Tesis sobre el tema "Glutamate"

The coenzyme B12-dependent enzyme glutamate mutase (E.C. 5.4.99.1) catalyses the rearrangement of (2S)-glutamic acid to (2S,3S)-3-methylaspartic acid. Each of the two components of the enzyme was purified to homogeneity using a combination of low and high performance chromatographic techniques. Component E and S displayed molecular weights of 53 KDa and 13 KDa respectively as determined by gel electrophoresis, contrary to literature reports. A large number of glutamate and 3-methylaspartate analogues were synthesised and tested as substrates for the enzyme from Clostridium tetanomorphum. No rearrangement products could be detected for (2S,3R)-3-methylaspartic acid, (2S,3S)-3-ethylaspartic acid, 3-methylglutamic acid, (2S,3R)-3-methylsuccinic acid or A/-methyl-3-methylaspartic acid. Five inhibitors were discovered for the enzyme. Four of them were typical competitive inhibitors: (2S,3S)- and (2S,3R)-3-methylglutamates (Ki = 1.0 mM and Ki = 1.5 mM respectively); (2S)-homocysteic acid, Ki = 5 mM; and 1-bromo-cis-1,2-cyclopropanedicarboxylic acid (Ki= 2.2+/-0.2 mM). Finally 1-bromo-trans-1,2- cyclopropanedicarboxylic acid prevented the enzyme from processing (2S)- glutamic acid for periods of times proportional to its concentration. Our results support a radical mechanism with a protein bound glycyl radical as an intermediate, and provide evidence for the existence of two distinct conformations of the holoenzyme, prior to and after the activation of the cofactor. (2S,3R)-3- and (2S,3S)-3-Methylglutamic acids were synthesised stereospecifically by extending Schollkopf's bis-lactim ether methodology. The attack of various carbon anions at C-5 of isopropyl N-benzyl-(4S,5R)-1,2,3- oxathiazolidone-5-methyi-4-carboxyiate S,S-dioxide was not a versatile pathway. Nevertheless, the reaction of the oxathiazolidone with an allylmagnesium lithium cuprate complex gave some promising results, but more research is necessary to optimise certain problematic steps. Several different routes were evaluated for the preparation of 1-amino-1,2-cyciopropanedicarboxylic acid, but either low yields or instability of intermediates thwarted any attempts to achieve this goal. Finally 1- bromo-cis-and trans-1,2-cyclopropanedicarboxylic acids were synthesised by reacting methyl acrylate with methyl dibromoacetate in the presence of sodium hydride. The two pairs of enantiomers, cis- ((2S,3S) and (2R,3R)) and trans- ((2R,3S) and (2S,3R)) were separated by selective ester formation.

