Telomerase is a highly regulated ribonucleoprotein complex that stabilizes eukaryotic genomes by replenishing telomeric repeats on chromosome ends. Defects in telomerase RNP components involving the catalytic subunit TERT or the RNA template TER lead to stem cell-related diseases such as dyskeratosis congenita and idiopathic pulmonary fibrosis, while inappropriate telomerase expression is a rate-limiting step in carcinogenesis. In this study we report the discovery of a novel negative regulatory mechanism for telomerase that stems from duplication and diversification of key components of the telomerase RNP in the flowering plant Arabidopsis thaliana. We show that Arabidopsis encodes three distinct TERs: TER1, TER2 and a processed form of TER2 termed TER2S. Although all three RNAs can serve as templates for telomerase in vitro, in vivo they have different expression patterns, assemble into distinct RNPs with different protein binding partners, and play opposing roles in telomere maintenance. The TER1 RNP is analogous to the telomerase enzyme previously described in other eukaryotes, but the TER2 RNP is a negative regulator of telomerase activity and telomere maintenance in vivo. Furthermore, we demonstrate that the Protection Of Telomeres (POT1) paralogs in Arabidopsis (POT1a, POT1b and POT1c) are novel TER binding proteins. This finding is striking because in yeast and vertebrates, POT1 is an essential component of the telomere capping complex and functions to distinguish the chromosome terminus from a double-strand break. Thus, our data argue that Arabidopsis POT1 proteins have migrated off of the chromosome terminus and onto the telomerase RNP, indicating that duplication and diversification of Arabidopsis telomerase may be the end result of the co-evolution of the TER and POT1 RNP components. Additionally, given the dire consequences of misregulating telomerase in human cells, our discovery of a novel negative regulatory mechanism for telomerase in plants strongly suggests that additional modes of telomerase control remain to be elucidated in vertebrates.

Texte remanié de: Diss.--Würzburg--Universität Würzburg, 1985.

Made available in DSpace on 2015-07-22T20:50:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-06-28. Added 1 bitstream(s) on 2015-08-11T03:25:41Z : No. of bitstreams: 1 Publico-091.pdf: 651136 bytes, checksum: 35401eae81afb55be34c6f47a332e47e (MD5) / 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

Taste impacts the palatability and intake of food, which is influenced by several factors such as cultural and genetic factors. Individual variations in taste perception may be important risk factors for poor eating habits and development of obesity. The differences in taste perception which impact dietary intake may lead to better understanding of obesity development and prevention of diet-related diseases. Obesity is one of the main causes for various health conditions in the United States as well as in the world. Genetic inheritance plays an important role in individual variations to taste and food choices. This study explored associations between two single nucleotide polymorphisms (SNPs, rs713598 and rs10246939) in the TAS2R38 bitter taste receptor gene, dietary intake, and body fat percentage. Five hundred presumably healthy students aged 18-25 years, including 86 (17%) males and 414 (83%) females from Mississippi State University participated in the study. Saliva was collected for genetic analysis, participants completed dietary history questionnaires and body composition was measured using bioelectrical impedance analysis. All statistical analysis of data was conducted using SPSS software to examine associations between SNPs, food intake, and percentage of body fat. Our results did not show a significant association between the SNPs; rs713598 and rs10246939 in the TAS2R38 bitter taste receptor gene and dietary intake of vegetables and fruits as well as percentage of body fat in this group of participants. However, alcohol and caffeine intakes were significantly different between genotypes in rs713598; p< 0.01, p< 0.05, respectively.

