This thesis focuses on the regulation of the ubiquitous second messenger Ca2+ in neuronal physiology and disease. Ca2+ signalling in neurons is regulated by ion channels located in the plasma membrane, as well as in the endoplasmic reticulum (ER) and mitochondrial membranes. Ca2+ signalling is essential for numerous cellular processes, including neuronal excitability, neurotransmitter release, synaptic plasticity, and induction of cell death. Age-related disruptions in Ca2+ signalling may contribute to decline of cognitive function and motor control associated with aging. Furthermore, disruption in neuronal Ca2+ signalling is implicated in several neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). In this thesis, I studied neuronal Ca2+ signalling and how it is affected in neurodegenerative disease. First, I examined the role of the ER Ca2+ binding protein Calreticulin (CRT) in AD. CRT is involved in regulation of ER Ca2+ signalling and modulation of susceptibility to cell death. I found that there was an increase in the expression of CRT in in vitro and in vivo models of AD. However, increased levels of CRT did not alter susceptibility of neuronal cells to death induced by AD-related stressors. Second, I examined the role of X-Linked Inhibitor of Apoptosis Protein (XIAP) in the modulation of neuronal Ca2+ signalling. I found that overexpression of XIAP in neuronal cells modified Ca2+ signalling by decreasing Ca2+ flux through multiple plasma membrane and ER channels. These effects appear to be independent of caspase inhibition, which is one of the ways that XIAP can inhibit apoptosis. Third, I examined a compound found in green tea, L-theanine, a glutamate receptor antagonist that is protective in models of excitotoxic neuronal injury. I found that 24 hour L-theanine treatment reduces the amount of Ca2+ released from neuronal intracellular stores in response to both glutamate stimulation and passive leak through ER channels. An acute 30 minute L-theanine treatment had similar effects. In conclusion, these observations further the understanding of the regulation of Ca2+ signalling in neurons and may lead to novel therapeutic strategies in neurodegenerative disease. / October 2008

This thesis focuses on the regulation of the ubiquitous second messenger Ca2+ in neuronal physiology and disease. Ca2+ signalling in neurons is regulated by ion channels located in the plasma membrane, as well as in the endoplasmic reticulum (ER) and mitochondrial membranes. Ca2+ signalling is essential for numerous cellular processes, including neuronal excitability, neurotransmitter release, synaptic plasticity, and induction of cell death. Age-related disruptions in Ca2+ signalling may contribute to decline of cognitive function and motor control associated with aging. Furthermore, disruption in neuronal Ca2+ signalling is implicated in several neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). In this thesis, I studied neuronal Ca2+ signalling and how it is affected in neurodegenerative disease. First, I examined the role of the ER Ca2+ binding protein Calreticulin (CRT) in AD. CRT is involved in regulation of ER Ca2+ signalling and modulation of susceptibility to cell death. I found that there was an increase in the expression of CRT in in vitro and in vivo models of AD. However, increased levels of CRT did not alter susceptibility of neuronal cells to death induced by AD-related stressors. Second, I examined the role of X-Linked Inhibitor of Apoptosis Protein (XIAP) in the modulation of neuronal Ca2+ signalling. I found that overexpression of XIAP in neuronal cells modified Ca2+ signalling by decreasing Ca2+ flux through multiple plasma membrane and ER channels. These effects appear to be independent of caspase inhibition, which is one of the ways that XIAP can inhibit apoptosis. Third, I examined a compound found in green tea, L-theanine, a glutamate receptor antagonist that is protective in models of excitotoxic neuronal injury. I found that 24 hour L-theanine treatment reduces the amount of Ca2+ released from neuronal intracellular stores in response to both glutamate stimulation and passive leak through ER channels. An acute 30 minute L-theanine treatment had similar effects. In conclusion, these observations further the understanding of the regulation of Ca2+ signalling in neurons and may lead to novel therapeutic strategies in neurodegenerative disease.

