Les canaux ioniques pentamériques activables par un ligand (pLGIC) sont l'une des principales familles de canaux transmembranaires. Ils permettent la transduction rapide du signal dans le système nerveux central et périphérique via la liaison de neurotransmetteurs. Les pLGIC sont également présents chez les archées et les bactéries. Seuls deux pLGIC bactériens ont été caractérisés biochimiquement et structurellement jusqu'à présent (GLIC et ELIC). Ils servent de modèle d’étude à de nombreux scientifiques et ont été largement étudiés aussi bien au niveau fonctionnel que structural. Dans la première partie de mon travail de thèse, j'ai purifié, cristallisé et résolu la structure cristalline d'un nouveau pLGIC originaire d'un symbiote de gamma-protéobactérie de Tevnia jerichonana (sTeLIC). Des expériences fonctionnelles montrent que sTeLIC est activé par un pH alcalin, est sélectif pour les ions cationiques monovalents et inhibé par les cations divalents. La structure cristalline résolue à pH 8,0 présente un pore largement ouvert qui est le premier de ce type à être caractérisé dans cette famille pLGIC. De plus, nous avons identifié un modulateur fortement positif qui se lie au "site vestibulaire" dans le domaine extracellulaire, et nous avons résolu la structure cristalline de ce complexe. Des expériences fonctionnelles montrent également que sTeLIC partage de nombreuses fonctionnalités avec ELIC. ELIC et sTeLIC constitutent les archétypes d’une nouvelle classe de pLGICs, dont la forme active se caractérise par un pore largement plus ouvert que les autres pLGICs.Dans la deuxième partie de mon travail de thèse, les résidus senseurs de protons dans GLIC ont été cartographiés, afin de déterminer comment la liaison du proton stabilise l'état ouvert de GLIC. Tous les résidus titrables de GLIC ont été cartographiés par mutagenèse dirigée afin de découvrir des capteurs de protons impliqués dans le processus de déclenchement. Nous avons ainsi démontré que la résidu E35 est un résidu clé, dont la forme chargée stabilise l’état de repos, et la forme protonée l'état actif. Nous avons également démontré que la réponse au proton dépend de deux réseaux distincts à l'interface ECD-TMD qui stabilisent l'état ouvert de GLIC. Dans la troisième partie, j'ai cloné, purifié, cristallisé et déterminé les structures cristallines des formes ouvertes et fermées de DeCLIC, un pLGIC de la protéobactérie Desulfofustis. Chaque sous-unité contient un grand domaine additionnel N-terminal constitué de deux sous-domaines (NTD1 et NTD2). Il s’agit de la première structure d’un pLGIC qui contient un domaine supplémentaire extracellulaire non-canonique. / Ligand-gated pentameric ion channels (pLGIC) are one of the major families of transmembrane receptors. They allow rapid signal transduction in the central and peripheral nervous systems via neurotransmitters binding. PLGICs are also present in archaea and bacteria. Only two bacterial pLGICs have been biochemically and structurally characterized so far (GLIC and ELIC). They serve as working models for many scientists and have been extensively studied both at the functional and structural levels. In the first part of my thesis, I purified, crystallized and solved the crystal structure of a new pLGIC from gamma-proteobacterial symbionts of Tevnia jerichonana (sTeLIC). Functional experiments show that sTeLIC is activated by alkaline pH, and is selective for monovalent cationic ions and inhibited by divalent cations. The crystal structure solved at pH 8.0 displays a widely open pore that is the first of this kind to be characterized in the pLGIC family. In addition, we identified a strongly positive modulator that binds to the "vestibule site" in the extracellular domain, and we solved the crystal structure of this complex. Functional experiments show that sTeLIC shares many features with ELIC. ELIC and sTeLIC are the archetypes of a new class of pLGICs, whose active form is characterized by a much more open pore than other pLGICs. In the second part of my thesis, the proton sensor residues in GLIC have been mapped. All titratable GLIC residues were tested by site-directed mutagenesis to discover proton sensors involved in the triggering process. We have demonstrated that the residue E35 is a key residue, whose charged form stabilizes the resting state, and the protonated form the active state. We have also demonstrated that the proton response is dependent on two distinct networks at the ECD-TMD interface, which stabilize the open state of GLIC.In the third part of my thesis, I cloned, purified, crystallized and determined the crystal structures of the open and closed forms of DeCLIC, a pLGIC of Desulfofustis proteobacterium. Each subunit contains a large N-terminal additional domain consisting of two subdomains (NTD1 and NTD2). This is the first structure of a pLGIC which contains a non-canonical additional extracellular domain.

Submitted by Milena Rubi (milenarubi@ufscar.br) on 2017-06-01T13:24:35Z No. of bitstreams: 1 PEREIRA_Tamyris_2015.pdf: 24726845 bytes, checksum: cad6e1bb20698eeb16bd6280b89c6c35 (MD5) / Approved for entry into archive by Milena Rubi (milenarubi@ufscar.br) on 2017-06-01T13:24:52Z (GMT) No. of bitstreams: 1 PEREIRA_Tamyris_2015.pdf: 24726845 bytes, checksum: cad6e1bb20698eeb16bd6280b89c6c35 (MD5) / Approved for entry into archive by Milena Rubi (milenarubi@ufscar.br) on 2017-06-01T13:24:59Z (GMT) No. of bitstreams: 1 PEREIRA_Tamyris_2015.pdf: 24726845 bytes, checksum: cad6e1bb20698eeb16bd6280b89c6c35 (MD5) / Made available in DSpace on 2017-06-01T13:25:07Z (GMT). No. of bitstreams: 1 PEREIRA_Tamyris_2015.pdf: 24726845 bytes, checksum: cad6e1bb20698eeb16bd6280b89c6c35 (MD5) Previous issue date: 2015-02-27 / Não recebi financiamento / This work focuses on the production of nanostructured films of poly (ethyleneimine) PEI together with MAO-b free and encapsulated in liposomes constructed by the layer-bylayer technique (LbL), for application in biosensors. Liposomes are microscopic vesicles composed of concentric lipid bilayers separated by aqueous medium, capable of encapsulating hydrophilic and/or hydrophobic molecules, which are, respectively, included in the aqueous compartment and the membrane or adsorbed. Enzymes encapsulated in liposomes have advantages in stability and catalytic activity compared to the free enzyme. Liposomes were synthesized by using a rotary evaporator and subsequently the enzyme was immobilized. The phospholipids dipalmitoyl phosphatidyl glycerol (DPPG) and palmitoyl oleoyl phosphatidyl glycerol (POPG) and the mixture of the two were used in this study in the ratio (1:1) and (1:4) (m/m). To build a biosensor in neurotransmitter monoamine oxidase b on, thin films were set up on poly (ethyleneimine) PEI together with monoamine oxidase b enzyme (MAO-B) immobilized or not in liposomes and deposited on ITO electrodes, being