Magister Commercii (Industrial Psychology) - MCom(IPS) / In the current recessionary cycle in which individuals finds themselves, it is interesting to see whether organisations and individuals have changed their strategies or whether they pursued their tried and tested inherent mechanisms of recruitment/work selection. In recessionary times, organisations would usually have a bigger pool to select from whilst employees, in turn, would try to position themselves in a stable work environment. From an organisational perspective, organisations have also undergone major transitions such as downsizing, merges and acquisitions, right sizing, restructuring, and reengineering. These changes have a direct impact on employees level of motivation and job satisfaction (Ellison Schreuder, 2000). The concept of a traditional career that an employee occupies for a lifetime performing one type of work in an organisation no longer exists. Instead, employees now work for more than one organisation in their lifetime. These changes entail that employees need to be flexible and adaptive in making career decisions (Schreuder Coetzee, 2006). Career anchors can be operationalized as a representation of self- perceived talents, motives, values and abilities that guide employees to make career decisions. Schreuder and Coetzee (2006), are of the opinion that if employees are not familiar with their career anchors, they could find themselves trapped in work environments that are not satisfactory and would continually be questioning themselves. Suutari and Taka (2004) emphasize the fact that there needs to be a fit between the careers of employees and the work environment. If there is no fit between the career anchors of employees and the work environment then employees are likely to become dissatisfied which may result in a high turnover of staff with a corresponding low productivity rate. This study investigates and explores the phenomenon of career anchors based on Scheins 1978 career anchor theory and how these career anchors affect employees level of job satisfaction. The Career Anchor Inventory and the Job Descriptive Index were administered to a sample of 154 employees at a leading retail organisation who completed the questionnaires. The results of this research study indicate that there are significant relationships between biographical factors and career anchors as well as between biographical factors and job satisfaction and similarly between typology of career anchors and dimensions of job satisfaction. / South Africa

A Doença Hepática Gordurosa Não Alcoólica (DHGNA) é atualmente uma das formas mais comuns de doença hepática, e está diretamente relacionada com a obesidade. Estudos indicam uma prevalência por volta de 30% na população ocidental e 11% na população oriental. A DGHNA possui um largo espectro abrangendo desde casos de esteatose simples sem inflamação, até casos com esteatohepatite e fibrose, podendo evoluir para cirrose e carcinoma hepatocelular. A fisiopatogênese da DHGNA é baseada em múltiplos eventos: resistência insulínica, aumento da lipólise periférica, aumento da síntese de novo de lipídeos, estresse oxidativo, endotoxemia crônica no desencadeamento da inflamação e fibrose entre outros. A etapa inicial caracteriza-se pelo acúmulo de ácidos graxos no hepatócito, suplantando sua capacidade de metabolização e exportação conseqüente à ação lipogênica da insulina, desta forma o metabolismo de ácidos graxos está intimamente ligado ao desenvolvimento da DHGNA. O Yo Jyo Hen Shi Ko (YHK) é um composto natural usado na medicina japonesa para tratamento de doenças hepáticas e apresenta propriedades antioxidantes, antiinflamatórias e hipolipemiantes. O presente estudo teve como objetivo avaliar o efeito do YHK no metabolismo hepático de lipídeos. Para tanto, foram utilizados camundongos obesos (ob/ob) com esteatohepatite não alcoólica (ENA) induzida por dieta deficiente em colina e metionina (DCM) e foram analisados no tecido hepático, genes relacionados com a síntese de novo de lipídeos (SREBP1c, FASn), genes relacionados com a oxidação e exportação de lipídeos (CPT1a e SCD-1, MTP), assim como genes relacionados com o armazenamento de lipídeos (Perilipina e ADFP). O YHK apresentou um efeito