This thesis focuses on the regulation of the ubiquitous second messenger Ca2+ in neuronal physiology and disease. Ca2+ signalling in neurons is regulated by ion channels located in the plasma membrane, as well as in the endoplasmic reticulum (ER) and mitochondrial membranes. Ca2+ signalling is essential for numerous cellular processes, including neuronal excitability, neurotransmitter release, synaptic plasticity, and induction of cell death. Age-related disruptions in Ca2+ signalling may contribute to decline of cognitive function and motor control associated with aging. Furthermore, disruption in neuronal Ca2+ signalling is implicated in several neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). In this thesis, I studied neuronal Ca2+ signalling and how it is affected in neurodegenerative disease. First, I examined the role of the ER Ca2+ binding protein Calreticulin (CRT) in AD. CRT is involved in regulation of ER Ca2+ signalling and modulation of susceptibility to cell death. I found that there was an increase in the expression of CRT in in vitro and in vivo models of AD. However, increased levels of CRT did not alter susceptibility of neuronal cells to death induced by AD-related stressors. Second, I examined the role of X-Linked Inhibitor of Apoptosis Protein (XIAP) in the modulation of neuronal Ca2+ signalling. I found that overexpression of XIAP in neuronal cells modified Ca2+ signalling by decreasing Ca2+ flux through multiple plasma membrane and ER channels. These effects appear to be independent of caspase inhibition, which is one of the ways that XIAP can inhibit apoptosis. Third, I examined a compound found in green tea, L-theanine, a glutamate receptor antagonist that is protective in models of excitotoxic neuronal injury. I found that 24 hour L-theanine treatment reduces the amount of Ca2+ released from neuronal intracellular stores in response to both glutamate stimulation and passive leak through ER channels. An acute 30 minute L-theanine treatment had similar effects. In conclusion, these observations further the understanding of the regulation of Ca2+ signalling in neurons and may lead to novel therapeutic strategies in neurodegenerative disease.

La L-Théanine (L-5-N-éthylglutamine) est un acide aminé présent dans le thé vert et qui a une structure similaire à celle de l'acide glutamique, le neurotransmetteur majoritaire du système nerveux central, SNC. La L-théanine possède la capacité de traverser la barrière hémato-encéphalique en plus de ses nombreuses activités physiologiques et pharmacologiques, anxiolytique et relaxante. La théanine et ses dérivés sont donc considérés comme des outils indispensables pour la compréhension de la synapse glutamatergique plus précisément. Nous avons synthétisé la théanine énantiomériquement pure (L et D). Nous avons développé deux nouvelles méthodes de synthèse de la théanine optiquement pure. Ensuite, nous avons décrit la préparation des dérivés 5-N-alkylés de la théanine et les dipeptides -glutamique en tant qu'analogues 5-N-substitués de la théanine. Dans le but d'élargir l'éventail d'applications de la Théanine ayant un intérêt biologique, nous avons synthétisé des dipeptides contenant la Théanine. Nous présentons également un accès aux dérivés 4-arylés de la théanine, via une alkylation régio et stéréosélective de l'acide pyroglutamique jamais décrit dans la littérature. L'analyse rigoureuse des intermédiaires réactionnels et des produits finaux par les RX, la RMN 1H à 600 MHZ et l'HRMS a prouvé l'obtention d'un seul diastéréoisomère (2S, 4R)-4-aryle théanineLes résultats des tests biologiques, utilisant la technique de l'imagerie calcique, montrent que les deux énantiomères L et D de la théanine possède un effet agoniste vis-à-vis les récepteurs NMDA et que cet effet est beaucoup plus important dans le cas de l'énantiomère (D). Parmi les analogues 5-N-alkylés de la théanine ayant un effet agoniste spécifique des récepteurs NMDA, la 5-N-Propyl-Gln (L et D) montre une activité en tant qu'agoniste beaucoup plus importante que la théanine même (naturelle ou synthétique) et aucun effet n'a été observé sur les récepteurs AMPA et métabotropiques. La L-théanine et ses dérivés pourraient donc être des structures intéressantes pour développer de nouveaux outils pharmacologiques nécessaires à l'étude des récepteurs glutamatergiques (métabotropiques et/ ou ionotropiques). / L-Theanine (5-N-L-ethylglutamine) which is an amino acid found in green tea, it has a structure similar to that of glutamic acid. L-theanine has the ability to cross the blood-brain barrier in addition to its physiological and pharmacological activities.Given the importance of this molecule as essential for the investigation of physiological roles of CNS tools, we synthesized the enantiomerically pure theanine (L and D). A serie of 5-N-substituted theanine were also synthesized. In order to broaden the range of applications of theanine, we synthesized dipeptides containing Theanine for the purpose of obtaining products that have biological significance. The regio and stereoselectively synthesized analogs of L-theanine in the 4-position substituted with an aryl group has been developed to be tested at the level of glutamate receptors.The results of biological tests, using calcium imaging technique, show that theanine with its two enantiomers (D and L) has an agonistic effect vis-à-vis the NMDA receptors and that this effect was much greater with the enantiomer (D). Among the 5-N-alkylated analogs of theanine which they had only an agonistic effect on the NMDA receptor, 5-N-Propyl-Gln (L and D) has activity as an agonist much larger than the theanine and no effects were scored on AMPA receptors and metabotropic. L-theanine and its derivatives could be interesting structures to develop new pharmacological tools to study glutamate receptors (metabotropic and / or ionotropic).

