Дисертації з теми "Amino acids Metabolism Disorders"

The 2-methylbranched chain acyl-CoA dehydrogenase (BCAD) is a mitochondrial enzyme that catalyzes the third reaction in isoleucine and valine metabolism, the oxidation of 2-methylbutyryl-CoA and isobutyryl-CoA, respectively. BCAD deficiency would result in the accumulation of branched chain acyl-CoAs or their derivatives. Three patients with a putative defect in BCAD have been reported. This study consists of a molecular examination of one such patient as well as the characterization of the BCAD gene. In Northern blot analysis of human fibroblast RNA, the BCAD cDNA hybridized to two RNA species of 2.7 and 6.5 kb. The 2.7 kb band corresponds to the size of the BCAD cDNA, which consists of the entire coding region of 1.3 kb and a 3$ sp prime$ untranslated region of 1.4 kb. The coding regions of the BCAD gene span approximately 21 kb and consist of 12 exons and 11 introns. The exons range in size from 39 to 108 bp. In the analysis of the putative BCAD-deficient patient, no significant difference was observed at the level of DNA (Southern), RNA (Northern) or protein (Western) when compared to controls, suggesting that the BCAD gene in this patient did not contain any large insertions or deletions, or a frameshift mutation. The single strand conformation polymorphism (SSCP) technique and sequencing of the entire coding region did not reveal any disease-causing mutations but two polymorphisms were identified: one in exon 6 and the other in exon 10.

Multiple carboxylase deficiency (MCD) results from a decreased activity of holocarboxylase synthetase (HCS) which is responsible for the biotinylation of the four biotin-dependent carboxylases found in humans. The disease can be treated with pharmacologic doses of oral biotin (biotin-responsiveness). The cDNA for HCS contains a biotin-binding domain deduced by analogy with the sequence and crystal structure of the E. coli BirA biotin ligase. E. coli birA$ sp-$ mutations causing biotin-auxotrophy all localize to this region. Of six point mutations I have identified in MCD patients, four localize to the biotin-binding region. In order to assess the HCS activity associated with patient mutations, I used an assay based on the expression of mutant HCS in E. coli. The method is based on the ability of mutant HCS to biotinylate the biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase in a temperature-sensitive birA$ sp-$ E. coli strain using 3H-biotin as tracer. I have shown that all of the mutations cause a severe decrease in HCS activity. In addition, I have shown that five of the mutant HCS are biotin-responsive. These findings are a major contribution to the understanding of the mechanism of biotin-responsiveness.

The objective of this thesis was to determine the molecular basis of neonatal multiple carboxylase deficiency (MCD) produced by an impairment in holocarboxylase synthetase (HCS) activity and the origin of the biotin-responsiveness that characterizes this disease. To determine HCS activity, I developed a peptide substrate and used the biotinylation system of E: coli to determine its properties. C-terminal fragments of the $ alpha$ subunit of human propionyl-CoA carboxylase (PCC-$ alpha$) were expressed in E. coli and site-directed mutagenesis was used to define the residues required for biotinylation by the bacterial biotin ligase, BirA. These experiments showed that the biotin region of PCC-$ alpha$ can act as an autonomous domain for biotinylation and suggested its use as substrate for human HCS. For the molecular characterization of MCD, I isolated several cDNA clones encoding human HCS by functional complementation of an E. coli mutant with a temperature-sensitive BirA. Comparison of the predicted amino acid sequence of HCS with bacterial biotin ligases allowed the identification of the putative biotin-binding domain of this protein. Mutation analysis of DNA from HCS deficient patients showed that most of the changes in the HCS sequence are clustered in the biotin-binding domain. All the patients tested in this study showed deficiency of HCS activity as determined using the PCC-$ alpha$ peptide as substrate for biotinylation. The biotin-responsiveness was demonstrated by obtaining a stimulation of HCS activity of MCD cells at high biotin concentrations while remaining unstimulated in extracts of normal cells. Together with the mutation studies, these results showed that neonatal MCD is caused by mutations in the biotin binding domain of HCS which reduce the affinity of the enzyme towards biotin. This change in the kinetic properties of HCS results in the inefficient biotinylation of carboxylases at physiological concentrations of biotin. The defect can be over

Short/branched chain acyl-CoA dehydrogenase (SBCAD), a member of the acyl-CoA dehydrogenase (ACD) family of enzymes, catalyzes the oxidation of branched chain fatty acids and the branched chain amino acids isoleucine and valine. This research project focuses on expression studies of the SBCAD gene. Northern blot analysis detected two SBCAD mRNA species of 2.7 and 6.5 kb in various human tissues and cell types. A single 4.1 and 2.0 kb SBCAD message was detected in rat and pig tissues, respectively, revealing a species difference in SBCAD mRNA size. Studies of human and rat SBCAD tissue-specificity and relative abundance, at both the RNA and protein levels, identified liver and kidney as the tissues with the highest levels of SBCAD expression, establishing a unique tissue-specific expression pattern that is not seen among the other members of the ACD family. Furthermore, a fetal and adult difference in SBCAD expression was observed in human kidney, suggesting that the SBCAD gene may be developmentally regulated in some tissues. Finally, an attempt was made to isolate and characterize the SBCAD promoter region in order to provide valuable data for future SBCAD promoter studies.

Postprandial urea production in subjects with insulin dependent diabetes mellitus (IDDM) on conventional insulin therapy is normal when the previous diet is high in protein, but there is an incomplete adaptive reduction in urea production following protein restriction. To evaluate the nutritional implications of restricted protein intake in human diabetes mellitus, it is first necessary to establish a reliable method to measure changes in urea production and amino acid catabolism in response to changes in dietary protein intake. We therefore tested (1) the accuracy of the urea production rate (Ra) to depict changes in urea production, (2) whether sulfate production can be accurately depicted using tracer or nontracer approaches, after establishing the use of electrospray tandem mass spectrometry to measure sulfate concentrations and 34SO4 enrichments following administration of the stable isotope tracer sodium [34S]sulfate, (3) the reproducibility of urea and sulfate measurements following a test meal low in protein (0.25 g/kg) in subjects previously adapted to high (1.5 g/kg.d) and low (0.3 g/kg.d) protein intakes, and compared the metabolic fate of [ 15N]alanine added to the test meal with that of [15N] Spirulina platensis, a 15N-labeled intact protein, and (4) whether we could identify the differences in postprandial urea and sulfate productions between normal subjects and persons with IDDM receiving conventional insulin therapy previously adapted to high protein intake, when the test meal was limiting in protein. Under basal conditions, steady state urea Ra is an accurate measure of urea production. Following changes in urea production, both the tracer and nontracer methods seriously underestimated total urea Ra. The tracer method overestimated sulfate production by 20%, but the nontracer method provided an accurate measure of sulfate production and, hence, sulfur amino acid catabolism. Postprandial changes in urea and sulfate productions following normal ada

The primary purpose of newborn screening is to quickly identify children that are at risk of having a specific disorder in order to start treatment, prevent early death and reduce the chances of permanent physical or mental damage. The current and widely accepted approach used for identification of metabolism disorders involves a flow injection analysis with mass spectrometry detection of acylcarnitines and amino acids. Although this approach is widely accepted and has shown to be sufficient for identification of multiple metabolism disorders the method is not fully quantitative and results often have to be confirmed by second-tier tests. The primary focus of this research was to improve the accuracy and selectivity of this screening method by employing a high resolution chromatographic separation for the combined analysis of twelve acylcarnitines and seven amino acids. This method is an improvement over the current methodology allowing for separation of key isomers that are diagnostic for different metabolism disorders, reducing the need for multiple second-tier tests to confirm results and shortening the time to diagnosis. In order to further improve the efficiency of newborn screening we developed an in-line desorption device, which allows for direct analysis of DBS eliminating the need for punching disks from the filter paper cards. Our device was the first published paper that demonstrated the ability to directly analyze dried blood spots, without the need for any offline sample processing. Using this device, we validated a method to quantify biomarkers related to Maple Syrup Urine Disease, a disorder that requires a second-tier test for confirmation. To further improve the accuracy of dried blood spot analysis we evaluated a technique to correct the sample volume in low and high hematocrit samples. The level of hematocrit in blood spotted on filter paper cards affects the volume of sample analyzed, leading to errors in accuracy. Diffuse reflectance was used to relate differences in sample hematocrit on dried blood spots. We validated our technique with eighteen donor samples at various levels of hematocrit. Correcting sample volume for hematocrit showed improved precision and accuracy over the standard approach, ultimately reducing the potential to misidentify samples.

