Nesta tese foi empregada a iodociclofuncionalização de β-enamino ésteres para obtenção de análogos conformacionalmente restritos do ácido γ-aminobutírico (GABA) e de 4-aril-1,4-di-hidropiridinas (DHP\'s). Foram preparados ciclopentanos trissubstituídos a partir de b-enamino ésteres cíclicos, com o objetivo de investigar o mecanismo desta reação. Utilizando ESI(+)-MS/MS, foi possível identificar e caracterizar os intermediários bicíclicos catiônicos, confirmando que a reação se processa através de uma SN2 intramolecular. A biorredução do grupo acetila existente nos ciclopentanos, utilizando raízes de Daucus carota e células íntegras de Aspergillus terreus CCT 3320 e Rhizopus oryzae CCT 4964, forneceu novos análogos oticamente ativos do GABA, com excelente enantiosseletividade. Também foram preparados iodo-β-enamino ésteres cíclicos de seis membros, contendo um grupo arila na posição 4 do anel tetra-hidropiridínico, cuja desidroiodação forneceu os respectivos derivados 1,4-di-hidropiridínicos. / This thesis presents a study of iodo-cyclization of β-enamino esters in order to obtain conformationally restricted analogues of γ-amino butiric acid (GABA) and of 1,4-dihydropyridines (DHP\'s). Trisubstituted cyclopentanes were obtained from iodo-β-enamino esters and the mechanism of their formation was proposed and corroborated by (+)-ESI-MS/MS through the interception and structural characterization of the key bicyclic iminium ion intermediates. Optically active GABA analogues have been prepared in high enantiomeric excesses (up to >99%) by bioreduction of the keto group in cyclopentane derivatives using whole fungal cells of Aspergillus terreus CCT 3320, Rhizopus oryzae CCT 4964 and Daucus carota root. 4-aryl-1,4,5,6-tetrahydropyridine derivatives were synthesized by iodo-cyclization of a-alkenyl-b-enamino esters and 4-phenyl-1,4-dihydropyridine derivatives were obtained after base-promoted dehydroiodination of the corresponding cyclic iodo-b-enamino esters.

Les objectifs de ma thèse ont été de caractériser le développement du gradient en ions chlorures dans les cellules de Purkinje dans un modèle d'étude de l'autisme : les souris exposées de façon prénatale au valproate de sodium. A cette fin, j'ai effectué des mesures éléctrophysiologiques de courants GABAergiques au cours du développement post-natal de ces animaux et des observations histologiques de la densité linéaire en cellules de Purkinje. D'autre part, j'ai participé à une étude comportementale visant à étudier l'influence d'un composé modulant le gradient en ions chlorures dans deux modèles génétiques d'étude de l'autisme : les souris Oprm1-/- et les souris Fmr1-/-. Au cours de ma thèse, j'ai mis en évidence un retard du développement du gradient en ions chlorures. J'ai également montré qu'une exposition prénatale au valproate de sodium induisait une perte post-natale en cellules de Purkinje. Enfin, le composé avec lequel j'ai travaillé améliore le phénotype autistique et laisse entrevoir un fort potentiel translationnel. / The aims of my PhD were to characterize the development of the chloride gradient in Purkinje cells in a model of autism: mice prenatally exposed to sodium valproate. To this end, I measured GABAergic currents along the post-natal development of these mice and made histological observations of the Purkinje cells linear density. Secondly, I took part of a behavioral study to test the influence of a compound acting on the chloride gradient in two genetic models of autism: Oprm1-/- and Fmr1-/- mice. During my thesis I showed a delay in the development of the chloride gradient. I also observed that a prenatal exposition of sodium valproate induced a post-natal Purkinje cells loss. Finally, the compound I worked with improves the autistic phenotype and opens the perspective for translational potential.

