Orientador: Antônio Carlos Cicogna / Resumo: Introdução: Embora existam contradições, de uma maneira geral, os dados da literatura mostram que em modelos experimentais de hipertrofia cardíaca por sobrecarga pressórica com disfunção ventricular e fração de ejeção preservada, o metabolismo de carboidratos e ácidos graxos está normal. Na fase em que há redução da capacidade de ejeção, com ou sem insuficiência cardíaca, ocorre um desvio para a programação fetal, aumento na utilização de glicose e diminuição na oxidação de ácidos graxos, por redução da expressão e atividade de proteínas envolvidas no metabolismo de lipídios. Não foram encontrados trabalhos que avaliaram o efeito de dieta hiperlipídica no metabolismo energético e função cardíaca após o estabelecimento da hipertrofia ventricular com disfunção diastólica isolada. Objetivo: Testar a hipótese de que o aumento na oferta de ácidos graxos insaturados, proveniente de dieta hiperlipídica, atenua a queda no metabolismo lipídico e a piora do desempenho cardíaco em ratos com hipertrofia ventricular e disfunção diastólica por sobrecarga pressórica. O mecanismo responsável seria o estímulo do PPARα pelos ácidos graxos, atenuando a queda na expressão de genes e proteínas envolvidas na regulação do metabolismo energético lipídico. Métodos: Ratos Wistar machos (80g) foram separados em dois grupos: controle operado (Sham) e estenose aórtica supravalvar (EAo). Após 6 semanas do procedimento cirúrgico, os animais Sham e EAo foram redistribuídos em novos grupos: tratados com diet... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Made available in DSpace on 2016-12-08T15:59:04Z (GMT). No. of bitstreams: 1 Tese de doutorado Lourenco Sampaio de Mara.pdf: 1379341 bytes, checksum: baa0c8e2efbdb57ed99a15951be025d1 (MD5) Previous issue date: 2013-03-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundamentação: A insuficiência cardíaca (IC) cursa com disfunções em diversos sistemas, repercussões na qualidade de vida (QV) e função sexual. A hipotestosteronemia é relevante na IC e fator contribuinte para o desbalanço catabolismo/anabolismo que integra a síndrome. Há pouco conhecimento a respeito dos efeitos do exercício de alta intensidade e terapia de suplementação de testosterona (TST) neste contexto. Hipótese: Exercício físico de alta intensidade e TST têm efeito sinérgico para um tratamento mais eficiente em pacientes com IC e baixos níveis de testosterona, inseridos em programa de reabilitação cardíaca (RC). Objetivo: Investigar os efeitos do exercício de alta intensidade e TST em pacientes com IC e baixos níveis de testosterona sérica participantes de programa de RC. Método: Dezenove pacientes portadores de IC (idade média de 58 anos; ± 10; fração de ejeção de 34 ±8%) foram randomizados para o grupo exercício alta intensidade (EAI) ou controle, (n=9) e grupo exercício de alta intensidade com suplementação de testosterona (EAIS) ou intervenção, (n=10). Pacientes exercitaram-se por 12 semanas e o grupo intervenção recebeu testosterona na primeira e sexta semanas. Antes e após o período de estudo foram obtidas medidas do teste cardiopulmonar, teste caminhada dos seis minutos (T6 ), ecocardiograma, função endotelial, erétil, perfil hormonal e da QV. Resultados: Houve aumento respectivamente intragrupos, do consumo máximo de oxigênio (12% e 15%; EAI e EAIS; p<0,05 e p<0,01), da distância percorrida no T6 (15% e 29%; EAI e EAIS; p<0,05 e p<0,01), da curva de eficiência de captação de oxigênio (22% e 14,2%; EAI e EAIS; p<0,05 em ambos os grupos), da velocidade máxima da onda E´ junto ao anel mitral septal (36%; EAI; p<0,05), da velocidade máxima da onda E´ junto ao anel mitral lateral (35%; EAI; p<0,05), do percentual de dilatação mediada pelo fluxo na artéria braquial (56% e 92%, EAI e EAIS, sem