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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
31

L-carnitina no tratamento da Doença da Urina do Xarope do Bordo : estudos em humanos e em modelo animal sobre o estresse oxidativo e o perfil inflamatório

Mescka, Caroline Paula January 2015 (has links)
A doença da urina do xarope do bordo (MSUD) é causada pela deficiência na atividade do complexo da desidrogenase dos U-cetoácidos de cadeia ramificada (BCKAD), promovendo o acúmulo dos aminoácidos de cadeia ramificada (BCAA) leucina (Leu), isoleucina (Ile) e valina (Val) e seus U-cetoácidos correspondentes (BCKA). A MSUD caracteriza-se por cetoacidose, ataxia, coma, retardo mental e psicomotor. Estudos em animais demonstraram que BCAA e BCKA estimulam a lipoperoxidação e reduzem capacidade antioxidante cerebral em ratos. Também há evidências de que o estresse oxidativo ocorra em pacientes com MSUD no diagnóstico e durante o tratamento e que devido à terapia com dieta restrita e hipoproteica eles possuam deficiência de L-carnitina (L-car), um importante composto para o metabolismo energético. Recentemente, estudos demonstraram o papel antioxidante e anti-inflamatório da L-car, através de sua ação antiperoxidativa, sequestradora de espécies reativas e efeito estabilizador de danos às membranas celulares. Considerando que a fisiopatologia da MSUD ainda é pouco compreendida e que existe um crescente número de estudos enfatizando o envolvimento do estresse oxidativo na doença, neste trabalho foi investigado o efeito in vitro e in vivo da L-car sobre o estresse oxidativo e o dano inflamatório na MSUD tendo como objetivos: A) estudar a indução ao dano oxidativo pelos metabólitos acumulados na MSUD, verificando o possível papel antioxidante da L-car sobre o dano ao DNA in vitro; B) avaliar o efeito in vivo da suplementação de 50 mg/kg/dia de L-car sobre: b.1) a indução do dano ao DNA em leucócitos de pacientes com a MSUD tratados com dieta de restrição proteica, correlacionando as concentrações dos principais metabólitos acumulados nesta doença e verificando o possível papel antioxidante da suplementação da Lcar; b.2) a concentração de citocinas pró-inflamatórias em plasma de pacientes com MSUD tratados com dieta de restrição proteica e a correlação com o estresse oxidativo; b.3) os parâmetros de dano oxidativo à biomoléculas em urina de pacientes com MSUD sob dieta de restrição proteica; C) avaliar o efeito da L-car sobre o estresse oxidativo causado pelos metabólitos acumulados na MSUD em córtex cerebral e cerebelo de ratos Wistar, através de um modelo crônico de indução química da doença. Verificou-se que a Leu e o seu - cetoácido correspondente, o ácido -cetoisocapróico (KIC), causaram danos ao DNA in vitro e L-car foi capaz de diminuir significativamente essas alterações, principalmente as causadas pelo KIC. Quando testado o efeito da suplementação de L-car sobre o dano ao DNA em pacientes MSUD, observou-se um aumento significativo de lesões ao DNA em pacientes com dieta de restrição proteica quando comparados aos controles e a terapia com L-car foi capaz de diminuir significativamente os níveis desses danos. Também foram verificadas correlações do tipo negativa entre as concentrações de L-car e os índices de dano ao DNA e do tipo positiva entre as lesões ao DNA e níveis de MDA, marcador de lipoperoxidação, explicitando uma relação entre o dano ao DNA observado nos pacientes com MSUD, estresse oxidativo e o benefício da suplementação de L-car. Também averiguou-se o efeito da terapia de L-car sobre as citocinas pró-inflamatórias interleucina 1Y (IL-1Y), interleucina 6 (IL-6) e interferon gama (INF- Z). Constatou-se aumentos significativos de IL-1Y, IL-6 e INF- Z no plasma de pacientes com MSUD antes da suplementação de L-car e uma reversão completa desses valores aos níveis dos controles para IL-1Y e INF- Z após a administração de L-car. Ainda, verificou-se que a L-car pode auxiliar na defesa celular contra a inflamação e o estresse oxidativo, observando-se uma correlação negativa entre todas citocinas testadas e as concentrações de L-car, e uma correlação positiva entre o conteúdo de MDA e níveis de IL-1Y e IL-6. Constatou-se também que as medidas de di-tirosina (dano oxidativo a proteínas) e isoprostanos (dano de lipoperoxidação) estavam aumentadas e a capacidade antioxidante total diminuída na urina de pacientes com MSUD sem terapia com L-car e a suplementação deste composto induziu efeitos benéficos sobre estes parâmetros, reduzindo os níveis de di-tirosina e isoprostanos e aumentando a capacidade antioxidante medida em urina. Foi também observado um aumento de KIC urinário após dois meses de tratamento com L-car, quando comparado com o grupo controle, demonstrando um incremento da excreção deste metabolito tóxico. Desta forma, esses resultados sugerem um efeito de reversão de dano oxidativo pela L-car e que a urina pode ser utilizada para monitorar este tipo de lesão em pacientes afetados pela MSUD. Por fim, foram analisados em córtex cerebral e cerebelo de ratos Wistar submetidos ao modelo crônico de MSUD: espécies reativas ao ácido tiobarbitúrico (TBARS), para avaliar lipoperoxidação, conteúdo de carbonilas (dano oxidativo proteico), oxidação de diclorofluoresceína (DCF), para quantificar produção de espécies reativas teciduais, conteúdo de glutationa reduzida (GSH) que é um importante antioxidante não enzimático e a atividade das enzimas antioxidantes catalase (CAT), superóxido dismutase (SOD), glutationa peroxidase (GPx) e glicose-6-fosfato-desidrogenase (G6PD). Os resultados mostraram que a administração crônica de BCAA estimulou a lipoperoxidação, o dano oxidativo proteico, aumento de espécies reativas e diminuição das defesas antioxidantes enzimáticas e não enzimáticas, especialmente em córtex cerebral e o tratamento com L-car foi capaz de prevenir estes efeitos, exceto o dano oxidativo a proteínas. Em conjunto, estes resultados demonstram que os metabólitos acumulados na MSUD induzem dano oxidativo a biomoléculas (lipídios, proteínas e DNA), diminuem o status antioxidante e promovem aumento de processos inflamatórios. Ainda, estes dados podem contribuir para a compreensão dos mecanismos de ação dos efeitos citotóxicos dos metabólitos acumulados na MSUD e evidenciar o papel do estresse oxidativo e da inflamação na neuropatofiosiologia desta doença, além do efeito protetor da L-car sobre este processo. O estudo de antioxidantes, como a L-car, pode propor uma abordagem terapêutica adicional ao que é empregado atualmente para pacientes com MSUD, que é essencialmente dietética e, portanto, de difícil manejo. / Maple syrup urine disease (MSUD) is caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain U-ketoacid dehydrogenase (BCKAD). The metabolic defect leads to accumulation of the branched chain amino acids (BCAA) leucine (Leu), isoleucine (Ile) and valine (Val) and the corresponding branched-chain U-keto acids. The clinical features of MSUD include ketoacidosis, seizures, coma, psychomotor delay and mental retardation. Treatment consists in Leu, Val and Ile restricted diet. Studies in animals have demonstrated that lipid peroxidation is stimulated by BCAA and BCKA in brain of rats and these metabolites reduce in vitro and in vivo the cerebral capacity to modulate the damage associated to increased free radical production. Also, there is evidence that oxidative stress occurs in MSUD patients at diagnosis and during treatment and that due to terapy with protein restricted diet they present L-carnitine (L-car) deficiency, an important compound for energy metabolism. Recent studies have demonstrated the antioxidant and anti-inflammatory role of L-carnitine (L-car), through its action against peroxidation in different tissues by various mechanisms, a scavenger of reactive oxygen species and the stabilizing effect of damage to cell membranes. Considering that the pathophysiology of MSUD is still poorly understood, and that there is an increasing number of studies emphasizing the oxidative stress involvement in the disease, this study investigated the in vitro and in vivo effect of L-car on oxidative stress and inflammatory damage in MSUD with the following purposes: A) to study the induction of damage by accumulated metabolites in MSUD, analyzing the possible antioxidant role of L-car on DNA damage in vitro; B) to evaluate the in vivo effect of 50 mg/kg/day of L-car supplementation about: b.1) the induction of