the Prussian blue a mediator of electrons. The obtained films were characterized by fluorescence spectroscopy, UV-visible and circular dichroism. The results indicated that the film is deposited successfully on quartz and ITO. The amperometric measurements were performed to detect the film sensitivity and the detection limits and check which architecture is best suited for the best composition of the biosensor. The results achieved until now show that the liposome retain the conformational structure of the enzyme and films composed of PEI / MAO-B + POPG:DPPG in proportion (1:4) (w/w) indicate better performance of the biosensor, due to the sensitivity value of 0.33 ± 0.02 µA.cm-2 mM-1 found and a good selectivity. / Este trabalho é centrado na produção de filmes nanoestruturados de poli (etilenoimina) PEI em conjunto com MAO-B livre e encapsulada em lipossomos, construídos através da técnica layer-by-layer (LbL), camada por camada, para aplicação em biossensores. Lipossomos são vesículas microscópicas compostas de bicamadas lipídicas concêntricas, separadas por um meio aquoso, capazes de encapsular moléculas hidrofílicas e/ou hidrofóbicas, que se encontram, respectivamente, no compartimento aquoso e inserida ou adsorvidas na membrana. Enzimas encapsuladas em lipossomos apresentam vantagens quanto à estabilidade e atividade catalítica em comparação com a enzima livre. Os lipossomos foram sintetizados por rotaevaporação e a enzima foi imobilizada na sequência. Os fosfolipídios dipalmitoil fosfatidil glicerol (DPPG) e palmitoil oleil fosfatidil glicerol (POPG) e a mistura dos dois foram utilizados neste trabalho nas proporções de (1:1) e (1:4) (m/m). Para construir um biossensor de dopamina baseado em monoamina oxidase b, filmes finos foram montados de poli (etilenoimina) PEI juntamente com a enzima Monoamina oxidase b (MAO-b) imobilizada ou não em lipossomos e depositados sobre eletrodo de ITO, tendo como mediador de elétrons o Azul da Prússia. Os filmes obtidos foram caracterizados por espectroscopias de fluorescência, UV-visível e dicroísmo circular. Os resultados indicaram que o filme é depositado com sucesso sobre quartzo e ITO. As medidas amperométricas foram realizadas nos filmes para detectar sensibilidades e limite de detecção e verificar qual arquitetura é mais adequada para a melhor composição do biossensor. Os resultados obtidos mostram que os lipossomos conservam a estrutura conformacional da enzima e os filmes compostos por PEI/MAO-B+POPG:DPPG na proporção (1:4) (m/m) indicam um melhor desempenho do biossensor, devido ao valor de sensibilidade encontrado de 0,33±0,02 ?A.cm- 2mM-1 e uma boa seletividade.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A terapia antirretroviral revolucionou o tratamento dos pacientes portadores do vÃrus da imunodeficiÃncia humana (HIV) porque melhorou o estado clÃnico destes pacientes. à essencial que no mÃnimo uma das drogas utilizadas nesta terapia atravesse bem a barreira hemato-encefÃlica, jà que o HIV alcanÃa o sistema nervoso central (SNC), causando danos. O Efavirenz à um antirretroviral capaz de atravessar bem a barreira hemato-encefÃlica, sendo bastante utilizado no tratamento para HIV. Entretanto, pacientes tratados com Efavirenz vÃm apresentando alteraÃÃes neuropsiquiÃtricas como depressÃo, ansiedade, mania, pensamentos suicidas, psicoses e alucinaÃÃes. Baseado nestes fatos, o presente estudo objetivou avaliar se as alteraÃÃes comportamentais ansiedade, depressÃo e convulsÃo ocorrem em animais tratados com Efavirenz com uma Ãnica dose e por um perÃodo de 15 dias, atravÃs de modelos padronizados de comportamento animal, bem como verificar se o tratamento com o fÃrmaco à capaz de causar alteraÃÃes de monoaminas e de aminoÃcidos. Foram utilizados ratos Wistar machos (35 dias, n=6-8). Os animais foram tratados uma Ãnica vez com efavirenz, no caso do tratamento agudo, ou por 15 dias, para o tratamento subcrÃnico. Os animais tratados e controles foram submetidos aos testes comportamentais de Labirinto em Cruz Elevado (Plus Maze), Nado ForÃado, Campo Aberto e ConvulsÃo induzida por pilocarpina. Para a avaliaÃÃo neuroquÃmica, as cobaias foram sacrificadas e o cÃrebro foi removido para anÃlise das seguintes Ãreas de interesse: corpo estriado, cÃrtex prÃ-frontal e hipocampo. Foram dosadas as monoaminas e seus metabÃlitos, os aminoÃcidos e os nÃveis de malonildialdeÃdo (MDA). Os resultados mostraram que o Efavirenz ocasionou, nos testes comportamentais, ansiedade apÃs administraÃÃo aguda, causou depressÃo no teste comportamental apÃs tratamento subcrÃnico, nÃo interferiu na latÃncia de convulsÃo e alterou monoaminas e metabÃlitos, alÃm de elevar os nÃveis de MDA. Desse modo, conclui-se que o efavirenz, de acordo com este estudo, promove alteraÃÃes neuroquÃmicas no sistema nervoso central causando distÃrbios neuropsiquiÃtricos. / Antiretroviral therapy has revolutionized the treatment of patients infected with human immunodeficiency virus (HIV) because it improved the clinical status of these patients. It is essential that at least one of the drugs used and in this therapy cross the blood-brain barrier, since HIV reaches the central nervous system (CNS), causing damage. Efavirenz is an antiretroviral widely used in HIV and able to cross the blood-brain barrier. However, patients treated with efavirenz are presenting neuropsychiatric disorders such as depression, anxiety, mania, suicidal thoughts, psychosis and hallucinations. Based on these facts, this study aimed to assess whether the behavioral changes anxiety, depression and seizures induced by pilocarpine occur in animals treated with efavirenz with a single dose and for a period of 15 days, using standard models of animal behavior as well as check if the treatment with drug can cause changes in monoamines and amino acids. We used male Wistar rats (35 days, n = 6-8). The animals were treated once with efavirenz in the case of an acute treatment, or 15 days for a subchronic treatment. The treated animals and controls were tested for behavioral elevated plus maze (Plus Maze), Forced Swim, Open Field and seizure induced by pilocarpine. To assess neurochemistry, mice were sacrificed and the brain was removed for the analysis of the following areas of interest: striatum, prefrontal cortex and hippocampus. Were measured monoamines and their metabolites, amino acids and the levels of malondialdehyde (MDA). The results showed that efavirenz caused anxiety and motor abnormalities similar to psychogenic drugs in behavioral tests after acute administration, caused depression in the behavioral test after subchronic treatment, had no effect on the latency of convulsion, altered monoamine metabolites and increased levels MDA. Thus, it is conclude that efavirenz, according to this study, promotes neurochemical changes in the central nervous system, causing neuropsychiatric disorders.

