Diversos estudos demonstraram que a administração aguda de leucina é capaz de atenuar a perda de massa muscular que ocorre no envelhecimento. Contudo, poucos estudos verificaram o efeito crônico dessa suplementação na composição corporal e em parâmetros metabólicos. O presente estudo analisou o efeito da suplementação com leucina durante 40 semanas na composição corporal e nos parâmetros bioquímicos e teciduais de ratos envelhecidos. Para isso, foram utilizados 40 animais Sprague-Dawley, com 6 meses de idade, os quais foram distribuídos em três grupos experimentais. Grupo adulto (n=10), ratos eutanaziados no início do experimento; Grupo Controle, recebeu ração contendo uma mistura de aminoácidos não essenciais por 40 semanas, e Grupo Leucina, recebeu ração contendo 4% de leucina por 40 semanas. Durante o experimento foi avaliado o consumo alimentar e o peso corporal. Após as 40 semanas de experimento os animais foram eutanaziados com intervalo de 5 horas de jejum (período pós-prandial) para coleta de soro sanguíneo e tecidos. Avaliaram-se: a composição química da carcaça; a massa de órgãos e dos músculos gastrocnêmio e sóleo; as concentrações de proteína e RNA no fígado e no músculo gastrocnêmio; o aminograma sanguíneo; as concentrações séricas de IGF-1, insulina, proteínas totais, albumina, glicose e perfil lipídico. Em relação ao peso corporal, o grupo leucina apresentou menor massa corporal, quando comparado ao grupo controle (p<0,005). No entanto, não houve diferença entre o grupo leucina e o controle em relação ao consumo alimentar. Ambos os grupos de animais envelhecidos apresentaram aumento da gordura corporal em comparação aos animais adultos, porém o grupo leucina apresentou menor conteúdo de gordura corporal (somatória dos depósitos de gorduras e porcentagem de gordura total) do que o grupo controle. Os animais envelhecidos dos grupos leucina e controle apresentaram maiores concentrações de glicose, triglicérides, colesterol, HDL, LDL, VLDL e insulina quando comparados ao grupo adulto. Os resultados obtidos permitem sugerir que a suplementação crônica com leucina não atenua a perda de massa muscular que ocorre durante o envelhecimento, porém é eficaz em reduzir a gordura corporal de ratos. Entretanto, essa redução de gordura não foi acompanhada de melhora em parâmetros indicativos da homeostase glicêmica e lipídica. / Several studies have demonstrated that acute administration of leucine is able to attenuate muscle mass loss which occurs during aging. However, few studies have assessed the chronic effects of this supplementation on the body composition and metabolic parameters. The present study analyzed the effect of leucine supplementation during 40 weeks on body composition, biochemical and tissue parameters in aged rats. For that, 40 Sprague-Dawley rats, 6 months old, were distributed into three experimental groups. In the Adult Group (n=10) rats were sacrificed at the beginning of the experiment; the Control Group received a diet containing a mix of non-essential amino acids during 40 weeks, and the Leucine Group received a diet containing 4% leucine during 40 weeks. Food consumption and body weight were evaluated during the experiment. After 40 weeks of experiment, the animals were sacrificed after a 5-hour fast (post-prandial period) for serum and tissue sampling. The following parameters were evaluated: carcass chemical composition; mass of organs and gastrocnemius and soleus muscles; concentration of protein and RNA in the liver and in the gastrocnemius muscle; blood profile of amino acids; serum concentrations of IGF-1, insulin, total proteins, albumin, glucose and lipid profile. Concerning body weight, the leucine group showed a lower body when compared to control group (p<0,005). There was no difference between the leucine group and the control group concerning food consumption. Both groups of aged animals showed increased body fat compared to adult animals, but the leucine group showed a lower body fat content (sum of fat depositions and percent of total fat) than the control group. Both in the leucine and the control groups showed higher concentrations of glucose, triglycerides, cholesterol, HDL, LDL, VLDL, and insulin compared to the adult group. The results obtained in this work allow us suggest that chronic leucine supplementation did not attenuate the loss of muscle mass that occurs during aging, but it is effective in reducing body fat in rats. However, this reduction of fat was not accompanied by improvement in parameters indicative of glucose homeostasis and serum lipid profile.