The researcher studied the role of amino acid metabolism in osteoarthritis progression. The study suggests that this abnormal amino acid metabolism aids in the development of the disease. This data further suggests that amino acids could be potential circulatory markers for diagnosing OA and therapeutic strategies of amino acids supplementation could be considered as a potential treatment.

Thesis (Ph.D.) - University of Glasgow, 2007.
Ph.D. thesis submitted to the Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, 2007. Includes bibliographical references.

A model was developed in growing piglets to study the use of urinary total sulfur excretion as an indicator of sulfur amino acid (SAA) catabolism and the nitrogen (N)/sulfur (S) balance ratio as an indicator of non-protein SAA storage. The recovery of administrated methionine as urinary total S over 48 hours was 106% in well-nourished piglets, but only 69% in protein malnourished piglets. The N/S balance ratio of protein malnourished piglets was lower than that of well-nourished piglets, and this ratio further decreased after methionine administration. We conclude that in a protein malnourished state, relatively more S than N is retained and a significant portion of the S derived from administrated methionine is retained in non-protein pools. These results demonstrate that urinary total S excretion can provide an accurate measure of SAA catabolism; and the N/S balance ratio can provide valuable information about non-protein SAA storage in growing piglets.

A very versatile chemioenzymatic synthesis of stereospecifically labelled amino acids first developed in our laboratory has been altered and improved so that only chemical steps are involved. Unlike the original synthesis, monodeuteriated compounds can be obtained so that isotope effects can be clearly identified. The synthesis has been used to prepare (2R,3R)-[3-2H1]-p-chloroalanine, (2R,3S)-[3-2H1]-p-chloroalanine, (2S,3R)[ 3}Hd-p-chloroalanine and (2S,3S)-[3-2Hd-p-chloroalanine. The first two compounds were incubated with D-amino acid aminotransferase, and the second two compounds were incubated with L-aspartate aminotransferase. The stereochemical outcome of these reactions indicated retention of stereochemistry in the processes, casting light on similarities between the evolutionary families of PLP-dependent enzymes. A synthesis of stereospecifically labelled (2S)-propargylglycine was attempted. The key intermediates (2S,3R)-[3-2H1]-N-2-nitrobenzenesulfonylaziridine-2-carboxylate and (2S,3S)-[3-2H d-N -2-nitrobenzenesulfonylaziridine-2-carboxylate were synthesised and regiospecific ring opening of the intermediates was achieved. Time did not permit full deprotection of these products

La consommation d'un régime hyper protéique (HP) améliore l'homéostasie glucidique, le gain de poids, l'adiposité, en réduisant le tissus adipeux blanc et la taille des adipocytes. Les adaptations métaboliques dues à l'augmentation de l'apport protéique sont au moins caractérisées, au niveau du foie, par la diminution de la lipogenèse et l'augmentation de la conversion des acides aminés (AA) en glycogène. Cependant, le rôle des acides aminés dans le contrôle de ces adaptations métaboliques et des voies de transduction responsables de la transmission du signal " acides aminés " n'ont pas encore été élucidés. L'objectif de notre étude a été de déterminer l'effet de l'augmentation de l'apport en acides aminés sur la traduction et la protéolyse, et d'identifier les voies de signalisation impliquées dans la détection des acides aminés ainsi que l'acide aminé ou le groupe d'acide aminés responsable de ces effets, en utilisant des approches in vivo et in vitro. Les extraits protéiques ont été analysés par western blots pour examiner l'état de phosphorylation des protéines impliquées dans les voies de signalisation qui participent à la détection des AAs et à la régulation de la traduction, à savoir les voies: " mammalian target of rapamycin " (mTOR), " adenosine monophosphate-activated protein kinase " (AMPK) et " general control non-depressible kinase 2 " (GCN2). Cette étude a montré que l'adaptation à un régime de HP est caractérisée par la stimulation de la traduction dans le foie, au moins au niveau de l'étape d'initiation. Cette activation requiert à la fois la présence de fortes concentrations en AA (au moins la leucine ou des AAs à chaîne branchée) et d'insuline, comme l'indique l'augmentation de la phosphorylation de mTOR, 4E-BP1 et S6 et la diminution de la phosphorylation de l'AMPK et GCN2. L'utilisation de l'AICAR (activateur de l'AMPK) et de la rapamycine (inhibiteur de mTOR) nous a permis de montrer qu'en présence de fortes concentrations en AA et d'insuline, mTOR n'est pas le seul régulateur de 4E-BP1 et de la S6K1 (cibles de mTOR) et que l'AMPK peut également jouer un rôle important dans la régulation de leur état de phosphorylation. En outre, l'augmentation de l'apport protéique provoque une inhibition de la dégradation des protéines dans le foie et une diminution de l'expression des gènes codant les principales protéines du système autophagie et de l'ubiquitine-protéasome. En conséquence, les protéines sont moins ubiquitinées, donc moins dégradées. Les AAs et l'insuline semblent être les principaux régulateurs de la voie de protéolyse ubiquitine-protéasome et les voies mTOR et AMPK seraient les médiateurs des effets acides aminés et de l'insuline. Ces résultats suggèrent que le contrôle des voies cataboliques et anaboliques du métabolisme des protéines sont régulées par les mêmes signaux et font intervenir les mêmes voies de signalisation
High Protein (HP) intake improves glucose homeostasis and reduces weight gain, body fat mass, white adipose tissue and adipocyte size in rats. The metabolic adaptation is characterized by at least a decrease in hepatic lipogenesis and an increase in hepatic amino acid (AA) conversion into glycogen. However, the role of amino acids (AAs) in the control of these metabolic adaptations has not been studied, and the transduction pathways involved in the sensing of the increase in AA supply remain unclear. Therefore, the aim of our study was to understand the effect of AAs on translation and on proteolysis, to identify the transduction pathways involved in AA signaling and the AA or the groups of AAs involved in these effects, using both in vivo and in vitro approaches. Western blot analysis was performed on protein extracts to examine the phosphorylation state of the mammalian target of rapamycin (mTOR), adenosine monophosphate-activated protein kinase (AMPK) and general control non-depressible kinase 2 (GCN2) transduction pathways which may be involved in AA sensing and in the control of translation in liver. This study demonstrated that adaptation to HP diet was characterized by the stimulation of translation, at least for the initiation step in the liver. Using primary culture of hepatocytes, we showed that this activation required both high AA levels (at least for leucine alone or a branched-chain AA mixture) and insulin, as indicated by the increase of mTOR, 4E-BP1 and S6 phosphorylation and the decrease of AMPK and GCN2 phosphorylation. Using AICAR (AMPK activator) and rapamycin (mTOR inhibitor), we demonstrated that mTOR might not be the only regulator of 4E-BP1 and S6K1 (downstream targets of mTOR) in high AA conditions and that AMPK may also play an important role in their control. Moreover, the HP diet induced the inhibition of protein breakdown in the liver and these results were concomitant with a decrease of gene expression of the major components for both autophagy and the ubiquitin-proteasome system in liver. Subsequently, ubiquitinated protein in the liver was lower and both AAs and insulin were required for the down-regulation of ubiquitination. Indeed, mTOR and AMPK were also involved in the control of the ubiquitin proteasome system in the liver in response to the increase in AA and insulin concentrations. These results suggested that the control of the catabolic and anabolic pathways of protein metabolism was regulated by the same set of signals and mediated by the same transduction signaling pathways