Die nichtproteinogene Aminosäure GABA (γ-Aminobuttersäure) gilt als der wichtigste inhibitorische Neurotransmitter im Zentralnervensystem von Vertebraten sowie Invertebraten und vermittelt ihre Wirkung u. a. über die metabotropen GABAB-Rezeptoren. Bisher sind diese Rezeptoren bei Insekten nur rudimentär untersucht. Für die Amerikanische Großschabe als etablierter Modellorganismus konnte pharmakologisch eine modulatorische Rolle der GABAB-Rezeptoren bei der Bildung von Primärspeichel nachgewiesen werden. Ziel dieser Arbeit war eine umfassende Charakterisierung der GABAB-Rezeptor-Subtypen 1 und 2 von Periplaneta americana. Unter Verwendung verschiedenster Klonierungsstrategien sowie der Kooperationsmöglichkeit mit der Arbeitsgruppe von Prof. Dr. T. Miura (Hokkaido, Japan) in Hinsicht auf eine dort etablierte P. americana EST-Datenbank gelang die Klonierung von zwei Rezeptor-cDNAs. Die Analyse der abgeleiteten Aminosäuresequenzen auf GB-spezifische Domänen und konservierte Aminosäure-Reste, sowie der Vergleich zu bekannten GB Sequenzen anderer Arten legen nahe, dass es sich bei den isolierten Sequenzen um die GABAB-Rezeptor-Subtypen 1 und 2 (PeaGB1 und PeaGB2) handelt. Für die funktionelle und pharmakologische Charakterisierung des Heteromers aus PeaGB1 und PeaGB2 wurden Expressionskonstrukte für die Transfektion in HEK-flpTM-Zellen hergestellt. Das Heteromer aus PeaGB1 und PeaGB2 hemmt bei steigenden GABA-Konzentrationen die cAMP-Produktion. Die Substanzen SKF97541 und 3-APPA konnten als Agonisten identifiziert werden. CGP55845 und CGP54626 wirken als vollwertige Antagonisten. Das in vitro ermittelte pharmakologische Profil im Vergleich zur Pharmakologie an der isolierten Drüse bestätigt, dass die GABA-Wirkung in der Speicheldrüse tatsächlich von GBs vermittelt wird. Für die immunhistochemische Charakterisierung konnte ein spezifischer polyklonaler Antikörper gegen die extrazelluläre Schleife 2 des PeaGB1 generiert werden. Ein weiterer Antikörper, welcher gegen den PeaGB2 gerichtet ist, erwies sich hingegen nicht als ausreichend spezifisch. Western-Blot-Analysen bestätigen das Vorkommen beider Subtypen im Zentralnervensystem von P. americana. Zudem wird der PeaGB1 in der Speicheldrüse und in den Geschlechtsdrüsen der Schabenmännchen exprimiert. Immunhistochemische Analysen zeigen eine PeaGB1-ähnliche Markierung in den GABAergen Fasern der Speicheldrüse auf. Demnach fungiert der PeaGB1 hier als Autorezeptor. Weiterhin konnte eine PeaGB1-ähnliche Markierung in nahezu allen Gehirnneuropilen festgestellt werden. Auch die akzessorischen Drüsen der Männchen, Pilzdrüse und Phallusdrüse, sind PeaGB1-immunreaktiv. / The non-proteinogenic amino acid GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the central nervous system of vertebrates and invertebrates, and GABA mediates its action among others via metabotropic GABAB receptors (GBs). So far, these receptors are only rudimentary characterized in insects. In the American Cockroach, which is an established model organism, pharmacological studies have pointed out a modulatory role of GBs in the production of primary saliva in the salivary gland. Therefore, the aim of this study is the profound characterization of the GABAB receptor subtypes 1 and 2 of Periplaneta americana. Diverse cloning strategies and the access to a Periplaneta EST-database enabled the cloning of two receptor-cDNAs. The analysis of the deduced amino acid sequences for GB-specific domains and conserved amino acid-residues, and the comparison to known GB-sequences of other species revealed that the sequences correspond to GABAB receptor subtypes 1 and 2 (PeaGB1 and PeaGB2). Next, we functionally and pharmacologically characterized the receptor-heteromer of PeaGB1 and PeaGB2. Therefore, we established expression constructs for the transfection of HEK-flpTM-cells. The PeaGB1/PeaGB2 heteromer inhibits dose-dependently the production of cAMP. The substances SKF97541 and 3-APPA imitate the GABA effect. In contrast, CGP54626 and CGP55845 are considered to be proper antagonists. The comparison of the in vitro with the known