Pannexin 1 (Panx1) is a metabolite channel enriched in the brain and known to localize to the cell surface, where it is involved in a variety of neuronal processes including cell proliferation and differentiation. The mechanisms through which Panx1 is trafficked or stabilized at the surface, however, are not fully understood. The proximal Panx1 C-terminus (Panx1CT), upstream of a caspase-cleavage site has been demonstrated to be required for Panx1 cell-surface expression. We discovered a previously unreported putative leucine-rich repeat (LRR) motif within the proximal Panx1CT. I investigated the involvement of this putative LRR motif on Panx1 localization and oligomerization. Deletion of the putative LRR motif or uniquely the highly conserved segment of the putative LRR motif resulted in a significant loss of Panx1 cell surface expression. Finally, ectopic expression of Panx1-EGFP in HEK293T cells increased cell proliferation, which was not recapitulated by a Panx1 deletion mutant lacking the putative LRR motif. Overall the findings presented in this thesis provide new insights into the molecular determinants of Panx1 trafficking and oligomerization. / Graduate / 2020-02-14

Neste trabalho, avaliamos os efeitos da suplementação crônica de leucina e do treinamento de força sobre a miopatia diabética. 40 ratos machos da linhagem Wistar Hannover foram distribuídos em 5 grupos: controle, não diabético (C), diabético não tratado (D), diabético treinado (DT), diabético suplementado com leucina e treinado (DLT). O início das intervenções ocorreu na 4ª semana de vida dos animais, e perdurou por 8 semanas. Foram avaliados: massa corporal, consumo de ração e água, concentrações sanguíneas de glicose, insulina e perfil lipídico; capacidade funcional muscular voluntária através de testes de força de preensão e de ambulação; conteúdo intracelular de proteínas relacionadas à via anabólica mTOR e p70S6K, totais e fosforiladas, no músculo extensor longo dos dedos. Os ratos diabéticos não tratados (grupo D) apresentaram hiperglicemia e hipoinsulinemia moderada, menor massa corporal, maior consumo de ração e água, menor peso absoluto dos músculos extensor longo dos dedos e gastrocnêmio, menor força de preensão, menor capacidade de ambulação e menor atividade das proteínas mTOR e p70S6K comparado ao grupo C, o que caracteriza o quadro de miopatia diabética. O peso relativo do músculo gastrocnêmico (peso absoluto/100g de peso do animal) foi maior nos grupos DT e DLT comparado com o grupo D, e maior no grupo DLT comparado com o grupo DL (p < 0,05). Não houve diferença estatística entre os grupos DL e D sobre os pesos relativos dos músculos, ou seja, a suplementação crônica de leucina não afetou este parâmetro nos ratos diabéticos. Interessantemente, houve diferença estatística entre os grupos DL e D sobre a força muscular (p < 0,05), sem haver diferença entre grupos DL e C quanto à glicemia; ou seja, a dieta suplementada com leucina foi capaz de controlar a glicemia e atenuar a perda de força muscular. O treinamento de força também controlou a glicemia, recuperou a força muscular e melhorou a capacidade de ambulação, bem como a regulação da via mTOR-p70S6K. A fosforilação da via mTOR-p70S6K foi maior nos grupos DT e DLT comparado com o grupo D (p < 0,05), e sem diferença entre estes grupos treinados e o grupo C, sugerindo que o treinamento de força combinado com a suplementação de leucina recuperou a atividade da via do mTOR-p70S6K nos animais diabéticos, que pode refletir em maior síntese proteica muscular. O colesterol total do grupo D foi maior comparado com o do grupo C; e nos grupos diabéticos treinados (DT e DLT), este parâmetro foi menor do que no grupo D (p < 0,05). Adicionalmente, o HDL-c aumentou nos grupos treinados (DT e DLT) quando comparado com o grupo D, mas não alterou no grupo que recebeu apenas a suplementação de leucina (grupo DL). Portanto, neste estudo, a suplementação crônica de leucina por si só normalizou a glicemia e melhorou a força muscular dos animais diabéticos. Além disso, o treinamento de força foi responsável pelo maior aumento de força e da massa muscular, bem como pela normalização da glicemia, pela elevação da concentração de HDL-c e pela redução do colesterol total dos animais diabéticos e ambas foram capaz de recuperar a via mTOR-p70S6K. / In this study, we evaluated the effects of chronic supplementation with leucine and resistance training on diabetic myopathy. 