Sistemas químicos capazes de produzir radicais livres OH• e O2•-, responsáveis por danos no DNA, foram estudados em diversos tipos de eletrodos de carbono previamente modificados. Nitrofural, RNO2, foi reduzido mono eletronicamente ao seu respectivo nitro ânion radical, RNO2•-, em eletrodo de pasta de carbono modificado superficialmente com um filme de guanina. O nitro ânion radical atacou a guanina imobilizada na superfície do eletrodo e, esta interação in situ, resultou na formação de cátions radicais de guanina (G+•), que ao interagirem com a guanina (G), foram identificados pelo pico de oxidação em voltametria pulso diferencial, VPD, nas formas diméricas de guanina na região positiva de potencial. Os outros sistemas estudados se referem ao desenvolvimento de novos eletrodos de carbono modificados ou ainda a utilização procedimentos de modificações desenvolvidas anteriormente, para a detecção de várias moléculas de importância biológica. O ácido ascórbico (AA), ácido úrico (AU), xantina (XA) e hipoxantina (HX) foram detectados simultaneamente em eletrodo de grafite pirolítico, previamente modificado em solução de dopamina (EGPD), utilizando VPD. Os picos de oxidação, obtidos por voltametria cíclica foram detectados em 51; 393; 765 e 1080 mV vs Ag / AgCl, KCl(sat) para AA, AU, XA e HX, respectivamente. O limite de detecção para XA em presença de 5,0 x10-5 mol L-1 de HX foi 2,3 x10-6 mol L-1 (com sensibilidade de 2,8 A mol-1 L cm2), enquanto que o limite de detecção para HX na presença de 5,0 x10-5 mol L-1 de XA foi de 5,6 x10-6 mol L-1 (com sensibilidade de 1,4 A mol-1 L cm-2). XA e HX foram determinadas em amostras de urina e os valores encontrados foram 0,47 µmol L-1 e 5,9 mol L-1, respectivamente. Serotonina (5-HT) e dopamina (DA) foram determinadas simultaneamente em eletrodo de carbono vítreo, previamente modificado em solução de serotonina (GCE - 5HT). A serotonina foi detectada em 379 mV vs Ag / AgCl, KCl(sat), 31 mV menos positivo daquele observado em eletrodo de carbono vítreo (410 mV), enquanto DA foi detectada a 200 mV. Nenhuma interferência foi observada na presença de (AA), tirosina (Tyr), epinefrina (EP) e noradrenalina (NE). Finalmente, na última etapa do presente trabalho lignina, extraída a partir de licor de Kraft, foi solubilizada em acetonitrila / H2SO4 e a solução resultante utilizada para dispersar nanotubos de carbono de parede múltipla, NTCPM. Esta suspensão foi empregada para modificar a superfície de um eletrodo de carbono vítreo, posteriormente utilizado na detecção de nitrito por VPD no intervalo de concentração de 4,0 x 10-6 ≤ [NO2-] ≤ 8, 0 x 10-5 mol L-1. O complexo formado entre neocuproina e Cu (I), um composto em potencial para geração de radicais livres e promoção de lesões no DNA, foi sintetizado e caracterizado. / Chemical systems, which are able to produce OH• and O2•- free radicals, responsible for damage in DNA, were studied at different carbon modified electrode surfaces. Guanine carbon paste modified electrode was used to promote the nitrofural (RNO2) monoelectronic reduction to its respective nitro anion radical, RNO2•-, which attacked guanine immobilized on the electrode surface. The interaction in situ promoted the formation of guanine cation radicals (G+•) between guanine (G), after a dimerization process, were detected in the positive potential range by Differential Pulse Voltammetry, DPV. The other studied systems refer to development of new carbon modified electrodes or utilization of carbon modified electrodes, previously described, for the detection of several important biological molecules. Ascorbic acid (AA), uric acid (UA), xanthine (XA) and hypoxanthine (HX) were simultaneously detected at pyrolytic graphite electrode, previously modified into dopamine solution (EGPD), using DPV. The oxidation peak potentials, were obtained by cyclic voltammetry at 51; 393; 0.765 and 1080 mV vs Ag/AgCl, KCl(sat) for AA, UA, XA and HX, respectively. The detection limit for XA in presence of 5.0 x10-5 mol L-1 HX was 2.3 x10-6 mol L-1 (with sensibility of 2.8 A mol-1 L cm-2), while the detection limit for HX in presence of 5.0 x10-5 mol L-1 XA was 5.6 x10-6 mol L-1 (with sensibility of 1.4 A mol-1 L cm-2). XA and HX were determined in urine samples and the values founded were 0.47 µM e 5.9 µM, respectively. Serotonin (5-HT) and dopamine (DA) were simultaneously detected at glassy carbon electrode, previously modified in serotonin solution (ECV - 5HT). Serotonin was detected at 379 mV vs Ag/AgCl, KCl (sat), 31 mV less positive potential than that observed at bare glassy carbon electrode (410 mV), while DA was detected at 200 mV. No interference was observed in presence of (AA), tyrosine (Tyr), epinephrine (EP) and noradrenaline (NE). Finally, in the last stage of this work, lignin, extracted from Kraft liqueur, was solubilized in acetonitrile/H2SO4 and used to disperse multi-wall carbon nanotubes, MWNTC. This suspension was used to modify the glassy carbon electrode surface and nitrite was detected, by DPV in the concentration range of 4,0 x 10-6≤ [NO2-] ≤ 8,0 x 10-5 mol L-1. The complex formed between neocuproina and Cu (I), a compound which can produce free radicals and thereby cause damage to DNA, was synthesized and characterized.