Stroke is one of the most common causes of disability and death worldwide. The most commonly experienced stroke in the clinical setting is focal ischaemia in which the middle cerebral artery (MCA) is occluded and leads to a complex series of various pathophysiological pathways that ultimately lead to neuronal cell death. Several studies have been conducted on various therapeutic agents in the search for a neuroprotective drug and various animal models have been used to carry out this research. While theanine, a component of green tea and minocycline, a tetracycline antibiotic, have been shown to possess some neuroprotective properties, the mechanisms by which these two agents carry out these effects still remains unclear. The objectives of this study were to investigate the mechanisms by which these drugs carry out these neuroprotective effects and their neuroprotective ability in a MCA occlusion model of focal ischaemia. Ischaemia leads to oxidative stress due to the imbalance of free radicals and the endogenous antioxidant defence system. An antioxidant assay using the stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH●) radical was used to assess the antiradical properties of each drug. It was found that minocycline showed superior antioxidant activity in vitro when compared to theanine. Further studies on the drugs‟ ability to attenuate the Fenton reaction (in which iron catalyses the formation of reactive species) were elucidated using electrochemical analysis, UV/VIS studies, ferrozine and ferritin assays. It was found that minocycline, in contrast to theanine, was able to bind to iron ions and thus potentially prevent the participation of iron in metal catalysed radical reaction. The antioxidant activity of both drugs was further investigated by assessing their effect on cyanide-induced superoxide generation and quinolinic acid (QA)-induced lipid peroxidation (LP). Experimental evidence shows that both drugs had no significant effect on the generation of superoxide in vitro and that there was a significant decrease in LP for minocycline in vitro and theanine in vivo. The metal binding and antioxidant properties were postulated to be a possible mechanism through which these agents reduced lipid peroxidation. A study was conducted to determine the effects of the drugs on the biosynthesis of the neurotoxin, QA and it was found that minocycline increases the levels of holoenzyme activity of tryptophan-2, 3-dioxygenase (TDO) in vitro and that theanine reduces the levels of the same enzyme in vivo after treatment for 10 days. TDO is the enzyme that converts tryptophan to other products that enable enzymatic activity to change it to QA. Minocycline was thought to bring about this effect as it has been shown from preceding experimental studies that it is an effective reducing agent. Theanine on the other hand is hypothesised to bring about a reduction in holoenzyme activity by changing the binding of tryptophan to the enzyme or affecting the radicals that participate in the enzymatic degradation of tryptophan. A focal ischaemic model of stroke was induced by occluding the MCA. Histological examination of the hippocampus post -ischaemia shows a reduction in the size of the infarct after pre-treatment with minocycline only. A further study into the effects of the drugs on the generation of superoxide and on the levels of the endogenous glutathione after a stroke was carried out. Pre-treatment of the animals with either theanine or minocycline showed no significant effects on the generation of the radical species or of the endogenous antioxidant which ruled out these as a mechanism of neuroprotection of both drugs, post-ischaemia.The findings of this study provide novel information on the possible mechanisms by which both theanine and minocycline act to bring about neuroprotection. In particular in this study, pre-treatment with minocycline has shown promise in the focal ischaemic model of stroke.

This thesis focused on optimization of separation methods for underivatized theanine enantiomers on two teicoplanin-based chiral stationary phases and separation methods for enantiomers derivatized by 9-fluorenylmethyloxycarbonyl chloride and dansylchloride. Using these optimized conditions the limits of detection and quantification were calculated. Under optimized conditions were also investigated the contents of theanine enantiomers in green tea and in selected dietary supplement containing L-theanine.