pharmacology of isolated glands reveals that GBs indeed mediate the effect of GABA in the salivary gland. For immunohistochemical localization, a specific polyclonal antibody was raised against the extracellular loop 2 of PeaGB1. A second antibody, which was raised against the analogous region of the PeaGB2, must be considered to be non-specific. Western blot analyses demonstrate the localization of both subtypes in the central nervous system of P. americana. Additionally, PeaGB1 is expressed in the salivary gland and in male accessory glands. Immunohistochemical analyses reveal the expression of PeaGB1 in GABAergic nerve fibers of the salivary gland. As a consequence, PeaGB1 must act as an autoreceptor in this organ. A widespread distribution of PeaGB1 in almost all neuropiles was detected in the cockroach brain. In the male accessory glands mushroom gland and phallic (conglobate) gland, an intense PeaGB1-like immunoreactivity was measured.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Thesis (M. Sc.)--Carleton University, 2004. / Includes bibliographical references (p. 40-50). Also available in electronic format on the Internet.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study demonstrated the sedative and anesthetic activity of the essential oil (EO) of Cymbopogon flexuosus in the silver catfish (Rhamdia quelen). The time for induction and recovery of this EO was compared with the EO of Aloysia triphylla, due to the similarity between their major components: α-citral (geranial) and β-citral (neral). Both EOs induced anesthesia at concentrations from 150 to 300 μL L-1 and sedation at 25 μL L-1. The EO of C. flexuosus was faster in inducing the initial stages of anesthesia, but there was no significant difference to reach deep anesthesia, and there was a significantly longer recovery time. Cooperative relations and the modulation of the benzodiazepine (BDZ) site of GABAa as a mechanism of action of both EOs was verified from the addition of diazepam and flumazenil to experiments (BDZ site of GABAa agonist and antagonist, respectively). The addition of diazepam (150 μM) induced potentiation in concentrations of 25, 150 and 300 μL L-1 both EOs without significant change in anesthesia recovery time. Flumazenil (10 μM) reversed the diazepam-induced anesthesia, but not the anesthesia induced by EOs at concentrations of 150 and 300 μL L-1, thus the EO of C. flexuosus induced effective sedation and anesthesia without short-term mortality and the modulation of the BDZ site of the GABAa in the anesthetic action mechanism of both EOs in this study was not demonstrated. / Este estudo demonstrou as atividades sedativa e anestésica do óleo essencial (OE) de Cymbopogon flexuosus em jundiás (Rhamdia quelen). Os tempos de indução e recuperação relatados para esse OE foram comparados com o OE de Aloysia triphylla devido à semelhança entre os seus principais componentes: α-citral (geranial) e β-citral (neral). Ambos os OEs induziram anestesia nas concentrações de 150 a 300 μL L-1 e sedação a partir de 25 μL L-1. O OE de C. flexuosus induziu mais rapidamente fases iniciais da anestesia, mas não houve diferença significativa para alcançar o estágio de anestesia profunda, além de induzir um tempo de recuperação significativamente mais longo. As relações cooperativas e a modulação do site benzodiazepínico (BDZ) do receptor GABAa como mecanismo de ação de ambos OEs foram testados a partir da adição de diazepam e flumazenil aos experimentos (agonista e antagonista do site BDZ do GABAa, respectivamente). A adição de diazepam (150 μM) potencializou o efeito de ambos os OEs nas concentrações de 25, 150 e 300 μL L-1, sem induzir alteração significativa nos tempos de recuperação anestésica. O flumazenil (10 μM) reverteu a anestesia induzida pelo diazepam, mas não a anestesia induzida pelos OEs nas concentrações de 150 e 300 μL L-1, portanto o OE de C. flexuosus induziu sedação e anestesia efetivas sem mortalidade a curto prazo e a modulação do site BDZ do GABAa como mecanismo de ação anestésica de ambos OEs não ficou demonstrada neste estudo.