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A transmissÃo de impulsos nervosos à feita por meio das sinapses, envolvendo neurotransmissores e receptores. Os receptores ionotrÃpicos glutamatÃrgicos (GluRs) sÃo importantes canais iÃnicos do sistema nervoso central, encontrados em sinapses de excitaÃÃo rÃpida, e estÃo relacionados a funÃÃes cerebrais importantes como aprendizado e memÃria. AlÃm disso, os GluRs tambÃm estÃo associados com certas doenÃas neurolÃgicas e psiquiÃtricas, como por exemplo: a doenÃa de Alzheimer, o mal de Parkinson, a epilepsia, o acidente vascular cerebral, a esclerose lateral amiotrÃfica e a esquizofrenia. Neste trabalho, tiramos vantagem dos dados disponÃveis na literatura da co-cristalizaÃÃo dos seguintes agonistas glutamato (C5H9NO4) e AMPA (C7H10N2O4), do agonista parcial cainato (C10H15NO4) e do antagonista DNQX (C8H2N4O6) com o receptor GluR2 com resoluÃÃo de 1,9 Ã, 1,7 Ã, 1,9 à e 1,8 Ã, respectivamente, para estudar a interaÃÃo destes quatro ligantes com a GluR2 por meio de mÃtodos computacionais ab initio. Os hidrogÃnios ausentes dos dados de difraÃÃo de raios-X foram colocados atravÃs de um processo semi-clÃssico de minimizaÃÃo da energia total GluR2-ligante. A seguir, as simulaÃÃes foram feitas usando a Teoria do Funcional da Densidade (DFT), tanto ao nÃvel da aproximaÃÃo da densidade local (LDA), como da aproximaÃÃo do gradiente generalizado (GGA), para descriÃÃo dos efeitos de troca e correlaÃÃo. A utilizaÃÃo do mÃtodo de fragmentaÃÃo molecular com capas conjugadas (MFCC) tornou possÃvel analisar a interaÃÃo dos ligantes com cada um dos resÃduos prÃximos e pÃs-prÃximos do GluR2. Considerou-se tambÃm a relevÃncia da blindagem dos resÃduos pÃs-prÃximos que interagem com os ligantes, bem como se fez uma anÃlise da energia de interaÃÃo dos resÃduos (prÃximos e pÃs-prÃximos) considerados com os Ãtomos dos ligantes (resultados apresentados nos grÃficos BIRD), sem e com mediaÃÃo das molÃculas de Ãgua existentes no sÃtio de ligaÃÃo (o que permite se determinar ab initio a relevÃncia da Ãgua na energÃtica da interaÃÃo ligante-GluR2). Obteve-se a energia total de interaÃÃo GluR2-ligante em funÃÃo da distÃncia dos centroides dos ligantes aos resÃduos, o que permitiu correlacionÃ-la à intensidade de ativaÃÃo e antagonismo dos neurotransmissores em questÃo. Demonstrou-se que ela segue a ordem AMPA > glutamato > cainato > DNQX somente quando um raio do sÃtio de ligaÃÃo suficientemente grande à considerado, o que explica dados experimentais publicados sobre a ativaÃÃo e antagonismo do receptor glutamatÃrgico GluR2, sugerindo que os resÃduos pÃs-prÃximos podem ser importantes para determinar o funcionamento do receptor. Para o glutamato, os resultados obtidos indicam que os resÃduos atrativos mais relevantes sÃo: Arg485, Lys730 (mediado pela Ãgua W39), Ser654, Leu650 mediado por W69, e Lys656 mediado por W22; os resÃduos repulsivos mais relevantes para o glutamato sÃo Glu402 (pÃs-prÃximo) mediado por W36, Glu657 e Asp651 (pÃs-prÃximos). Para o AMPA os resÃduos atrativos mais relevantes sÃo: Arg485, Thr655 mediado por W134, Lys730 mediado por W137, Lys656 mediado por W138, Lys449 e Arg684 (pÃs-prÃximos); os resÃduos repulsivos mais relevantes para o AMPA sÃo Glu402 mediado por W3, Asp651 mediado por W96 e W139 (pÃs-prÃximo), e Glu657 (pÃs-prÃximo) mediado por W140. Para o cainato os resÃduos atrativos mais relevantes sÃo Arg485, Ser654, Thr655 e Arg684 (pÃs-prÃximo); os resÃduos repulsivos mais relevantes para o Cainato sÃo Glu402, Glu657 mediado por W78 (pÃs-prÃximo) e Asp651. Para o DNQX os resÃduos atrativos mais relevantes sÃo Arg485, Glu705 e Tyr450 mediado por W26 e W137; o resÃduo repulsivo mais relevante para o DNQX à Leu498. Uma plÃiade de perspectivas relacionadas aos resultados obtidos reluz e dentre elas podemos destacar a possibilidade do desenvolvimento de agonistas e antagonistas glutamatÃrgicos com especificidades voltadas à diminuiÃÃo de efeitos colaterais quando utilizados no tratamento de doenÃas relacionadas à neurotransmissÃo glutamatÃrgica.
The transmission of nerve impulses occurs through the synapses, involving neurotransmitters and receptors. The ionotropic glutamate receptors GluRs are important ionic channels of the central nervous system, founded in rapid excitation synapses, and related to important cerebral functions like learning and memory. Besides this, GluRs are also associated with important neurological and psychiatric diseases like Alzheimer, Parkinson, epilepsy, cerebral ischemia, amyotrophic lateral sclerosis, and schizophrenia. In this work, we take advantage of the available data in the literature of co-crystallization of the following full agonists glutamate (C5H9NO4) and AMPA (C7H10N2O4), the partial agonist kainate (C10H15NO4) and the antagonist DNQX (C8H2N4O6) with the GluR2 receptor with resolution of 1.9 Ã, 1.7 Ã, 1.9 Ã and 1.8 Ã, respectively to study the interaction of these four ligands with GluR2 by means of ab initio computational methods. The absent hydrogens in the GluR2-ligand X-ray diffraction data were inserted through a semi-classical total energy minimization process. Next, the simulations were performed within the scope of the Density Functional Theory (DFT), both in the local density approximation (LDA) as generalized gradient approximation (GGA) for the description of exchange-correlation effects. The use of the molecular fragmentation method with conjugated caps (MFCC) allowed to analyze the interaction between the ligands with each one close and next-closed GluR2 residues. It was also considered the relevance of the screening of the next-closed residues with interact with the ligands, and it was performed an analysis of the interaction energy between the focused residues (close and next-closed) with the atoms of the ligands (results depicted in the BIRD panels), without and with the mediation of water molecules existing in the binding pocket (which allows to determine ab initio the relevance of water in the GluR2-ligands energetic). It was obtained the GluR2-ligand total energy interaction as a function of the distance between the ligand centroid and the residues, which allowed to correlate it to the activation strength and antagonism of the ligands focused. It was demonstrated that it follows the order AMPA > glutamate > kainite > DNQX only when a large enough binding pocket radius is taken into account, explaining the experimental data published on the activation and antagonism of the glutamatergic receptor GluR2 and suggesting the next-closed residues can be important to determine the receptor functioning. For the glutamate, the obtained results point that the most important attractive residues are Arg485, Lys730 (water W39 mediated), Ser654, Leu650 (water W69 mediated), and Lys656 (water W22 mediated); the most important repulsive residues for the glutamate are Glu402 (next-closed water W36 mediated), Glu657 and Asp651 (nex-closed). For AMPA, the most important attractive residues are Arg485, Thr655 (water W134 mediated), Lys730 (water W137 mediated), Lys656 (water W138 mediated), Lys449 and Arg684 (next-closed); the most important repulsive residues for AMPA are Glu402 (water W3 mediated), Asp651 (next-closed, water W96 and W139 mediated), and Glu657 (next-closed, water W140 mediated). For kainate the most important attractive residues are Arg485, Ser654, Thr655 and Arg684 (next-closed); the most important repulsive residues for kainite are Glu402, Glu657 (next-closed, water W78 mediated) and Asp651. For DNQX, the most important attractive residues are Arg485, Glu705 and Tyr450 (water W26 and W137 mediated); the most important repulsive residue for DNQX is Leu498. A pleiade of perspectives related with the obtained results shines, among which one can highlight the possibility to develop glutamatergic agonists and antagonists with specificities related to decrease side effects when used in the treatment of maladies related with the glutamatergic neurotransmission.

