Resumo: As plaquetas são fragmentos do citoplasma do megacariócito e possuem papel fundamental na manutenção da hemostasia. Ao aderirem umas às outras, ativam-se, mudam de forma e liberam grânulos que contêm fatores de crescimento, proteínas que atuam na divisão celular. Os grânulos α- plaquetários secretam fatores de crescimento que regulam eventos, tais como, síntese de DNA, quimiotaxia e citodiferenciação. A estocagem prolongada das plaquetas a 22ºC é prejudicial à viabilidade e à função, mesmo quando estocadas em condições adequadas nos bancos de sangue. Observa-se diminuição da concentração do glutation intraplaquetário, representando diminuição da proteção contra o stress oxidativo, do qual resulta a perda do grupo sulfidril. As plaquetas desempenham funções com consumo de energia, como exemplo a diapedese, que pode ser recuperada após cinco dias de armazenamento pelo azul de metileno. Durante a divisão celular há necessidade de bloqueio das funções citoplasmáticas com aumento da produção de proteínas de adesão para que ocorra a fixação da célula que vai se dividir. Os resultados mostraram que quando adicionamos azul de metileno, a quantidade de células viáveis diminui com aumento de apoptose e necrose, muito provavelmente devido à liberação de energia / Abstract: Platelets are megakaryocyte cytoplasmic fragments that play a fundamental role in maintaining hemostasis. When they adhere to each other, they become activated, change shape and release granules containing growth factors, proteins involved with cell division. Platelet α-granules secrete growth factors that regulate events such as DNA synthesis, chemotaxis and cytodifferentiation. Long platelet storage at 22ºC is harmful to their viability and function, even when they are stored under proper conditions at blood banks. A reduced concentration of intraplatelet glutathione is observed, representing decreased protection against oxidative stress, from which the loss of the sulfhydryl group results. Platelets perform actions with energy consumption, such as diapedesis, which can be recovered after five storage days by methylene blue. During cell division, cytoplasmic functions must be blocked by increased adhesion protein so that the fixation of the dividing cell can occur. Results showed that when methylene blue is added, the number of viable cells decreases with apoptosis and necrosis increase, which is very likely due to energy release / Orientador: Paulo Eduardo de Abreu Machado / Coorientador: Márjorie de Assis Golim / Banca: Izolete Aparecida Thomazini Santos / Banca: Sueli Gonçalves Saez / Mestre

Le retard de croissance intra-utérin (RCIU) reste une complication fréquente de la grossesse et expose non seulement à une mortalité néonatale plus élevée, mais également au risque d'un cortège de pathologies chroniques (cardio-métaboliques) à l'âge adulte. Étant un fardeau important pour la santé publique dans le monde entier, il n'existe pas encore à ce jour de traitement curatif autre qu'une extraction plus. précoce avec éventuellement une prématurité. Une étude récente de notre équipe a montré que l'administration de citrulline pendant la gestation améliore la croissance et la synthèse protéique fœtales dans un modèle de RCIU induit chez la rate par la restriction sévère en protéines. L'objectif de ce travail de thèse était donc d'explorer les mécanismes d'action de la supplémentation en citrulline pendant la période périnatale (gestation et allaitement) dans le même modèle. Notre première partie de ce travail confirme que la citrulline stimule la croissance en nous montrant que la supplémentation anténatale en citrulline agit dès le milieu de la gestation sur des gènes codant pour des facteurs de croissance, d’angiogenèse et de transporteurs d'acides aminés pour aboutir à une efficacité placentaire renforcée jusqu'à la fin de la gestation fœtale. Dans la deuxième partie, nous n'avons pas mis en évidence d'effet bénéfique de cette supplémentation postnatale chez des ratons nés dénutris, puis soumis à un régime déséquilibré riche en fructose, ni en termes de croissance, ni sur le métabolisme glucido-lipidique à l'âge adulte jeune. La vigilance impose davantage d'explorations mécanistiques avant d'envisager une étude translationnelle clinique à ce stade sensible de développement qu'est la période néonatale. En revanche, dans le cas de la période gestationnelle, nos résultats incitent à réfléchir à la mise en place d'essais cliniques pertinents pour cette pathologie de la croissance fœtale. / Intra-uterine growth restriction (IUGR) remains a common pregnancy-related complication resulting not only in a significant neonatal mortality, but in an increased risk of chronic cardio-metabolic diseases in adulthood as well. This condition represents a serious burden to public health across the world due to lack of a curative treatment except early cessation of gestation with induced prematurity. Developing alternative strategies aimed towards targeted therapy would thus be highly desirable. In recent studies, we showed that citrulline supplementation during gestation in rats under severe dietary protein restriction enhanced fetal growth and protein synthesis. The objective of this work was to further investigate the mechanisms mediating the effect of citrulline during the perinatal period, i.e. gestation and nursing in the same model. In the first part, we confirmed that citrulline improved fetal growth, and further demonstrated that citrulline activated placental genes coding for growth factors, angiogenesis and amino acid transporters early from mid-gestation, resulting in improved fetal weight. However, in the second part of the current work, we failed to observe any beneficial effect of neonatal citrulline supplementation neither on growth, nor on the prevention of alterations of glucose and lipid metabolism in IUGR rats that were later exposed to an unbalanced, fructose-enriched diet. Therefore, further explorations are needed for a better mechanistic understanding before postnatal citrulline supplementation can be considered in translational trials. Otherwise, the results obtained in the gestational period in this work suggest clinical trails should be envisioned for prenatal citrulline supplementation in targeted populations of patients.