DNA damage in leukocytes of MSUD patients treated with protein-restricted diet, correlating this damage with the concentrations of the major metabolites accumulated in this disorder and checking the possible antioxidant role of L-car supplementation; b.2) plasma inflammatory cytokines in treated MSUD patients with protein-restricted diet and the correlation with oxidative stress; b.3) oxidative damage parameters in urine of MSUD patients with protein-restricted diet supplemented with L-car; C) to investigate the BCAA effect on some oxidative stress parameters and evaluate the L-car efficacy against these possible pro-oxidant effects in cerebral cortex and cerebellum of rats submitted to a chronic chemically-induced model of MSUD. DNA damage index (DI) showed that Leu and -ketoisocaproic acid (KIC) groups was significantly higher than that of the control group, and that L-car was able to significantly prevent this damage, especially that due to KIC. Accordingly, DNA DI in MSUD patients under BCAA-restricted diet was significantly increased as compared to controls and L-car supplementation was able to significantly decrease this parameter. It was also verified a significant positive correlation between DNA DI and MDA content, a marker of lipid peroxidation. Furthermore, we found an inverse significant correlation between DI and L-car levels. These results strengthen a relationship between DNA damage observed in MSUD patients, oxidative stress and the L-car supplementation benefit. The role of L-car on plasma inflammatory cytokines interleukin-1Y (IL-1Y), interleukin-6 (IL-6) and interferon-gamma (INF- Z) was also evaluated in these patients. Significant increases of IL-1Y, IL-6, and INF- Z were observed before the treatment with L-car. Moreover, there is a negative correlation between all cytokines tested and L-car concentrations and a positive correlation among the MDA content and IL-1Y and IL-6 values after L-car supplementation. It was also demonstrated that the oxidative stress parameters di-tyrosine (oxidative protein damage) and isoprostanes (lipid peroxidation assay) were increased and the antioxidant capacity was reduced in urine of MSUD patients without L-car therapy and that the supplementation of this compound induced beneficial effects on these parameters, so reducing the di-tyrosine and isoprostanes levels and increasing the antioxidant capacity. It was also showed a significant increase in urinary KIC after 2 months of L-car treatment compared to control group, demonstrating an increased excretion of this toxic metabolite. In conclusion, these results suggest a reversion effect of the oxidative damage by L-car and that urine can be used to monitorize oxidative damage in patients affected by this disease. The following parameters were analysed in cerebral cortex and cerebellum of Wistar rats submitted to MSUD chemically-induced chronic model: thiobarbituric acid reactive species (TBA-RS), to evaluate lipid peroxidation, carbonyl content to evaluate protein oxidative damage, DCF oxidation to quantify reactive species production, reduced glutathione (GSH), an important non-enzymatic antioxidant and the activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PD). The results showed that the chronic administration of BCAA was able to promote both lipid and protein oxidation, increase of reactive species production and decreased brain antioxidant defenses, especially in cerebral cortex and that L-car was able to prevent these effects, except for oxidative damage to proteins. Taken together, these results demonstrate that the metabolites accumulated in MSUD cause oxidative damage to biomolecules (lipids, proteins and DNA), decrease antioxidant status and promote increased inflammatory processes. These results may contribute to the understanding of the mechanism of action of the cytotoxic effect of the metabolites accumulated in MSUD and the role of oxidative stress and inflammation in the MSUD neuropathophysiology besides the protective effect of L-car on this process. The study of antioxidants like L-car can opens an additional therapeutic approach to that currently employed for MSUD patients, which is primarily dietary and therefore difficult to handle.