Introdução: A Leucemia Linfóide Aguda (LLA) e o Linfoma não-Hodgkin (LNH) são os tipos de câncer mais incidentes em crianças e a ingestão alimentar pode ser diminuída pela quimioterapia. A sensação do gosto é resultante da detecção e resposta ao estímulo doce, salgado, azedo, amargo e umami. Esse último, identificado pelo glutamato monossódico (MSG), é relacionado ao aumento da palatabilidade, o que pode colaborar para a melhora da aceitação alimentar em crianças com câncer. Objetivo: Identificar os limiares de detecção do gosto umami e a qualidade da alimentação em crianças portadoras de LLA e LNH. Metodologia: Foi aplicado teste de sensibilidade de Threshold para determinar o limiar do gosto umami, com 6 concentrações crescentes de água deionizada e MSG. Aplicou-se recordatório 24 horas e questionário de frequência alimentar para avaliar o consumo alimentar. O peso e altura foram mensurados e IMC utilizados para classificação do estado nutricional, segundo o National Center for Health Statistics (2000). Caracterizou-se a amostra através da distribuição de frequência das variáveis, com auxílio do pacote estatístico Epinfo Versão 6.0. As análises estatísticas e gráficas foram feitas no software R, versão 2.6.2. Foi realizado teste de Cluster para caracterizar a amostra. Resultados: Dos 102 pacientes, 94 eram sensíveis ao umami. 54,3 por cento do sexo masculino e 45,7 por cento do feminino. 78,4 por cento portadores de LLA e 21,6 por cento de LNH. 91,0 por cento em fase de manutenção. Quanto à idade, 38,3 por cento entre 6 e 7 anos; 20,6 por cento entre 8 e 9; 15,7 por cento entre 10 e 11; 15,7 por cento entre 12 e 13 e 9,8 por cento 14 anos. 8,5 por cento apresentaram baixo peso, 66,0 por cento eutrofia e 25,5 por cento sobrepeso ou obesidade. O produto rico em glutamato monossódico mais consumido foi macarrão instantâneo. O molho inglês e de soja foram os menos consumidos. Os alimentos preferidos foram salgadinhos e macarrão instantâneo, e o que menos gostavam era a mostarda. Não houve diferença estatisticamente significante entre os limiares de sensibilidade ao umami e as variáveis em estudo. O agrupamento da amostra caracterizou 4 grupos: Grupo 1, composto por crianças mais sensíveis ao umami, mais jovens, maioria eutrófica e do sexo masculino; Grupo 2, maioria eutrófica, do sexo feminino, com menor consumo de carboidrato e maior de proteína e lipídeo; Grupo 3, composto por crianças mais velhas, maioria eutrófica e do sexo masculino, com maior consumo calórico e de carboidrato; Grupo 4, com crianças menos sensíveis ao umami, eutróficas e com sobrepeso, maioria do sexo masculino e com ingestão calórica mais baixa. Conclusão: As crianças são sensíveis ao gosto umami e essa característica independe do sexo, idade, estado nutricional, fase de tratamento, ingestão calórica e de macronutrientes. A qualidade da alimentação e a idade foram variáveis determinantes das similiradades entre os grupos. O teste de sensibilidade para detecção do gosto umami é de grande interesse para conhecer o comportamento alimentar e auxiliar na melhora da aceitação dos alimentos
Introduction: The Acute Lymphoblastic Leukemia (ALL) and non-Hodgkin Lymphoma (NHL) are the most frequent cancers in children and food intake can be reduced by chemotherapy. The sense of taste is a result of the detection and response to the sweet, salty, sour, bitter and umami stimulus. The latter is identified by monosodium glutamate (MSG) and is related to the increase of palatability, which may contribute to improve food acceptance in children with cancer. Objective: identification of the thresholds of detection of umami taste and food quality in children with LLA and LNH. Methodology: the threshold sensitivity test was applied in order to determine the threshold of the umami taste using six increasing concentrations of deionized water and MSG. A 24-hour recall and food frequency questionnaire were applied to check food intake. Weight and height were measured and the BMI was used to determine the nutritional status, according to the National Center of Health Statistics (2000). The sample was characterized by the distribution of the frequency of the variables, with the help of the Epinfo Version 6.0 statistical package. The statistical and graphical analyses were done using the R statistical software, version 2.6.2. The Cluster test was applied to characterize the sample. Results: from the 102 patients, 94 were sensitive to umami taste. 54,3 per cent were male and 45,7 per cent were female. 78,4 per cent had ALL and 21,6 per cent had NHL. 91 per cent were in the maintenance stage. Regarding age, 38,3 per cent were between 6 and 7 years old; 20,6 per cent were between 8 and 9 years old; 15,7 per cent were between 10 and 11 years old; 15,7 per cent were between 12 and 13 years old and 9,8 per cent were 14 years old. 8,5 per cent were underweight, 66 per cent were eutrophic and 25,5 per cent were overweight or had obesity. The most consumed product was instant noodles, rich in monosodium glutamate. The tabasco and soy sauces were the least consumed. The favorite food was snacks and instant noodles and mustard was the food they liked the least. There was no statistically significant difference between the thresholds of sensitivity to umami and the variables in study. The gathering of the sample characterized four groups: Group 1 formed by younger children, most male and eutrophic, more sensitive to the umami taste; Group 2 formed by most eutrophic and female children, showing lower intake of carbohydrates and higher intake of proteins and lipids; Group 3 formed by older children, most eutrophic and male, showing higher caloric and carbohydrate intake; Group 4 formed by eutrophic and overweight children, most male and with lower caloric intake, less sensitive to the umami taste. Conclusion: children are sensitive to the umami taste and this characteristic does not depend on the sex, age, nutritional status, treatment stage, caloric and macronutrients intake. The food quality and age were determinant variables of the similarities among the groups. The test of sensitivity for the detection of the umami taste is of great interest in the knowledge of food intake behavior and in the increase of food acceptance

L'etude d'un cycle complet de developpement chez le colza a montre qu'il existait un fort potentiel de reduction du nitrate au moment de la reprise de la croissance au printemps. Au moment de l'initiation de la floraison, la reduction du nitrate se delocalise des feuilles vers les tiges et enfin vers les siliques. Une telle capacite des siliques a reduire le nitrate constitue une originalite. Par ailleurs, nous avons mis en evidence que la dynamique de la biomasse foliaire, croissance et senescence, est un facteur cle de l'efficacite d'utilisation de l'azote (leleu et al. 2000, plant biology and biochemistry 2000 38 : 639-645). Des plantules de colza cultivees in vitro presentent dans leur partie aerienne (cotyledons et hypocotyle), une activite nitrate reductase (anr) independante de la source d'azote. Quand l'ammonium constitue la seule source d'azote disponible pour le colza, l'anr est fortement augmentee par rapport a celle obtenue en presence de nitrate. Le taux de transcrits nitrate reductase (nr) et le contenu en proteine nr sont egalement augmentes. Cette regulation particuliere se trouve dans les cotyledons aussi bien en presence d'ammonium qu'en absence totale d'azote. L'anr semble proportionnelle au contenu endogene en ammonium. L'assimilation de l'ammonium et la production de glutamate puis de glutamine paraissent avoir un role fondamental dans la regulation de l'anr des parties aeriennes de plantules de colza. C'est la premiere fois que l'on montre une telle deregulation de la nr en presence d'un stress ammoniacal. L'elucidation des voies metaboliques en cause pourrait apporter des idees nouvelles quant a la regulation de la nr en conditions naturelles.