significância), dos escores da função erétil (150% e 59%, EAI e EAIS; p<0,01 e p<0,05), da testosterona total (78%, EAIS,p<0,01), da testosterona livre (89%; EAIS; p<0,01), da testosterona biodisponível (89%. EAIS; p<0,01), do hematócrito (8%, EAIS; p<0,01), do antígeno prostático específico (33%; EAIS; p<0,01). Houve diminuição da curva do equivalente ventilatório de dióxido de carbono (5%; EAIS; p<0,05), da relação da velocidade máxima da onda E com a velocidade máxima da onda E´ junto ao anel mitral septal (29%; EAI; P<0,05), do hormônio luteinizante (96%; EAIS; p<0,01), do hormônio folículo estimulante (84%; EAIS; p<0,01), do fator de necrose tumoral-&#945; (42% e 47%, EAI e EAIS; p<0,05 em ambos os grupos), do escore global do Minnesota Living with Heart Failure Questionnaire (54% e 54%;EAI e EAIS; p<0,01 e p<0,05), do domínio físico (52% e 54%; EAI e EAIS; p<0,01 e p<0,05), do domínio emocional (60%; EAIS; p<0,05), do domínio das questões remanescentes (58%; EAI; p<0,01). Entre grupos houve aumento a favor do EAIS nos níveis de testosterona total, testosterona livre e testosterona biodisponível (54%, p<0,05; 48%, p<0,05 e 48%, p<0,05 respectivamente), e diminuição a favor do EAIS nos níveis do hormônio folículo estimulante e luteinizante (83%, p<0,01 e 97%, p<0,01 respectivamente). Conclusões: Pacientes com IC e baixos níveis de testosterona submetidos a programa de exercícios de alta intensidade e TST apresentam melhora da capacidade funcional, dos índices de eficiência ventilatória, da função cardíaca, da QV e função erétil, contudo o estudo não corroborou a hipótese que a TST tem efeito sinérgico associado ao exercício físico de alta intensidade no tratamento destes pacientes.

Submitted by Loreta Casquel de Tomasi null (loretacasquel@hotmail.com) on 2017-09-15T21:44:29Z No. of bitstreams: 1 tese doutorado final.pdf: 1729009 bytes, checksum: a58f06348f3ca04d50ce35f533fd920a (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-09-19T17:57:56Z (GMT) No. of bitstreams: 1 tomasi_lc_dr_bot.pdf: 1729009 bytes, checksum: a58f06348f3ca04d50ce35f533fd920a (MD5) / Made available in DSpace on 2017-09-19T17:57:56Z (GMT). No. of bitstreams: 1 tomasi_lc_dr_bot.pdf: 1729009 bytes, checksum: a58f06348f3ca04d50ce35f533fd920a (MD5) Previous issue date: 2017-02-17 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introdução: Embora existam contradições, de uma maneira geral, os dados da literatura mostram que em modelos experimentais de hipertrofia cardíaca por sobrecarga pressórica com disfunção ventricular e fração de ejeção preservada, o metabolismo de carboidratos e ácidos graxos está normal. Na fase em que há redução da capacidade de ejeção, com ou sem insuficiência cardíaca, ocorre um desvio para a programação fetal, aumento na utilização de glicose e diminuição na oxidação de ácidos graxos, por redução da expressão e atividade de proteínas envolvidas no metabolismo de lipídios. Não foram encontrados trabalhos que avaliaram o efeito de dieta hiperlipídica no metabolismo energético e função cardíaca após o estabelecimento da hipertrofia ventricular com disfunção diastólica isolada. Objetivo: Testar a hipótese de que o aumento na oferta de ácidos graxos insaturados, proveniente de dieta hiperlipídica, atenua a queda no metabolismo lipídico e a piora do desempenho cardíaco em ratos com hipertrofia ventricular e disfunção diastólica por sobrecarga pressórica. O mecanismo responsável seria o estímulo do PPARα pelos ácidos graxos, atenuando a queda na expressão de genes e proteínas envolvidas na regulação do metabolismo energético lipídico. Métodos: Ratos Wistar machos (80g) foram separados em dois grupos: controle operado (Sham) e estenose aórtica supravalvar (EAo). Após 6 semanas do procedimento cirúrgico, os animais Sham e EAo foram