A ocorrência de potenciais de ação e neurotransmissores, componentes principais do sistema nervoso animal, em plantas, bactérias e fungos mostra a universalidade dos princípios de sinalização e transmissão de informações na forma de sinais químicos e elétricos em todos os organismos. Esses tópicos de estudo, juntamente com inteligência em plantas e transporte vesicular de auxina, constituem as linhas de pesquisa principais da recém-criada Neurobiologia Vegetal. Entre os neurotransmissores encontrados em plantas, a acetilcolina atua, entre outras situações, no crescimento e desenvolvimento controlado pelo fitocromo e na permeabilidade iônica de membranas. Nesse contexto, foi sugerido que a acetilcolina pode desempenhar um papel importante na regulação do movimento estomático, tendo efeito estimulatório na abertura dos estômatos além de poder atuar na sinalização entre raiz e parte aérea. Dessa forma, foi proposto identificar a presença deste neurotransmissor em plantas de milho hidratadas e submetidas a estresse hídrico, com o objetivo de correlacionar a presença de acetilcolina com as respostas estomáticas de tais plantas. Além disso, foi objetivo do trabalho avaliar parâmetros fisiológicos como potencial hídrico, condutância estomática, transpiração e fotossíntese líquida e suas possíveis relações com a acetilcolina em três folhas das plantas hidratadas e estressadas. Para tanto, foi montado um experimento em blocos casualizados em esquema fatorial (2x3). Os fatores foram: água, nos níveis de hidratação e estresse, e idade das folhas nos níveis folha 4 (mais velha), folha 5 (idade intermediária) e folha 7 (mais jovem). As plantas foram divididas em 20 blocos, contendo uma planta hidratada e uma sob estresse cada e as análises fisiológicas feitas nas três folhas. As plantas foram colocadas em câmara de crescimento tipo BOD com controle de iluminação e temperatura. Após análises fisiológicas, as folhas foram utilizadas para extração e determinação de acetilcolina. Os extratos purificados e secos foram submetidos à pirólise e cromatografia gasosa e as substâncias identificadas por espectrometria de massas. Não foi detectada acetilcolina nas plantas, apesar de estudos anteriores demonstrarem sua ocorrência em folhas e sementes de milho. Hipóteses foram levantadas para explicar tal fato. Quanto as variáveis fisiológicas, o déficit hídrico reduziu em aproximadamente 59% a transpiração, em 65% a condutância estomática e em 59% a fotossíntese das plantas. Condutância estomática e transpiração, condutância e fotossíntese, e transpiração e fotossíntese apresentaram intensa correlação. Já o potencial hídrico teve baixa correlação com essas variáveis. Quanto ao fator idade, folhas 7 apresentaram maiores valores de fotossíntese, condutância e transpiração que as folhas 4 e 5. / The occurrence of action potential and neurotransmitters, the major components of animal nervous system, in plants, bacteria and fungi, shows the universality of signaling principles and information transmission in the way of chemical and electrical signals in all organisms. These study topics, along with plant intelligence and vesicular-based auxin transport, constitute the major research lines of the newly created Plant Neurobiology. Among the neurotransmitters found in plants, the acetylcholine plays a role in phytochromecontrolled growth and development and in membrane ion permeability. In this context, it was suggested that acetylcholine can play an important role in the regulation of stomatal movements, having stimulatory effect in the stomatal opening. In addition it can play a role in root-to-shoot signaling process. Therefore, it was proposed to identify the presence of this neurotransmitter in maize plants hydrated and under water stress, with the aim of correlating the presence of acetylcholine with the stomatal responses of such plants. Moreover, another aim of the study was to evaluate physiological parameters like water potential, stomatal conductance, transpiration rate and net photosynthetic rate and their possible relationship with the acetylcholine in three leaves of hydrated and stressed plants. Therefore, an experiment was set up in randomized block design in 2x3 factorial. The factors were: water, in the levels of hydration and stress, and leaves age in the levels leaf 4 (older), leaf 5 (intermediary age) and leaf 7 (younger). The plants were divided in 20 blocks, and each one has had one hydrated plant and one stressed plant and the physiological analysis was made in three leaves. The plants were placed in B.O.D. growth chamber under controlled conditions of light and temperature. After the physiological analysis, the leaves were used to extraction and determination of acetylcholine. The dried and purified extracts were subjected to pyrolysis and gas chromatography and the substances identified by mass spectrometry. The acetylcholine was not detected in plants, although earlier studies have had demonstrated its occurrence in maize leaves and seeds. Hypotheses were elaborated to explain such fact. Regarding the physiological variables, water stress reduced the plants transpiration rate in 59%, stomatal conductance in 65% and net photosynthesis in 59%. Stomatal conductance, transpiration and photosynthesis were strongly related. On the other hand, the water potential showed weak correlation with that variable. As for the age factor, leaves 7 had higher photosynthetic rates, conductance and transpiration than the leaves 4 and 5.

A neuroimunologia é o ramo da imunologia que estuda a relação entre sistema imune e o sistema nervoso. Muitos estudos têm demonstrado a capacidade direta de neurotransmissores em modular a resposta imune, assim como de citocinas em influenciar funções cognitivas. Neste contexto, o glutamato possui papel de destaque, por se tratar do neurotransmissor excitatório mais importante e mais abundante no sistema nervoso central dos mamíferos. Sua função é exercida através de dois tipos de receptores principais: i) os receptores ionotrópicos (iGluR) e ii) os receptores metabotrópicos (mGluR). A descoberta da expressão de receptores de glutamato em células do sistema imune tem despertado interesse científico, levantando questões acerca de sua expressão e função. No presente trabalho, avaliamos parâmetros como viabilidade celular, linfoproliferação e ativação de MAP quinase pelo receptor NMDAR esplenócitos totais e linfócitos cultivados in vitro. Nossos resultados demonstram que linfócitos em repouso e ativados apresentam diferentes perfis de expressão do receptor NMDAR. O uso do antagonista deste receptor, o MK801, foi capaz de reduzir a proliferação de linfócitos T CD4 e T CD8 estimulados com anti-CD3 em cultura de esplenócitos. Tal redução pode ser explicada por um aumento na taxa de morte celular, o que foi avaliado através de marcação com anexina-V, indicador de apoptose, ou 7-AAD, indicador de necrose. Para entendermos um pouco a respeito da sinalização do receptor NMDAR no sistema imune, avaliamos a fosforilação da MAP quinase ERK 1,2 em linfócitos T CD4 ativados na presença do agonista (NMDA) ou do antagonista (MK801) do receptor. Observamos um aumento na ativação desta quinase na presença de NMDA, o que é revertido na presença do MK801. Ao avaliar o papel do receptor NMDAR in vivo, verificamos uma redução significativa na gravidade da encefalomielite experimental auto-imune em animais tratados com MK801. Mais interessante, esta redução se correlaciona também com uma redução na fosforilação de ERK 1,2 em esplenócitos totais obtidos ao dia 7 pós-imunização. Em resumo, nossos dados sugerem que o receptor NMDA possui o papel de ativador de vias intracelulares importantes, como as da MAP quinase ERK 1,2; e que o seu bloqueio resulta em morte celular in vitro. Logo, isso indica a importância do glutamato como modulador da intensidade da resposta e viabilidade de linfócitos T CD4 e T CD8 in vitro e in vivo. Sendo assim, nossos resultados contribuem para um melhor entendimento dos fenômenos de imunoregulação, especialmente aqueles no campo da neuroimunologia ou neuroimunomodulação. / Neuroimmunology is a field within immunology which studies the relationship between the nervous system and the immune system. Several studies have demonstrated the direct ability of neurotransmitters in modulating the immune response, as for cytokines in influencing cognitive functions. In this context, glutamate stands out for being the most important and abundant neurotransmitter in the mammal central nervous system. Its role is exerted through two main types of receptor: i) ionotropic receptors (iGluR) and ii) metabotropic receptors (mGluR). The discovery of glutamate receptor expression in immune cells has led to scientific interest, raising issues concerning its expression and function. In the present study, we evaluated parameters such as cell viability, lymphoproliferation, and activation of the MAP quinase pathway by the NMDA receptor on total splenocytes and lymphocytes cultured in vitro. Our results demonstrate that naive and activated lymphocytes present different profiles of NMDA receptor expression. The use of MK801, an antagonist for this receptor, was able to reduce the T CD4 and T CD8 lymphocyte proliferation stimulated with anti-CD3 in splenocyte culture. Such reduction may be explained by the increase of the cellular death rate, evaluated by annexin-V staining, indicator of apoptosis or 7-AAD, indicator of necrosis. With the intent of understanding part of the NMDA receptor signaling in the immune system, we evaluated the ERK 1,2 MAP quinase phosphorylation in T CD4 lymphocytes activated in the presence of the agonist (NMDA) or the antagonist (MK801) of the receptor. We observed an increase in this quinase activation in the presence of NMDA, which is reversed by the MK801. When evaluating the role of the NMDA receptor in vivo, we verified a significant reduction in the degree of experimental auto-immune encephalomyelitis in animals treated with MK801. More interesting, this reduction also correlates to a reduction on the phosphorilation of ERK 1,2 in total splenocytes obtained at the seventh day post-immunization. In sum, our data suggest that the NMDA receptor has the role of activating important intracellular pathways, such as the MAP quinases ERK 1,2; and that its blockage results in cellular death in vitro. As so, this indicates the importance of glutamate as a modulator of the intensity of response and the viability of T CD4 e T CD8 lymphocytes in vitro e in vivo. Thus, our result contribute for a better understanding of the immunoregulation phenomena, especially those in the neuroimmunology ou neuroimmunomodulation field.