Os nossos resultados demonstraram a atividade enzimática prolina desidrogenase (PRODH) do produto do gene anotado como codificante de uma prolina oxidase na base dados do genoma de Trypanosoma cruzi. A atividade da proteína codificada por esse gene foi avaliada inicialmente por complementação de uma linhagem de S. cerevisiae deficiente na expressão funcional da PRODH endógena, e posteriormente mediante a obtenção da enzima recombinante ativa expressa em um sistema bacteriano. Nos estudos feitos tanto com leveduras complementadas com o gene PRODH, quanto com as formas epimastigotas de T. cruzi, observamos que nesses organismos há uma correlação positiva entre o nível intracelular de prolina livre e a resistência ao estresse oxidativo. Através dos ensaios de RT-PCR quantitativo observamos que o RNAm do gene da PRODH é regulado ao longo do ciclo de vida do T. cruzi, com maior nível de expressão no estágio epimastigota intracelular, perfil apresentado também em experimentos de Western Blotting, o que é concordante com o papel fundamental desse aminoácido na diferenciação dessa forma para as forma tripomastigota. Nos diferentes ensaios de localização subcelular observou-se que a proteína PRODH está presente na mitocôndria. A seqüência da PRODH apresenta um sítio EF-hand de ligação a Ca2+. Mostramos experimentalmente a funcionalidade desse sítio e a sua função na regulação da atividade por Ca2+. Em ensaios de respiração mitocondrial, utilizando prolina como substrato, foi mostrado que a prolina fornece elétrons à cadeia respiratória através da PRODH, transferindo elétrons e gerando FADH2 no mesmo nível e com a mesma eficiência que o complexo II (succinato desidrogenase). O análogo de prolina T4C, inibidor do transporte de prolina, causa uma diminuição do conteúdo de prolina livre intracelular, o que foi relacionado com uma diminuição significativa da sobrevivência do parasito em combinação com o estresse nutricional e oxidativo, mostrando a participação do metabolismo de prolina na resistência a esses estresses. Todos esses dados sugerem ademais que o transportador de prolina do parasita pode ser um alvo para drogas terapêuticas de interesse quando combinada com outras drogas que agem via geração de espécies reativas de oxigênio.
In the present work, we demonstrated the proline dehydrogenase enzymatic activity (PRODH) for the protein encoded by a gene annotated as a proline oxidase in the T. cruzi genome data base. This activity was shown firstly through complementation of a S. cerevisiae lineage lacking its endogenous PRODH gene. The PRODH gene was also expressed in a bacterial system and the active recombinant protein was obtained. Experiments performed with both, complemented yeasts and T. cruzi epimastigotes, showed a correlation between the intracellular free proline levels and the oxidative stress resistance. Quantitative RT-PCR assay revealed that the PRODH gene is differentially expressed across T. cruzi life cycle, being the highest expression level shown by the intracellular epimastigote form, this result was confirmed by Western blotting. Both results are in accordance with the fact that proline is essential for the differentiation of the intracellular epimastigote into trypomastigote. Subcellular localization assays showed that PRODH is present preferentially in the mitochondria. In silico analyses of the PRODH peptidic sequence indicated the presence of an EF-hand domain, wich is, usually, involved in Ca2+ binding. In fact, our results confirm not only the ability of such domain of binding Ca2+ but also its function in the activity regulation. Mitochondrial respiration assays using proline as substrate showed that PRODH transfers electrons and generates FADH2, with an eficience comparable to that of the complex II (Succinate dehydrogenase). Experiments using the T4C, an analogue of proline that inhibits the proline uptake, caused the depletion of the intracellular free proline, which was followed by the significantly decrement of the cellular viability of the parasites under nutritional and oxidative stresses. Taken together, this data suggest that proline transporters are promising drug targets when combined with other drugs that act by generating reactive oxygen species.