Tem sido proposto que vias serotonérgicas distintas originárias do núcleo dorsal da rafe (NDR) modulariam diferentes tipos de reações de defesa a estímulos aversivos. Deakin e Graeff (1991) propuseram que a ativação da via ascendente do NDR, que inerva a amígdala e o córtex frontal, facilitaria comportamentos defensivos aprendidos em resposta a perigo potencial ou distal, enquanto que a ativação da via periventricular, também originária no NDR e que inerva a matéria cinzenta periaquedutal dorsal (MCPD) inibiria reações de fuga e luta inatas em resposta a perigo proximal. No presente trabalho pretendeu-se explorar esta hipótese investigando o efeito da inibição do NDR, da MCPD e do núcleo amigdalóide basolateral, em ratos submetidos a dois modelos animais de ansiedade: o labirinto em T elevado (LTE) e o modelo de transição claro-escuro (MTCE). Os testes com o LTE incluíram uma variação metodológica: um dia antes do experimento, os animais foram pré-expostos por 30 minutos a um dos braços abertos do modelo. Tem sido demonstrado que a pré-exposição leva a uma diminuição do tempo gasto em atividade exploratória no braço aberto, assegurando que durante as medidas de fuga, os animais estão realmente fugindo do estímulo aversivo. No presente trabalho, a administração intra-NDR do agonista gabaérgico muscimol diminuiu as latências da esquiva inibitória (EI), efeito ansiolítico, e da fuga dos braços abertos do LTE, efeito ansiogênico. Além disso, o muscimol intra-NDR aumentou o tempo gasto no compartimento claro (TGCC) do MTCE, efeito ansiolítico. Por outro lado, a droga não apresentou efeito no modelo do labirinto em T fechado. Já a microinjeção de muscimol intra-MCPD aumentou a latência de fuga do braço aberto do LTE, efeito ansiolítico, não apresentando qualquer efeito na EI do labirinto em T elevado ou no MTCE. Finalmente, a administração de muscimol intra-amígdala basolateral prejudicou a esquiva inibitória do LTE e aumentou o TGCC do MTCE, efeitos ansiolíticos, sem alterar a fuga dos braços abertos. Em conjunto, os dados do presente trabalho corroboram alguns dos pressupostos da teoria do papel dual da 5-HT na ansiedade proposta por Deakin e Graeff (1991). É interessante salientar, ainda, a importância da pré-exposição sobre as medidas de fuga do braço aberto do LTE. / It has been proposed that distinct 5-HT pathways modulate different types of anxiety reaction to aversive stimuli. Deakin and Graeff (1991) proposed that the activation of the ascending dorsal raphe (DR)-5-HT pathways innervating the amygdala and frontal cortex would facilitate learned defensive behaviors in response to distal or potential threat, while activation of the DR-periventricular 5-HT pathways, which innervates the dorsal periaqueductal gray matter (DPAG) would inhibit innate flight or fight reactions in response to proximal threat. In an attempt to explore this hypothesis, we investigated the effects of reversible inhibition of the DRN, the DPAG and of the basolateral amygdala nucleus in rats exposed to 2 animal models of anxiety: the elevated T maze (ETM) and the light-dark model (LDM). An important methodological difference was included to the ETM tests. The animals were exposed for 30 minutes to one of the ETM open arms, 24 h before the tests. Pre-exposure seems to improve escape measurements in the ETM, assuring that during escape, animals are really escaping from the aversive threat and not simply ambling. In the present study, muscimol administered intra-DRN impaired both the avoidance, anxyolitic effect, and the escape task, anxyogenic effect, of the ETM and increased the time spent in the light compartment (TSLC) in the LDM, an anxyolitic effect. On the other hand, muscimol microinjected intra-DPAG enhanced escape latencies in the ETM, an anxyolitic effect, without altering inhibitory avoidance or LDM measurements. Finally, muscimol administered intra-basolateral amygdala impaired avoidance in the ETM and increased the TSLC in the LDM, revealing an anxyolitic effect in both tests, without altering the escape task. Taken together the obtained data seem to corroborate the dual role of 5-HT in anxiety, proposed by Deakin and Graeff (1991). At last, the importance of pre-exposure to the open arms of the LTE should be emphasized as a procedure capable of improving escape measurement.