redistribuídos em novos grupos: tratados com dieta normolipídica insaturada (Sham-N, n=13 e EAo-N, n=11) ou com dieta hiperlipídica insaturada (Sham-H, n=12 e EAo-H, n=14) por 12 semanas, até a 18ª semana. A remodelação cardíaca foi caracterizada pelas análises estrutural e funcional por ecocardiograma na 6ª e 18ª semana e estudo macroscópico post mortem. O metabolismo energético lipídico cardíaco foi analisado pela expressão gênica e proteica do PPARα, PGC1α, FAT/CD36, CPT1β, MCAD, pela atividade da beta-hidroxiacilCoA desidrogenase (OHADH) e pelo conteúdo de triacilglicerol (TAG). Foi analisada a atividade da hexoquinase (HK) e da fosfofrutoquinase (PFK), envolvidas na via glicolítica, e da citrato sintase (CS), referente ao ciclo de Krebs. A expressão proteica da lactato desidrogenase (LDH), da piruvato desidrogenase (PDH) e dos sensores metabólicos, AMPK total, AMPK fosforilada na treonina 172 e SIRT1 foram também analisadas, bem como as relações intracelulares AMP/ATP e NAD+/NADH. Resultados: Na 6ª semana, ao iniciar o tratamento dietético, os animais EAo apresentavam hipertrofia ventricular esquerda concêntrica, disfunção diastólica e melhoria da função sistólica. Na 18ª semana os grupos EAo mantiveram a disfunção diastólica e melhoria do desempenho sistólico, ou seja, não houve piora da performance cardíaca durante o período experimental de 12 semanas. Não houve diferença na estrutura e função cardíaca entre os grupos EAo-N e EAo-H. Houve diminuição da expressão dos genes relacionados à captura e oxidação de lipídios (CD36, CPT1β, MCAD) nos grupos EAo em comparação com os Sham, e não houve diferença entre os grupos EAo. A atividade da hexoquinase e fosfofrutoquinase foi maior nos EAo comparado com os Sham, e foi semelhante entre EAo-N e EAo-H. A atividade da OHADH não foi diferente entre EAo-N e Sham-N e também não diferiu entre EAo-N e EAo-H. O conteúdo de TAG miocárdico foi menor no grupo EAo-N vs Sham-N e não foi diferente entre os grupos EAo. A expressão das proteínas PGC1α, PPARα, CPT1β, MCAD, LDH, PDH, dos sensores SIRT1, AMPK, pAMPK Thr172, e a relação NAD+/NADH não foram diferentes entre os quatro grupos. Conclusão: Em contraste com a nossa hipótese, durante a evolução da remodelação cardíaca, os animais com estenose aórtica apresentaram alteração parcial no metabolismo lipídico miocárdico e não tiveram piora da função cardíaca. A dieta hiperlipídica insaturada não teve efeito no processo metabólico e na função cardíaca desses animais. Os mecanismos no qual a dieta hiperlipídica não foi capaz estimular o PPARα nos animais com estenose aórtica são desconhecidos. / Introduction: Although controversial, in general, data from literature show that in experimental models with ventricular dysfunction and preserved ejection fraction, the carbohydrates and fatty acids metabolism is normal. At the stage of reduced ejection capacity, with or without heart failure, there is a switch for fetal programming, increased glucose utilization, and decrease in fatty acid oxidation due to downregulation of proteins involved in lipids uptake and oxidation. There are no studies that evaluated the effects of high-fat diet on energy metabolism and cardiac function after the establishment of ventricular hypertrophy with isolated diastolic dysfunction. Objective: To test the hypothesis that increased unsaturated fatty acid supply, from a high-fat diet, attenuate the downregulation of lipid metabolism and the impairment of cardiac function in rats with left ventricular hypertrophy and diastolic dysfunction by stimulating genes and proteins involved in the regulation of lipid energy metabolism. Methods: Male Wistar rats (80g) underwent aortic stenosis (AS) or Sham surgery. After 6 weeks, rats received either normolipid diet (N, 17% calories from fat) or