L’envelliment progressiu de la població pot implicar una major incidència de malalties neurodegeneratives, el que suposa un problema social important que requereix d’investigació. Les regions cerebrals especialment sensibles als efectes de l’envelliment són l’escorça prefrontal (CPF) i l’hipocamp (HPC), amb especial implicació dels seus receptors d’àcid N-metil-D-aspartat (NMDAr) i α-amino-3-hidroxi-5-metil-4-isoxazolpropionic (AMPAr). Donada la implicació d’aquests receptors glutamatèrgics en els processos cognitius, diversos estudis els han utilitzat com a diana per a tractaments destinats a la millora cognitiva. Així doncs, creiem que l’administració d’agonistes glutamatèrgics podrien revertir dèficits cognitius associats a l’envelliment, o induïts per fàrmacs. En la present tesi hem estudiat en rates l’administració intracerebral al còrtex prelímbic (CPL), d’una banda, de l’ampaquina S18986 per reduir els dèficits de memòria produïts per: a) la hipofunció colinèrgica deguda a l’administració d’escopolamina (SCOP) al CPL (Experiment 1) i b) la inactivació amb muscimol (agonista GABAèrgic) del nucli parafascicular del tàlem (PFn) (Experiment 2). S’han avaluat els efectes dels tractaments en paradigmes conductuals de memòria implícita (Discriminació Simple d’Olors, DSO) i relacional (Transmissió Social de Preferència d’Aliment, TSPA). D’altra banda, també hem estudiat la capacitat del tractament amb d-cicloserina (DCS), un agonista parcial dels NMDAr, per a potenciar en rates envellides la memòria de treball (MT) i la flexibilitat cognitiva,dues funcions cognitives clarament minvadesdurant l’envelliment. Els efectes de l’administració intracerebral de DCS al CPL en aquestes funcions i en la memòria a curt i llarg termini, s’han avaluat utilitzant el paradigmes de DelayedMatching / Non-Matching to Position (DMTP/DNMTP),en un laberint en Y i en una tasca operant de respostes alternes(Experiment 3). Els resultats dels experiments 1 i 2 indiquen que l’SCOP al CPL produeix un efecte bloquejadorde la memòria de la DSO en el record a les 24 hores i que la inactivació del PFn impedeix l’adquisició d’aquesta tasca associativa olfactiva. La hipofunció colinèrgica i l‘alteració de la influència talàmica sobre el CPF podrien explicar aquests resultats. L’S18986 va reduir els errors comesos durant l’adquisició de la tascaDSO, respecte el grup control i el grup tractat amb SCOP. Però, en canvi, aquests efectes facilitadors no van observar-se ni en les sessions de retenció, ni en d’altres models conductuals com la TSPA. Diferents aspectes metodològics, com les dosis administrades o el mitjà de dilució del fàrmac, podrien explicar les divergències observades respecte d’altres antecedents experimentals. Els efectes procognitiusmés destacables els hem observat en l’experiment 3, on l’administració intracerebral de DCS ha revertitles alteracions deMT associades a l’envelliment natural. L’administració pre-entrenament de DCS al CPL ha igualat l’execució de les rates velles tractades a la de les rates joves, amb o sense tractament amb DCS. Aquests resultats concorden amb d’altres estudis que mostren com l’efecte beneficiós de la DCS sembla estarassociat a l’existència d’algun tipus de dèficit cognitiu. En general, l’efecte de la DCS ha estat més evident en la MT (DMTP) que en la flexibilitat cognitiva (DNMTP) o en la memòria a curt termini (laberint en Y). Aquestes divergènciespodrien ser conseqüència d’un efecte de sensibilització dels receptors degut a l’administració crònica del tractament ode la finestra temporal d’aplicació. En canvi, en el test de memòria als set dies de l’aprenentatge de la tasca de respostes alternes, el tractament de DCS en els animals vells va facilitar l’execució, igualant-la a la dels grups d’animals joves. L’administraciódels diferents tractaments aplicats en aquesta tesino va tenir cap efecte significatiu en altres variables que podrien haver intervingut en els resultats, com la capacitat olfactòria, la l’activitat motora ola motivació per menjar. Amb els resultats obtinguts en la present tesi aportem coneixement sobre potencials tractaments dirigits a la modulació glutamatèrgica per a revertir dèficits cognitius resultants de l’envelliment, o d’altres patologies associades a disfuncions cerebrals. Tant l’administració de l’S18986 com de la DCS promouen mecanismes de plasticitat sinàptica, com la potenciació a llarg termini,la qual a mésde facilitar la memòria a llarg termini també podria estar implicada en la memòria a curt termini,prodouint una facilitació de tasques amb un component atencional rellevant. Addicionalment, el fet que l’ús d’agonistes parcials redueixi la possibilitat de toxicitat, i que aquests fàrmacs promoguin mecanismes neuroprotectors, converteix a aquests dos fàrmacs en potencials substàncies nootròpiques, les quals podrien servir com a tractament de dèficits cognitius, tenint en compte que es requereix investigació addicional sobre els possibles efectes dosi-dependents, el perfil agonista i d’altres aspectes.
The progressive aging of the population may involve a high incidence of neurodegenerative diseases, which is an important social problem that requires research. The cerebral regions especially sensitive to the effects of aging are the prefrontal cortex (PFC) and the hippocampus (HPC), with special involvement of its receptors N-methyl-D-aspartate (NMDAr) and α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic (AMPARr). Given the involvement of these glutamatergic receptors in the cognitive processes, several studies have used them as targets for treatments aimed at cognitive improvement. Therefore, we believe that the administration of glutamatergic agonists could reverse cognitive deficits associated with aging, or induced by drugs. In the present thesis we have studied in rats the intracerebral administration in the prelimbic cortex (CPL), on the one hand, of the ampakine S18986 to reduce the memory deficits produced by: a) cholinergic hypofunction due to the administration of scopolamine (SCOP) into the CPL (Experiment 1) and b) inactivation of the parafascicular nucleus of the thalamus (PFn) by muscimol (GABAergic agonist) (Experiment 2). The effects of these treatments have been evaluated in behavioural paradigms of implicit memory (Odour Discrimination Task, ODT) and relational memory (Social Transmission of Food Preference, STPF). On the other hand, we have studied the capacity of the treatment with d-cycloserine (DCS), a partial agonist of the NMDAr, to enhance working memory (WM) and cognitive flexibility in aged rats, two cognitive functions clearly depleted during the aging process. The effects of intracerebral administration of DCS in the CPL on these functions and on short- and long-term memory have been evaluated using the paradigms of Delayed Matching / Non-Matching to Position (DMTP / DNMTP), in a Y-maze and in an operant alternate response task (Experiment 3). The results of experiments 1 and 2 indicate that SCOP into the CPL produces a memory-blocking effect on the DSO in the 24-h recall test and the PFn inactivation prevents the acquisition of this associative olfactory task. The cholinergic hypofunction and the alteration of the thalamic influence on the PFC could explain these results. Moreover, S18986 reduced the number of errors committed in the DSO acquisition, respect to the control group and the group treated with SCOP. However, these facilitating effects are not observed, neither in the retention sessions nor in other behavioural models such as the TSPA. Different methodological aspects, such as the administered doses or the dilution medium of the drug, could explain the divergences observed with respect to other experiments. The most remarkable procognitive effects have been observed in the Experiment 3, where the intracerebral administration of DCS has reversed the WM alterations associated with natural aging. The pre-training administration of DCS to the CPL has matched the performance of the treated old rats to the young rats. These results agree with other studies showing that the beneficial effect of DCS seems to be associated with the existence of some kind of cognitive deficit. In general, the effect of DCS has been more evident in WM (DMTP) than in cognitive flexibility (DNMTP) or in short-term memory (Y-maze). These divergences could be a consequence of a sensitization effect of the receptors due to the chronic administration of the treatment or the temporal administrationwindow. On the other hand, in the 7-d memory test of the alternate response task, the DCS treatment in aged animals facilitated itsperformance, equalling it to the young animals. The administration of the different treatments applied in this thesis did not have any significant effect in other variables that could have influenced the results, such as the olfactory capacity, the motor activity or the motivation to eat. With the results obtained in this thesis we provide knowledge about potential treatments aimed at glutamatergic modulation to reverse cognitive deficits resulting from aging, or other pathologies associated with brain dysfunction. Both the administration of S18986 and the DCS promote mechanisms of synaptic plasticity, such as long-term potentiation, which facilitate long-term memory and also short-term memory, providing facilitation of tasks with a relevant attentional component. Additionally, the fact that the use of partial agonists reduces the possibility of toxicity and that these drugs promote neuroprotective mechanisms, turns these two drugs into potential nootropic substances, which could be used as treatment of cognitive deficits, taking into account that additional research is required on the possible dose-dependent effects, the agonist profile and other issues.

Les récepteurs métabotropiques du glutamate ont fait l’objet d’une attention particulière ces dernières années au vu de leur intérêt dans de multiples aspects de la transmission glutamatergique. Leur expression étendue à travers le système nerveux central en fait des cibles thérapeutiques particulièrement prometteuses et de récentes études corroborent l’utilité des mGluR dans des troubles neurologiques ou psychiatriques tels que les maladies d’Alzheimer ou de Parkinson, l’anxiété, la dépression et la schizophrénie. C'est dans ce contexte, que nous nous sommes intéressés à la synthèse d’analogues du glutamate, ligands des mGluR. La première partie de ce travail a été consacrée à la construction du squelette de l’acide glutamique. L'approche développée est basée sur l’addition de b-énaminoesters chiraux sur des accepteurs de Michael a-substitués via une réaction de Michael asymétrique. Dans la deuxième partie, nous avons développé deux voies devant mener aux quatre diastéréoisomères de nos analogues. Les étapes-clés de notre approche font intervenir une cyclisation intramoléculaire puis l’ouverture de ces cycles
Metabotropic glutamate receptors have received considerable attention over the past decade in view of their relevance in multiple aspects of glutamatergic transmission. The widespread expression of mGluRs through the central nervous system makes these receptors particularly attractives drug targets, and recent studies validate the therapeutic utility of mGluR ligands in neurological and psychiatric disorders such as Alzheimer’s disease, Parkinson’s disease, anxiety, depression and schizophrenia. It is in this context that we were interested in the synthesis of glutamic acid analogues. The first part of this thesis describes the construction of the glutamic acid’s framework. The synthetic approach involves the asymmetric Michael addition of chiral b-enaminoesters to a-substituted Michael acceptors. In the second part, two synthetic pathways to the four diastereomers of our analogues are described. In this approach, the intramolecular cyclization and then the opening of the cycle are the key-steps