citoprotetor hepático com melhora dos parâmetros histológicos neste modelo experimental de ENA. Associadamente, houve redução na expressão de genes relacionados à síntese de novo como SREBP e da FASn, quando se comparou o grupo tratado com DCM+YHK com o grupo não tratado DCM. Em contrapartida, houve aumento na expressão da MTP e da SCD-1 ocasionando uma maior exportação de triglicerídeos hepáticos nos animais que utilizaram o YHK. Ainda, o YHK modulou as proteínas Perilipina e ADFP. Por outro lado, não houve modificação na oxidação de lipídeos.. Conclui-se que o YHK pode ser uma droga promissora no tratamento da DHGNA, já que age modulando genes envolvidos na síntese e exportação de lipídeos hepáticos, reduzindo o acúmulo de gordura no hepatócito / Nonalcoholic fatty liver disease (NAFLD) is currently one of the most common forms of liver disease, related directly to the increase of obesity in the world. Previous studies indicate a prevalence of around 30% in the western population and 11% in the eastern. This disease covers cases from simple steatosis without inflammation to cases of steato hepatitis (NASH) and fibrosis and it may lead to liver cirrhosis and hepatocellular carcinoma. The pathophysiology of NAFLD is based on multiple events: insulin resistance, increased peripheral lipolysis, increased de novo lipogenesis, oxidative stress, endotoxemia in triggering chronic inflammation and fibrosis and others. The initial stage of the disease is characterized by the accumulation of fatty acids in hepatocytes caused by the lipogenic action of insulin. This condition results in the surpassing of the cells ability to metabolize and export. This way, the metabolism of fatty acids is closely linked to the development of NAFLD. The Yo Jyo Hen Shi Ko (YHK) is a natural compound used in Japanese medicine for the treatment of liver disease and it has antioxidant, anti-inflammatory and lipid lowering properties. This study aimed to evaluate the effect of YHK in the hepatic metabolism of lipids. An experimental model was designed using obese mice (ob/ob) affected by NASH induced by a deficient diet in choline and methionine (MCD). Then, genes from its hepatic tissue related to de novo lipogenesis (SREBP1c, FASN), to lipid oxidation and exportation (CPT1A and SCD-1, MTP) as well as genes related to lipid storage (Perilipin and ADFP) were analyzed. The YHK presented a cytoprotective effect, improving the hepatic histological parameters in this experimental model. Additionally, when comparing the group treated with MCD + YHK (diet + YHK) to the untreated MCD group (diet) there was a reduction in the expression of genes related to de novo synthesis like SREBP1c and FASn. However, an increased expression of MTP and SCD-1 were observed leading to a greater liver exportation of fatty acids in animals that were treated with YHK. Moreover, the YHK modulated the proteins Perilipin and ADFP. On the other hand, there was no changing in lipid oxidation. In summary, the YHK can be a promising drug for the treatment of NAFLD, as it acts by modulating genes involved in the synthesis and exportation of hepatic lipids, reducing the accumulation of fat in hepatocytes

As atividades intestinais são coordenadas pelo sistema nervoso entérico e, disfunções na motilidade intestinal são observadas em indivíduos obesos. O ATP é um co-transmissor e seus receptores estão distribuídos nos neurônios entéricos constituindo a família P2X e P2Y. O presente estudo tem como objetivo analisar a expressão do receptor P2X2 e o código químico nos neurônios mioentéricos na obesidade. Foram utilizados íleo de camundongos obesos machos (OBM) e fêmeas (OBF) (C57BL/6J ob/ob), e controles (CF; CM) (+/+), que foram submetidos à técnica imuno-histoquímica de duplas marcações em neurônios imunomarcados ao receptor P2X2 com NOS (inibitórios), ChAT (excitatórios) ou Calr (excitatórios e sensoriais). Resultados dos grupos CF e OBF: Foi verificado a imunomarcação ao receptor P2X2, NOS, ChAT e Calr no citoplasma e nas membranas celular e nuclear dos neurônios mioentéricos de ambos os grupos. As duplas marcações dos neurônios imunorreativos (-ir) ao receptor P2X2 com NOS, ChAT ou Calr em CF foram 24±4%, 24±2% e 24±4%, e em OBF foram 19±4%, 25±4% e 