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Fundação de Amparo à Pesquisa do Estado de Minas Gerais
Mitocondrias vegetais estão envolvidas em vários processos chaves da célula, vão além da produção de energica, tais como morte celular programada, amadurecimento de frutos, ou mesmo aqueles processos dependente de luz como fotossíntese e fotorrespiração. Dessa forma, aquisição mitocondrial pela célula hospedeira trouxe avanços para as atuais células vegetais: desde a manutenção de diversas vias metabólicas que incluem o metabolismo energético bem como processos de bissíntese de lipidios, nucleotidios e vitaminas. No tocante ao metabolismo energ- etico, destaca-se a herança do ciclo do ácido tricarboxílico. Este ciclo é uma via essencial relacionada com a produção de poder redutor (NADH e FADH 2 ), assimilação de nitrogênio e otimização da fotssíntese. Acredita-se que o ciclo do ácido tricarboxílico oprerasse como passos isoladados antes do processo endossimbiótico e somente após a aquisição da mitocôndria resultou que aquele organizar-se e atuasse como uma via cíclica. O cíclo do ácido tricarboxílico é composto por oito enzimas. Contudo, cada enzima é codificada por vários genes os quais são endereçados para diversos compartimetos celulares e, não somente, mitocôndrias. Essas enziimas locallizadas em diferentes comparimentos subcelulares acarretaram em uma possível ampla conecção entre mitocôndrias e outras organelas (peroxissomos e cloroplastos) permitindo fluxos alternativos dos intermediários do ciclo cujo resultado alterou seu funcionamento para um não convencional modo não cíclico. É bastante aceito que sob estresses, no quais reduzem os níveis de carboidratos. O ciclo do ácido tricarboxílico pode funcionar no modo não cíclico, devido a perda de esquelos carbônico que entram se fazendo necessário ser alimentado por reações anapleuróticas. Portanto, aminoácidos tornam-se fundamentais para suprir a respiração e síntese de ATP sob tais situações. Fortes evidencias demonstraram que aminoácidos de cadeia ramificada (BCAA) e lisina podem fornecer elétrons para o sistema a cadeia de transporte de elétrons mitocondrial pela ação do sistema flavoproteína de transferência de elétrons (ETF)- ETF: ubiquinona oxidorredutase (ETF/ETFQO). Em plantas, duas enzimas: Isovaleril-CoA desidorgenase (IVDH) e (D)-2-hidroxidoglutarato desidrogenase (D2HGDH) foram caracterizadas como doadores de elétrons para o pool de ubiquinone através do sistema ETF/ETFQO a partir da degradação de BCAA e lisina, respectivamente. Na verdade, o catabolismo de BCAA mostra-se de uma importância fundamental para nutrir o ciclo do ácido tricarboxílico, principalmente, em situações de estresse enquanto lisina mostra uma estreia associação com o ciclo do ácido tricarboxílico sendo importante para fazer um elo da degradação de aminoácido com a geração de energia. A transferência de elétrons através da fdoacadeia transportadora de elétrons mitocondrial acopla a síntese de ATP a partir da regeneração de NADH e FADH 2 para fosforilar ADP a ATP. Contudo, o conhecimento com relação a organização do sistema de fosforilação oxidativa (OXPHOS) e sua via alternativa sob limitação energética permanece escasso. Assim, essa tese, a qual se concentra no funcionamento da respiração em um contexto que o ciclo do ácido tricarboxílico e via alternativa como doador de elétrons para cadeia transportadora de elétrons mitocondrial, é composta por três independentes capítulos centrados no metabolismo energético e respiração alternativa em Arabidopsis thaliana. Por isso, para se obter uma visão global de como ocorre o envolvimento e interação do ciclo do ácido tricarboxílico juntamente da via alternativa para coordenar o ajustamento das necessidades metabólicas e celulares, três abordagens experimentais foram usadas (i) uma abordagem in silico, nós investigamos a história evolucionária dos genes do ciclo do ácido tricarboxílico gerando um modelo para a origem dos genes do ciclo em plantas bem como seu comportamento submetido a uma série de estresse; (ii) a importância da biossíntese de lisina foi investigado usando mutante de Arabidopsis com reduzida atividade da enzima L,L-diaminopimelato aminotransferase (dapat) da via biossintética de lisina; (iii) reprogramação metabólica do sistema OXPHOS associado a limitação de carbono foi investigado. Rapidamente, os resultados apresentados aqui forneceram resultados que permitiu, no mínimo um prévio, a elaboração de mecanismo do metabolismo energético junto a vias alternativas. Primeiramente, permitiu a elucidação da origem evolutiva dos constituintes do ciclo do ácido tricarboxílico em plantas fornecendo elemento para a origem das isoformas presentes nos diferentes compartimentos subcelulares os quais que devem ser associados com eventos de transferência gênica ou com novas cópias geradas por duplicação genômica. Ademais, análises de co-expressão dos genes do ciclo em diferentes condições estressantes em ambos tecidos parte aérea e raiz demonstrou a presença de plasticidade molecular e forneceu uma explicação para o funcionamento do ciclo do ácido tricarboxílico em plantas. Após isso, o uso de Arabidopsis mutante com reduzida atividade para biossíntesi de lisina L,L-diaminopimelato aminotransferase (dapat) foi demonstrada que biossíntese de lisina simula condições de estresse e impacta no crescimento e metabolismo foliar. Por fim, uma avaliação de como o comportamento do sistema OXPHOS sob limitação de carbono e como vários aminoácidos podem impactar os complexos respiratórios foi possível demonstrar que o sistema OXPHOS tem sua função afetada por diferentes fontes de carbono e que vias alternativas são induzidas sob essas condições. Ademais, imunoensaios revelaram que é mais provável ser regulado por modificações pós traducionais. Juntos, esses resultados realçam a complexidade e especificidade da respiração vegetal durante evolução e que é differentemente afetado por linitações energéticas e pelo uso de substratos alternativos. Os resultados discutidos aqui suportam que ETF/ETFQo é uma via essencial capaz de doa elétrons para a cadeia transportadora de elétrons e que amioácidos são substratos alternativos para manter a respiração sob limitação de carbono. Os resultados obtidos são discutidos em um contexto de evolução metabólica mostrando estreia associação da metabolismo energético com metabolismo de aminoácidos e onde possível mcanísticos são devidamente discutidos. Palavras chaves: ciclo do ácido tricarboxílico; escassez de energia; evolução mitochondrial; fosforilação oxidativa; genes parálogos, metabolismo mitocondrial; neofuncionalização; respiração; resposta a estresse; substratos alternativos
Plant mitochondrion are involved in several key cellular processes that goesbeyond energy production being also associated with programmed cell death, fruit ripening and even light- associate process including phorespiration and photosynthesis. In this context, mitochondria acquisition by host cell brought evolutionary advances for the existing plant cell by the preservation of diverse metabolic pathways including both those related to energy metabolism as well as those associated with lipids, nucleotides and vitamin biosynthesis. The most notorious heritage is related to the tricarboxylic acid (TCA) cycle. The TCA cycle is an essential pathway which is related to reducing power (NADH and FADH2) generation, nitrogen assimilation and photosynthesis optimization. It has been suggested that the TCA cycle operated as isolated steps prior endosymbiosis events and that only after mitochondria acquisition it was possible for it to be organized and function as a cycle. The TCA cycle is composed by a set of eight enzymes. However, each enzyme is encoded by several genes which are targeting not just to mitochondria, but that are also imported into others subcellular compartments. These TCA enzymes located in other subcellular compartiments result in likely a broader connection between mitochondria and other organelles (e.g. peroxissome and chloroplast) allowing a bypass of the intermediates of the cycle switching his operation to an unusual in non-cyclic modes flux. It is also currently accepted that under stress conditions, which leads to decreases in carbohydrate levels, the TCA cycle can function in non-cyclic flux mode due to diminishing of carbon skeleton the enter it making required that be fed by anauplerotic reactions. Therefore, amino acids become essential to support respiration and ATP synthesis under such situations. Compelling evidence have demonstrated that branched chain amino acids (BCAA) and lysine can supply electrons to the mitochondrial electron transport chain (mETC) by the action of the electron transfer flavoprotein (ETF)-ETF: ubiquinone oxidoreductase (ETF/ETFQO) system and associated dehydrogenases. In plants, only isovaleryl- CoA dehydrogenase (IVDH) and (D)-2-hydroxyglutarate dehydrogenase (D2HGDH) have been characterized as electron donnor to the ubiquinol pool via this system so far by the degradation of BCAA and lysine, respectively. In fact, BCAA catabolism is of pivotal importance to provide intermediates to TCA cycle, particularly under stress situations, whereas lysine shows a strict association with the TCA cycle being required to couple amino acid degradation and energy generation. The electron transfer through the mETC is tightly coupled to ATP synthesis and use electron donates by NADH and FADH 2 to phosphorylate ADP to ATP. However, our knowledged regarding the organization of the mitochondrial oxidative phosphorylation (OXPHOS) system and its alternatives pathways under energy limitation remains elusive. Thus, this thesis, which is focused on the function of respiration within the context of the role of the TCA cycle as well as the function of alternative electron donors to the mETC, iscomprised by three independent stand-alone chapters focusing on energy metabolism and alternative respiration in Arabidopsis thaliana. Hence to obtain a compreenhesive picture of how the TCA cycle evolved and to which extend its alternative pathways interact to adjust to different cellular and metabolic requirements, three experimental approaches were used: (i) by using bioinformatic approaches we investigated the evolutionary history of TCA cycle genes allowing the generation of a model for the origin of the TCA cycle genes in plants and connected its evolution with TCA cycle behavior under a range of stress; (ii) the importance of lysine deficiency were investigated by using an Arabidopsis mutant with reduced activity of the lysine biosynthesis enzyme L,L-diaminopimelate aminotransferase (dapat), and (iii) the metabolic reprograming associated with the OXPHOS system were investigated following carbon limitation.. In brief, the results presented here provided several novel findings and allowed, at least preliminarly, mechanistic interpretation thereof. First, it facilitate the elucidation of the evolutionary origem of the TCA cycle in land plants providing support to the contention that the origin of isoforms present in different subcellular compartments might be associated either with gene-transfer events which did not result in correct targeting or with new gene copys generated by genome duplication and horizontal transfer gene. Additionally, coexpression analyses of TCA cycle genes following different stress conditions in both shoot and root tissues demonstrated the presence of a large molecular plasticity and provided an explanation for the modular operation of the TCA cycle in land plants. Secondly, by using an Arabidopsis mutant with reduced activity of the Lys biosynthesis enzyme L,L-diaminopimelate aminotransferase (dapat) it was demonstrated that lysine biosynthesis deficiency mimics stress situation and impacts both plant growth and leaf metabolism.Thirdly, by evaluating OXPHOS system behavior following carbon starvation and how a range of amino acids can impact respiratory complexes it was possible to further demonstrate that OXPHOS is affected in function of the carbon source and that alternative pathways are induced under this condition.In addition, immunoblotting assays revealed that OXPHOS system is most likely regulated by posttranslational modification. When considered together these results highlight the complexity and specificity of plant respiration during evolution and that it is differently affected following energy limitation by the usage of alternative substrates. The results discussed here support the contention that ETF/ETFQO is an essential pathway able to donate electrons to the mETC and that amino acids are alternative substrates maintaining respiration under carbon starvation.The results obtained are discussed in the context of current models of metabolic evolution showing the strict association of energy metabolism with amino acids metabolism, and where possible, mechanistic insights are properly discussed. Key-words: alternative substrate respiration; energy deprivation; mitochondria evolution; mitochondria metabolism; neofunctionalization; OXPHOS; paralogous genes; stress response; TCA cycle;