high-fat diet (H, 40% calories from fat) for 12 weeks yielding 4 groups: Sham-N (n=13), AS-N (n=11), Sham-H (n=12), AS-H (n=14). Cardiac structure and function was assessed by echocardiography at 6 and 18 weeks after surgery. Cardiac lipid energy metabolism was analyzed by gene and protein expression of PPARα, PGC1α, FAT/CD36, CPT1β, MCAD, the activity of beta-hydroxy-acyl CoA dehydrogenase (OHADH) and TAG content. We analysed the activities of hexokinase (HK) and phosphofructokinase (PFK), involved in the glycolytic pathway, and citrate synthase (CS), from Krebs cycle. The protein expression of lactate dehydrogenase (LDH), pyruvate dehydrogenase (PDH) and the metabolic sensors, total AMPK, AMPK phosphorylated on threonine 172 and SIRT1 were also evaluated, as well as the intracellular AMP/ATP and NAD+ /NADH ratios. Results: In the 6th week, prior to dietary treatment, the AS animals had left ventricular hypertrophy, diastolic dysfunction and improved systolic function. In the 18th week, AS animals kept diastolic dysfunction and improved systolic function, that is, there was no worsening of cardiac performance. There was no difference in cardiac structure and function between the AS-N and AS-H groups. There was a decrease in the expression of genes related to lipid uptake and oxidation (CD36, CPT1β, MCAD) in the AS groups compared to the Sham, and there was no difference between the AS groups. The activity of hexokinase and phosphofructokinase was higher in AS compared to Sham, and was similar between AS-N and AS-H. The activity of OHADH was not different between AS-N and Sham-N and also did not differ between AS-N and AS-H. TAG content was reduced in AS-N vs Sham-N and was not different between AS groups. Expression of the proteins PGC1α, PPARα, CPT1β, MCAD, LDH, PDH, the sensors SIRT1, AMPK, pAMPK Thr172, and the NAD+ /NADH ratio were not different among the four groups. Conclusion: In contrast to our hypothesis, during the progression of cardiac remodeling, animals with aortic stenosis showed partial alterations in myocardial lipid metabolism and did not present worsening of cardiac function. The high-unsaturated fat diet had no effect on the metabolic process and cardiac function of these animals. The mechanisms in which the high-fat diet was unable to stimulate PPARα in animals with aortic stenosis is unknown. / FAPESP: 2012/19679-0

Heart disease is the leading cause of death in women in the United States. Premenopausal women appear to have better cardiac function and lower risk of heart disease compared to male postmenopausal female counterparts. Ovarian hormone loss influences blood pressure homeostasis and causes systemic inflammation, which may result in chronic stress on the heart. Two key physiological changes in cardiac dysfunction are reemergence of the fetal gene pattern and myocardial remodeling. Physical activity has been linked to improved cardiac function. The purpose of this study was to investigate the effects of ovariectomy on early markers of cardiac dysfunction and fibrosis and to determine if voluntary physical activity alters expression patterns in ovariectomized mice. We investigated the effects of ovariectomy and exercise on cardiac expression of fetal genes and markers and mediators of fibrosis in two cohorts of 8-10 week old female mice. Ovariectomized mice had greater expression of cardiac fetal genes and real time-PCR (RT-PCR) results indicated activation of the fibrosis pathway. Exercise was able to influence the expression of some markers of cardiac dysfunction. We concluded that ovarian hormone loss and associated physiological changes such as increased adiposity and systemic inflammation trigger early changes in cardiac gene expression that precede overt cardiac dysfunction.

No description available.

