with conformations close to (±90°, 0°) are seen to have high solvent accessibility. Analysis of a sub-set of non-homologous structures with very high resolution (1.5A or better) shows that water molecules are indeed present at distances suitable for hydrogen bond interaction with glycyl residues possessing conformations close to (±90°, 0°). It is concluded that water molecules play a key role in determining and stabilising these conformations of glycyl residues and explains the aberration in the symmetry of glycyl conformations in proteins. Chapter 4 discusses an analysis of backbone mimicry performed by polar side-chains in protein structures. Backbonemimicry bythe formation of closed loop C7, C10, C13 (mimics of γ-, β- and α-turns) conformations through side-chain main-chain hydrogen bonds by polar groups is found to be a frequent observation in protein structures. A data-set of 250 non-homologous and high-resolution protein structures was used to analyse these conformations for their characteristic features. Seven out of the nine polar residues (Ser, Thr, Asn, Asp, Gin, Glu and His) have hydrogen bonding groups in their side-chains which can participate in such mimicry and as many as 15% of all these polar residues engage in such conformations. The distributions of dihedral angles of these mimics indicate that only certain combinations of the involved dihedral angles aids the formation of these mimics. The observed examples have been categorised into various classes based on these combinations resulting in well-defined motifs. Asn and Asp residues show a very high capability to perform such backbone secondary structural mimicry. The most highly mimicked backbone structure is of the Cio conformation by the Asx residues. The mimics formed by His, Ser, Thr and Glx residues are also discussed. The role of such conformations in initiating the formation of regular secondary structures during the course of protein folding seems significant. Chapter 5 presents a description of deterministic features of side-chain main-chain hydrogen bonds as observed in protein structures. A total of 19,835 polar residues from the data set of 250 non-homologous and highly resolved protein crystal structures were used to identify side-chain main-chain (SC-MC) hydrogen bonds. The ratio of the total number of polar residues to the number of SC-MC hydrogen bonds is close to 2:1, indicating the ubiquitous nature of such hydrogen bonds. Close to 56% of the SC-MC hydrogen bonds are local involving side-chain acceptor/donor (‘i’) and a main-chain donor/acceptor within the window i-5 to i+5. These short-range hydrogen bonds form well defined conformational motifs characterised by specific combinations of backbone and side-chain torsion angles. Some of the salient features of such hydrogen bonds are as follows, (a) The Ser/Thr residues show the greatest preference in forming intra-helical hydrogen bonds between the atoms Oyi and Oi-4 Such hydrogen bonds form motifs of the form αRαRαRαR(g") and are most commonly observed at the middle of α-helices. (b) These residues also show great preference to form hydrogen bonds between OYi and Oi-3, which are closely related to the previous type and though intra-helical, these hydrogen bonds are more often found at the C-termini of helices than at the middle. The motif represented by αRαRαRaR(g+) is most preferred in these cases, (c) The Ser, Thr and Glu (between the side-chain and main-chain of the same residue), (d) The side-chain acceptor atoms of Asn/Asp and Ser/Thr residues show high preference to form hydrogen bonds with acceptors two residues ahead in the chain, which are characterised by the motifs β(tt’)αR and β(t)αR, respectively. These hydrogen bonded segments referred to as Asx turns, are known to provide stability to type I and type I’ β-turns. (e) Ser/Thr residues often form a combination of SC-MC hydrogen bonds, with the side-chain donor hydrogen bonded to the carbonyl oxygen of its own peptide backbone and the side-chain acceptor hydrogen bonded to an amide hydrogen three residues ahead in the sequence. Such motifs are quite often seen at the beginning of a-helices, which are characterised by the β (g+)αRαR motif. A remarkable majority of all these hydrogen bonds are buried from the protein surface, away from the surrounding solvent. This strongly indicates the possibility of side-chains playing the role of the backbone, in the protein interiors, to satisfy the potential hydrogen bonding sites and maintaining the network of hydrogen bonds which is crucial to the structure of the protein. Chapter 6 provides a detailed characterisation of isolated β-strands. Reason for the formation of β-strands in proteins is often associated with the formation of β -sheets. However β-strands, not part of β-sheets, commonly occur in proteins. This raises questions about the structural role and stability of such isolated β-strands. Using a data set consisting of 250 proteins, 518 isolated β-strands have been identified from 187 proteins. The two important features that distinguish isolated β-strands from p-strands occurring in β-sheets are (i) the high preponderance of prolyl residues to occur in isolated β-strands and (ii) their high solvent exposure. It is shown that the high propensity for proline residues to occur in isolated β-strands is not due to the occurrence of polyproline type segments in the data-set. The propensities of other amino acids to occur in isolated β-strands follows the same trend as those for β-sheet forming β-strands. Isolated β-strands are characterised often by their main-chain amide and carbonyl groups involved in hydrogen bonding with polar side-chains or water. They are often flanked by irregular loop structures indicating that they are part of long of loops. Analysis of the conservation of such strands among families of homologous protein structures indicates that a sizeable fraction of them are highly conserved. It is suggested that though the formation of isolated β-strands are driven by the intrinsic preferences of amino acid residues, they have many characteristics like loop segments but with repetitive (Φ,Ψ) values falling within the β-region of the Ramachandran map. In addition of the material described in the six chapters above, the thesis also contains the details of work carried out on an aspect slightly different from the main theme of the thesis. This pertains to the comparative analysis of the members of a family of cytokine receptors to derive information to model new members of the family. The three dimensional modelling of the leptin receptor has been used as a case study and the details are included as an appendix. Appendix describes the 3-dimensional model of the satiety factor receptor (the leptin receptor) modelled using principles of homology modelling. Recessive mutations in the mouse obese (ob) and diabetes (db) genes result in obesity and diabetes in a syndrome resembling human obesity. Data from parabiosis (cross circulation) experiments suggested that the ob gene coded, and was responsible for the generation of a circulating factor called leptin which regulated energy balance and the db gene encoded the receptor for this factor. While the structure of the leptin has been determined that of its cognate receptor is as yet unknown. The leptin receptor shows low but clear sequence similarity to the members of the interleukin type 6 family of receptors. The structures of the members of this family are characterised by two p-sandwich like domains connected by a short 4-residue helical linker. The 3-dimensional models for the N- and C-terminal domains of the leptin receptor was generated using the corresponding structures of the signal transducing component of gpl30, the erythropoetin receptor and the prolactin receptor. Further using the evidence that the leptin binds to its receptor with a stoichiometry of 1:1, the relative orientation of the two domains was modelled based on the structure of the human growth hormone receptor, which also binds its ligand with similar stoichiometry. The complex of leptin with its receptor was also modelled based on the structure of human growth hormone/receptor complex. The final energy minimised model of the complex elucidates the mode of interaction between the leptin and its receptor.