The different reproductive strategies of males and females underlie differences in behavior that may also lead to differences in nutrient use between the two sexes. We studied sex differences in the utilization of two essential amino acids (EAAs) and one non-essential amino acid (NEAA) by the Carolina sphinx moth (Manduca sexta). On day one post-eclosion from the pupae, adult male moths oxidized greater amounts of larva-derived AAs than females, and more nectar-derived AAs after feeding. After 4 days of starvation, the opposite pattern was observed: adult females oxidized more larva- derived AAs than males. Adult males allocated comparatively small amounts of nectar-derived AAs to their first spermatophore, but this allocation increased substantially in the second and third spermatophores. Males allocated significantly more adult-derived AAs to their flight muscle than females. These outcomes indicate that adult male and female moths employ different strategies for allocation and oxidation of dietary AAs.

Improving our understanding of milk protein production regulation and AA transport is important for successfully formulating diets for AA and improving N efficiency. The objectives were to study protein synthesis regulation and AA transport using in vitro and in vivo models. In the first experiment, the objective was to evaluate the ability of five distinct AA profiles and balancing Lys to Met ratio to 3:1 to stimulate protein translation. No single AA profile uniquely stimulated phosphorylation of translational machinery related proteins suggesting identification of a single optimal AA profile as unlikely. In the second experiment, an in vitro method using three different AA isotopes was developed to trace AA movement. The method assesses bi-directional transport of multiple AA simultaneously enabling evaluation of unidirectional uptake kinetics. This method was used to evaluate AA concentrations representing 16, 100, 186, and 271% of cow plasma AA concentrations. Amino acid uptake was not saturable within the in vivo range for eleven AA. Arginine, Val, and Pro exhibited saturation with the Michaelis-Menten km being 95, 49, and 65% of in vivo concentrations. Results suggest that AA transport is generally non-saturable and that high bi-directional transport exists which enables a mechanism for mitigating AA shortages. In experiment 3, the objective was to evaluate milk protein production and regulation from infusing Met, Lys, and His (MKH) or Ile and Leu (IL). The two EAA groups independently and additively increased milk protein yield. This finding contradicts the single limiting AA theory that a single nutrient will limit milk protein yield. Changes in udder AA extraction and blood flow from supplemental EAA reveal flexible delivery mechanisms. The phosphorylation state of proteins associated with the mTOR pathway was impacted by both EAA treatments. Changes in the udder proteome suggest negative feedback on mTOR pathway activation when milk protein yield was increased by the EAA groups separately but when supplemented together, negative feedback was lessened. Results indicate that multiple EAA can stimulate milk protein production, the ability of AA transport to match intracellular needs, and that the single limiting AA theory or existence of a unique optimal AA profile is likely irrelevant in dairy cows.
Doctor of Philosophy

Thesis (Ph.D.)--University of Missouri-Columbia, 2006.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on May 1, 2009) Vita. Includes bibliographical references.