22±1%, respectivamente. As duplas marcações dos neurônios NOS-ir, ChAT-ir ou Calr-ir com os neurônios receptor P2X2-ir foram 100% nos dois grupos. A densidade dos neurônios P2X2-ir aumentou em 62%, enquanto que a dos neurônios NOS-ir e ChAT-ir reduziram em 49% e 57%. A morfometria demonstrou aumento na área dos neurônios NOS-ir (CF 234±63, OBF 312±67); ChAT-ir (CF 210±24, OBF 253±14) e Calr-ir (CF 203±41, OBF 315±47), no entanto, neurônios P2X2-ir não apresentaram alterações (CF 325±23, OBF 336±67). A histoquímica demonstrou não haver diferença estatística entre os grupos quanto á densidade e a morfometria dos neurônios NADH-diaforase positivos. Resultados dos grupos CM e OBM: A expressão do receptor P2X2 foi identificada somente em CM e a imunomarcação a NOS, ChAT e Calr nos dois grupos. Em CM as duplas marcações dos neurônios P2X2-ir com NOS-ir, ChAT-ir ou Calr-ir foram 23±3%, 34±5% e 32±6%, respectivamente, e nos neurônios NOS-ir, ChAT-ir e Calr-ir com o receptor P2X2 foram 100%. A densidade dos neurônios NOS-ir e Calr-ir reduziram em 31% e 16%, enquanto que a densidade da população ChAT-ir aumentou 31%. A morfometria demonstrou que neurônios NOS-ir (CM 390±49, OBM 350±22); ChAT-ir (289±18, OBM 312±44), Calr-ir (CM 375±49, OBM 360±38) e P2X2-ir (CM 437±190) não apresentaram diferenças entre os grupos. A expressão protéica pela técnica do Western Blotting mostrou que houve uma redução de 36,5% no grupo OBM. Concluímos que neste modelo experimental tanto a obesidade (C57BL/6J ob/ob) como os gêneros podem contribuir com alterações: na expressão do receptor P2X2, na densidade e na morfologia dos neurônios NOS-ir, ChAT-ir e Calr-ir, promovendo mudanças nas atividades intestinais. / The intestinal activities are coordinated by enteric nervous system, and dysfunctions in intestinal motility were observed in obese individuals. The enteric neurons have been demonstrated to express different purinergics receptors. This study aims to analyze the expression of P2X2 receptor and the chemical code in the myenteric neurons in obesity. Sample from ileum of obese male (OBM) and females (OBF) (C57BL/6J ob/ob) and controls mice (CF, CM) (+/+) were used. Immunohistochemistry for double labelling in neurons P2X2 receptor immunostained with NOS (inhibitory), ChAT (excitatory) or Calr (excitatory and sensory) was performed. Results of CF and OBF groups: immunostaining was verified for P2X2 receptor, NOS, ChAT and Calr in the cytoplasm and in cell and nuclear membranes of myenteric neurons of both groups. The double-labelling for P2X2 receptor/NOS, ChAT or Calr in CF were 24 ±4%, 24 ±2% and 24 ±4%, respectively, and in OBF were 19 ±4%, 25 ±4% and 22±1%. Inversely, NOS, ChAT or Calr/P2X2 receptor immunoreactives neurons were 100% in both groups. The density of P2X2 neurons showed an increase of 62%, whereas NOS and ChAT neurons decreased by 49% and 57%. In morphometric analysis showed an increase in NOS (CF 234 ± 63, 312 ± 67 OBF), ChAT (CF 210 ± 24, 253 ± 14 OBF) and Calr (CF 203 ± 41, 315 ± 47 OBF) neuronal area, however, P2X2 neurons no changed in this feature (FC 325 ± 23, 336 ± 67 OBF). The histochemistry for NADH-diaphorase showed no statistical difference to density and morphometry of neurons between the groups. Results of CM and OBM groups: The P2X2 receptor expression was identified only in CM. Immunostaining for NOS, ChAT and Calr were observed in both groups. The double-labelling observed in CM for P2X2 receptor and NOS, ChAT or Calr were 23±3%, 34 ±5% and 32 ±6%, and inversely, NOS, ChAT or Calr/P2X2 receptor immunoreactives neurons were 100%. The NOS and Calr neuronal density were reduced about 31% and 16%, while population ChAT neuronal density increased about 31%. The morphometry showed that NOS (CM 390 ± 49, 350 ± 22 OBM), ChAT (289 ± 18, 312 ± 44 OBM), Calr (CM 375 ± 49, 360 ± 38 OBM) and P2X2 (CM 437 ± 190) neurons no differ between groups. The P2X2 receptor protein expression by Western blotting showed that there was a reduction in 36.5% in OBM. We concluded that in this experimental model, both obesity (C57BL/6J ob/ob) and genders may contribute to changes in P2X2 receptor expression, density and morphology of neurons NOS, ChAT and Calr, promoting alterations in intestinal activities.