El estrés oxidativo es un desequilibrio entre la producción de radicales libres y los sistemas antioxidantes encargados de su neutralización. El resultado de este desequilibrio es la acumulación de daños en diversas estructuras celulares incluyendo el DNA. El cáncer se acompaña de un mayor estrés oxidativo a nivel celular, por este motivo, muchos de los tratamientos terapéuticos van dirigidos a aumentar los niveles citotóxicos de ROS, conduciendo a la célula tumoral a la apoptosis. Sin embargo, durante la tumorigénesis las células van adquiriendo una serie de características que permiten mantener la homeostasis de los ROS y desarrollar resistencia a los tratamientos antineoplásicos. El cáncer de mama es un tipo de neoplasia en la que el factor endocrino juega un papel relevante tanto en la etiología como en la evolución de la enfermedad. En esta tesis nos planteamos como objetivo estudiar los efectos del 17β-estradiol (E2), la genisteína y la leptina, como factores hormonales, sobre la modulación del estrés oxidativo en la carcinogénesis de mama. E2 es uno de los principales factores de riesgo en la iniciación y progresión de la enfermedad; la genisteína es uno de los fitoestrógenos de mayor actividad estrogénica y, por su parte, la leptina también ha mostrado efectos potenciadores sobre el cáncer de mama, considerándose a nivel epidemiológico como un posible enlace entre obesidad y cáncer. Para alcanzar estos objetivos se estudió: i) el efecto dual de E2 y genisteína sobre el estrés oxidativo en función de la dotación de ERα y ERβ en líneas celulares de cáncer de mama (MCF-7 y T47D), y el estrés oxidativo en muestras de carcinomas ductales infiltrantes en función de la ratio de receptores estrogénicos; ii) la influencia de la leptina crónica sobre el estrés oxidativo y su respuesta al fármaco antineoplásico cisplatino en la línea celular MCF-7; y iii) la importancia de la UCP2 en la regulación del estrés oxidativo endógeno e inducido en las células MCF-7, así como, en líneas celulares de cáncer de páncreas con p53 mutado. Los resultados indican que E2 induce estrés oxidativo de forma dependiente de la dotación de ERα/ERβ. Así, el aumento del estrés oxidativo, debido en parte a un descenso de los sistemas antioxidantes, está mediado por ERα. En cambio, la activación de ERβ por la genisteína implica un menor estrés oxidativo y una mejor función mitocondrial, promovido por una respuesta antioxidante. En consonancia con el estudio in vitro, los carcinomas mamarios con una menor ratio ERα/ERβ también mostraron una mayor respuesta detoxificante, favoreciendo la supervivencia celular. Por su parte, la leptina parece disminuir el nivel de estrés oxidativo basal, lo que podría jugar un papel en la adquisición de resistencia a los tratamientos anticancerígenos. El desacoplamiento mitocondrial mediado por UCP2 protege a la célula cancerosa del daño oxidativo, lo que posiblemente podría conferir citoprotección a través de la adquisición de quimioresistencia. En células de cáncer de páncreas, p53 mutado induce la producción de ROS debido a una disminución de UCP2, contribuyendo al crecimiento celular. En conclusión, los resultados de la presente tesis sugieren que tanto ERβ como UCP2 podrían ser biomarcadores interesantes para conseguir una mejor caracterización del tumor en relación a su nivel de estrés oxidativo y la posible respuesta al tratamiento.