A regulação dependente de Ca2+ da atividade ATPásica da acto-miosina em concentrações fisiológicas de actina, tropomiosina e troponina ocorre exclusivamente na presença de troponina T (TnT). Nosso grupo demonstrou que um polipeptídeo correspondente aos primeiros 191 aminoácidos da TnT ativa a atividade ATPásica da acto-miosina na presença de tropomiosina e na ausência das outras duas subunidades do complexo troponina (TnI/TnC). Com o objetivo de mapear e caracterizar esse domínio ativatório da TnT, construímos fragmentos de TnT correspondentes às regiões compreendidas entre os resíduos de aminoácidos: 1-157 (TnTl-157), 1-76 (TnTl-76), 77-157 (TnT77-57), 77-191 (TnT77-191) e 158-191 (TnT158-191). Estudos das interações desses fragmentos com actina e tropomiosina demonstraram que: i) o fragmento TnTl-76 não se liga à tropomiosina ou a actina; ii) a região da TnT correspondente aos resíduos 158-191 liga-se à actina cooperativamente, mas não se liga à tropomiosina; iii) a região correspondente seqüência de aminoácidos 77-157 é necessária para a interação da TnT com o resíduo de aminoácido 263 da tropomiosina; iv) TnT77-191 ativa a atividade ATPásica da acto-miosina com a mesma intensidade que TnTl-191. Também observamos que TnTl-157, TnTl-76, TnT77-157, TnT158-91 e combinações de TnT158-191 com TnTl-157 e TnT77-157 não afetam a atividade ATPásica da acto-miosina. Concluímos que a região da TnT delimitada pelos aminoácidos 77 e 191 é essencial para a ativação da atividade ATPásica da actomiosina e que essa ativação é mediada pelas interações dessa região da TnT com a tropomiosina e a actina.
The Ca2+-regulation of the actomyosin ATPase activity at physiological ratios of actin, tropomyosin and troponin occurs only in the presence of troponin T. Our group has previously demonstrated that a recombinant polypeptide corresponding to the first 191 amino acids of TnT (TnTl-191) activates the aetomyosin Mg2+-ATPase activity in the presence of tropomyosin and in the absence of TnI/TnC. In order to further map and characterize this activation domain, we constructed a set of recombinant or synthetic TnT fragments, corresponding to amino acids 1-157 (TnTl-157), 1-76 (TnTl-76), 77-57 (TnT77-157), 77-191 (TnT77-191) and 158-191 (TnT158-191). Binding assays using these fragments demonstrated that: i) amino acids 1-76 of TnT do not bind to tropomyosin or actin; ii) amino acids 158-191 bind to actin cooperatively, but not to tropomyosin; iii) the sequence 77-157 is necessary for TnT\'s interaction with residue 263 of tropomyosin; iv) TnT77-191 on its own activates de actomyosin ATPase activity to the same extent as previously described for TnTl-191. TnT1-157; TnTl-76; TnT77-157; TnT158-191 and combinations of TnT158-191 with TnTl-157 or TnT77-157 showed no effect on the ATPase activity. We conclude that interactions of amino acids 77-191 of TnT with tropomyosin and actin are essential for the activation of actomyosin ATPase activity, and that this activation may be mediated in part by a direct interaction between TnT residues 158-191 and actin.

Na maioria dos estudos de digestibilidade de aminoácidos em suínos usam-se animais de peso igual ou superior a 20 kg, isso se deve a menor dificuldade na implantação de cânulas íleo-cecais e a melhor recuperação pós-cirúrgica na fase do crescimento. No entanto, avaliar ingredientes com leitões mais jovens torna-se importante, visto que há limitações fisiológicas do trato gastrointestinal nesse período que podem afetar o seu desenvolvimento. Assim, 175 leitões desmamados, divididos em 7 ensaios experimentais com 25 animais cada, foram usados para determinar o balanço de nutrientes e da energia, a digestibilidade ileal aparente (AID) e estandardizada (SID) dos aminoácidos (AA) de sete ingredientes usualmente utilizados em dietas de leitões, com ou sem suplementação de multi-carboidrase (MC) e fitase (F). Os leitões foram desmamados aos 23 dias de idade e alojados em gaiolas para estudo de digestibilidade e metabolismo, permanecendo no experimento até os 45 dias de idade. Adaptação, coleta total de fezes e urina ocorreram do 10° ao 20° dia do período experimental e as amostragens do conteúdo ileal se deu ao abate, no 22° dia (45 dias de idade). Foi utilizado o delineamento experimental inteiramente casualisado com 4 tratamentos e cinco repetições. O leitão foi considerado como unidade experimental. As dietas experimentais consistiram de ingrediente teste sem enzimas e combinado a MC, F ou MC + F. Dois tipos de dieta referência foram usados como base para os cálculos do balanço nutricional e os coeficientes de digestibilidades aparente e estandardizada dos aminoácidos. Óxido de cromo (0,3%) foi usado como marcador indigestível nas avaliações dos aminoácidos. A enzima MC apresentava 10% de galactomananase, 10% de xilanase, 10% de β-glucanase, 60% de cevada maltada e 10% de α-galactosidase. A F era proveniente da fermentação de Saccharomyces cerevisiae com atividade de 10.000 FTU/g. Os ingredientes testados foram: farelo de arroz integral, soja integral micronizada, farelo de trigo, quirera de arroz, sorgo, milho e farinha de soja texturizada. Os resultados indicaram efeitos da combinação ingrediente e enzima exógena e as evidencias se deram de acordo com as características bromatológicas de cada um
Most of the studies, involving AA digestibility in pigs are used animals with 20 kg minimal weight or higher than. This is due difficulty to implant a simple T-cannula on distal ileum of younger pigs, besides a better post-surgical recovery in growing phase. However, ingredient evaluations with young pigs become important because there are physiological limitations in gastrointestinal tract that may affect its performance. Thus, 175 weaned pigs, divided into seven experimental trials with 25 animals each, were used to determine the nutrients and energy balance, the apparent (AID) and standardized (SID) ileal digestibility of amino acids (AA) from seven ingredients usually used in pig diets, with or without multi-carbohydrase (MC) and phytase (Phy) supplementation. Weaned piglets at 23 d old were housed in cages to studies digestibility and metabolism, remaining in the experiment until 45 d of age. Pig adaptation to feces and urine collection was 10 to 20 d experimental period and ileal content sampling at slaughter at 22 d (45 d old). A completely randomized experimental design was used with 4 treatments and 5 replicates. The pig was considered as an experimental unit. The experimental diets consisted of test ingredient as the sole source of protein with or without MC, Phy or MC+Phy. Two kind of reference diet were used to calculate the nutrient balance or AID and SID of AA. Titanium dioxide (0.3%) was used as indigestible marker. The MC enzyme had: galactomannanase, 10%; xylanase, 10%; beta-glucanase, 10%; malted barley, 60% and α-galactosidase, 10%. The Phy was obtained from the Saccharomyces cerevisiae fermentation with 10,000 FTU/g activity. The ingredients used were: rice bran, micronized full fat soybean meal, wheat bran, broken rice, sorghum, corn and texturized soybean meal. The results indicated effects from ingredients and enzymes combinations and the evidences occurred according to bromatological characteristics of each one