PhD / The overall objective of this thesis is to provide additional data to assist clinicians and experimental neurologists alike in the quest for better understanding, more accurately diagnosing and more successfully treating patients suffering from Parkinson’s disease (PD). The general theme of the thesis is the interaction between certain environmental stimuli, including the exposure to adverse events during early central nervous system (CNS) development and the manifestation of elements of neurodegeneration, whether by means of neurochemical changes or expressed as a dysfunctional voluntary motor system. The first chapter provides a general introduction to the research theme of the thesis. This includes, in particular, a discussion on current understanding concerning the etiology and clinical profile of PD, the relative contribution made by genetic factors compared to environmental ones, and current treatment strategies for treating the disease. Mention is also made of the failure of these therapeutic applications for reversing or protecting against the disease, due to the side-effects associated with them. The material covered in chapter 1 provides the basis for the more complete discussion concerning these various aspects, contained in the chapters to follow. The overall aim was also to characterise the effects of commonly used toxin-induced animal models of PD, and the extent of vulnerability that the CNS displays towards them. The destruction of dopaminergic neurons following the administration of 6-OHDA at targeted points along the nigrostriatal tract is used extensively to model PD pathology in rats and is an established animal model of the disease. However, mature or even aged animals are mainly used in these studies, while the effects that the toxin might have on the developing CNS remain unclear. The study reported in chapter 4 aimed to elucidate some of 6-OHDA’s actions on the young adolescent (35 days-old) CNS by comparing the motor and biochemical effects of a unilateral infusion of the toxin into two anatomically distinct basal ganglia loci: The medial forebrain bundle (MFB) and the striatum. Animals were randomly assigned to receive either a direct delivery of 6-OHDA (12μg/4μl) into the MFB or an indirect injection, into the striatum. Although both lesion types were used, the MFB model is considered a more accurate portrayal of end-stage PD, while the striatum-model better reflects the long-term progressive pathology of the disease. The different lesions’ effects on motor function were determined by observing animal’s asymmetrical forelimb use to correct for weigh shifting during the vertical exploration of a cylindrical enclosure. Following the final behavioral assessment, the concentration of dopamine (DA) and DA metabolites remaining in the post-mortem brains were determined using 4 HPLC electrochemistry (HPLC-EC) and the levels compared between the two groups. The HPLC-EC results revealed a compensatory effect for DA production and DA turnover on the lesioned hemisphere side of the toxin-infused animal group. Thus, following 6-OHDA treatment, there appears to be extensive adaptive mechanisms in place within the remaining dopaminergic terminals that may be sufficient for maintaining relatively high extracellular and synaptic concentrations of DA. However, since substantial changes in motor-function were observed, it is suggested that the capacity of the remaining dopaminergic neurons to respond to increased functional demands may be limited. In addition, the behavioral results indicate that the distinct indices relating to different functional deficits depend on the lesioning of anatomically distinct structures along the nigrostrial tract. It has long been known that far fewer women are diagnosed with PD than men are. This seeming protection offered to females against degenerative disease of the CNS may relate to estrogen, although the hormone’s mechanism of action on the dopaminergic system is poorly defined. With an estimated 10-15 million women using oral contraceptives (OCs) in the United States alone, the aim of chapter 2 was to examine the evidence for a possible relationship between PD and the female reproductive hormone estrogen. A review of the current literature available on the topic was performed by consulting Medline, and by performing a search of the case-reports contained within the World Health Organization’s (WHO) International Drug Monitoring database, for possible PD-related symptoms that may arise from estrogen replacement therapy (ERT). The results, whilst conflicting, seem to suggest that estrogen protects women from obtaining the disease, or at least some features of it. Intensive research efforts are called for, with sufficient power to establish the relationship between ERT and the onset and development of parkinsonism. Chapter 3 reports on the results obtained from an experiment that subjected young Sprague-Dawley rats, 35 days of age, to a lower and a higher dose of 6-OHDA delivered to the MFB. Control rats received equivalent saline infusions. At 14 days post-surgery, the rats were evaluated for forelimb akinesia. For the higher dose of 6- OHDA the female rats were less impaired than males in making adjustment steps in response to a weight shift and in the vibrissae-evoked forelimb placing test. In addition, Tyrosine hydroxylase (TH) immunoreactivity was significantly higher for the female rats. Early gender differences in cell survival factors and/or other promoters of neuroplasticity may have contributed to the beneficial outcome seen in the females. For example, nerve growth factor (NGF) was found to be higher in the female rats following administration of the DA neurotoxin. It is unclear whether gonadal steroids are involved, and, if so, whether female hormones