L'estrès oxidatiu és un desequilibri entre la producció de radicals lliures i els sistemes antioxidants encarregats de la seva neutralització. El resultat d'aquest desequilibri és l'acumulació de danys a diverses estructures cel•lulars incloent el DNA. El càncer va acompanyat d'un major estrès oxidatiu a nivell cel•lular, per aquest motiu, molts dels tractaments terapèutics van dirigits a augmentar els nivells citotòxics de ROS, conduint a la cèl•lula tumoral a la apoptosi. No obstant això, durant la tumorigènesi les cèl•lules van adquirint una sèrie de característiques que permeten mantenir l'homeòstasi dels ROS i desenvolupar resistència als tractaments antineoplàstics. El càncer de mama és un tipus de neoplàsia en la qual el factor endocrí juga un paper rellevant tant en la etiologia como en l'evolució de la malaltia. En aquesta tesi ens vàrem plantejar com a objectius estudiar els efectes del 17β-estradiol (E2), la genisteïna i la leptina, com a factors hormonals, sobre la modulació de l'estrès oxidatiu en la carcinogènesi de mama. E2 és un dels principals factors de risc en la iniciació i progressió de la malaltia, la genisteïna és un dels fitoestrògens de major activitat estrogènica i, per la seva part, la leptina també ha mostrat efectes potenciadors sobre el càncer de mama, considerant-se a nivell epidemiològic un possible enllaç entre obesitat i càncer. Per a assolir aquests objectius es va estudiar: i) l'efecte dual de E2 i genisteïna sobre l'estrès oxidatiu en funció de la dotació de ERα i ERβ en línies cel•lulars de càncer de mama (MCF-7 i T47D), i l'estrès oxidatiu a mostres de carcinomes ductals infiltrants en funció de la ràtio de receptors estrogènics; ii) la influència de la leptina crònica sobre l'estrès oxidatiu i la seva resposta al fàrmac antineoplàstic cisplatí en la línea cel•lular MCF-7; i iii) la importància de la UCP2 en la regulació de l'estrès oxidatiu endogen i induït en les cèl•lules MCF-7, així como, en línies cel•lulars de càncer de pàncrees amb p53 mutat. Els resultats indiquen que E2 indueix estrès oxidatiu de forma depenent de la dotació de ERα/ERβ. Així, l'augment de l'estrès oxidatiu, causat en part per un descens dels sistemes antioxidants, està mediat per ERα. En canvi, l'activació de ERβ per la genisteïna implica un menor estrès oxidatiu i una millor funció mitocondrial, promogut per una resposta antioxidant. En consonància amb l'estudi in vitro, els carcinomes mamaris amb una menor ràtio ERα/ERβ també varen mostrar una major resposta detoxificant, afavorint la supervivència cel•lular. Per la seva part, la leptina sembla disminuir el nivell d'estrès oxidatiu basal, la qual cosa podria jugar un paper en l'adquisició de resistència als tractaments anticancerígens. El desacoblament mitocondrial mediat per UCP2 protegeix a la cèl•lula cancerosa del dany oxidatiu, fet que possiblement podria conferir citoprotecció a través de l'adquisició de quimioresistència. En cèl•lules de càncer de pàncrees, p53 mutat indueix la producció de ROS a causa d'una disminució de UCP2, contribuint al creixement cel•lular. En conclusió, els resultats de la present tesi suggereixen que tant ERβ com UCP2 podrien ser biomarcadors interessants per a aconseguir una millor caracterització del tumor en relació al seu nivell d'estrès oxidatiu i la possible resposta al tractament.
Oxidative stress is an imbalance between free radical production and the antioxidant systems responsible for counteracting them. The result of this imbalance is accumulation of damage in several cellular structures, including DNA. Cancer is associated with increased oxidative stress, therefore, many therapeutic treatments are targeted to raise cytotoxic ROS levels, which would lead to tumor cell apoptosis. However, during tumorigenesis, cells acquire several features that maintain ROS homeostasis and develop resistance to anticancer treatments. Breast cancer is a type of neoplasia in which the endocrine factor plays an important role in etiology and disease progress. In the preset thesis we set out to study the effects of hormonal factors: 17β-estradiol (E2), genistein and leptin, on oxidative stress modulation in breast carcinogenesis. E2 is one of the main risk factors for breast cancer initiation and progression; genistein is one of the phytoestrogens with greater estrogenic activity and, while, leptin has also shown enhancing effects on breast cancer, epidemiologically it is also considered to be a possible link between obesity and cancer. The aim of this work was to study: i) the dual effect of E2 and genistein on oxidative stress depending on the ERα and ERβ ratio in breast cancer cell lines (MCF-7 and T47D), and oxidative stress in invasive ductal carcinoma samples depending on estrogen receptor ratio; ii) the influence of chronic leptin on oxidative stress and response to anticancer drug cisplatin in MCF-7 cell line; iii) the significance of UCP2 in the regulation of endogenous and induced oxidative stress in MCF-7 cells as well as in mutant p53 pancreatic cancer cell lines. Results indicate that E2 induces oxidative stress in a ERα/ERβ ratio-dependent manner. Thus, the increase in oxidative stress, due to in part to a decrease in antioxidant systems, is mediated by ERα. In contrast, ERβ activation by genistein involves a lower oxidative stress and better mitochondrial function, which is promoted by an antioxidant response. In agreement with the in vitro study, breast tumors with a lower ERα/ERβ ratio showed a higher detoxifying response, which also improved cellular survival. In turn, leptin appears to decrease the basal level of oxidative stress, which could play a role in the acquisition of resistance to anticancer treatments. UCP2-mediated mitochondrial uncoupling protects the cancer cell from oxidative damage, which may possibly confer cytoprotection through chemoresistance acquisition. In pancreatic cancer cells, p53 mutant induces ROS production due to a decrease in UCP2, contributing to cell growth. In conclusion, the results of this thesis suggest that both ERβ and UCP2 may be interesting biomarkers for a better characterization of the tumor in relation to its level of oxidative stress and possible treatment response.