Made available in DSpace on 2015-07-22T20:50:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-26 / Obesidade, hipertensão, dislipidemia e diabetes tipo 2 são os principais fatores de risco que caracterizam a síndrome metabólica quando presentes simultaneamente. Com o aumento dos níveis de leptina em quadros de obesidade e a correlação desta patologia com a hipertensão, objetivou-se neste trabalho o estudo das possíveis implicações do hormônio leptina na hipertensão de forma central e periférica e a interação com o principal sistema no controle da pressão arterial (PA), o sistema renina-angiotensina (SRA). Neste trabalho nós mostramos que camundongos com mutação espontânea no gene da leptina, ob/ob, ou no receptor de leptina, db/db apresentam deficiências no controle da PA e ritmo circadiano, alterações no SRA e disfunção autonômica. Em camundongos ob/ob, a atividade da ECA, uma das principais enzimas do SRA, apresentou-se reduzida em condições basais e foi restabelecida após o tratamento agudo com leptina. Cronicamente, a administração de leptina aumentou a atividade dessa enzima e a concentração de fatores que compõem o SRA, como o peptídeo vasoconstritor angiotensina II em camundongos ob/ob. No entanto, não houve aumento da PA. Os camundongos ob/ob apresentaram menor baroreflexo, diminuição da ativação parassimpática e aumento da ativação simpática. Os camundongos db/db também apresentaram o mesmo fenótipo. O tratamento com leptina provocou diminuição do peso e restabeleceu a disfunção autonômica em camundongos ob/ob. Surpreendentemente, o tratamento com enalapril também restabeleceu o tônus autônomo, simpático e parassimpático, assim como o baroreflexo em ob/ob e db/db sugerindo a importância da angiotensina II no controle autônomo concomitante com a ausência na sinalização de leptina. Nossos dados sugerem uma nova interação da via de leptina com o SRA, sendo a angiotensina II um possível fator necessário para a manutenção da ativação autonômica e sobrevivência desses camundongos. / The leptin deficient ob/ob mice are insulin resistant and obese. However, the control of blood pressure in this model is not well defined. The goal of this study was to evaluate the role of leptin and the renin-angiotensin system (RAS) in the cardiovascular abnormalities observed in obesity using a model lacking leptin. Leptin is a hormone related to metabolism. It also influences blood pressure, but the mechanisms triggered in this process are not yet elucidated. Angiotensin-I converting enzyme (ACE) regulates cardiovascular functions and recently has been associated with metabolism control and obesity. Here we tried to answer the question whether ACE and leptin could influence blood pressure control being a link between renin-angiotensin system and obesity in ob/ob mice, a model lacking leptin. These mice are obese and diabetic, but have normal 24h mean arterial pressure. Our results show that plasma and lung ACE activities as well as ACE mRNA expression were significantly decreased in ob/ob mice. In agreement with these findings, the hypotensive effect produced by enalapril administration was attenuated in the obese mice. Plasma renin, angiotensinogen, angiotensin I, bradykinin and angiotensin 1-7 were increased, whereas plasma angiotensin II concentration was unchanged in obese mice. Leptin chronic infusion increased renin activity and angiotensin II concentration in both groups and increased ACE activity in ob/ob mice. Acute leptin infusion could restore ACE activity