40 Wistar Hannover rats were divided into 5 groups: control, non-diabetic (C), untreated diabetic (D), trained diabetic (DT), diabetic supplemented with leucine and trained (DLT). The beginning of the interventions occurred in the 4th week of life of the animals, and lasted for 8 weeks. Were evaluated: body weight, food and water intake, blood concentrations of glucose, insulin and lipid profile; voluntary muscle functional capacity through grip strength and ambulation test; intracellular content of proteins related to the anabolic mTOR and p70S6K pathway, total and phosphorylated in the extensor digitorum longus muscle. Diabetics untreated mice (group D) had hyperglycemia and moderate hypoinsulinemia, lower body mass, food and water intake, reduced absolute weights of the muscles of the long extensor digitorum, and gastrocnemius, the lower grip strength, lower ambulation capacity and lower activity of mTOR and p70S6K protein compared the C group, featuring diabetic myopathy. The relative weight of the gastrocnemius muscle (absolute weight / 100g of body weight) was greater in DT and DLT groups compared with group D, and higher in the DLT group compared to the DL group (P < 0.05). No statistical difference between the DL and D groups on the relative weights of the muscles, that is, chronic supplementation of leucine did not affect this parameter in diabetic rats. Interestingly, there was statistical difference between the DL and D groups on muscle strength (P < 0.05), with no difference between groups DL and C on the blood glucose; that is, the diet supplemented with leucine was able to control glycemia and avoid loss of muscle strength of diabetic animals. Resistance training also controlled glycemia, recovered muscle strength and improved the capacity of ambulation of diabetic animals and the regulation of the mTOR-p70S6K pathway. The phosphorylation of mTOR-p70S6K pathway was higher only in the DT and DLT groups compared with the D group (P < 0.05), and no difference between the DT and C groups, suggesting that the training recovered muscle mass in diabetic animals. Total cholesterol was greater in Group D compared to the group C; and trained diabetic groups (DLT and DT), this parameter was lower than that of the D group (P < 0.05). In addition, HDL-C increased in trained groups (DT and DLT) as compared to group D, but had no effect the group that received only leucine supplementation (DL group). Therefore, in this study, chronic supplementation of leucine alone normalized glucose and improved muscle strength of diabetic animals. In addition, resistance training was responsible for the largest increase in strength and muscle mass, as well as the normalization of glucose, elevated concentrations of HDL-C and reduction in total cholesterol of animals diabetics and both were able to recover mTOR- p70S6K pathway.

A hiperglicemia crônica no diabetes está relacionada com distúrbios nas vias de sinalização da insulina e do mTOR, e com o desbalanço na secreção de adipocinas pelo tecido adiposo branco (TAB). Em longo prazo, esta disfunção metabólica pode causar uma perda severa de massa adiposa, o que agrava a resistência à insulina (RI). Estudos têm destacado o potencial efeito da suplementação com leucina no tratamento de doenças metabólicas como o diabetes tipo 2 e obesidade. No entanto, os efeitos da leucina sobre a homeostase glicêmica e a sensibilidade à insulina em doenças em que ocorre perda severa de gordura ainda necessitam melhores esclarecimentos. Portanto, foi investigado se a suplementação crônica com leucina pode afetar a adiposidade de ratos diabéticos com perda intensa de TAB, e melhorar a RI e outras desordens metabólicas relacionadas com TAB. Ratos recém-desmamados foram distribuídos em 3 grupos: i) Grupo controle (C) - não diabético e recebiam ração controle; ii) Grupo diabetes (D) - diabético e recebiam ração controle; iii) Grupo diabetes Leucina (DL) - diabético e recebiam ração suplementada com 5% de L-leucina. Após 8 semanas, foram analisados: glicemia e insulinemia de jejum, HOMAIR, citocinas pro- e anti-inflamatórias no soro e tecido adiposo branco, expressão de proteínas (mTOR, p-MTOR, p70S6K1, p-p70S6K1, PPAR&#947, C/EBP&#945;, ACC1, FAS, AKT, p-AKT) nos tecidos adiposos subcutâneo (SC) e retroperitoneal (RP), bem como a expressão de RNAm da adiponectina e leptina no TAB. In vivo, foram realizados testes de tolerância oral à glicose (OGTT) e de sensibilidade à insulina (ITT), glicemia pós prandial e consumo de ração. O tratamento crônico com leucina reverteu a perda de massa adiposa dos coxins subcutâneo e viscerais neste modelo experimental, o que pode ser explicado pelo aumento da expressão da p-p70S6K1, PPAR&#947;, ACC1 e FAS, proteínas que estimulam a adipogênese e lipogênese de novo nos adipócitos. Além