32

Différenciation du sirop d'érable à défaut de saveur de type bourgeon (√R5) du sirop d'érable à saveur sans défaut (REG) à l'aide de SPME-GC/MS et UPLC-QTOF-MS/MS

Beniani, Issraa 01 1900 (has links)
L’industrie des produits de l’érable est particulièrement importante en Amérique du Nord. Elle est touchée par un phénomène naturel qui se produit en fin de saison de récolte. Il s’agit de l’altération du goût du sirop ; un goût fort désagréable se développe et se nomme goût de bourgeon vu qu’il coïncide au bourgeonnement des feuilles d’érable. Des coûts et du temps sont impliqués à sa récolte et sa production. Cependant, un sirop d’érable avec un goût de bourgeon est vendu à moindre prix. Il constitue donc une perte potentielle d'argent pour les producteurs. Au fil du temps, ils ont appris à se fier à des signes distincts de la nature pour déterminer le début du bourgeonnement. Néanmoins, il est important de le détecter hâtivement pour les producteurs et de mieux comprendre les changements moléculaires associés au défaut de saveur de type bourgeon. Plusieurs travaux sur les produits de l’érable ont été effectués durant le dernier siècle, mais ce n’est que récemment que des projets de recherche sont centrés sur le goût de bourgeon. La détection de celui-ci est certes importante, mais il y est aussi question de mieux connaître la variation de la composition du sirop ou de la sève d’érable, afin de mieux comprendre le phénomène. Tel que décrit dans l’introduction de ce mémoire, on connaît maintenant mieux la composition du sirop d’érable, même que quelques solutions ont été proposées à notre problème, mais beaucoup de questions restent sans réponse que ce mémoire tentera de résoudre. C'est dans ce contexte que cette étude se pose. En visant à identifier les molécules responsables du défaut de goût de type bourgeon que l’on peut retrouver dans le sirop d’érable, nous espérons aider à mieux cibler le problème et à mieux comprendre le métabolisme et, éventuellement, comprendre comment ce goût se développe. Dans un second temps, la découverte de molécules pas encore rapportées, au meilleur de notre connaissance, comme étant présentes dans le sirop d’érable est aussi visée, sans égard à la classification du sirop. Les travaux de ce mémoire contribueront, ultimement, à trouver une façon d’empêcher la formation de ce goût et surtout un moyen de l’éliminer. Pour ce, une première partie correspondant à l’analyse des composés volatils a été effectuée par SPME et GC-MS sur 78 échantillons de sirop d’érable. L’analyse statistique des résultats par PCA cible 42 composés et associe chacun d’entre eux au sirop d’érable avec le défaut de saveur ou alors au sirop sans défaut de saveur. Parmi elles, seules 36 ont pu être identifiées par leurs spectres de masse. La seconde partie correspond à l’analyse des composés non volatils par UPLC-QTOF et UPLCMS/MS. La méthode a été optimisée pour cibler les molécules relativement apolaires par rapport aux sucres et aux acides aminés présents dans le sirop d’érable. Au total, 20 échantillons de sirop ont été analysés, dont 10 à défaut de saveurs de type bourgeon et 10 sans défaut. Pour cette partie, l’analyse de composantes principales a encore une fois été utilisée pour classer les types de sirop à l’étude selon les composés détectés. Ainsi, le mémoire avance les connaissances moléculaires liées à l’émergence du défaut de bourgeon dans le sirop d’érable. / The maple products industry is particularly important in North America. It is affected by a natural phenomenon that occurs at the end of the harvest season. It’s the alteration of the taste of the syrup; a very unpleasant taste develops and is called buddy flavour since it coincides with the budding of maple leaves. Costs and time are involved in its harvest and production. When maple syrup is associated with the buddy taste, its commercial value is lower. Therefore, it contributes to a loss of money for producers. Over time, they have learned to rely on distinct signs from nature to determine the onset of budding. However, methods are needed to detect it and research is needed to better understand the molecular changes in maple syrups. Several research projects on maple products have been carried out during the last century, but it is only recently that research projects have focused on the buddy flavour. They focus on determining the variation in the composition of maple syrup or sap, but also on better understanding of the phenomenon. We now know more than ever about the composition of maple syrup, on top of that some solutions have been proposed to our problem, but many questions remain unanswered and it is obvious that there is still so much to discover. It is in this context that this study arises. By aiming to identify the molecules responsible for the buddy flavour defects that can be found in maple syrup, we hope to help on targeting the problem and better understanding the metabolism of maple trees and, eventually, how the buddy taste develops. Secondly, it is aimed at discovering molecules not yet reported, to the best of our knowledge, as being present in maple syrup regardless of the classification of the syrup. This will contribute, ultimately, in finding a way to prevent the formation of this taste and a way to eliminate it. Thus, the first part of this master’s thesis corresponds to the analysis of volatile compounds by SPME and GC-MS on 78 maple syrup samples. Statistical analysis of the results targets 42 compounds and associates them with either maple syrup with flavour defects or with maple syrup with regular flavour maple syrup. Among these, 36 molecules could be identified by mass spectrometry. To extract correlations from our data, the principal component analysis (PCA) ensued. The second part corresponds to the analysis of non-volatile compounds by UPLC QTOF and UPLC-MS/MS. The method has been optimized to target molecules that are relatively apolar compared to sugars and most of the amino acids present in maple syrup. A total of 20 syrup samples were analyzed, including 10 with bud-like flavour defects and 10 with no defects. For both parts, PCA was once again used to determine which types of syrup under study are the compounds associated with. Taken together, the results presented in this master’s thesis advances knowledge on the molecular origins of the buddy flavour defect in maple syrups.

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