in leptin deficient mice. Moreover, the effect of ACE inhibitor on blood pressure during leptin treatment was not changed in lean, but increased four times in obese mice. In a second part of the study we measured blood pressure in ob/ob and control animals by radiotelemetry combined with fast Fourier transformation before and after both leptin and enalapril treatment. Autonomic function was assessed pharmacologically. Blood pressure during daytime was slightly higher in the ob/ob compared to control mice while no difference in heart rate was observed. Blood pressure response to trimetaphane and heart rate response to metoprolol were greater in ob/ob mice than in control littermates indicating an activated sympathetic nervous system. Heart rate response to atropine was attenuated. Baroreflex sensitivity and heart rate variability were blunted in ob/ob mice, while low frequency of systolic blood pressure variability was found increased. Chronic leptin replacement reduced blood pressure and reversed the impaired autonomic function observed in ob/ob mice. Inhibition of ACE by enalapril treatment had similar effects prior loss of weight. These findings suggest that the RAS is involved in the autonomic dysfunction caused by the lack of leptin in ob/ob mice. In summary, our findings show that the RAS is altered in ob/ob mice, with markedly reduced ACE activity, which suggests a possible correlation between RAS and leptin. These results point to an important interplay between the angiotensinergic and the leptinergic systems and may contribute to clarify the role played by these systems in the pathogenesis of obesity, hypertension, and metabolic syndrome. / TEDE / BV UNIFESP: Teses e dissertações

As atividades intestinais são coordenadas pelo sistema nervoso entérico e, disfunções na motilidade intestinal são observadas em indivíduos obesos. O ATP é um co-transmissor e seus receptores estão distribuídos nos neurônios entéricos constituindo a família P2X e P2Y. O presente estudo tem como objetivo analisar a expressão do receptor P2X2 e o código químico nos neurônios mioentéricos na obesidade. Foram utilizados íleo de camundongos obesos machos (OBM) e fêmeas (OBF) (C57BL/6J ob/ob), e controles (CF; CM) (+/+), que foram submetidos à técnica imuno-histoquímica de duplas marcações em neurônios imunomarcados ao receptor P2X2 com NOS (inibitórios), ChAT (excitatórios) ou Calr (excitatórios e sensoriais). Resultados dos grupos CF e OBF: Foi verificado a imunomarcação ao receptor P2X2, NOS, ChAT e Calr no citoplasma e nas membranas celular e nuclear dos neurônios mioentéricos de ambos os grupos. As duplas marcações dos neurônios imunorreativos (-ir) ao receptor P2X2 com NOS, ChAT ou Calr em CF foram 24±4%, 24±2% e 24±4%, e em OBF foram 19±4%, 25±4% e 22±1%, respectivamente. As duplas marcações dos neurônios NOS-ir, ChAT-ir ou Calr-ir com os neurônios receptor P2X2-ir foram 100% nos dois grupos. A densidade dos neurônios P2X2-ir aumentou em 62%, enquanto que a dos neurônios NOS-ir e ChAT-ir reduziram em 49% e 57%. A morfometria demonstrou aumento na área dos neurônios NOS-ir (CF 234±63, OBF 312±67); ChAT-ir (CF 210±24, OBF 253±14) e Calr-ir (CF 203±41, OBF 315±47), no entanto, neurônios P2X2-ir não apresentaram alterações (CF 325±23, OBF 336±67). A histoquímica demonstrou não haver diferença estatística entre os grupos quanto á densidade e a morfometria dos neurônios NADH-diaforase positivos. Resultados dos grupos CM e OBM: A expressão do receptor P2X2 foi identificada somente