disso, houve um aumento da expressão de AKT total no coxim SC no grupo DL, mas não foi alterada no coxim RP, indicando que a leucina também pode melhorar a resistência à insulina por ativar a AKT, que é considerada enzima limitante da cascata de fosforilação da insulina. Por outro lado, a leucina melhorou o perfil de adipocinas secretadas pelo coxim RP, pois aumentou a secreção de adiponectina e IL-10. Estas citocinas, direta ou indiretamente, reduzem a RI em tecidos como fígado, TAB e músculo esquelético. Isto sugere que a ação da leucina sobre a sensibilidade à insulina no coxim subcutâneo parece estar mais relacionada com a recuperação da via de sinalização da insulina, ao passo que, no coxim RP está indiretamente relacionada com a melhora do perfil de adipocinas secretadas por este tecido. Estes dados corroboram com os resultados de HOMAIR, glicemia de jejum e pós prandial, OGTT e ITT, nos quais foi observada uma significativa melhora do quadro de intolerância à glicose e resistência à insulina em ratos diabéticos tratados com leucina. Em conclusão, a suplementação crônica com leucina aumentou a adiposidade corporal em ratos diabéticos induzido por estreptozotocina no período neonatal, o que foi relacionado com a melhora da intolerância à glicose e da resistência à insulina. Isto demonstra que a recuperação trófica do tecido adiposo branco é fundamental para a melhora dos distúrbios metabólicos relacionados ao metabolismo da glicose neste modelo experimental. / The chronic hyperglycemia in diabetes is associated with disturbances in insulin and in mTOR pathways, and changes in adipokine secretion in white adipose tissue (WAT). Long-term, this metabolic dysfunction can cause a severe loss of fat mass, which increases insulin resistance (IR). Studies have highlighted the effect of leucine supplementation in treatment of metabolic diseases as type 2 diabetes and obesity. However, the effects of leucine on glucose homeostasis and insulin sensitivity in diseases with intense fat loss remain unknown. Therefore, was investigated whether chronic leucine supplementation can affect the adiposity of diabetic rats with severe adipose tissue loss, and to improve the IR and other metabolic disorders associated with WAT. After weaning, rats were distributed in 3 groups: i) control group (C) - no diabetic and received control chow ; ii) diabetes group (D) - diabetic and received control chow; iii) Leucine Diabetes Group (DL) - diabetic and received diet supplemented with 5% L-leucine. After 8 weeks, were analyzed: fasting glycaemia and insulin, HOMA>IR, antiinflammatory and proinflammatory cytokines in serum and in WAT, protein expression of mTOR, p-MTOR, p70S6K1, p-p70S6K1, PPAR&#947;, C/EBP&#945;, ACC1, FAS, AKT, p-AKT in subcutaneous (SC) and retroperitoneal adipose tissue, as well as the RNAm expression of adiponectin and leptin in WAT. In vivo, were realized oral glucose tolerance test (OGTT) and insulin sensitivity test (ITT), postprandial glycaemia and chow ingestion. O chronic treatment with leucine recovered the adipose mass in subcutaneous and visceral fat pad in this experimental model, this was explicated by increase of protein expression of p-p70S6K1, PPAR&#947;, ACC1 and FAS that stimulate the adipogenesis and de novo lipogenesis in adipocytes. Moreover, had an increase of protein expression of total AkT in subcutaneous fat pad in group DL, but don\'t change in RP fat pad, indicating that the leucine can to activate the AKT, which is limiting enzyme of phosphorylation cascade of insulin, and improve the insulin resistance. On the other hand, leucine improved the profile of adipokines secreted in RP fat pad, because increased the secretion of adiponectin and IL-10. This cytokines reduced the insulin resistance in tissues as liver, WAT and skeletal muscle. This suggest that action of leucine on insulin sensitivity in subcutaneous fat pad is more related to recovery of insulin signaling, and in RP fat pad is indirectly related to improve of profile of adipokines secretion in this tissue. This data corroborates with results of HOMAIR, postprandial and fasting glycaemia, OGTT and ITT, which showed significant improve of glucose intolerance and insulin resistance in diabetic rats treated with leucine. In conclusion, the chronic leucine supplementation increased adiposity in streptozotocin-induced diabetic rats in neonatal period, which was related to improve of glucose intolerance and insulin resistance. This show that trophic recovery of white adipose tissue is important for improve of metabolic disturbances related to glucose metabolism in this experimental model.