As reações de alerta e defesa compreendem ajustes cardiovasculares proporcionando um fluxo sangüíneo muscular adequado nas situações de \"luta ou fuga\". As vias centrais e os possíveis neurotransmissores envolvidos nestes ajustes permanecem ainda, em grande parte, desconhecidas. Neste estudo buscamos analisar a participação da neurotransmissão purinérgica e glutamatérgica no núcleo do trato solitário (NTS) na gênese da vasodilatação muscular durante reações de defesa e o papel das vias glutamatérgicas do NTS para o núcleo rostroventrolateral (RVL) nestas respostas. Ratos Wistar machos (250-350 g) foram anestesiados (uretana 600 mg/kg + alpha-chloralose 50 mg/kg, i.v.), paralisados (d-Tubocurarina, 0,5 mg/kg, i.v.) e ventilados artificialmente. Registramos a pressão arterial média (PAM), a freqüência cardíaca (FC) e o fluxo sangüíneo dos membros posteriores (FSMP). A condutância vascular dos membros posteriores (CVMP) foi determinada como a razão FSMP/PAM e expressa como percentagem do valor basal. A estimulação elétrica (EE; 150 MuA; 0,6 ms; 100 Hz; 6 s) do hipotálamo lateral provocou hipertensão, taquicardia e vasodilatação nos membros posteriores. A microinjeção bilateral de suramin (100 pmol/50 nl), um antagonista não específico de receptores P2x no NTS, reduziu a vasodilatação nos membros posteriores durante a EE do hipotálamo (173±19,0 vs 28±14,1% do basal) sem alterar as respostas pressora e taquicárdica. A microinjeção do agonista P2x alpha, beta-methylene ATP (100 pmol/50 nl) no NTS produziu hipotensão, bradicardia e vasodilatação dos membros posteriores. A microinjeção de suramin (100 pmol/50 nl) bloqueou a vasodilatação muscular (76±15,2 vs 9±2,1% do basal) e a hipotensão (-47±4,5 vs -6±2,0 mmHg). A microinjeção de ácido quinurênico (4 nmol/50 nl), um antagonista glutamatérgico ionotrópico não seletivo no NTS bloqueou, de forma semelhante ao suramin, a vasodilatação durante a EE do hipotálamo (134±21,5 vs 27±12,7% do basal) sem alterar as respostas pressora ou taquicárdica. O bloqueio bilateral no RVL com microinjeções de ácido quinurênico reduziu intensamente a resposta hipotensora (-60±6,1 vs -9±3,7 mmHg) e vasodilatadora (126±16,9 vs 17±4,6% do basal) provocada pelas microinjeções de alpha, beta-methylene ATP (100 pmol/50 nl) no NTS. O agonista purinérgico A2a, CGS21680 (20 pmol/50 nl) no NTS, evocou hipotensão, bradicardia e vasodilatação muscular de longa duração. O bloqueio do RVL com ácido quinurênico (4 nmol/50 nl) reduziu a hipotensão (- 41±4,7 vs -7±1,9 mmHg), a bradicardia (-33±9 vs -10±3,1 bpm) e a vasodilatação nos membros posteriores (81±5,6 vs 8±1,5% do basal). Estes resultados sugerem que a vasodilatação muscular nas repostas de defesa depende da ativação de receptores P2x e receptores glutamatérgicos no NTS. Ajustes cardiovasculares por ativação dos receptores purinérgicos P2x e A2a no NTS provocam vasodilatação muscular que depende da liberação de glutamato no RVL, provavelmente ativando interneurônios inibitórios ali presentes. / The electrical stimulation (ES) of the hypothalamus in the rat produces a well- defined pattern of cardiovascular adjustments including hypertension, tachycardia and skeletal muscle vasodilation. These hemodynamic responses can also be observed in natural conditions during fight and/or flight behaviors. However the neural pathways and possible neurotransmitters involved remain largely unknown. In this study we sought to determine the role of purinergic and glutamatergic receptors into the nucleus tractus solitarius (NTS) in the cardiovascular responses induced by hypothalamic ES, also we aimed to analyze the role of glutamatergic neural pathways from the NTS to the rostral ventrolateral medulla (RVLM) in these responses. Male Wistar rats (250-350 g) were anesthetized (urethane 600 mg/kg + alpha-chloralose 50 mg/kg, iv), paralyzed (d-tubocurarine 0.5 mg/kg, iv) and artificially ventilated. Mean arterial blood pressure (MAP), heart rate (HR) and hindquarter blood flow (HQBF) were recorded. Hindquarter vascular conductance (HQVC) was calculated as the ratio HQBF/MAP and expressed as percentage of baseline. Hypothalamic ES (6s trains, 0.6 ms square pulses, 100 Hz, 150 MuA) evoked a transitory hypertension, tachycardia and hindlimb muscle vasodilation. After bilateral microinjections of suramin (100 pmol /50 nl), a non-specific P2x receptor antagonist, into the NTS the hindlimb vasodilation was reduced (173±19.0 vs 28±14.1% of baseline), even so the transitory hypertension and tachycardia remained unchanged. A similar vasodilation reduction (134±21.5 vs 27±12.7% of baseline) was observed after microinjections of kynurenic acid bilaterally at the same NTS sites. Microinjections of the P2x receptor agonist alpha, beta-methylene ATP (100 pmol/50 nl) into the NTS produced hypotension, bradycardia and hindlimb muscle vasodilation. Bilateral microinjections of suramin at the same NTS site reduced the hypotension (-47±4.5 vs -6±2.0 mmHg) and the vasodilation (76±15.2 vs 9±2.1% of baseline). After bilateral microinjection of kynurenic acid into the RVLM, both hypotension (-60±6.1 vs -9±3.7 mmHg) and the vasodilation response (126±16.9 vs 17±4.6% of baseline) induced by alpha, beta- methylene ATP into the NTS were reduced. The A2a agonist CGS21680 (20 pmol/50 nl) into the NTS produced a long-lasting hypotension, bradycardia and hindlimb vasodilation. Bilateral RVLM glutamatergic blockade reduced the hypotension (-41±4.7 vs -7±1.9 mmHg), the tachycardia (-33±9.0 vs -10±3.1 bpm) and the muscle vasodilation (81±5.6 vs 8±1.5% of baseline) when CGS21680 was injected into the NTS. Therefore the results suggest that in alerting defense reaction, hindquarter vasodilation is mediated by NTS P2x and also by glutamatergic receptors into the intermediate NTS. Cardiovascular responses evoked by either P2x or A2a receptors stimulation in the NTS are mediated by glutamatergic synapses into the RVLM probably through activation of inhibitory interneurones in this area.