No description available.

Fipronil é um inseticida fenilpirazol de amplo espectro, desenvolvido para inibir seletivamente receptores GABA associados a canais de cloreto de insetos; tanto em Medicina Veterinária, como em Agricultura tem sido utilizado para o controle de pragas. Embora vários estudos procurem compreender os mecanismos da toxicidade neuronal dos praguicidas em mamíferos, há poucos relacionados aos efeitos neurocomportamentais. Assim, o presente estudo investigou os efeitos da exposição prolongada de ratos ao fipronil, observando-se alguns comportamentos ligados ao sistema GABAérgico, como atividade no campo aberto, no labirinto em cruz elevado (LCE), bem como na convulsão induzida por picrotoxina e pentilenotetrazol; avaliou-se ainda os níveis cerebrais de alguns neurotransmissores, como GABA, dopamina, noradrenalina, serotonina e seus respectivos metabólitos. Além disso, foram realizadas análises anátomo-histopatológicas em fígado, cérebro e rins dos animais. Ratos Wistar receberam, por via intragástrica (gavage), durante 28 dias um dos seguintes tratamentos: fipronil (0,1; 1,0 ou 10,0 mg/Kg/dia) ou água destilada 1 mL/Kg/dia. Após uma hora da última administração do fipronil ou água destilada, os ratos tiveram seu comportamento avaliado no campo aberto e no LCE com auxílio do sistema computadorizado EthoVision®. Observou-se no campo aberto apenas redução significante do nº de levantamentos nos animais tratados com 10,0 mg/Kg de fipronil, quando comparados com os ratos do grupo controle. No LCE, a exposição prolongada de ratos ao fipronil nas doses de 1,0 e 10,0 mg/Kg provocou redução na distância percorrida pelos animais e o tempo de permanência dos animais nos braços abertos, além de aumentar o tempo de permanência nos braços fechados. Para o cálculo da dose convulsivante mínima, seguiu-se o mesmo protocolo de tratamento, sendo observada diminuição significante na dose de picrotoxina e de pentilenotetrazol necessária para a indução da convulsão em animais tratados com 10,0 mg/Kg fipronil. A determinação dos níveis de neurotransmissores e seus respectivos metabólitos mostrou aumento dos níveis de dopamina no córtex frontal de ratos tratados com 10,0 mg/Kg de fipronil, redução nos níveis de GABA no striatum de ratos tratados com 1,0 mg/Kg de fipronil. Em relação à serotonina houve aumento de seus níveis no córtex frontal de ratos tratados com 1,0 mg/Kg e seu metabólito, ácido 5 (5HIAA), foi observado redução de seus níveis no córtex frontal de ratos tratados com 0,1 e 1,0 mg/kg p.c. de fipronil. No striatum também houve redução de seus níveis em ratos tratados com 0,1 e 1,0 mg/Kg de fipronil. O estudo anátomo-histopatológico revelou que a exposição prolongada ao fipronil pode causar hepatotoxicidade, caracterizada por aumento dos pesos absoluto e relativo do fígado e tumefação celular de hepatócitos. Em conclusão, os resultados mostraram que a exposição de ratos por 28 dias, via gavage, ao fipronil pode causar efeitos comportamentais relacionados a alterações em sistemas centrais de neurotransmissão e também hepatotoxicidade. / Fipronil is a phenylpyrazole insecticide with broad spectrum, developed to selectively inhibit insect GABA-gated chloride channels; both in Veterinary Medicine, and in Agriculture it has been used for the control of nuisances. Although several studies try to understand the mechanism of neuronal toxicity of pesticide in mammalian, there are few studies related to neurobehavioral effects. Thus, the present study investigated the effects of fipronil long term exposure in rats, being observed some behaviors connected to GABAergic system, as activity in open field, in elevated plus maze (EPM), as well as in the seizures induced by picrotoxinin and pentylenotetrazole. In addition, this study determinated the brain levels of some neurotransmitter as