As Stanniocalcinas (STC1 e STC2) são hormônios glicoproteicos originalmente encontrados em peixes teleósteos. Em mamíferos, esses hormônios são expressos em uma variedade de tecidos e estão envolvidos em processos como o transporte de cálcio e fosfato pelos rins e intestino, a carcinogênese, a reprodução e o crescimento. Recentemente, foram encontrados efeitos da STC1 e da STC2 no metabolismo intermediário. Sítios de ligação para a STC1 já foram identificados na membrana mitocondrial e resultados preliminares do nosso laboratório demonstraram que a STC1 possui um efeito inibitório sobre a gliconeogênese renal e tanto a STC1 quanto a STC2 diminuem a incorporação de 14C-glicose em 14CO2 no fígado e no músculo gastrocnêmio, respectivamente, de ratos. No entanto, o papel desses hormônios no metabolismo de aminoácidos permanece desconhecido. No presente trabalho, as ações da STC1 e da STC2 foram avaliadas no fígado e no músculo gastrocnêmio excisados de ratos machos (Rattus norvegicus, n=48 animais) de 300±50g, alimentados ad libitum. Os resultados obtidos mostram que a STC1, no fígado, diminuiu a captação do ácido 2-(metilamino)isobutírico, aumentou a atividade da bomba Na+/K+-ATPase, diminuiu a atividade da enzima malato desidrogenase mitocondrial e estimulou a síntese de glicogênio a partir da alanina. A STC2, no fígado, diminuiu a atividade da enzima malato desidrogenase mitocondrial, estimulou a síntese de proteínas a partir de leucina, e estimulou a síntese de glicogênio a partir de alanina. Já, no músculo, a STC2 estimulou a oxidação de leucina e a incorporação desse aminoácido em proteínas. Esses resultados confirmam a existência de ações das STC1 e STC2 no metabolismo de aminoácidos e sugerem, com exceção da ação da STC2 sobre a enzima malato desidrogenase, um papel anabólico para a STC2 em ambos os tecidos. A mesma afirmação não pode ser feita para a STC1, que apresentou efeitos antagônicos no tecido hepático. Por fim, o trabalho mostrou que as ações da STC1 e da STC2 sobre as vias metabólicas dos aminoácidos ocorrem com a utilização de doses muito baixas desses hormônios. / Stanniocalcins (STC) are glycoprotein hormones that were first discovered in teleostean fishes. In mammals, these hormones are expressed in a variety of tissues. Besides its role on the calcium and phosphate transport by the kidneys and intestine, they are involved in processes such as carcinogenesis, reproduction and growth. Recently it has been shown that STC1 and STC2 affect the control of intermediary metabolism. Binding sites for STC1 have been already identified in the mitochondrial membrane. Preliminary results of our laboratory showed that STC1 has an inhibitory effect on renal gluconeogenesis in rats and both STC1 and STC2 decrease the 14C-glicose incorporation into 14CO2 in the liver and the gastrocnemius muscle, respectively. Despite these evidences that STC1 and STC2 have a role in the control of glucose and lipids metabolism, the function of these hormones in amino acids metabolism remains unknown. In the present study, the STC1 and STC2 actions were evaluated in livers and gastrocnemius muscles excised from male rats (Rattus norvegicus, n=48 animals). The rats weighted 300±50g and were fed ad libitum. The results show that STC1 decreased 2-(metilamine)isobutyric acid uptake, increased Na+/K+-ATPase activity, decreased mitochondrial malate dehydrogenase activity and stimulated glycogen synthesis from alanine. All actions of STC1 were shown in the hepatic tissue and this hormone did not affect any parameter in muscular tissue. In the liver, STC2, decreased the mitochondrial malate dehydrogenase activity, stimulated protein synthesis from leucine and stimulated glycogen synthesis from alanine. In muscle, STC2 stimulated leucine incorporation into CO2 and proteins. These results confirm the regulatory role of STC1 and STC2 on amino acid metabolism in muscle and liver of rats. They suggest, with exception to the STC2 action into the hepatic malate dehydrogenase, an anabolic role for STC2 in both tissues. However this cannot be stated for STC1, which show antagonistic effects in the hepatic tissue. Lastly, another important finding of this study is that STC1 and STC2 actions on amino acid metabolism occur with low hormone concentrations.