A moxidectina (MOX) é um antiparasitário utilizado na clínica veterinária. Em mamíferos seu mecanismo de ação envolve o ácido ?-aminobutírico (GABA), um neurotransmissor que tem papel relevante na regulação dos comportamentos sexual e motor. Dados anteriores por nós obtidos mostraram que a MOX prejudicou o comportamento sexual e a coordenação motora de ratos machos avaliados na trave elevada. Assim, dando continuidade a esse estudo, o objetivo deste trabalho foi avaliar os efeitos da administração da dose terapêutica de MOX (0,2 mg/kg) na motivação sexual e ereção peniana de ratos machos, bem como estudar seu envolvimento em diferentes sistemas de neurotransmissão central. Em todos os experimentos os ratos do grupo experimental receberam a MOX por via subcutânea (SC); e os ratos do grupo controle receberam 1 ml/kg de óleo de amêndoas pela mesma via, e foram avaliados após 72 h. A motivação sexual foi avaliada em um aparelho constituído de uma arena e dois compartimentos separados desta por tela de arame; num compartimento foi colocado um rato macho experiente e no outro uma fêmea sexualmente receptiva. Neste aparelho foi medido o tempo que o rato permaneceu nas proximidades de cada compartimento. Os resultados obtidos neste experimento não mostraram diferenças significantes entre os grupos. A ereção peniana foi induzida pela administração SC de 80 ?g/kg de apomorfina, sendo avaliadas a latência e a freqüência de ereção. Os resultados mostraram aumento da latência e redução da freqüência de ereção peniana dos animais tratados com MOX, enquanto que a administração dos antagonistas GABAérgicos (biculina e faclofen) não alterou estes parâmetros. Por outro lado, observou-se que a biculina (antagonista GABAA) reverteu os efeitos da MOX na ereção peniana, enquanto o faclofen aumentou a freqüência de ereção peniana em ratos tratados com a MOX. Quanto aos níveis hipotalâmicos e estriatais de neurotransmissores e metabólitos, observou-se que a MOX reduziu os níveis estriatais de dopamina e de seu metabólito ácido homovanílico (HVA) e também os níveis hipotalâmicos de GABA. Estes dados sugerem que a MOX embora não interfira na motivação sexual, prejudica o desempenho sexual avaliado pela ereção peniana. Esse efeito da MOX pode ser atribuído a sua ação em receptores GABAA, os quais modulam receptores tipo B, aumentando a liberação de GABA, e 72 h depois, conseqüente redução dos níveis deste neurotransmissor no hipotálamo (uma das áreas centrais envolvidas com o comportamento sexual) e também dos níveis de dopamina e seu metabólito HVA no estriado, área do sistema nervoso central relacionada com a função motora e na qual neurônios GABAérgicos modulam a atividade de neurônios dopaminérgicos. / The moxidectina (MOX) is an antiparasitic drug used in veterinary clinic. In mammals its mechanism of action involves GABA, neurotransmitter that has an important role in the regulation of the sexual and motor behaviors. Previous data showed that MOX impair male rat\'s sexual behavior and motor coordination observed at wooden dowel. The objective of the present work was to evaluate the effects of therapeutic dose of MOX (0.2 mg/kg) in sexual motivation and penile erection of male rats, as well as to study its involvement in different central systems of neurotransmission. In all experiments the rats of experimental groups received MOX subcutaneous (SC), and the rats of control groups received 1.0 mL/kg of almonds oil (SC), and were observed after 72h. Sexual motivation test was performed in an arena with two cages, separate from the arena with a wall of wire screen; in one cage was put an intact male rat and in the other one, a sexually receptive female. In this test was measured the time that the rats stayed near of each cage. The data obtained in this experiment didn\'t show any significant differences among the groups. The penile erection (PE) was induced by 80 ?g/kg of Apomorphine (SC), being evaluated the latency to and frequency of PE. The results showed increased latency and reduction of the frequency of PE of animals treated with MOX, while the GABAergic antagonists\' administration (Biculline and Phaclofen) didn\'t change these parameters. On the other hand, it was observed that the Biculline (GABAA antagonist) reversed the effects of MOX in PE, while the Phaclofen increased the frequency of PE in rats treated with MOX. About Hypothalamic and Striatal neurotransmitters levels and their metabolites, was observed that MOX reduced Dopamine (DA) and its metabolite homovanillic acid (HVA) striatal levels and hypothalamic GABA levels. These data suggest that MOX although doesn\'t interfere in sexual motivation, impair sexual performance evaluated by penile erection. This effect of MOX can be attributed to its action in GABAA receptors, which modulate type B receptors, increasing GABA release, and consequent reduction of its levels in the Hypothalamus (one of the central areas involved with sexual behavior) and also, reduction of the DA and its metabolite HVA striatal levels. Striatum is a central nervous system area related with motor function in which GABAergic neurons modulate the activity of dopaminergic neurons.