Alzheimer’s disease (AD), the most common form of dementia in the elderly, is a global issue affecting about 24 million individuals. Because AD is a systemic pathology, dementia is not the only leading factor contributing to loss of independence in AD patients. AD may also impair skeletal muscle metabolism and function. Creatine (CR) supplementation may enhance skeletal muscle hypertrophy/mass and function in sarcopenia and muscular dystrophies, but has yet to be studied in AD. This study examined the effect of oral CR on muscle metabolism in a triple-transgenic (3xTg) AD mouse model. Twenty-four, 3×Tg AD mice (~8 month-old) were randomly assigned to control (CON) or CR (3% w/w) diet. Bodyweights and feed intakes were measured throughout the 8-week study. Lower limb (quadriceps muscle; QM and gastrocnemius; GM) and upper limb muscles (triceps; TM) were collected to analyze levels of CR, total protein, DNA, RNA, amino acids (AA), adenosine triphosphate (ATP), adenosine diphosphate (ADP), total and phosphorylated p70 ribosomal S6 kinase (p70S6K). Data (mean ± SEM) were assessed by analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) post hoc test. In comparison to the CON group, CR supplementation increased CR content in both GM (p=0.002) and QM (p=0.037), with higher (p=0.032) ATP/ADP ratio in CR in comparison with CON in QM. A higher protein concentration (p<0.0001) was notable in GM of CR supplemented group vs. CON. Total branched-chain AA levels in QM increased 2-fold (p< 0.0001) in CR groups. Additionally, CR resulted in a higher (p<0.05) protein/DNA ratio; an index of muscle cell size, in both QM and GM for CR groups. The index of cell capacity for protein synthesis (RNA/DNA ratio) in GM was also higher (p=0.001) in CR groups. However, phosphorylation (activation) level of p70S6K, an integral component in protein synthesis signalling pathway, did not show any significant differences in female (p=0.161) and male (p=0.292) CR supplemented groups compared with CON. To conclude, CR supplementation is capable of inducing muscle hypertrophy/growth parameters in the 3×Tg AD mouse model, thereby enhancing protein synthesis capacity in skeletal muscles, thus possibly promoting muscle function in AD.
May 2016

The effects of denervation, non-weight bearing (unloading) or immobilization on hindlimb muscle growth, protein and amino acid metabolism were studied. In the first 3 days after denervation or unloading, atrophy of the soleus was caused by a suppression of protein synthesis and an acceleration of protein degradation. Thereafter, further atrophy, up to 6 days was due to depressed protein synthesis only. The changes in both protein synthesis and degradation in the first three days accounted for 69% and 65%, respectively, of the total loss of protein and mass in 6 days of unloaded or denervated soleus. Over the 6-day period, denervated soleus lost more mass and protein than the unloaded muscle owing to the earlier onset and greater extent of proteolysis. In denervated soleus, both lysosomal and non-lysosomal proteolysis may be enhanced, whereas in the unloaded muscle possibly only non-lysosomal proteolysis was enhanced. In both cases non-lysosomal proteolysis may be mediated by Ca²⁺-activated neutral protease, partially as a result of Ca²⁺ release from sarcoplasmic reticulum. Possibly due to the lack of lysosomal proteolysis, the insulin receptor did not show apparent increased turnover with unloading, as suggested by increased insulin sensitivity of in vitro protein turnover in the unloaded soleus. In contrast, denervated soleus showed a normal response to insulin for in vitro protein turnover. These findings suggested a mechanistic difference of unloading and denervation atrophy of soleus. A decreased ratio of glutamine/glutamate in fresh muscle suggested that the synthesis of glutamine in soleus may be diminished by denervation just as by unloading. This diminution of glutamine synthesis was probably due to reduced availability of ammonia, as evidenced by the slow disappearance of ATP in incubated denervated soleus. Similiar to unloading, denervation led to a decrease in aspartate concentration. This decreased concentration apparently resulted in decreased rather than increased utilization of aspartate. Effects of stretch on unloaded soleus were particularly pronounced in the first two days. Thereafter, in the stretched, unloaded soleus protein degradation increased to nearly the same extent as did protein synthesis. Hence after two days, stretch seems to lose its effectiveness in mitigating the effects of unloading so that it may not be an adequate preventive measure of muscle wasting under non-weight bearing condition.

Au cours de ma thèse, nous avons utilisé la truite arc-en-ciel, un poisson carnivore et modèle potentiellement pertinent du diabète, pour étudier des mécanismes de régulation du métabolisme intermédiaire hépatique par les nutriments (acides aminés (AA) et le glucose). Nous nous sommes plus particulièrement intéressés aux voies de signalisation de l’insuline et des acides aminés (Akt et mTORC1). Grâce à l’utilisation de rapamycine, un inhibiteur pharmacologique de mTORC1, nous avons montré que l'activation de mTORC1 stimule l'expression de gènes de la lipogenèse, de la glycolyse et du catabolisme des acides aminés, tandis que la voie de signalisation Akt inhibe celle des gènes impliqués dans la néoglucogenèse. Ces études ont été conduites dans le foie de truite ou en culture primaire d’hépatocytes de truite arc-en-ciel. En outre, nous avons démontré lors de stimulations à court terme in vivo et in vitro que l'expression hépatique des gènes de la lipogenèse est plus sensible à l'apport de protéines alimentaires ou d’AA qu’à l'apport de glucides ou de glucose. De plus, nous avons observé que des taux élevés d’AA conduisent, par le biais de l’activation de la voie de signalisation mTORC1, à une augmentation de l'expression des gènes lipogéniques mais surtout à une répression de l’inhibition de l’expression des gènes de la néoglucogenèse induite par l’insuline. Cet effet s’accompagne d’une augmentation de la phosphorylation de IRS-1 sur le résidu Ser302 qui pourrait être responsable de la baisse de phosphorylation d'Akt et par conséquent d’une inhibition de l’action de l'insuline. Enfin, en réalisant un test de tolérance au glucose chez des truites préalablement traitées avec de la rapamycine, nous avons conclu que la néoglucogenèse hépatique joue un rôle probablement majeur dans le contrôle de l'homéostasie glucidique chez la truite. Ainsi, une absence d’inhibition de la néoglucogenèse pourrait contribuer au maintien de l'hyperglycémie prolongée et au phénotype d’intolérance au glucose caractéristique des poissons carnivores. Cette thèse met en avant le rôle des protéines/AA dans la régulation du métabolisme intermédiaire de la truite et identifie certaines voies de signalisation cellulaire sollicités par les acides aminés pour réguler le métabolisme. Elle permet ainsi d’éclaircir certaines particularités nutritionnelles de la truite
During my doctoral study, we used rainbow trout, a representative carnivorous fish and relevant diabetic model, to study the mechanisms underlying the regulation of hepatic intermediary metabolism by nutrients (amino acids (AAs) and glucose), and determine the potential involvement of insulin/Akt and mTORC1 signaling pathways in these regulations. Using acute administration of rapamycin, a pharmacological inhibitor of TOR, we first identified that mTORC1 activation promotes the expression of genes related to fatty acid biosynthesis, glycolysis and amino acid catabolism, while Akt negatively regulates gluconeogenic gene expression in rainbow trout liver and primary hepatocytes. Furthermore, we demonstrated hepatic fatty acid biosynthetic gene expression is more responsive to dietary protein intake/AAs than dietary carbohydrate intake/glucose during acute stimulations in vivo and in vitro. Moreover, we further showed that high levels of AAs up-regulate hepatic fatty acid biosynthetic gene expression through an mTORC1-dependent manner, while excessive AAs attenuate insulin-mediated repression of gluconeogenesis through elevating IRS-1 Ser302 phosphorylation, which in turn impairs Akt phosphorylation and dampens insulin action. Finally, using glucose tolerance test and acute inhibition of rapamycin, we concluded that hepatic gluconeogenesis probably plays a major role in controlling glucose homeostasis, which maybe account for the prolonged hyperglycemia and glucose intolerance phenotype of carnivorous fish. The present thesis brings forward our understandings about the roles of protein/AAs in the regulation of hepatic intermediary metabolism in trout and identifies relevant cellular signaling pathways mediating the action of amino acids on metabolism. It also clarifies some nutritional characteristics of the trout