Since its discovery in 1994, the adipose tissue hormone leptin has been well established as a key regulator of energy balance in mammals. However, little is known about the molecular evolution of the hormone and its function in non-mammalian vertebrates. This project builds on the recent identification of leptin in an amphibian, the tiger salamander, to investigate the leptin signalling system in a laboratory salamander, the axolotl. The overall aim of the project was to obtain cDNA sequences of the axolotl leptin and leptin receptor (LEPR) genes, to analyse their expression and to study their expression due to nutritional state. Cloning the axolotl LEPR was a key component of the work because no sequence information was previously available. Semi-degenerate primers were used to clone a 248 bp fragment of the LEPR, which shared 62% identity with human leptin at the amino acid level. Attempts to obtain the full-length cDNA sequence were unsuccessful. However, the sequence grouped in proximity to a Xenopus LEPR in a phylogenetic tree, and Northern hybridization revealed a transcript size of approximately 3 kb, which corresponded with that of other vertebrate LEPRs. To establish the expression pattern of leptin and the LEPR between tissues, quantitative real-time PCR was performed in two different age groups of animals. In adults, the highest expression of leptin was observed in the fat, brain and heart whereas in juveniles leptin expression was significantly higher in the fat body compared to all other tissues. The highest expression of LEPR was found in the brain and skeletal muscle. These findings agree with the main sites of leptin and LEPR expression in mammals, Xenopus, and fish providing further evidence that the gene fragments cloned represents the axolotl leptin and LEPR. In order to understand the possible role(s) of leptin in the regulation of food intake and energy metabolism in amphibians, changes in leptin and LEPR expression due to nutritional state were investigated. Short-term fasting did not result in any significant changes in leptin expression in the fasted animals, nevertheless it showed a tendency towards a lower leptin and LEPR expression of fasted axolotls. These findings indicate that the regulation of leptin expression by nutritional state more closely resemble the situation in other ectotherms such as teleost fish. This work provides the opportunity to explore how the physiological functions of leptin have changed during evolutionary history.

The objectives of the present study were to examine the effects of dietary fat type and level of energy intake on hypothalamic leptin binding affinity and membrane fatty acid composition, circulating leptin levels, and body weight homeostasis in rats. Animals were fed diets containing tallow, safflower oil, or menhaden oil (20% wt/wt) for 10 wks, ad libitum or at 60% of ad libitum intakes. Specific leptin binding could not be detected in hypothalamic membrane homogenates; hypothalamic leptin levels were unaffected by diet or energy intake. Levels of tracer exceeding assay reference values were found in homogenates, suggesting intra-membrane binding. Excess tracer levels were weakly associated (p < 0.07) with the sum of hypothalamic phospholipid monounsaturates. Restriction lowered weight gain and food intakes (p < 0.0001 for both). In hypothalamic phospholipids, energy restriction lowered levels of 18:3(n-3) and increased levels of 20:1(n-9), 20:4(n-6), and 22:4(n-6) (p < 0.05, p < 0.02, p < 0.05, and p < 0.04, respectively). Fat type and energy level interactively affected hypothalamic levels of 20:4(n-6), 22:5(n-3) and 22:6(n-3) (p < 0.05, p < 0.006, and p < 0.05, respectively). Restriction lowered circulating leptin levels (p < 0.0001); overall plasma leptin levels were marginally associated (p < 0.07) with hypothalamic 16:0 concentrations. The results of the study support previous findings suggesting that leptin binding at the level of the hypothalamic membrane may not be detectable. The results also support the lack of a dietary fat effect on plasma leptin levels and levels of certain hypothalamic fatty acids, such as 20:4(n-6), 22:4(n-6), and 22:5(n-3), with energy restriction. The findings of the study suggest a link between increased membrane fluidity, increased binding affinity, and lower circulating leptin levels, promoting the possibility that the biological actions of leptin can be controlled through dietary effects on

Leptin is a peripheral adiposity signal and a key hormone in energy balance regulation in mammals, acting as a link between nutritional status and the endocrine reproductive axis. If this is also the role of leptin in fish is not fully understood. This thesis investigates how different components of the leptin system are affected by sexual maturation and seasonal changes in energy balance in male Atlantic salmon (Salmo salar L.) parr under fully fed and feed-restricted conditions. Moreover, the role of sex steroids as being one of the possible mechanisms by which sexual maturation interacts with leptin is explored. The salmon leptin-a genes, lepa1 and lepa2, were expressed mainly in liver and the leptin receptor (lepr) in brain and ubiquitously in peripheral tissues. Seasonal characterization of the lepa genes and lepr during the growth and reproductive season in one-year old males showed that hepatic lepa1 and lepa2 mRNA levels and plasma leptin levels were down-regulated concomitantly with an increase in weight and body fat. Feed restriction up-regulated hepatic leptin, and pituitary lepr expression as well as plasma leptin levels. Correlation between leptin levels and body lipid stores were either lacking or negative. These findings show that leptin and lepr are sensitive to changes in energy balance, but that leptin might not reflect adiposity in juvenile salmon. Hepatic lepa1 and lepa2, and testicular lepr expression increased during mid- to late spermatogenesis in early maturing males. This up-regulation was preceded by rapid gonadal growth and elevated pituitary follicle-stimulating hormone gene expression levels, whereas peak leptin levels coincided with peak pituitary luteinizing hormone expression and the presence of running milt in the testes. The sex steroids testosterone (T), 11-ketotestosterone and 17-β estradiol stimulated lepa1 and lepa2 gene expression in Atlantic salmon hepatocytes in vitro differentially depending on developmental stage. T was also able to stimulate hepatic lepa1 and pituitary lepa1 and lepr gene expression in immature male salmon in vivo. These results suggest that leptin plays a role in male fish reproduction during later stages of the maturational process and that the elevation of leptin expression during spermatogenesis could be caused by androgen stimulation.