Nosso objetivo foi investigar o efeito da suplementação de leucina na via intracelular acionada por miostatina, uma proteína reguladora negativa da massa muscular, em um modelo de atrofia gerada por imobilização em ratos. Nossa hipótese inicial contemplava que a expressão de miostatina se elevaria com a imobilização e que a leucina poderia atenuar esse aumento. Nossos resultados, entretanto, mostraram uma regulação gênica no sentido de suprimir a via da miostatina. A suplementação com leucina, associada à imobilização, também provocou queda da expressão gênica de miostatina. Em ambos os grupos a expressão proteica permaneceu inalterada. Em contrapartida, a folistatina, inibidor endógeno da miostatina, apresentou acentuado aumento de expressão no grupo imobilizado e imobilizado/suplementados com leucina. Nossos resultados mostraram que o efeito protetor da leucina no modelo atrófico utilizado não se dá através da sinalização da miostatina. Os dados também sugerem uma resposta biológica coordenada entre o ligante, miostatina, e seu inibidor endógeno, a folistatina. / Our purpose was to investigate the effect of leucine supplementation upon the intracellular pathway triggered by myostatin, a negative regulator of muscle mass, in an atrophic model driven by immobilization in rats. Our initial hypothesis contemplated that myostatin expression would be elevated under immobilization and that leucine supplementation would be able to attenuate this raise. However our results showed a strong downregulation of myostatin gene expression in atrophy induced by cast. The immobilization associated with leucine supplementation also caused mRNA myostatin downregulation. Protein levels were unchanged. On the other hand, follistatin, a myostatin endogenous inhibitor, was markedly upregulated in both immobilized and immobilized/supplemented groups. Our results revealed that leucine protective effect in this atrophic model is not through myostatin signailing. Furthermore, these data suggest a coordinated biological response between ligand, myostatin, and its endogenous inhibitor, follistatin.

Diversos estudos demonstraram que a administração aguda de leucina é capaz de estimular a síntese protéica. Contudo, poucos estudos verificaram os efeitos crônicos dessa suplementação em parâmetros que refletem o estado nutricional protéico (ENP). O presente estudo avaliou o efeito da suplementação crônica com leucina na composição corporal e no estado protéico de ratos submetidos à restrição alimentar (RA) e a período de recuperação nutricional (RN). Foram realizados dois experimentos com 88 ratos Wistar machos e adultos que foram mantidos em gaiolas individuais, em ambiente climatizado e com ciclo biológico de 12 h claro/12 h escuro. No experimento I, 28 animais foram distribuídos em dois grupos: CON (ração AIN93-M) (n = 15) e LEU (ração suplementada com 0,59 % de L-leucina) (n = 13) e submetidos à RA de 50 % por seis semanas. No experimento II, 60 animais foram distribuídos em dois grupos: CON (ração AIN93-M) (n = 30) e LEU (ração suplementada com 0,59 % de L-leucina) (n = 30) e submetidos à RA de 50 % durante uma semana. Findo o período, 15 animais de cada grupo foram sacrificados; o restante dos animais (n = 15 por grupo) foi submetido a duas semanas de RN com suas respectivas rações. No período de RN, as rações foram consumidas ad libitum. Avaliou-se: a composição química da carcaça; a massa de órgãos e do músculo gastrocnêmio; os conteúdos e as concentrações de proteína, RNA e DNA no fígado e no músculo gastrocnêmio; as concentrações séricas de IGF-1, corticosterona, leptina, proteínas totais, albumina e globulina; e as concentrações plasmáticas de uréia e glicose. No experimento I, a suplementação reduziu a gordura corporal e aumentou as concentrações de proteína hepática e de RNA muscular. No experimento II, não houve alterações na massa do intestino e nos conteúdos de proteína e de RNA no fígado dos animais suplementados durante os períodos de RA e RN. A suplementação com leucina aumentou a uremia dos animais do grupo LEU, em relação ao grupo CON, após RN. Não houve diferença significativa na concentração de corticosterona sérica e no conteúdo protéico da carcaça e de tecidos específicos nos animais suplementados. Os resultados obtidos permitem sugerir que a suplementação crônica com leucina aumenta a perda de gordura e melhora alguns parâmetros do ENP na RA em longo prazo, sem alterar o conteúdo protéico da carcaça e de tecidos específicos. Também permitem sugerir que essa suplementação melhora alguns parâmetros do ENP durante variação de peso corporal. / Several