Mass spectrometry imaging (MSI) has been extensively used to produce qualitative maps of distributions of proteins, peptides, lipids, neurotransmitters, small molecule pharmaceuticals and their metabolites directly in biological tissue sections. Moreover, during the last 10 years, there has been growing demand to quantify target compounds in tissue sections of various organs. This thesis focuses on development and application of a novel instrument- and manufacturer-independent MSI software suite, msIQuant, in the open access format imzML, which has been developed specifically for quantitative analysis of MSI data. The functionality of msIQuant facilitates automatic generation of calibration curves from series of standards that can be used to determine concentrations of specific analytes. In addition, it provides many tools for image visualization, including modules enabling multiple interpolation, low intensity transparency display, and image fusion and sharpening. Moreover, algorithms and advanced data management modules in msIQuant facilitate management of the large datasets generated following rapid recent increases in the mass and spatial resolutions of MSI instruments, by using spectra transposition and data entropy reduction (at four selectable levels: coarse, medium, fine or superfine) before lossless compression of the data. As described in the thesis, implementation of msIQuant has been exemplified in both quantitative (relative or absolute) and qualitative analyses of distributions of neurotransmitters, endogenous substances and pharmaceutical drugs in brain tissue sections. Our laboratory have developed a molecular-specific approach for the simultaneous imaging and quantitation of multiple neurotransmitters, precursors, and metabolites, such as tyrosine, tryptamine, tyramine, phenethylamine, dopamine, 3-methoxytyramine, serotonin, gamma-aminobutyric acid (GABA), and acetylcholine, in histological tissue sections at high spatial resolution by matrix-assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI) MSI. Chemical derivatization by charge-tagging primary amines of analytes significantly increased the sensitivity, enabling mapping of neurotransmitters that were not previously detectable by MSI. The two MSI approaches have been used to directly measure changes in neurotransmitter levels in specific brain structures in animal disease models, which facilitates understanding of biochemical mechanisms of drug treatments. In summary, msIQuant software has proven potency (particularly in combination with the reported derivatization technique) for both qualitative and quantitative analyses. Further developments will enable its implementation in multiple operating system platforms and use for statistical analysis.

Katecholamine (Dopamin, Adrenalin, Noradrenalin) und Serotonin sind wichtige Monoamin-Neurotransmitter im menschlichen zentralen Nervensystem, deren quantitative Bestimmung von großem medizinischen Interesse ist, weil damit Aussagen zum Verlauf von Nervenkrankheiten und zur Tumorgefährdung des sympathoadrenalen bzw. neuroendokrinen Systems möglich sind. Ascorbinsäure und Harnsäure finden sich in vielen Körperflüssigkeiten. Ihre Bestimmung ist klinisch ebenfalls bedeutend, da deren Konzentration als Indikatoren bekannter Krankheitsbilder dienen. Etablierte Standardmethoden, wie die Hochleistungsflüssigkeitschromatographie (HPLC) und immunologische Nachweisverfahren (ELISA) werden im klinischen Bereich zur Bestimmung der Neurotransmitter genutzt. Diese sind kostenintensiv und zeitaufwändig und daher für die Anwendung in den Arztpraxen, vor allem in Entwicklungsländern nicht geeignet. Elektrochemische Verfahren, insbesondere voltammetrische Messmethode haben den Vorteil, solche Bestimmungen in einfacher Weise zu ermöglichen. In der Literatur finden sich Angaben zu eingesetzten Elektroden auf Kohlenstoffbasis mit hoher Sensitivität für die Katecholamine. Allerdings wurden diese Elektroden meist einzeln hergestellt. Der kommerzielle Durchbruch ist deshalb bisher, hauptsächlich infolge der mangelnden Reproduzierbarkeit der Elektrodeneigenschaften und der Verfügbarkeit einfacher elektronischer Geräte ausgeblieben. Es war daher Ziel dieser Arbeit, durch industrienahe Herstellungsverfahren Graphitelektroden mit reproduzierbaren Eigenschaften zu entwickeln und diese auf ihre Eignung für den quantitativen Nachweis bioaktiver Stoffe zu erproben. Dazu waren Verfahrensschritte zu optimieren, die es erlauben, diese siebgedruckten Graphitelektroden reproduzierbar und kostengünstig zu fertigen und sie auf verschiedene Weise, z.B. durch halbleitende Polymere und nanoskalige Metalle zu modifizieren. Neben den Neurotransmittern enthalten Körperflüssigkeiten unter anderem Ascorbinsäure und Harnsäure in hohen Konzentrationen. Daher waren zunächst Modellanalyten unter Verwendung dieser Stoffe herzustellen. Die voltammetrischen Methoden, wie die zyklische Voltammetrie (CV), die Differentielle Puls-Voltammetrie (DPV) und die Square-Wave-Voltammetrie (SWV) sollten auf ihre Eignung zum Nachweis der bioaktiven Substanzen erprobt werden. Schließlich waren die Elektroden in realen Analyten zu testen. Insgesamt konnte in der vorliegenden Arbeit gezeigt werden, dass ausgewählte Neurotransmitter, Ascorbinsäure und Harnsäure sich mit differentiellen voltammetrischen Verfahren an industrienah hergestellten modifizierten Dickschichtelektroden bestimmen lassen. Es ist erstmalig gelungen, eine modifizierte Dickschichtelektrode zu entwickeln, mit der es möglich ist, Katecholamine unabhängig von Ascorbinsäure (3 mM) und Harnsäure (2 mM) quantitativ nachzuweisen. Damit eröffnen sich neue Wege für den Einsatz von elektrochemischen Sensoren für die einfache Bestimmung der Neurotransmitter vor Ort. Die beschriebenen modifizierten Dickschichtelektroden sind ohne Verlust an