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Objetivo: Estudar o efeito da obesidade na regulação do sistema calicreína-cininas por meio da expressão dos receptores B1 e B2 de cininas em humanos e camundongos, e as alterações na síntese e funcionalidade dos receptores em tecidos murinos. Métodos: Foram coletados tecido adiposo branco humano e diferentes tecidos de camundongo. Desses tecidos foi extraído o RNA e analisada a expressão dos receptores de cinina por meio da reação em cadeia da polimerase em tempo real. Tecidos como estômago e aorta de camundongos ob/ob e selvagens foram utilizados para extração de proteínas e estudos fisiológicos. Por Western Blotting estudou-se a quantidade de receptor produzida nos animais. O fundus de estômago e aorta abdominal foram utilizados para se obter registros de contrações isométricas para determinação da potência e eficácia dos agonistas em camundongos obesos e magros. Foram aplicadas doses crescentes cumulativas dos agonistas peptídicos dos receptores B1 e B2, bradicinina e des-Arg9-bradicinina. Resultados: Nos experimentos de PCR em tempo real, a expressão gênica dos receptores B1 e B2 de cininas foi mostrada alterada em alguns tecidos dos animais deficientes na produção do hormônio leptina em relação ao controle. Nos tecidos: adiposo branco, aorta, fígado, hipotálamo e estômago, a expressão do receptor B1 apresentou-se aumentada, porém em tecido cardíaco e tecido adiposo marrom, essa estava diminuída. O receptor B2 teve expressão diminuída em tecido adiposo branco e hipotálamo. Nos demais tecidos estudados não houve alteração da expressão do receptor B2. Em humanos, esses receptores apresentaram-se alterados em indivíduos obesos. O estudo foi realizado em tecido adiposo humano de duas regiões de depósito diferentes, visceral e subcutâneo. Foi observada diferença na expressão do mesmo tecido, porém de regiões distintas. Nos tecidos dos camundongos obesos a resposta aos agonistas dos receptores B1 e B2, bradicinina e des-Arg9-bradicinina, respectivamente, foi mostrada diminuida. Em fundus de estômago foi observada diminuição significativa na resposta ao agonista BK em animais obesos e tratados com dieta hiperlipídica. Tais efeitos podem ser devido às conseqüências do aumento excessivo de peso, como inflamação crônica apresentada nesses animais, ou mesmo devido a diabetes tipo II, a qual consiste em uma patologia diretamente relacionada à obesidade, sugerida neste trabalho como fator capaz de alterar a ação do sistema calicreína-cininas em determinados tecidos. Conclusão: Análises de expressão gênica mostraram que a obesidade afeta os receptores de cinina em diversos tecidos de camundongo, assim como em humanos. Análises fisiológicas funcionais mostraram diminuição na resposta ao agonista de B1 em animais obesos. Os dados deste trabalho sugerem que a obesidade afeta a modulação do sistema calicreína-cininas em modelo murino e humano. Dessa forma, uma possível interação entre obesidade e sistema calicreína-cininas poderia estar envolvida nesta patologia, assim como ser um fator para desenvolvimento a sindrome metabólica.
Objectives: To study the effect of obesity on the kallikrein-kinin system through the expression of receptors B1 and B2 on humans and mice, and alterations in the synthesis and functionality of the receptor in murine tissues. Methods: white human adipose tissue and different kinds of mice tissues were collected. RNA was extracted and the kinin receptors expression analyzed through a real-time polymerase chain reaction. Tissues and organs such as stomach and aorta were used for protein extraction and physiological studies. By Western Blotting, receptor quantitation was studied. Stomach fungus and abdominal aorta were used to register isometric contractions to determine the potency and effectiveness of the agonists on obese and control mice. Increasing accumulating doses of bradykinin and des-Arg9-bradykinin, B2 and B1 receptors agonists respectively, were applied. Results: In the real-time PCR experiments, the gene expression of the B1 and B2 receptors were altered in some tissues of the animals deficient for leptin, when compared to the control. In the white adipose tissue, aorta, liver, hypothalamus and stomach, the B1 receptor expression was increased, but in cardiac tissues and brown adipose tissue, it was decreased. The expression of B2 receptor was decreased in white adipose tissue and hypothalamus. In the other studied tissues, no changes was detected in the B2 receptor expression. In humans, these receptors were altered in obese individuals. The study was performed in human adipose tissue from two different regions of depots, visceral and subcutaneous. There was a tendency of different expression in the same tissue, but from different areas. In tissues from obese mice the response to the B2 and B1 agonists, bradykinin and des-Arg9-bradykinin, respectively, had a decreasing tendency. A significant decrease was observed in stomach fundus in response to the BK agonist. Such effects can be due to the increased weight and its consequences, such as chronic inflammation or diabetes type II, which is a pathology directly related to obesity. Conclusion: expression and functional analysis show that obesity affects kinin receptors in many different mouse tissues as well as in humans.