Made available in DSpace on 2015-04-17T15:02:59Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1361043 bytes, checksum: 4455c38a98b7073e2147348a625e880c (MD5) Previous issue date: 2013-03-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Anxiety is characterized by a state of tension, apprehension and discomfort that arise from internal or external danger imminent and may be a response to stress or environmental stimuli, and can measured by psychological and physiological changes of the individual. The L-theanine (gamma-L-glutamic acid ethylamide) or theanine is an amino acid found in Camellia sinensis, commonly known as "green tea". This amino acid has been used to reduce mental and physical stress, improving memory function and for treating anxiety. Because the anxiety disorders are among the most common disorders seen in both the general population and in the services of primary health care, it is relevant to the investigation of non-pharmacological methods for their treatment. The aim of this study was to investigate the psychophysiological effects of L-theanine on a model of anxiety in healthy humans. The study was an experimental, kind of randomized controlled clinical trial. Thirtie was composed of healthy students, male or female, which were inserted into one of three groups (control, L-theanine 1 or L-theanine 2). The participants of the experimental groups received 200 mg of L-theanine by a capsule as a single dose. The human anxiety was induced by Simulated Public Speaking Test - SPST and was assessed by physiological parameters (blood pressure, heart rate, ending temperature and electrical conductance of the skin) and psychological parameters (anxiety Inventory, trait - STAI-T and state - STAI-E). Statistical tests were considering parametric data the ANOVA (one way) and ANOVA for repeated measures data and considering the nonparametric Kruskal-Wallis and Friedman. Results were considered significant when showed a significance level of 95% (p < 0,05). There was a reduction in the levels of systolic blood pressure and heart rate in group L-Theanine 2 (p <0,05) and the electrical conductance of the skin in group L-theanine 1. There was also a reduction in the score of the STAI-E in both experimental groups since the score remained within the range of anxiety low throughout the test (<40 points). These results suggest that L-theanine has an anxiolytic effect on the physiological and psychological parameters that change in anxiety. Thus, this amino acid may be a non-pharmacological strategy in the treatment thereof. / A ansiedade caracteriza-se por um estado de tensão, apreensão e desconforto, que se originam de um perigo interno ou externo iminente, podendo ser resposta a estresse ou a estímulo ambiental e pode ser mensurada através de medidas psicológicas e fisiológicas do individuo. A L-teanina (ácido gama-etilamida L-glutâmico) ou teanina é um aminoácido encontrado na Camellia sinensis, popularmente conhecida como chá verde . Este aminoácido vem sendo utilizado para reduzir o estresse mental e físico, melhorar a função da memória e no tratamento da ansiedade. Visto que os transtornos da ansiedade estão entre os transtornos mais comumente observados tanto na população geral quanto nos serviços de atenção primária à saúde, é relevante a averiguação de métodos não farmacológicos para o seu tratamento. O objetivo deste estudo foi investigar os efeitos psicofisiológicos da L-teanina sobre um modelo de ansiedade em humanos saudáveis. O estudo teve caráter experimental, do tipo ensaio clínico randomizado e controlado. Foi composto por trinta estudantes saudáveis, do gênero masculino ou feminino, os quais foram inseridos em um dos três grupos experimentais (controle, L-teanina 1 ou L-teanina 2). Os participantes dos grupos experimentais receberam 200 mg de L-teanina na forma de cápsula e em dose única. A ansiedade humana experimental foi induzida pelo Teste de Simulação de Falar em Público (TSFP) e foi avaliada por meio de parâmetros fisiológicos (pressão arterial, frequência cardíaca, temperatura de extremidade e condutância elétrica da pele) e de parâmetros psicológicos (inventário de ansiedade traço IDATE-T e estado IDATE-E). Os testes estatísticos utilizados considerando os dados paramétricos foram o ANOVA (one way) e ANOVA para medidas repetidas e considerando os dados não paramétricos o teste de Kruskal-Wallis e de Friedman. Os resultados foram considerados significativos quando apresentaram um nível de significância de 95% (p < 0,05). Observou-se uma redução nos níveis da pressão arterial sistólica e frequência cardíaca no grupo L-teanina 2 (p < 0,05) e da condutância elétrica da pele no grupo L-teanina 1. Ocorreu também uma redução no escore do IDATE-E nos dois grupos experimentais, uma vez que a pontuação manteve-se dentro da faixa de ansiedade baixa durante todo o teste (< 40 pontos). Estes resultados sugerem que a L-teanina teve um efeito ansiolítico sobre os parâmetros fisiológicos e psicológico que se alteram na ansiedade. Sendo assim, este aminoácido pode ser uma estratégia não farmacológica no tratamento da mesma.