studies have demonstrated that the acute administration of leucine is capable of stimulating protein synthesis. However, few studies have investigated the chronic effects of this supplementation as to the parameters that reflect the protein nutritional state (PNS). With this in mind, the aim of this study was o evaluate the effect of the chronic supplementation with leucine on body composition and protein state of rats submitted to food restriction (FR) and a subsequent nutritional recovery period (NR). Two experiments were carried out, using 88 adult male Wistar rats that were kept in individual metabolic cages, in an acclimatized environment with a 12-hour dark-light cycle. In experiment I, 28 animals were distributed into 2 groups: CON (fed an AIN93-M ration, n=15) and LEU (fed a ration supplemented with 0.59% L-leucine, n=13), both groups being submitted to 50% FR with their respective rations, during 6 weeks. In experiment II, 60 animals were distributed into two groups: CON (fed an AIN93-M ration, n=30) and LEU (fed a ration supplemented with 0.59% L-leucine, n=30), both groups being submitted to 50% FR with their respective rations, during 1 week. After this period, 15 animals belonging to each group were sacrificed. The remaining animals (n=15 per group) were submitted to a further 2 weeks of NR each with their respective rations. The period of NR was characterised by ration consumption ad libitum. The chemical composition of the carcass; the mass of the viscera and of the gastrocnemius muscle; protein, RNA and DNA concentration and content of the liver and gastrocnemius muscle; the serum concentrations of IGF-1, corticosterone, leptin, total protein, albumin and globulin; and the plasma concentrations of urea and glucose were determined. The results of experiment I showed that supplementation with leucine reduced the amount of body fat and increased the concentrations of liver protein as well as that of muscle DNA after 6 weeks of FR. The supplementation with leucine also increased uremia of the animals belonging to the LEU group with respect to those of the CON group. However, there was no significant difference in the concentration of serum corticosterone and in the protein content of the carcass and specific tissues of the animals subjected to supplementation. We can therefore suggest that supplementation with leucine increases the loss of body fat and improves parameters of the PNS in FR in the long run, without, however, altering the protein content of the carcass and specific tissues. It can also be said that this supplementation improves some of the parameters of the PNS throughout the variation of body weight.

The homeodomain leucine zipper (HDZip) proteins constitute a plant-specific family of transcription factors, that based on sequence criteria have been grouped into four classes, HDZip I-IV. This thesis describes the phylogeny, function, expression patterns and regulation of the HDZip class I genes in the model species Arabidopsis thaliana. The phylogenetic analyses, traced duplication history and exon/intron organisation of the 17 class I genes in Arabidopsis show that the genes form six monophyletic groups, clades, with an origin in early plant evolution. All genes are expressed in broad tissue distribution patterns and the majority are responsive to water availability and/or light conditions. The expression of the genes show different patterns and dependence on environmental stress conditions, indicating evolutionary changes within and between clades. Ectopic expression of the genes suggest that they regulate genes in part by conserved mechanisms. Therefore, different functional roles seem to have evolved by a divergence mainly in the regulatory properties of the genes. Detailed expression analyses of the paralogous HDZip I genes ATHB7 and ATHB12 show that they have essentially overlapping patterns of activity in response to abscisic acid, ABA, or water deficit in leaves, stems and roots. The water deficit response of ATHB7 and -12 is mediated by ABA and depends on the protein phosphateses ABI1 and ABI2. Transgenic plants with ectopic expression of ATHB7 and/or -12, and athb7 and athb12 mutants, reveal that the genes in roots mediate the growth inhibitory effects of ABA. In this aspect of their function they do not overlap. In leaves and stems, the genes might act as growth regulators redundantly with other factors. Taken together these data suggest that ATHB7 and -12 regulate growth in response water deficit and that other HDZip I genes have related functions in response to environmental stress conditions.