TEDE
BV UNIFESP: Teses e dissertações

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Leptin, a cytokine produced by adipose cells, is the target of the scientific community for believing that it has an impact on the reproduction of the animals promoting puberty, folliculogenesis and oogenesis, estrous cycle and aiding in fertilization. The understanding of the mechanisms controlling the reproductive activity of Spix's Yellow-toothed Cavy (Galea spixii) plays a relevant role in the preservation of the species. Thus, the present work proposed to analyze the immunolocalization of leptin receptors (Ob-R) in the ovary of cavies. Ovaries from 20 adult, non-pregnant, healthy females were collected. The samples were fixed in 4% phosphate buffered paraformaldehyde, embedded in paraffin and sectioned for immunohistochemistry. The sections were photomicrographs and intensity of the reaction was measured. Strong immunoreaction was observed in oocyte and theca cells, moderate in ovarian stromal cells and large luteal cells and weak stained in granulosa, endothelial, perivascular and small luteal cells. When compared to receptor expression along follicular development it was observed that the oocyte and the theca cells remained with expression at the same intensity. However, the granulosa cells presented strong stained in the preantral stages, whereas in the antral follicles it presented low intensity. We conclude that in the ovaries of Galea spixii there is the presence of Ob-R in the main structures of the ovary sugesting that this hormone plays a fundamental role in the reproduction of this species
A leptina, uma citocina produzida pelas células adiposas, possui ação na reprodução dos animais promovendo a puberdade, foliculogênese e oogênese, ciclo estral e auxiliando na fecundação. A compreensão dos mecanismos que controlam a atividade reprodutiva de preás (Galea spixii) possui papel relevante para a preservação da espécie. Desta forma, o presente trabalho propôs analisar a imunolocalização dos receptores de leptina (Ob-R) no ovário de preás. Coletaram-se os ovários de 20 fêmeas adultas, não prenhes e saudáveis. As amostras foram fixadas em paraformaldeído a 4% em tampão fosfato, incluídas em parafina e seccionadas para a realização de imunohistoquímica. As secções foram fotomicrografadas e avaliadas quanto à intensidade da reação. Observou-se forte imunorreação no oócito e nas células da teca, moderada nas células do estroma ovariano e nas células luteínicas grandes e fracamente coradas nas células da granulosa, endoteliais, perivasculares e células luteínicas pequenas. Quando comparado a expressão de receptores ao longo do desenvolvimento folicular foi observado que o oócito e as células da teca se mantiveram com expressão na mesma intensidade. Entretanto, as células da granulosa apresentaram forte marcação nos estádios pré-antrais enquanto que nos folículos antrais apresentou fraca intensidade. Concluímos que em ovários de Galea spixii existe a presença de Ob-R nas principais estruturas do ovário sugerindo que este hormônio desempenhe papel fundamental na reprodução desta espécie
2017-03-30

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Made available in DSpace on 2017-05-09T15:47:00Z (GMT). No. of bitstreams: 1 Nubia Michelle Vieira da Silva.pdf: 1519635 bytes, checksum: 28fb82a2e5ac497c9fefcd231c2cf135 (MD5) Previous issue date: 2010-06-23
Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
This study aimed to avaluate the relationship the polymorphism from the leptin gene (LEP), specifically exon 2, and from the microsatellite of the growth hormone receptor (GHRSSR) with the weight and weaning characteristics of animal breeds Anglo-Nubian, and Boer, to identify useful markers for selecting goats of high genetic merit. It was obtained the allele frequencies and heterozygosity with the Toolkit (PARK, 2001). The test for the Hardy-Weinberg equilibrium was performed with GenePop program according to Rousset (2008), and showed that the markers were in desequilibrium in populations. Observed heterozygosity values for LEP were greater than expected and all animals showed the same electrophoretic pattern with two alleles (150 and 152 bp). It was detected with GHR locus five alleles ranging from 90 to 125 bp examined in populations. The genotypes influence of polymorphic fragments of GHR and leptin on animals development was evaluated using the birth weight (BW) and weaning (PD), by analysis of variance and mean test with the GLM procedure of (SAS, 1999). The genotypes showed a significant effect on birth weight (BW) and weaning (PD). It is necessary to study of these polymorphisms on a larger sample of animals to confirm the effect on growth characteristics.
Objetivou-se estudar a relação entre o polimorfismo no gene da Leptina (LEP), especificamente o éxon 2, e o microssatélite do receptor do hormônio do crescimento (SSRGHR) com as características de peso ao nascer e desmame em caprinos das raças Anglo- Nubiana e Boer, a fim de identificar marcadores que possam ser úteis na seleção desses animais de elevado mérito genético. Foram obtidas as frequências alélicas e a heterozigosidade com auxílio do programa Toolkit (PARK, 2001). O teste para o equilíbrio de Hardy-Weinberg foi feito com auxílio do GENEPOP,conforme Rousset (2008), no qual os marcadores se mostraram em desequilíbrio para as populações. Para a LEP, os valores de heterozigosidade observada foram bem maiores do que os esperados e todos os animais apresentaram o mesmo padrão eletroforétrico com dois alelos (150 e 152 pb). No loco do ghr observaram-se cinco alelos com tamanho variando de 90 a 125 pb. Para verificar a influência dos genótipos dos fragmentos polimórficos do ghr e da leptina sobre o desenvolvimento dos animais foram utilizados os pesos ao nascer (PN) e ao desmame (PD), para os quais foi feito análise de variância e teste de médias com auxílio do procedimento GLM do programa (SAS, 1999). Observou-se um efeito significativo dos genótipos do ghr sobre os pesos ao nascer (PN) e à desmame (PD). Sugere-se então, um estudo destes polimorfismos em maior número de animais para confirmação do efeito sobre características de crescimento.