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Pathological cardiac hypertrophy (PCH) occurs in response to pathological stimuli affecting the heart such as coronary artery disease, myocardial infarction, or hypertension. PCH is also be independent risk factor for cardiac events and/or sudden death. Despite therapeutic advancements in the treatment of cardiovascular diseases (CVD) and heart failure, deaths due to CVD remain the leading cause of mortality worldwide. Furthermore, treatment of these cardiovascular diseases slows their progression, but individuals eventually progress to heart failure, which has a 5-year survival rate of approximately 50 percent. There is a clear need for development of new therapies that can reverse PCH and the associated damage to the heart. As healthcare improves, populations are living longer, and illness due to age increases. One issue that occurs with aging is loss of normal cardiac function leading to heart failure. This functional decline is accompanied by morphological changes in the heart, including hypertrophy. Although it is well documented that myocardial remodeling occurs with aging, the mechanisms underlying these changes are poorly understood. Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor β (TGF-β) superfamily of proteins, which regulate a number of cellular processes. Shared circulation of a young mouse with an old mouse or a single daily intraperitoneal (IP) injection of GDF11 for 30 days was shown to reverse aging-induced pathological cardiac hypertrophy. This molecule is highly homologous with another TGF-β family member, myostatin, which is a known negative growth regulator of skeletal muscle. We began by attempting to validate published data claiming that a single daily intraperitoneal (IP) injection of 0.1 mg/kg/day of GDF11 could reverse aging induced cardiac hypertrophy. We performed a blinded study during which treated 24-month-old C57BL/6 male mice with a single IP injection of 0.1 mg/kg/day of GDF11for 28 days and monitored changes in cardiac function and structure using echocardiography (ECHO). We also looked for differences in fibrosis, myocyte size, markers of pathological hypertrophy and heart weight. We were unable to find any differences between vehicle treated age mice and GDF11 treated aged mice in any of the measured parameters. While we did find an increase in heart weight between 8-week-old mice and the 24-month-old mice, there was no difference in the heart weight to body weight ratios of these groups of animals. From these data we concluded that our aged- mice did not have pathological hypertrophy and the dose of GDF11 used in this study did not have any effect on cardiac structure or function. Hypertensive heart disease results in changes in cardiac structure and function including left ventricular hypertrophy, systolic and/or diastolic dysfunction. It is also a leading cause of heart failure. Members of the TGF-β superfamily of proteins have been shown to be involved in many of the processes that occur in the heart in response to hypertension, such as the fibrotic response. Although it was previously shown that treatment with 0.1 mg/kg of GDF11 did not prevent pressure overload induced cardiac hypertrophy, we found this dose was too low to alter cardiac structure in our aging study. In addition, a single GDF11 dose is insufficient to fully address this issue. We therefore performed a blinded dose-ranging study to investigate the effects of GDF11 on pressure overload induced cardiac hypertrophy using transverse aortic constriction (TAC) which mimics the effects of chronic hypertension on the heart. In this study, animals received TAC surgery and were assigned to treatment groups so that there were no differences in wall thickness, cardiac function, or pressure gradients across the aortic constriction at the start of the treatments 1 week after TAC. Mice were given 0.5 mg/kg/day of GDF11, 1.0 mg/kg/day GDF11, 5.0 mg/kg/day of GDF11, or vehicle via a single daily IP injection for 14 days. Using these higher doses, we found that GDF11 had dose dependent effects on both cardiac structure and function following TAC. Myocyte cross sectional area was dose-dependently decreased compared to vehicle treated mice in both sham and TAC conditions. Cardiac function was preserved in the 1.0 and 5.0 mg/kg groups treatment groups after TAC. Left ventricular internal chamber dimensions were preserved with the 1.0 mg/kg treatment group. Treatment with GDF11 caused a dose dependent decrease on both body weight and heart weight in both normal and TAC mice, but with an effect on heart weight in the TAC mice that was independent of body weight. However, the 5.0 mg/kg dose caused large reductions in body weight (cachexia) and death. Our results show that GDF11 can reduce pathological hypertrophy and cardiac remodeling after pressure overload, but there is a narrow therapeutic range. / Biomedical Sciences

Thesis (M.C.E.)--University of Delaware, 2008. / Principal faculty advisor: Babatunde Ogunnaike, Dept. of Chemical Engineering. Includes bibliographical references.

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