'Excitotoxicity' represents the excitatory amino acid mediated degeneration of neurons. Glutamate is the major excitatory neurotransmitter in the brain. Glutamate excitotoxicity has been implicated in a number of neurodegenerative disorders like Stroke, Epilepsy, Alzheimer's disease and traumatic brain injury. This neurotoxicity is summed up by the 'glutamate hypothesis' which describes the cause of neuronal cell death as an excessive release of glutamate causing over excitation of the glutamate receptors and subsequent increase in influx of calcium leading to cell death. An effort to counteract this neurotoxicity has lead to the development of glutamate receptor antagonists that can effectively serve as neuroprotective agents. Nevertheless, the downside to these drugs has been the side effects observed in clinical trial patients due to their disruptive action on the physiological function of these receptors like learning and memory. This work was undertaken to identify targets that can effectively be used to treat excitotoxicity without affecting any normal physiological functions. In one approach, (chapter I) we have identified the KATP channels as an effective modulator of epileptogenesis. In another approach, (Chapter II) we show that targeting the AMPA receptor subunit GluR2 is a practical strategy for stroke therapy. KATP channels that are gated by intracellular ATP/ADP concentrations are a unique subtype of potassium channels and play an essential role in coupling intracellular metabolic events to electrical activity. Opening of KATP channels during energy deficits in the central nervous system (CNS) induces efflux of potassium ions and in turn hyperpolarizes neurons. Thus, activation of KATP channels is thought to be able to counteract excitatory insults and protect against neuronal death. Here, we show that, functional Kir6.1 channels are located at excitatory pre-synaptic terminals as a complex with type-1 Sulfonylurea receptors (SUR1) in the hippocampus. The mutant mice with deficiencies in expressing the Kir6.1 or the SUR1 gene are more vulnerable to generation of epileptic form of seizures, compared to wild-type controls. Whole-cell patch clamp recordings demonstrate that genetic deletion of the Kir6.1/SUR1 channels enhances glutamate release at CA3 synapses. Hence, expression of functional Kir6.1/SUR1 channels inhibits seizure responses and possibly acts via limiting excitatory glutamate release. In addition to epilepsy, ischemic stroke is a leading cause of death in developed countries. A critical feature of this disease is a highly selective pattern of neuronal loss; certain identifiable subsets of neurons, particularly CA1 pyramidal neurons in the hippocampus are severely damaged, whereas others remain intact. A key step in this selective neuronal injury is Ca2+/Zn2+ entry into vulnerable neurons through [alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels, a principle subtype of glutamate receptors. AMPA receptor channels are assembled from glutamate receptor (GluR) -1, -2, -3, and -4 subunits. Circumstance data have indicated that the GluR2 subunits dictate Ca2+/Zn2+ permeability of AMPA receptor channels and gate injurious Ca2+/Zn2+ signals in vulnerable neurons. Here we show that ischemic insults induce toxic Ca2+ entry through AMPA receptors into vulnerable neurons by modification of GluR2 RNA editing. Thus, targeting of GluR2 subunit can be considered as a promising target for stroke therapy.
Ph.D.
Department of Biomolecular Science
Burnett College of Biomedical Sciences
Biomolecular Sciences PhD

Microglia, the immune cells of the central nervous system (CNS), are important in the protection of the CNS, but may be implicated in the pathogenesis of neuroinflammatory disease. Upon activation, microglia produce reactive oxygen and nitrogen species; intracellular antioxidants are therefore likely to be important in their self-defence. Here, it was confirmed that cultured microglia contain high levels of glutathione, the predominant intracellular antioxidant in mammalian cells. The activation of microglia with lipopolysaccharide (LPS) or LPS + interferon-gamma was shown to affect their glutathione levels. GSH levels in primary microglia and those of the BV-2 cell line increased upon activation, whilst levels in N9 microglial cells decreased. Microglial glutathione synthesis is dependent upon cystine uptake via the xc- transporter, which exchanges cystine and glutamate. Glutamate is an excitatory neurotransmitter whose extracellular concentration is tightly regulated by excitatory amino acid transporters, as high levels cause toxicity to neurones and other CNS cell types through overstimulation of glutamate receptors or by causing reversal of xc- transporters. Following exposure to LPS, increased extracellular glutamate and increased levels of messenger ribonucleic acid (mRNA) for xCT, the specific subunit of xc-, were observed in BV-2 and primary microglial cells, suggesting upregulated GSH synthesis. An activation-induced decrease in N9 GSH levels suggests that this cell line is more susceptible to oxidative damage, and may be less able to upregulate GSH synthesis. Albumin, to which microglia may be exposed following blood-brain barrier damage, increased iNOS expression, glutamate release, xCT mRNA levels and intracellular levels of GSH and ATP in BV-2 and primary microglia. Primary and BV-2 microglial conditioned medium contained low levels of GSH, suggesting that microglia may release GSH. Modulation of microglial metabotropic glutamate receptors (mGluRs) may alter microglial activation and neurotoxicity. Here, stimulation of the neuroprotective mGluR5 and group III mGluRs caused a decline in GSH levels in BV-2 and N9 microglia, respectively. In contrast mGluR1 stimulation may increase BV-2 GSH levels. The work presented in this thesis therefore extends current knowledge regarding microglial GSH and its regulation, and contributes to the understanding of microglial neurotoxicity and neuroprotection.

本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである
Kyoto University (京都大学)
0048
新制・論文博士
博士(農学)
乙第9225号
論農博第2056号
新制||農||722(附属図書館)
学位論文||H8||N2934(農学部図書室)
UT51-96-F462
(主査)教授 左右田 健次, 教授 加藤 暢夫, 教授 木村 光
学位規則第4条第2項該当