are protective or whether male hormones are prodegenerative. Determining the mechanisms for the improved outcome seen in the young female rats may lead to potential treatment strategies against PD. 5 Many studies have shown that early life stress may lead to impaired brain development, and may be a risk factor for developing psychiatric diseases, including clinical depression. However, few studies have investigated the impact that early stress may have on the onset and development of neurodegenerative disorders such as PD. The study reported on in chapter 5 conjointly subjected rat pups to a maternal separation (MS) paradigm that is a well characterised model of adverse early life events, and a unilateral, intrastriatal injection of 6- OHDA. The combined effects of these models on motor deficits and brain protein levels were investigated. Specifically, the animals were assessed for behavioral changes at 28 days postlesion with a battery of tests that are sensitive to the degree of DA loss sustained. The results show that animals that had been subjected to MS display poorer performance in the vibrissae and single-limb akinesia test compared to non-MS control animals (that had also been subjected to the toxin exposure). In addition, there was a significant increase in the loss of TH staining in MS rats compared to non-MS ones. The results from this study therefore suggest that exposure to adverse experiences during the early stages of life may contribute towards making dopaminergic neurons more susceptible to subsequent insults to the CNS occurring during mature stages of life. Therefore, taken together, early exposure to stress may predispose an individual towards the onset and development of neurodegenerative disease, which especially becomes a threat during the later stages of adult life. Moreover, within the framework of these characteristics, the capacity of a widely-used pharmacological agent (statins) was tested for possible future therapeutic application in PD (chapter 7). Although the precise cause of sporadic PD remains an enigma, evidence suggests that it may associate with defective activity of complex I of the mitochondrial electron transport chain. Mitochondrial DNA transmit and express this defect in host cells, resulting in increased oxygen free radical production, depressed antioxidant enzyme activities, and greater susceptibility to apoptotic cell death. Simvastatin is a member of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) group of drugs that are widely used for lowering cholesterol levels in patients who display elevated concentrations of low-density lipoprotein cholesterol. The study aimed to investigate the effects that statin-treatment have on motor-function and at the mitochondrial-protein level, using rotenone, a mitochondrial complex I inhibitor, as a rat-model of PD. Adult male Sprague-Dawley rats were treated either with simvastatin (6mg/day for 14 days) or with a placebo. Two different tests to assess motor function were used: The apomorphine-rotation test, and the vibrissae-elicited forelimb placement test. Following the drug administration protocol, the nigrostriatal tract was unilaterally lesioned with either rotenone (3 μg/4 μl) or, for the controls, were sham-operated by infusing the vehicle (DMSO:PEG) only. Five days later the rats were killed and a highly purified concentration of isolated mitochondria was prepared from the substantia nigra (SN) sections. 2- 6 Dimensional electrophoresis (2-DE) with subsequent identification of the spots using electronspray ionization quadruple time-of-flight mass spectrometrical (ESI-Q-TOF MS) was performed and the results BLAST-searched using bio-informatics tools for naming the identified peptides. The motor test results indicate that while unilateral rotenone causes behavioral asymmetries, treatment with simvastatin improved motor function relative to the rotenoneinduced ones. Mass Spectroscopy identified 23 mitochondrial proteins that differ significantly in protein expression (p < 0.05) following simvastatin treatment. The altered proteins were broadly classified according to their cellular function into 6 categories, with the majority involved in energy metabolism. This study effectively illustrated how neuroproteomics, with its sophisticated techniques and non-biased ability to quantify proteins, provides a methodology with which to study the changes in neurons associated with neurodegeneration. As an emerging tool for establishing disease-associated protein profiles, it also generates a greater understanding as to how these proteins interact and undergo post-translational modifications. Furthermore, due to the advances made in bioInformatics, insight is created concerning their functional characteristics. Chapter 4 summarises the most prominent proteomics techniques and discuss major advances made in the fast-growing field of neuroproteomics in PD. Ultimately, it is hoped that the application of this technology will lead towards a presymptomatic diagnosis of PD, and the identification of risk factors and new therapeutic targets at which pharmacological intervention can be aimed. The final chapter (chapter 8) provides a retrospective look at the academic work that had been performed for the purpose of this thesis, recaps on the main findings, and also highlights certain aspects of the project and provides relevant suggestions for future research. Lastly, the appendix provides a detailed overview of the methods followed for the experiments described in this thesis. It provides not only a comprehensive description of the techniques that had been followed, but provides information concerning the care taken with the animals (i.e. post-surgery) in order to control for the potential influence of experimental variables on the results.