D-amino acids (D-AAs) are the α-carbon enantiomers of L-amino acids (L- AAs), the building blocks of proteins in known organisms. It was largely believed that D-AAs are unnatural and must be toxic to most organisms, as they would compete with the L-counterparts for protein synthesis. Recently, new methods have been developed that allow scientists to chromatographically separate the two AA stereoisomers. Since that time, it has been discovered that D-AAs are vital molecules and they have been detected in many organisms. The work of this dissertation focuses on their place in bacterial metabolism. This specific area was selected due to the abundance of D-AAs in bacteria-rich environments and the knowledge of their part in several processes, such as peptidoglycan synthesis, biofilm disassembly, and sporulation. We focused on the bacterium Pseudomonas putida KT2440 which inhabits the densely populated plant rhizosphere. Due to its versatility and cosmopolitan character, this bacterium has provided an excellent system to study D-AA metabolism. In the first chapter, we have developed a new approach to identify specific genes encoding enzymes acting on D-AAs, collectively known as amino acid racemases. Using this novel method, we identified three amino acid racemases encoded by the genome of P. putida KT2440. All of the enzymes were subsequently cloned and purified to homogeneity, followed by a complete biochemical characterization. The aim of the second chapter was to understand the specific role of the peculiar broad-spectrum amino acid racemase Alr identified in chapter one. After constructing a markerless deletion of the cognate gene, we conducted a variety of phenotypic assays that led to a model for a novel catabolic pathway that involves D-ornithine as an intermediate. The work in chapter three identifies for the first time numerous rhizosphere-dwelling bacteria capable of catabolizing D-AAs. Overall, the work in this dissertation contributes a novel understanding of D-AA catabolism in bacteria and aims to stimulate future efforts in this research area.

Manufacturers of amino acid supplements claim that they can act as natural stercoids. Eighteen experienced male weight lifters were studied to test this hypothesis for the amino acid arginine.
Master of Science

Peroxinitrito (ONOO- + ONOOH), o produto da rápida reação do óxido nítrico com o ânion radical superóxido, tem recebido muita atenção como possível mediador dos efeitos deletérios associados a uma superprodução de •NO. O peroxinitrito é um potente oxidante que é capaz de oxidar e nitrar várias biomoléculas por mecanismos que contribuímos para esclarecer no decorrer desta tese. Especificamente, estudamos a oxidação de urato e tirosina por peroxinitrito. Demonstramos que o urato é oxidado por peroxinitrito a alantoina, aloxana e ao radical aminocarbonila. Como a reação direta entre urato e peroxinitrito tem uma constante de velocidade relativamente baixa (k= 4,8 x 102 M -1.s-1) em comparação com àquelas de outras biomoléculas, sugerimos que o urato é um potente sequestrador dos radicais derivados do peroxinitrito (•NO2 e CO3•-, na maioria dos ambientes biológicos; a pH ácido, o radical •OH também pode se tornar relevante). No caso da tirosina, confirmamos que ela não reage diretamente com o peroxinitrito mas com os radicais dele derivados. Como antecipado, o rendimento relativo dos produtos (3-nitrotirosina, 3,3-bitirosina e 3-hidroxitirosina (DOPA)) variou com o pH e a presença de CO2. Esses estudos nos levaram a propor a co-localização de proteínas nitradas e hidroxiladas como um possível biomarcador de peroxinitrito. Para testar essa hipótese, um anticorpo monoclonal anti-DOPA foi desenvolvido e utilizado em modelos de infecção por Leishmania amazonenses (macrófagos (J774), e camundongos resistentes (C56Bl/6) e suscetíveis (BALB/c). A co-localização de proteínas hidroxiladas e nitradas ficou evidênciada em todos os modelos testados e ocorreu concomitantemente a máxima produção de •NO. Infelizmente, o anticorpo obtido perdeu a atividade e ainda não pudemos confirmar esses dados.
Peroxynitrite (ONOO- + ONOOH), which is formed by the fast reaction between nitric oxide and superoxide anion, has been receiving increasing attention as a mediator of the deleterious effects associated with an overproduction of •NO. The compound is a strong oxidant that is able to oxidize and nitrate a variety of biotargets by mechanisms that this work has contributed to establish. Specifically, we studied the oxidation of urate and tyrosine by peroxynitrite. Urate oxidation produced allantoin, alloxan and the amiocarbonyl radical. Since the rate constant of the direct reaction between urate and peroxynitrite (k= 4,8 x 102 M-1.s-1) is low in comparison with those of other biotargets, we proposed that urate is an efficient scavenger of peroxynitrite-derived radicals (•NO2 and CO3•- in most biological environments; at acid pH, the •OH radical may also become relevant). ln the case of tyrosine, we confirmed that it does not react directly with peroxynitrite but, instead, with the radicals derived form it. As anticipated, the relative yield of the products (3-nitrotyrosine, 3,3-bityrosine and 3-hydroxytyrosine (DOPA)) varied with the pH and CO2 presence. These results led us to propose that co-localization of nitrated and hydroxylated proteins could be a peroxynitrite biomarker. To test this hypothesis, a monoclonal anti-DOPA antibody was developed and tested in Leishamnia amazonensis infection models (macrophages (J774), and resistant (C56Bl/6) and susceptible mice (BALB/c). It was possible to evidence co-localization of hydroxylated and nitrated proteins in all tested models in a time when •NO synthesis was maximum. Unfortunetly, we were unable to confirm these results due to antibody inactvation; new antibody baches are being obtained.