The plant glutamate receptors (GLRs) are homologs of mammalian ionotropic glutamate receptors (iGluRs) and are hypothesized to be potential amino acid sensors in plants. Since their first discovery in 1998, the members of plant GLRs have been implicated in diverse processes such as C/N ratio sensing, root formation, pollen germination and plant-pathogen interaction. However, the exact properties of these channels, such as the spectrum of ligands, ion specificities, and subunit compositions are still not well understood. It is well established that animal iGluRs form homo- or hetero-tetramers in order to form ligand-gated cation channels. The first aspect of this research was to determine if plant GLRs likewise require different subunits to form functional channels. A modified yeast-2-hybrid system approach was initially taken and applied to 14 of the 20 AtGLRs to identify a number of candidate interactors in yeast. Forster resonance energy transfer (FRET), which measures the transfer of energy between interacting molecules, was performed in mammalian cells to confirm interaction between a few of those candidates. Interestingly, despite an abundance of overlapping co-localization between heteromeric combinations, only homomeric interactions were identified between GLRs 1.1 and 3.4 in HEK293 cells. Further, amino acids have been implicated in signaling between plants and microbes, but the mechanisms for amino acid perception in defense responses are far from being understood. Recently it was demonstrated that calcium responses initiated by bacterial and fungal microbe-associated molecular patterns (MAMPs) were diminished in seedlings treated with known agonists and antagonists of mammalian iGluRs, suggesting potential roles of GLRs in pathogen responses. Analysis of publicly available microarray data shows altered gene expression of a sub-fraction of GLRs in response to pathogen infection and bacterial elicitors. Thus, the second goal of my PhD research was aimed at determining whether GLRs are involved in the interaction between plants and pathogens. Gene expression changes of a number of candidate GLRs as well as pathogen growth was examined in response to the plant pathogen Pseudomonas syringae pv. tomato DC3000. Interestingly, single gene and multi-gene deficient plants responded differently with regards to pathogen susceptibility, likely as a result of functional compensation between GLRs.
Ph. D.

The gene gdhA from Escherichia coli, that encodes a NADPH-dependent glutamate dehydrogenase (GDH), directs a novel pathway in transgenic plants that potentially allows an increase in ammonium assimilation. Glufosinate leads to plant death by the irreversible inhibition of glutamate synthetase (GS) leading to a disruption of subsequent GS-related processes resulting in elevated ammonium and disruption of photorespiration. Therefore, it was speculated that the gdhA-transformed plants may exhibit a novel mechanism of resistance to glufosinate by altered activity of the GDH pathway and subsequently related processes. Studies were conducted in the greenhouse to evaluate 1) whole plant tolerance to glufosinate, 2) changes in absorption, translocation and metabolism of glufosinate, and 3) metabolic fingerprint changes in response to glufosinate treatment in tobacco plants containing the gdhA gene. Whole plant tolerance experiments showed that tobacco transformed with the gdhA gene expressed up to six fold increased resistance (GR50) to glufosinate compared with the non-gdhA control line. GDH enzyme activity among gdhA-transformed tobacco lines was highly correlated (r2 = 0.9903) with the amount of herbicide resistance. Thus, use of the E. coli gdhA gene in plant transformations can provide an additional mechanism for resistance to glufosinate. Foliar absorption and translocation of 14C from glufosinate was not altered to any large extent in gdhA-transformed plants which suggests these factors cannot fully explain the mechanism for whole-plant resistance to glufosinate. However, the metabolic fingerprint resulting from glufosinate treatment was significantly altered in gdhA tobacco. It was also shown that metabolic perturbation induced by glufosinate was lower in the high GDH activity tobacco line, +gdhA 9, than in the non-gdhA control tobacco line as evidenced by the reduced number of altered peaks recorded in leaves of these two tobacco lines. Thus, gdhA-transformed tobacco plants with low and high expression of GDH activity, exhibited greater overall stability of metabolism following the application of glufosinate, than recorded in non-gdhA control plants. This greater metabolic stability during GS inhibition was likely due to the amelioration of amino acid production through the increased activity of GDH. Therefore, the hypothesized mechanism of increased resistance to glufosinate in gdhA-transformed tobacco lines is by maintenance of amino acid production and maintenance of photorespiratory activity.

Introduction: L'injection ip de lipopolysaccharide (LPS) d'E.coli à la rate gestante aboutit à un phénotype cognitivo-comportemental de pathologies neuropsychiatriques chez la progéniture mâle. L'objectif principal était de vérifier l'hypothèse d'une atteinte structurelle et d'un déséquilibre entre excitation et inhibition dans l'hippocampe. L'objectif secondaire était de dégager des stratégies thérapeutiques ciblées.Méthodes: 500 μg/kg de LPS d'E.coli de sérotype O55:B5 ou 2 ml/kg de sérum physiologique étaient injectés ip à la rate au 19e jour de gestation. La progéniture mâle était étudiée à différents stades du développement. L'étude structurelle reposait sur de l'immunohistochimie, l'étude fonctionnelle sur des enregistrements électro-physiologiques de l'activité des cellules pyramidales de l'aire CA1. L'effet protecteur de la N-acétylcystéine (NAC) donnée po à la rate gestante après l'injection de LPS était testé. Résultats: Les animaux soumis à un stress prénatal par le LPS présentaient une désorganisation durable de la couche pyramidale de l'aire CA3, un déficit transitoire de neurones exprimant la reeline, une altération de la dépression à long terme des synapses glutamatergiques (LTDe) liée à un déficit des récepteurs NMDA et du système GABAergique. Un inhibiteur de la recapture du GABA parvenait à corriger les anomalies de la LTDe. La NAC prévenait les anomalies cyto-architecturales.Conclusion: Cette thèse confirme l'impact d'un stress immuno-inflammatoire maternel sur la structure et la fonction hippocampique. Elle démontre l'intérêt d'un traitement prénatal par la NAC et de la modulation du tonus GABAergique pour corriger les troubles cognitifs associés
Introduction: A late gestational exposure to lipopolysaccharide (LPS) leads to a behavioral and cognitive phenotype of neuropsychiatric disorders in male offspring. The main goal was to test the hypothesis of structural damage and imbalance between excitation and inhibition in the hippocampus. The secondary goal was to identify targeted therapeutic strategies.Methods: Pregnant rats were ip injected with either 500 μg/kg LPS from E.coli O55:B5 or 2 ml/kg saline vehicle on gestational day 19. Male offspring were studied at different developmental stages. The structural study was based on immunohistochemistry, the functional study on electrophysiological recordings of the activity of pyramidal cells in the CA1 area. The protective effect of N-acetylcysteine (NAC) given to pregnant rats after LPS injection was tested.Results: In male offspring, LPS induced late gestational immune challenge led to sustainable disarray of the pyramidal layer in the CA3 area, transient deficit of reelin expressing neurons, impaired long term depression of glutamatergic synapses (LTDe), due to NMDA receptor and GABAergic system dysfunction. An inhibitor of GABA reuptake completely restored plasticity lost after prenatal stress. NAC prevented cyto-architectural abnormalities.Conclusion: This thesis confirms the impact of a late prenatal immune challenge on hippocampal structure and function. It demonstrates that prenatal treatment with NAC and GABAergic tone modulation are valuable therapeutic strategies for the cognitive impairment associated with prenatal immune challenge