GABA, dopamine, noradrenaline, serotonin and metabolic levels and investigated liver, brain and kidney damage. Wistar rats received by intragastric (gavage) way during 28 days one of this treatment: fipronil (0,1, 1,0 or 10,0 mg/Kg) or distilled water; the rats behavior were evaluated in open field and elevated plus maze apparatus with system computer EthoVision®. There was a significant reduction of rearing in open field in rats treated with 10,0 mg/Kg of fipronil when compared with control rats. In the EPM, there was a decrease of distance moved in open arms and increase of time spent in closed arms to rats treated with 1,0 and 10,0 mg/Kg of fipronil when compared to control. It was still noticed the reduction in the time spent in open arms EPM in rats treated with 1,0 and 10,0 mg/Kg of fipronil when compared to control group. To calculate the minimum convulsant dose, was used the same treatment protocol and it was observed a significant reduction in picrotoxinin and pentylenotetrazole necessary dose to induction of seizure in animals treated with fipronil 10,0 mg/Kg. The level determination of neurotransmitters and their metabolites, it was observed the dopamine increasing in forebrain from fipronil treated rats by 10,0 mg/Kg. GABA levels decreased in fipronil treated rats striatum by 1,0 mg/Kg. Serotonin neurotransmitter was increased in fipronil treated rats forebrain by 1,0 mg/Kg to and its metabolite, 5HIAA, it was observed a levels decrease in fipronil forebrain treated rats by 0,1 e 1,0 mg/kg. There was decrease of 5HIAA levels in fipronil treated rats striatum by 0,1 e 1,0 mg/Kg. According to organ analyses, it was observed dose-response liver damage such as swelling liver cells. In conclusion, the data showed that fipronil administered in rats by 28 days caused behavioral effects related to central neurotransmitter alterations and hepatotoxicity.

Nesta tese foi empregada a iodociclofuncionalização de β-enamino ésteres para obtenção de análogos conformacionalmente restritos do ácido γ-aminobutírico (GABA) e de 4-aril-1,4-di-hidropiridinas (DHP\'s). Foram preparados ciclopentanos trissubstituídos a partir de b-enamino ésteres cíclicos, com o objetivo de investigar o mecanismo desta reação. Utilizando ESI(+)-MS/MS, foi possível identificar e caracterizar os intermediários bicíclicos catiônicos, confirmando que a reação se processa através de uma SN2 intramolecular. A biorredução do grupo acetila existente nos ciclopentanos, utilizando raízes de Daucus carota e células íntegras de Aspergillus terreus CCT 3320 e Rhizopus oryzae CCT 4964, forneceu novos análogos oticamente ativos do GABA, com excelente enantiosseletividade. Também foram preparados iodo-β-enamino ésteres cíclicos de seis membros, contendo um grupo arila na posição 4 do anel tetra-hidropiridínico, cuja desidroiodação forneceu os respectivos derivados 1,4-di-hidropiridínicos. / This thesis presents a study of iodo-cyclization of β-enamino esters in order to obtain conformationally restricted analogues of γ-amino butiric acid (GABA) and of 1,4-dihydropyridines (DHP\'s). Trisubstituted cyclopentanes were obtained from iodo-β-enamino esters and the mechanism of their formation was proposed and corroborated by (+)-ESI-MS/MS through the interception and structural characterization of the key bicyclic iminium ion intermediates. Optically active GABA analogues have been prepared in high enantiomeric excesses (up to >99%) by bioreduction of the keto group in cyclopentane derivatives using whole fungal cells of Aspergillus terreus CCT 3320, Rhizopus oryzae CCT 4964 and Daucus carota root. 4-aryl-1,4,5,6-tetrahydropyridine derivatives were synthesized by iodo-cyclization of a-alkenyl-b-enamino esters and 4-phenyl-1,4-dihydropyridine derivatives were obtained after base-promoted dehydroiodination of the corresponding cyclic iodo-b-enamino esters.