em CM e a imunomarcação a NOS, ChAT e Calr nos dois grupos. Em CM as duplas marcações dos neurônios P2X2-ir com NOS-ir, ChAT-ir ou Calr-ir foram 23±3%, 34±5% e 32±6%, respectivamente, e nos neurônios NOS-ir, ChAT-ir e Calr-ir com o receptor P2X2 foram 100%. A densidade dos neurônios NOS-ir e Calr-ir reduziram em 31% e 16%, enquanto que a densidade da população ChAT-ir aumentou 31%. A morfometria demonstrou que neurônios NOS-ir (CM 390±49, OBM 350±22); ChAT-ir (289±18, OBM 312±44), Calr-ir (CM 375±49, OBM 360±38) e P2X2-ir (CM 437±190) não apresentaram diferenças entre os grupos. A expressão protéica pela técnica do Western Blotting mostrou que houve uma redução de 36,5% no grupo OBM. Concluímos que neste modelo experimental tanto a obesidade (C57BL/6J ob/ob) como os gêneros podem contribuir com alterações: na expressão do receptor P2X2, na densidade e na morfologia dos neurônios NOS-ir, ChAT-ir e Calr-ir, promovendo mudanças nas atividades intestinais. / The intestinal activities are coordinated by enteric nervous system, and dysfunctions in intestinal motility were observed in obese individuals. The enteric neurons have been demonstrated to express different purinergics receptors. This study aims to analyze the expression of P2X2 receptor and the chemical code in the myenteric neurons in obesity. Sample from ileum of obese male (OBM) and females (OBF) (C57BL/6J ob/ob) and controls mice (CF, CM) (+/+) were used. Immunohistochemistry for double labelling in neurons P2X2 receptor immunostained with NOS (inhibitory), ChAT (excitatory) or Calr (excitatory and sensory) was performed. Results of CF and OBF groups: immunostaining was verified for P2X2 receptor, NOS, ChAT and Calr in the cytoplasm and in cell and nuclear membranes of myenteric neurons of both groups. The double-labelling for P2X2 receptor/NOS, ChAT or Calr in CF were 24 ±4%, 24 ±2% and 24 ±4%, respectively, and in OBF were 19 ±4%, 25 ±4% and 22±1%. Inversely, NOS, ChAT or Calr/P2X2 receptor immunoreactives neurons were 100% in both groups. The density of P2X2 neurons showed an increase of 62%, whereas NOS and ChAT neurons decreased by 49% and 57%. In morphometric analysis showed an increase in NOS (CF 234 ± 63, 312 ± 67 OBF), ChAT (CF 210 ± 24, 253 ± 14 OBF) and Calr (CF 203 ± 41, 315 ± 47 OBF) neuronal area, however, P2X2 neurons no changed in this feature (FC 325 ± 23, 336 ± 67 OBF). The histochemistry for NADH-diaphorase showed no statistical difference to density and morphometry of neurons between the groups. Results of CM and OBM groups: The P2X2 receptor expression was identified only in CM. Immunostaining for NOS, ChAT and Calr were observed in both groups. The double-labelling observed in CM for P2X2 receptor and NOS, ChAT or Calr were 23±3%, 34 ±5% and 32 ±6%, and inversely, NOS, ChAT or Calr/P2X2 receptor immunoreactives neurons were 100%. The NOS and Calr neuronal density were reduced about 31% and 16%, while population ChAT neuronal density increased about 31%. The morphometry showed that NOS (CM 390 ± 49, 350 ± 22 OBM), ChAT (289 ± 18, 312 ± 44 OBM), Calr (CM 375 ± 49, 360 ± 38 OBM) and P2X2 (CM 437 ± 190) neurons no differ between groups. The P2X2 receptor protein expression by Western blotting showed that there was a reduction in 36.5% in OBM. We concluded that in this experimental model, both obesity (C57BL/6J ob/ob) and genders may contribute to changes in P2X2 receptor expression, density and morphology of neurons NOS, ChAT and Calr, promoting alterations in intestinal activities.