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O gene responsável pela codificação do hormônio leptina tem sido associado à produção de leite, e diversos polimorfismos encontrados nesse gene foram associados a características produtivas em bovinos. O objetivo do presente estudo foi a identificação do polimorfismo LEP-1620 (A/G) no gene bubalino da leptina e suas possíveis associações com as produções de leite, gordura, proteína e porcentagens de gordura e proteína. Foram coletadas amostras de pelo da cauda de 200 búfalas, e após a extração do DNA as amostras foram genotipadas pela técnica PCR-RFLP. Três amostras foram sequenciadas e foi encontrado um SNP na posição 70 do íntron 2 do gene da leptina, caracterizado pela substituição de um A por um G. As médias das produções mensais de leite, gordura, proteína e as porcentagens de gordura e proteína foram avaliadas em um modelo misto. Os genótipos encontrados (AA, AG, GG) foram positivamente associados às características porcentagem de gordura e de proteína (p<0,05), sendo que os animais AA apresentaram médias superiores para as características. As curvas de lactação para as características produção de leite e porcentagens de gordura e proteína apresentaram trajetórias semelhantes para os três genótipos. Esses resultados indicam que o polimorfismo LEP-1620 (A/G) pode ser utilizado futuramente como marcador molecular para as características porcentagem de gordura e proteína do leite de búfalas
The gene responsible for encoding the hormone leptin has been associated with milk production, and several polymorphisms of this gene were associated with production traits in cattle. The aim of the present study was to identify the LEP-1620 (A/G) polymorphism in the buffalo leptin gene and its possible associations with milk, fat and protein yield, and fat and protein percentages. Samples of tail hair from 200 buffalo cows were collected, and after DNA extraction the samples were genotyped by PCR-RFLP. Three samples were sequenced and an SNP was found at position 70 of intron 2 in the leptin gene, characterized by the substitution of an A for a G. The means from monthly milk, fat and protein yield and falt and protein percentages were evaluated by a mixed model. The genotypes found (AA, AG, GG) were positively associated with fat and protein percentages (p<0,005), and the AA animals showed the highest means for this traits. The lactation curves for milk yield and fat and protein percentages showed similar trajectories for the three genotypes. These results indicate that the LEP-1620 (A/G) polymorphism can be used in the future as a molecular marker for fat and protein percentages traits of buffalo cow’s milk

Orientador: Humberto Tonhati
Coorientador: Marcelo Cervini
Banca: Maria Elisabete Jorge Amaral
Banca: Joslaine Noely dos Santos Gonçalves Cyrillo
Resumo: O gene responsável pela codificação do hormônio leptina tem sido associado à produção de leite, e diversos polimorfismos encontrados nesse gene foram associados a características produtivas em bovinos. O objetivo do presente estudo foi a identificação do polimorfismo LEP-1620 (A/G) no gene bubalino da leptina e suas possíveis associações com as produções de leite, gordura, proteína e porcentagens de gordura e proteína. Foram coletadas amostras de pelo da cauda de 200 búfalas, e após a extração do DNA as amostras foram genotipadas pela técnica PCR-RFLP. Três amostras foram sequenciadas e foi encontrado um SNP na posição 70 do íntron 2 do gene da leptina, caracterizado pela substituição de um A por um G. As médias das produções mensais de leite, gordura, proteína e as porcentagens de gordura e proteína foram avaliadas em um modelo misto. Os genótipos encontrados (AA, AG, GG) foram positivamente associados às características porcentagem de gordura e de proteína (p<0,05), sendo que os animais AA apresentaram médias superiores para as características. As curvas de lactação para as características produção de leite e porcentagens de gordura e proteína apresentaram trajetórias semelhantes para os três genótipos. Esses resultados indicam que o polimorfismo LEP-1620 (A/G) pode ser utilizado futuramente como marcador molecular para as características porcentagem de gordura e proteína do leite de búfalas
Abstract: The gene responsible for encoding the hormone leptin has been associated with milk production, and several polymorphisms of this gene were associated with production traits in cattle. The aim of the present study was to identify the LEP-1620 (A/G) polymorphism in the buffalo leptin gene and its possible associations with milk, fat and protein yield, and fat and protein percentages. Samples of tail hair from 200 buffalo cows were collected, and after DNA extraction the samples were genotyped by PCR-RFLP. Three samples were sequenced and an SNP was found at position 70 of intron 2 in the leptin gene, characterized by the substitution of an A for a G. The means from monthly milk, fat and protein yield and falt and protein percentages were evaluated by a mixed model. The genotypes found (AA, AG, GG) were positively associated with fat and protein percentages (p<0,005), and the AA animals showed the highest means for this traits. The lactation curves for milk yield and fat and protein percentages showed similar trajectories for the three genotypes. These results indicate that the LEP-1620 (A/G) polymorphism can be used in the future as a molecular marker for fat and protein percentages traits of buffalo cow's milk
Mestre

Autonomic nervous system (ANS) activity is related to body weight regulation. Based on the hypothesis that Most Obesities kNown Are Low In Sympathetic Activity (MONA LISA), it has been suggested that most obese subjects and animals have low sympathetic nervous system activity. Leptin, leptin receptor, and ghrelin genes influence the ANS regulation of body weight and food intake. The aim of this study was to investigate whether there are differences in leptin, the leptin receptor, or ghrelin regulation between lines of chickens selected for high (HWS) or low body weight (LWS). Intraperitoneal injections of reserpine were administrated to chickens from the HWS and LWS lines. Body weight and food intake were then compared to evaluate ANS regulation. While reserpine caused a transitory decrease in food intake and body weight in both lines, the magnitude of the change was greater in the HWS than in the LWS chickens. However, chickens from the LWS line exhibited greater catecholamine and indoleamine level