elektrochemischer Aktivität an der Luft oder im Grundelektrolyten monatelang lagerfähig. Die Elektroden lassen sich im Gegensatz zu den in der Literatur beschriebenen Elektroden mit Einzelfertigung kostengünstig in großer Stückzahl mit hoher Reproduzierbarkeit herstellen.:Inhaltsverzeichnis I Abkürzungen V 1 Einleitung und Zielsetzung der Arbeit 1 2 Theoretischer Teil 5 2.1 Elektrochemische Verfahren in der Analytik 5 Klassifizierung elektroanalytischer Methoden 5 2.1.1 Voltammetrie 5 Cyclovoltammetrie (CV) 6 Differential-Puls-Voltammetrie (DPV) 9 Square-Wave-Voltammetrie (SWV) 10 2.1.2 Chronocoulometrie (ChrC) 11 2.1.3 Impedanzmessung (EIS) 12 2.1.4 Elektrochemische Quarzmikrowaage (EQCM) 14 2.2 Poly-3,4-Ethylendioxythiophen, ein leitfähiges Polymer 19 2.2.1 Leitfähige Polymere 19 2.2.2 Das Poly-3,4-ethylendioxythiophen 20 Elektrochemische Synthese und Dotierung 20 2.3 Bioaktive Stoffe 24 2.3.1 Katecholamine 24 Dopamin 25 Noradrenalin und Adrenalin 25 Abnorme Konzentration der Katecholamine 25 2.3.2 Serotonin 26 2.3.3 Interaktion von Katecholaminen und Serotonin 26 2.3.4 Ascorbinsäure und Harnsäure 27 2.3.5 Elektrochemisches Verhalten der bioaktiven Stoffe 28 Katecholamine 28 Serotonin 30 Ascorbinsäure 30 Harnsäure 30 3 Experimenteller Teil 32 3.1 Chemikalien 32 3.2 Lösungen 33 3.2.1 Ausgangslösungen 33 Grundelektrolyte 33 Lösungen der bioaktiven Stoffe 33 3.2.2 Lösungen für Elektrodenmodifizierungen 33 EDOT-haltige Lösungen 33 Neurotransmitter-Lösungen 34 HAuCl4-Lösungen 34 Goldkolloide 34 Eisenhexacyanoferrat(II)-Goldsäurehaltige Lösung 35 3.3 Elektrochemische Messmethoden 35 3.3.1 Voltammetrie, Chronocoulometrie und Impedanz 35 3.3.2 Elektrochemische Quarzmikrowaage 38 3.4 Elektroden und Präparation der Elektroden 39 3.4.1 Untersuchte Elektroden, deren Aktivierung und Konditionierung 39 3.4.3 Modifizierungen der Elektroden 41 Poly-3,4-Ethylendioxythiophen (PEDOT) 41 Goldnanopartikel 41 Komposite aus Goldnanopartikeln und Preußisch Blau (Au/PB) 42 Polymerfilme aus Monoamin-Neurotransmittern 42 3.5 Präparation der UP für Untersuchungen in realen Medien 43 3.6 Spektroskopische Methoden 43 4 Ergebnisse und Diskussion 45 4.1 Unmodifizierte Elektrodenoberflächen 45 4.1.1 Einfluss der Aktivierung der Elektrodenoberflächen auf das Messverhalten 45 4.1.2 Bestimmung bioaktiver Stoffe an unmodifizierten Elektroden 48 Ermittlung des Peakpotenzials 48 Messeffekte an Gold- und Graphitelektroden in Neurotransmitter-Lösungen hoher Konzentrationen 50 Bestimmung bioaktiver Stoffe im Gemisch 52 4.2 Au- und Au/PB-modifizierte Elektroden 54 4.2.1 Abscheidung 54 4.2.2 Untersuchungen bioaktiver Stoffe an Au-modifizierten Elektroden 56 4.3 PEDOT-modifizierte Elektroden 58 4.3.1 Abscheidungen der PEDOT-Schichten 58 CV-Abscheidungen der PEDOT-Schichten 59 ChrC-Abscheidungen der PEDOT-Schichten 62 4.3.2 Voruntersuchungen an PEDOT-modifizierten Elektroden 66 Ermittlung des optimalen Potenzialbereiches für voltammetrische Messungen an PEDOT-modifizierten Elektroden 66 Ermittlung der optimale PEDOT-Schichten für die Bestimmung bioaktiver Stoffe 68 Peakpotenziale bioaktiver Stoffe 71 Einfluss des pH-Wertes des Elektrolyten und der Scangeschwindigkeit auf voltammetrische Messsignale bioaktiver Stoffe 72 Einfluss der Messmethoden auf die Messsignale bioaktiver Stoffe an PEDOT-modifizierten Elektroden 74 4.3.3 Bestimmung bioaktiver Stoffe an PEDOT-modifizierten Elektroden 78 Bestimmung der Neurotransmitter (Dopamin, Adrenalin, Noradrenalin und Serotonin) 78 Bestimmung von Ascorbinsäure und Harnsäure 81 Bestimmung der Neurotransmitter mit Zusatz von Ascorbinsäure und Harnsäure 82 Stabiltität der PEDOT-modifizierten Elektroden 83 Vergleich der Ergebnisse an PEDOT-Elektroden mit Literaturangaben 84 4.3.4 Spektroskopische Untersuchungen der PEDOT-Oberflächen 85 4.3.5 Zusammenfassung der Ergebnisse an PEDOT-Elektroden 87 4.4 Au-PEDOT-modifizierte Elektroden 88 4.4.1 Abscheidungen der Goldnanopartikel auf PEDOT-Oberflächen 88 Abscheidung der Goldnanopartikel durch Adsorption aus Goldkolloiden 88 Abscheidung der Goldnanopartikel auf PEDOT-modifizierten Elektroden mittels Cyclovoltammetrie 92 4.4.2 Bestimmung bioaktiver Stoffe an Au-PEDOT-Elektroden 94 Peakpotenziale bioaktiver Stoffe an Au-PEDOT-Elektroden 94 Bestimmung von Neurotransmittern in 0,1 M Phosphatpufferlösungen 96 Bestimmung von Neurotransmittern mit Zusatz von Ascorbinsäure und Harnsäure 98 Bestimmung von Ascorbinsäure und Harnsäure 99 Stabilität der Sensitivitäten und Reproduzierbarkeit der Elektrodenherstellung 102 Vergleich der Ergebnisse an Au-PEDOT-Elektroden mit Literaturangaben 102 4.4.3 Zusammenfassung der Ergebnisse an Au-PEDOT-Elektroden 104 4.5 Polymonoamin-modifizierte Elektroden bzw. PEDOT-Elektroden 105 4.5.1 Abscheidungen der Polymerschichten aus Monoaminen an Graphitelektroden 106 4.5.2 Abscheidungen der Polymerschichten aus Monoaminen an PEDOT-Elektroden 106 CV-Abscheidung 106 SWV-Abscheidung 108 4.5.3 Bestimmung bioaktiver Stoffe an Polyserotonin-modifizierte PEDOT-Elektroden 111 Peakpotenziale bioaktiver Stoffe 111 Bestimmung der Neurotransmitter 112 Bestimmung von Ascorbinsäure und Harnsäure 114 Bestimmung der Neurotransmitter mit Zusatz von AS und HS 114 Bestimmung von Harnsäure in Gegenwart von Dopamin 116 4.5.4 Möglicher Einsatz der 5-HT-PEDOT-Elektroden als pH-Elektroden 117 4.5.5 Zusammenfassung der Ergebnisse an Polyserotonin-PEDOT-Elektroden 118 4.6 Bestimmung bioaktiver Stoffe in UM 119 4.6.1 Bestimmung von Harnsäure 119 Bestimmung von Harnsäure im Modellanalyten 119 Bestimmung von Harnsäure in präparierten UP 119 4.6.2 Bestimmung von Dopamin 120 DA-Bestimmung im Modellanalyten 120 Bestimmung von Dopamin in präparierten UP 121 5 Zusammenfassung und Ausblick 123 Zusammenfassung 123 Ausblick 126 Tabellenverzeichnis 127 Abbildungsverzeichnis 130 Anhang 138 Literaturverzeichnis 152 VERSICHERUNG 157