Introdução - A metabolômica permite determinar padrões de variação dos metabólitos entre indivíduos doentes e não doentes, com ou sem ingestão de um determinado alimento ou dieta. Compreender a relação entre metabólitos e doenças metabólicas pode ajudar a combater obesidade e doenças crônicas. Objetivo - Investigar a associação entre o perfil metabolômico de aminoácidos, a ingestão dietética e o estado nutricional em adultos participantes do Inquérito de Saúde no município de São Paulo, Brasil. Métodos - Foram avaliados dados de 168 indivíduos. A análise metabolômica para identificação de 21 aminoácidos foi realizada nas amostras de plasma, utilizando kit AbsoluteIDQTMp180 da Biocrates Life Science AG (Innsbruck, Austria). O consumo alimentar foi estimado por meio da aplicação do questionário de frequência alimentar. Os grupos de aminoácidos e de alimentos foram submetidos à análise fatorial por componente principal. A variável Metabolicamente Saudável foi construída considerando-se as Diretrizes da Sociedade Brasileira de Cardiologia para o diagnóstico e tratamento da síndrome metabólica. A regressão linear múltipla foi aplicada para avaliar as associações, tendo como variáveis de ajustes: etnia, idade, renda familiar, sexo, dieta, atividade física. A comparação entre grupos foi feita por MANOVA e a confirmação da diferença significativa pelo teste de Bonferroni. Resultados - A porcentagem de indivíduos com obesidade foi de 24%, sendo que a média de IMC correspondeu a 26 kg/m2. A população estudada apresentou média de idade de 50 anos, sendo a maioria de etnia branca (56%) e do sexo masculino (52%), com pouca adesão à atividade física (21%). Os parâmetros bioquímicos estavam, em média, abaixo das concentrações estabelecidas como normais, exceto a insulina, cuja média foi de 20,8 ?UI/mL. Todavia, ao estratificar pelo estado nutricional e metabolicamente saudável, os parâmetros bioquímicos se mostraram diferentes, tais como a glicemia, triglicerídeos e colesterol total. A ingestão dietética foi igual entre os grupos estudados. O perfil de aminoácido revelou potencial de diferenciar os estados nutricional e metabólico, destacando os aminoácidos de cadeia ramificada (Leu, Ile, Val). Foram identificados dois padrões de aminoácidos relacionados a beta oxidação e aos aminoácidos glicogênicos. O primeiro teve relação positiva para os indivíduos com obesidade e metabolicamente não saudável. O segundo, apresentou relação inversa para ambos os estados. O consumo de manganês pela população foi de 2,5 mg/dia, sendo infusão de mate a principal fonte (28mg Mn/porção). Os indivíduos obesos ingeriram menor quantidade de manganês, que apresentou relação inversa ao estado nutricional e ao padrão de aminoácidos relacionado ao metabolismo não saudável. Identificaram-se três padrões alimentares: perfil saudável, tradicional e moderno. A associação com o estado nutricional e metabolicamente saudável foi positiva para o padrão saudável. Conclusão - Os aminoácidos de cadeia ramificada se revelaram biomarcadores para identificar o estado nutricional e metabólico de indivíduos adultos. Os indivíduos que tiveram baixo consumo de manganês apresentaram maior adesão ao perfil metabolômico de aminoácidos relacionados com beta oxidação.
Introduction - The metabolomics allows to determine patterns of variation of the metabolites between people with or without illness, considering or not the food consumption. Understanding the relationship between metabolites and metabolic disorders, it is possible to deal with obesity and chronic diseases. Objective - To investigate the association between the metabolic profile of amino acids and dietary intake and nutritional status in adults from the household survey conducted in the city of São Paulo, Brazil. Method - The 21 amino acids were identified by metabolomic analysis, using Absolute IDQTMp 180 kit from Biocrates Life Science AG (Innsbruck, Austria). Food intake was estimated using the food frequency questionnaire. The amino acid and food groups were submitted to factorial analysis by main component. The Metabolically Healthy variable was constructed considering the Guidelines of the Brazilian Society of Cardiology for the diagnosis and treatment of the metabolic syndrome. Multiple linear regression was applied to evaluate the associations, adjusted by the variables: ethnicity, age, family income, sex, diet, physical activity. The comparison between groups was made by MANOVA, and the confirmation of the significant difference, by the Bonferroni test. Results - The percentage of people with obesity was 24%, although the mean BMI corresponded to 26 kg/m2. The population studied presented a mean age of 50 years, most of them white (56%) and male (52%), with little adherence to physical activity (21%). The biochemical parameters were, on average, below the established normal concentrations, except for insulin (20,8 ?UI/mL). However, when stratified by nutritional status and metabolically healthy, the biochemical parameters were statistically different. The dietary intake was the same among the groups studied. The amino acid profile revealed potential to differentiate the nutritional and metabolic states, highlighting the branched chain amino acids. Two amino acid patterns related to beta-oxidation and glycogenic amino acids had been identified. The former had a positive relationship for obese and metabolically unhealthy people. The second presented an inverse relation for both states. The manganese consumption by the population was 2.5 mg / day, and the mate infusion was the main source (28mg Mn/serving). Obese subjects consumed less manganese, which had an inverse relationship to nutritional status and to the amino acid pattern related to unhealthy metabolism. Three dietary patterns were identified: healthy, traditional and modern profiles. The association with nutritional and metabolically healthy status was positive for the healthy pattern. Conclusion - The branched-chain amino acids had been revealed to be biomarkers to identify the nutritional and metabolic status of adult individuals. The individuals that had low manganese consumption showed greater adhesion to the metabolic profile of amino acids related to beta-oxidation, and could be used as biomarker for the ingestion of this micronutrient.

Elevations in extracellular glutamate in the brain are implicated in the pathogenesis of several neurological conditions, including mesial temporal lobe epilepsy. The underlying mechanisms of this elevation are not completely understood, however, and there are no effective methods that reduce the elevation or limit its neurotoxic effects. Glutamate is normally cleared from the extracellular space and replenished in axon terminals by a series of compartmentalized processes collectively known as the glutamate-glutamine cycle. A critical step of the cycle is the conversion of glutamate to glutamine by the astrocyte-specific enzyme glutamine synthetase. In several neurological conditions including mesial temporal lobe epilepsy, studies have demonstrated that glutamine synthetase activity is pathologically low. Because glutamine synthetase is thought to be critical for glutamate metabolism, its deficiency has been postulated as a possible mechanism for the increased glutamate observed in the extracellular fluid of the epileptogenic areas of the brain.

The branched-chain amino acids valine, leucine, and isoleucine are thought to contribute to de novo synthesis of glutamate in the brain by transferring an amino nitrogen to the tricarboxylic acid intermediate alpha-ketoglutarate. The branched-chain amino acids have gained increasing attention in recent years for the important roles they play in cell signaling, immune modulation, protein metabolism, and glutamate synthesis. It was previously unknown, however, if increasing peripheral branched-chain amino acids concentrations can increase extracellular glutamate concentrations in the brain during physiological or in pathological conditions, particularly when glutamate metabolism is perturbed (i.e. in glutamine synthetase deficiency). Moreover, the effects of branched-chain amino acids on seizures and neuronal viability were unknown.

The objective of this thesis was to use state of-the-art methods in microdialysis, isotope tracing, mass spectrometry and video-intracranial electroencephalogram recordings to study the metabolism and functional effects of branched-chain amino acids and glutamate in naïve and glutamine synthetase-inhibited, epileptic rats. The central hypothesis was that increased extracellular concentrations of branched-chain amino acids in the brain, in combination with alterations in enzymatic processes of glutamate metabolism, are key pathogenic features that result in brain glutamate excess and seizures in mesial temporal lobe epilepsy. To achieve the objective of this thesis, we pursued 3 specific aims.

In Aim 1, we determined the effects of intravenous branched-chain amino acid administration on brain extracellular fluid concentrations of glutamate and glutamine in naïve rats. We found that the administration of a high-dose bolus of branched-chain amino acids significantly increased the concentrations of branched-chain amino acids and glutamine in the extracellular compartment of the brain. Glutamate concentrations transiently increased, but the elevation was not statistically significant. In Aim 2, we determined the effects of intravenous isotope-labeled leucine administration on brain extracellular fluid concentrations of glutamate and glutamine in glutamine synthetase-inhibited rats. We found that glutamine synthetase-inhibited rats, like normal rats, were remarkably efficient in handling glutamate. Moreover, we demonstrated that leucine influx across the blood brain barrier is highly dependent on glutamine levels in the extracellular fluid of the brain. In Aim 3, we investigated the effects of chronic oral branched-chain amino acid supplementation on spontaneous and induced seizures, and neuron loss in glutamine synthetase-inhibited epileptic rats. We found that the branched-chain amino acid supplementation was ineffective in reducing the frequency and severity of spontaneous seizures, but increased the threshold to pentylenetetrazole-induced seizures. Furthermore, chronic branched-chain amino acid supplementation resulted in increased loss of hippocampal hilar neurons. Future studies will explore the impact of glutamine and leucine dysregulation in the brain on cell signaling and immune modulation, which may play an important role in epilepsy as well as other disorders. We will also further explore the mechanisms underlying the branched-chain amino acid-induced neuron loss.