A Doença Hepática Gordurosa Não Alcoólica (DHGNA) é atualmente uma das formas mais comuns de doença hepática, e está diretamente relacionada com a obesidade. Estudos indicam uma prevalência por volta de 30% na população ocidental e 11% na população oriental. A DGHNA possui um largo espectro abrangendo desde casos de esteatose simples sem inflamação, até casos com esteatohepatite e fibrose, podendo evoluir para cirrose e carcinoma hepatocelular. A fisiopatogênese da DHGNA é baseada em múltiplos eventos: resistência insulínica, aumento da lipólise periférica, aumento da síntese de novo de lipídeos, estresse oxidativo, endotoxemia crônica no desencadeamento da inflamação e fibrose entre outros. A etapa inicial caracteriza-se pelo acúmulo de ácidos graxos no hepatócito, suplantando sua capacidade de metabolização e exportação conseqüente à ação lipogênica da insulina, desta forma o metabolismo de ácidos graxos está intimamente ligado ao desenvolvimento da DHGNA. O Yo Jyo Hen Shi Ko (YHK) é um composto natural usado na medicina japonesa para tratamento de doenças hepáticas e apresenta propriedades antioxidantes, antiinflamatórias e hipolipemiantes. O presente estudo teve como objetivo avaliar o efeito do YHK no metabolismo hepático de lipídeos. Para tanto, foram utilizados camundongos obesos (ob/ob) com esteatohepatite não alcoólica (ENA) induzida por dieta deficiente em colina e metionina (DCM) e foram analisados no tecido hepático, genes relacionados com a síntese de novo de lipídeos (SREBP1c, FASn), genes relacionados com a oxidação e exportação de lipídeos (CPT1a e SCD-1, MTP), assim como genes relacionados com o armazenamento de lipídeos (Perilipina e ADFP). O YHK apresentou um efeito citoprotetor hepático com melhora dos parâmetros histológicos neste modelo experimental de ENA. Associadamente, houve redução na expressão de genes relacionados à síntese de novo como SREBP e da FASn, quando se comparou o grupo tratado com DCM+YHK com o grupo não tratado DCM. Em contrapartida, houve aumento na expressão da MTP e da SCD-1 ocasionando uma maior exportação de triglicerídeos hepáticos nos animais que utilizaram o YHK. Ainda, o YHK modulou as proteínas Perilipina e ADFP. Por outro lado, não houve modificação na oxidação de lipídeos.. Conclui-se que o YHK pode ser uma droga promissora no tratamento da DHGNA, já que age modulando genes envolvidos na síntese e exportação de lipídeos hepáticos, reduzindo o acúmulo de gordura no hepatócito / Nonalcoholic fatty liver disease (NAFLD) is currently one of the most common forms of liver disease, related directly to the increase of obesity in the world. Previous studies indicate a prevalence of around 30% in the western population and 11% in the eastern. This disease covers cases from simple steatosis without inflammation to cases of steato hepatitis (NASH) and fibrosis and it may lead to liver cirrhosis and hepatocellular carcinoma. The pathophysiology of NAFLD is based on multiple events: insulin resistance, increased peripheral lipolysis, increased de novo lipogenesis, oxidative stress, endotoxemia in triggering chronic inflammation and fibrosis and others. The initial stage of the disease is characterized by the accumulation of fatty acids in hepatocytes caused by the lipogenic action of insulin. This condition results in the surpassing of the cells ability to metabolize and export. This way, the metabolism of fatty acids is closely linked to the development of NAFLD. The Yo Jyo Hen Shi Ko (YHK) is a natural compound used in Japanese medicine for the treatment of liver disease and it has antioxidant, anti-inflammatory and lipid lowering properties. This study aimed to evaluate the effect of YHK in the hepatic metabolism of lipids. An experimental model was designed using obese mice (ob/ob) affected by NASH induced by a deficient diet in choline and methionine (MCD). Then, genes from its hepatic tissue related to de novo lipogenesis (SREBP1c, FASN), to lipid oxidation and exportation (CPT1A and SCD-1, MTP) as well as genes related to lipid storage (Perilipin and ADFP) were analyzed. The YHK presented a cytoprotective effect, improving the hepatic histological parameters in this experimental model. Additionally, when comparing the group treated with MCD + YHK (diet + YHK) to the untreated MCD group (diet) there was a reduction in the expression of genes related to de novo synthesis like SREBP1c and FASn. However, an increased expression of MTP and SCD-1 were observed leading to a greater liver exportation of fatty acids in animals that were treated with YHK. Moreover, the YHK modulated the proteins Perilipin and ADFP. On the other hand, there was no changing in lipid oxidation. In summary, the YHK can be a promising drug for the treatment of NAFLD, as it acts by modulating genes involved in the synthesis and exportation of hepatic lipids, reducing the accumulation of fat in hepatocytes