Gliomas are the most common primary brain tumours, and their capacity to invade surrounding normal brain prevents complete removal of the tumour. Malignant glioma has still a poor prognosis. However, with the rapid development of molecular biology our understanding about glioma has increased dramatically. Among known growth factors, EGF and its receptor are frequently amplified and over expressed in malignant glioma. Therefore, it is of interest to find approaches to hamper the activity of EGF/EGFR. The aim of this thesis was to identify and characterize human analogues to a recently identified gene in Drosophilia, kekkon-1, which negatively regulates the activity of Drosophilia EGF receptor. In the first part, we set up a quantitative real-time RT-PCR assay, which showed good linearity, reproducibility and uniformity. We analyzed the expression of the most commonly used reference genes, and showed that 18S was the most reliable endogenous reference gene in this study. In the second part, we cloned, identified, and sequenced a gene family, which we named leucine-rich repeats and immunoglobulin–like domains family (LRIG). The LRIG gene family had three vertebrate paralogs and one homolog in ascidiacea. The proteins encoded by human LRIG genes shared an overall structure with a signal peptide, 15 tandems leucine-rich repeats with N- and C- terminal flanking regions followed by 3 immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. Northern blot showed the mRNA sizes to be 5.5 kb for LRIG1, 4.8 kb for LRIG2, and 5.1 kb for LRIG3. LRIG1-3 mRNAs were detected in all human and mouse tissues analyzed, however, at various levels. FISH and BLAST analysis showed that LRIG1 was located at 3p14, LRIG2 at 1q13, and LRIG3 at 12q13. LRIG1 was shown to be down-regulated in several cancer cell lines and proposed to be a tumour suppressor gene. In the third part, we analysed the expression of LRIG gene family in human astrocytic tumours. LRIG1-3 mRNAs were detected in all human glioma cell lines, in primary tumour tissues and control-matched normal brain tissues, at various levels. Subcellular localizations of LRIG1-GFP fusion proteins were visualized in nuclear, perinuclear, and cytoplasmic compartment. According to the predicted protein sequences, short peptides were synthesized and used to raise antibodies in rabbits. The antibodies were used for immunohistochemical analysis of LRIG1-3 in 404 human astrocytic tumours in a tissue micro array. The pattern of immunoreactivity of LRIG1-3 was heterogeneous with staining in nuclear, perinuclear and cytoplasmic compartment of positive tumour cells. Perinuclear staining of LRIG1-3 displayed a significant inverse correlation with WHO grade and especially positive LRIG3 perinuclear and cytoplasmic staining correlated with a low proliferation index. The LRIGs correlated with survival, and LRIG3 perinuclear staining was in addition to tumour grade an independent prognostic factor. The results suggest that LRIGs may play a role in normal tissue, and may be of importance in the pathogenesis and prognosis of tumours. The exact function of LRIG1-3 remains to be established.

PIDD has previously been described as a death domain (DD)-containing protein that is inducible upon p53 activation and plays a role in programmed cell death. It has previously been shown that PIDD interacts with RAIDD (RIP-associated ICE/CED3 homologous protein with a death domain) in a cytoplasmic complex known as the PIDDosome, which results in the activation of capsase-2 and ultimately in cell death in response to DNA damage. Despite earlier studies on PIDD, however, the physiological role of PIDD has not been elucidated. Thus, we have generated PIDD-deficient mice and examined its in vivo functions particularly in cell death and in antiviral innate immunity. The first major aim of the thesis is to determine whether or not PIDD is required in cell death. PIDD mice are developmentally normal and do not display a pronounced phenotype. Surprisingly, PIDD deficiency perturbed neither DNA damage-induced nor stress-induced cell death in a variety of cell types, suggesting that PIDD may not play a critical role in cell death. In addition, caspase-2 processing occurred normally in the absence of PIDD in response to ionizing irradiation or etoposide treatment, indicating that PIDD is dispensable in the cleavage of caspase-2. The second major aim is to examine the role of PIDD and RAIDD in LCMV-induced innate immunity. To study the role of PIDD and RAIDD in antiviral immune responses, I have generated PIDD/RAIDD double-deficient mice and challenged them with lymphocytic choriomeningitis virus (LCMV). Interestingly, I observed that ablation of both PIDD and RAIDD together resulted in defective viral clearance in the spleen, but not in other organs including the lung, liver, and kidney. In addition, the production of type I IFN was also decreased in the mice deficient in both PIDD and RAIDD. However, the cytotoxicity of the T lymphocytes was largely intact in the absence of both PIDD and RAIDD. Collectively, our results suggest that PIDD is dispensable in cell death, yet PIDD and RAIDD together have a synergistic effect in LCMV-induced antiviral innate immunity. The findings presented in this thesis provide a better understanding of the physiological role of PIDD and may ultimately contribute to the novel therapeutic strategies for the proper control of viral infection.