La maladie de Parkinson est une maladie neurodégénérative du système cerveau central conduisant à l'apparition des troubles moteurs caractéristiques de la maladie : akinésie, rigidité et tremblement de repos. La perte des neurones dopaminergiques de la voie nigro-striée va conduire à des modifications biochimiques au niveau du putamen. Notamment, les travaux réalisés en électrophysiologie, microdialyse et spectroscopie par résonance magnétique (SRM) suggèrent une hyperactivité de la voie glutamatergique cortico-striatale associée à un changement du microenvironnement glial au niveau du putamen. Ces observations conduisent à penser à une adaptation du cycle glutamate-glutamine ayant lieu entre les neurones et les astrocytes en réponse à la perte neuronale. Ainsi, dans ce travail de thèse, deux approches ont été développées afin de suivre par SRM les changement métaboliques impliqués dans la pathologie parkinsonienne, notamment les variations des concentrations en glutamate et glutamine dans le putamen. Une première approche de la quantification des métabolites cérébraux par spectroscopie du 1H, technique couramment utilisée en clinique, a été utilisée pour suivre l'évolution des métabolites d'intérêt chez des patients parkinsoniens à jeun ou suite à la prise d'un traitement dopaminergique. Si cette étude a révélé des changements de concentration en N-acetylaspartate, créatine et myoinositol chez les patiens parkinsoniens, aucun changement du métabolisme glutamatergique n'a pu être observé par cette technique, peut-être à cause d'un manque de sensibilité de la technique pour discriminer les pools de glutamate et de glutamine. De ce fait, une nouvelle approche de SRM du Carbone 13C a été développée pour le suivi du cycle glutamate-glutamine in vivo, c'est la polarisation dynamique nucléaire (PDN). Grâce à la haute sensibilité de cette technique, il est désormais possible de suivre des voies métaboliques in vivo en temps réel. La mise en place et l'optimisation de la PDN pour le suivi du cycle glutamate-glutamine a été un des objectifs au cours de ce projet de thèse. Validée sur un groupe d'animaux contrôle, cette technique offre un avenir prometteur pour l'analyse de ce flux dans les pathologies neurodégénératives. En conclusion, les stratégies diagnostiques en clinique par SRM du 1H restent, à l'heure actuelle, peu sensibles pour l'étude des modifications du cycle glutamate-glutamine in vivo chez l'homme. Les développements technologiques réalisés au cours de ce travail de thèse notamment avec la PDN du 13C laissent entrevoir une nouvelle approche pour le suivi en temps réel de ce métabolisme cérébral. Si la PDN est principalement utilisée dans des études précliniques, la disponibilité de nouveaux systèmes cliniques pourrait permettre son avènement en tant que nouvelle stratégie de diagnostic en imagerie clinique
Parkinson's disease is a neurodegenerative disorder characterized by motor troubles such as akinesia, rigidity and tremor. The loss of dopaminergic neurons from the nigro-striatal pathway will lead to biochemical changes in the putamen. Especially, works on electrophysiology, micro dialysis and magnetic resonance spectroscopy (MRS) suggests hyperactivity of the glutamatergic cortico-striatal pathway associated with glial microenvironment changes. These observations suggest a modification of the glutamate-glutamine cycle occurring between neurons and astrocytes in response to neuronal loss.In this thesis, two approaches have been developed in order to follow by MRS the metabolic changes occuring in Parkinson's disease. In particular, we want to follow the changes in glutamate-glutamine cycle inside the putamen.in a first study, a a 1H MRS approach was used to assess the metabolic changes inside the putamen of Parkinson's disease without or under dopaminergic treatment. In this study, changes in N-acetylaspartate, creatine and myo-inositol were observed in Parkinsonian patients, but no change in glutamatergic metabolism was observed. This could be due to the lack of sensitivity of the technique to differentiate glutamate and glutamine pools.Thus, we chose to use a new 13C carbon MRS approach in order to follow dynamically in vivo the glutamate-glutamine cycle inside the brain: dynamic nuclear polarization (DNP). Thanks to the high sensitivity of this technique, it is now possible to follow metabolic pathways in vivo in real time. The implementation of DNP was assessed under a control group of animals. This technique offers a new promising tool for the analysis of this flow under pathologic conditions.To conclude, the MRS strategies for clinical diagnostic strategies remain, at present, poorly sensitive for the study of glutamate-glutamine cycle in vivo in humans. The development of DNP opens the door to a new approach for real-time monitoring of this cerebral metabolism Even if DNP is mainly used in preclinical studies at present, the development of new clinical systems could lead to its emergence as a new diagnostic strategy in clinical imaging

O estudo investigou o efeito de diferentes competições de ciclismo de estrada e de um período de descanso na Creatina Kinase (CK), Lactato Desidrogenase (LDH), Interleucina-1 (IL-1), Interleucina-6 (IL-6), Fator de necrose tumoral (TNF-a), prostaglandina E2 (PGE2) e na razão Glutamina/Glutamato (Gln/Glu). Para isso, 12 ciclistas profissionais que completaram 3 diferentes competições. A primeira e a terceira (C1 e C3) competições foram de uma 1 etapa e a segunda foi uma volta ciclística (C2) de 7 dias. A C3 foi realizada após um período de 20 dias de descanso. Foram coletadas amostras de sangue 24 horas antes do início e 24 horas após o término de cada competição. Não houve diferenças significativas para nenhum dos mediadores inflamatórios (p<0,05). Aumentos (p<0,05) foram encontrados após o término da C2 e da C3 para CK e LDH e diminuição (p<0,05) na razão Gln/Glu C3. A conclusão foi a de que lesões musculares são diagnosticadas em ciclistas profissionais de estrada após o término de uma volta ciclística e de uma competição precedida de um período de descanso.
The objective of this research was to observe the effect of different road cycling competitions and a 20 days break period in Creatine Kinase (CK), Lactate Dehydrogenase (LDH), Interleukin-1(IL-1), Interleukin-6 (IL-6), Factor necrosis tumoral-alpha (TNF-a), prostaglandin E2 (PGE2) and in the ratio of Glutamine/Glutamate (Gln/Glu). The hypothesis tests were conducted with a sample of 12 professional cyclists which have completed 3 different road cycling races. The first and third ones (C1 and C3) were mass start races. The second one was a stage racing (C2). One analysis was made after a 20 days break period (C3). Blood samples were collected 24 hours before and 24 hours after each competition. There were no significant differences for none of the inflammatory mediators (p<0,05). Increases (p<0,05) were detected after the end of C2 and C3 for CK and LDH and low (p<0,05) in the ratio of Gln/Glu at C3. The conclusion of the research was that muscular damages are diagnostified in professional road cyclists 24 hours after the end of the stage racing and after mass start competition preceded by 20 days a break period.