changes in response to reserpine than those from the HWS line. Therefore, HWS chickens were more sensitive to the body weight-reducing effects of reserpine than LWS lines, while LWS chickens appeared to have greater sympathetic nervous system activity. Food and water intakes were differentially affected in HWS and LWS chickens in response to intracerebroventricular administration of human recombinant leptin. Leptin caused a linear decrease in food intake in the LWS line, but no effect on food intake in the HWS lines. The HWS chickens tended to have reduced water intake following leptin administration. These results suggest that the leptin receptor, or the down-stream neuropeptide regulation pathway mediating the effect of leptin; may be different between chickens from the HWS and LWS lines. Leptin, insulin like growth factor (IGF)-1, and IGF-2 concentrations in the plasma of HWS and LWS lines of chickens were evaluated. Leptin, IGF-1 and IGF-2 levels were significantly higher in the LWS than HWS chickens. The HWS female leptin concentrations were significantly lower than in HWS males or LWS females. Male chickens had greater IGF-1 concentrations in the plasma than female chickens. However, the concentration of IGF-2 did not differ between sexes. The difference in leptin concentrations in these lines and sexes may explain the differences in age of sexual maturity. Different IGF-1 and IGF-2 concentrations may be involved in the obese and anorexic conditions, fast and slow growth, and high and low food consumption found in these two lines of chickens. Differences in the gene sequence of the leptin receptor were observed in HWS and LWS lines of chickens. A single nucleotide polymorphism (SNP) in the intron between exon 8 and 9 introduced a restriction site for the enzyme Sel I in the HWS, but not the LWS line. Two SNP were detected in the leptin receptor cDNA region at nucleotides 189 and 234. At nucleotide 189, the LWS line has both a homozygous (T-T) and heterozygous (C-T), whereas the HWS line has only homozygous (T-T) form. The SNP found in nucleotide 234 introduces a restriction site Mse I in the HWS, but not the LWS line. These specific changes may be directly involved or closely linked to differences between the two lines in either the coding or regulatory domains of the leptin receptor. Differences in the leptin receptor gene expression between HWS and LWS lines of chickens in various organs and ages were observed. Leptin receptor expression in the whole brain was significantly different between sexes at 28 days-of-age in the HWS and LWS lines. The LWS line had higher leptin receptor gene expression in the liver at 2 days-of-age than at 56 and 363 days-of-age, but no differences were observed in the HWS line. In addition, at 2 days-of age, liver leptin receptor gene expression was higher in LWS than HWS chickens, but the reverse was observed at 363 days-of age. In adipose tissue, leptin receptor expression was higher in the LWS than HWS line. Leptin receptor expression in adipose tissue was greater at 363, than 28 and 56 days-of-ages. Our results showed that changes in the regulation of leptin and the leptin receptor were associated with sex, age, and growth. Differences in the ghrelin gene in the HWS and LWS lines under different feeding conditions were investigated. Both HWS and LWS chickens have six extra base pairs in the 5'-untranslated region. The LWS male ghrelin gene expression was significantly lower than in the LWS female and HWS male. The 84 day-old males had lower gene expression than 84 day-old females and 363 day-old males. When comparing different feeding methods, females allowed ad libitum feed consumption had a lower cycle threshold cycle number (CT) ratio than males allowed ad libitum feeding or fasted females. However, the inflection point cycle number of ad libitum fed females was lower than that of the ad libitum fed males, but greater than the fasted females. Ghrelin gene expression was different between the two lines of chickens, and the expression of ghrelin in chickens was influenced by body weight selection, sex, age, and feeding condition.
Ph. D.

La obesidad es uno de los factores más reconocidos en el desarrollo del cáncer de mama, y el tejido adiposo juega un papel muy importante en esta patología debida la secreción de sus diversas citosinas, principalmente Leptina y Adiponectina. La base molecular de esta asociación no es bien conocida y muy pocos son los que han estudiado a los receptores de Adiponectina (AdipoR1 y AdipoR2) y la influencia de la Leptina sobre ellos. Por lo que se requiere elucidar los mecanismos por los cuales niveles altos de Leptina en el organismo generan un aumento en el riesgo de desarrollo de cáncer de mama y si esta proteína tiene una influencia sobre AdipoR1 y AdipoR2, la cual podría afectar la acción anti-proliferativa de la Adiponectina.
Se ha documentado el papel que juega el tejido adiposo a través de Leptina y Adiponectina implicados en el desarrollo y progreso del cáncer de mama, pero muy pocos son los estudios sobre AdipoR1 y AdipoR2 y la influencia de la Leptina sobre ellos. Objetivo: Analizar la expresión de AdipoR1 y AdipoR2 modulada por concentracionesdiferenciales de Leptina en un modelo de obesidad asociado a cáncer de mama en las líneas celulares MCF-7, MDA-MB231 y HCC1937. Métodos: Se analizó la expresión de AdipoR1 y AdipoR2 por PCR en tiempo real utilizando sondas TaqMan®, mediado por concentraciones de Leptina (0 ng/mL, 10ng/mL, 100 ng/mL y 1000 ng/mL) en líneas celulares de cáncer de mama: MCF-7,MDA-MB231 y HCC1937. Se caracterizó cada línea celular por Inmunohistoquímica. Resultados: La Leptina generó un aumento de la población celular en MCF-7 (23.8%, 10 ng/mL, 48 h); en MDA-MB231 la población aumentó hasta un 17.02% (1000 ng/mL, 72 h) y en HCC1937 aumentó en un 17.24% (1000 ng/mL, 72h). En MCF-7 la expresión de AdipoR1 disminuyó (3.81%, 1000 ng/mL), excepto para 100 ng/mL (64.03%). Laexpresión de AdipoR2 aumentó hasta 13.74 veces (10 ng/mL) respecto al control. EnHCC1937 la expresión de AdipoR1 disminuyó hasta un 86.28% (10 ng/mL), mientras que la expresión de AdipoR2 tuvo una disminución hasta un 50.3% (100 ng/mL). Conclusiones: La concentración de normo-peso (10 ng/mL) de Leptina generó un aumento de la expresión de ambos receptores de Adiponectina.
Proyecto DSA/103.5/16/10569