Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on Mar. 2, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Notre Dame, 2005. / Thesis directed by Sunny K. Boyd for the Department of Biological Sciences. "March 2005." Includes bibliographical references (leaves 114-145).

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 146-173). Also available in electronic version.

No description available.

Uga3p, a member of the zinc binuclear cluster transcription factor family, is required for [gamma]-aminobutyric acid-dependent transcription of the UGA genes in Saccharomyces cerevisiae. Crystallographic data of some of the protein-DNA complexes of this family reveal that members of this family bind to CGG triplets. A conserved 19-nucleotide activation element in certain UGA gene promoter regions contains a CCG-N4-CGG everted repeat, proposed to be the binding site of Uga3p, UAS[subscript GABA]. The spacer region (N4) between the CGG triplets has been suggested to be the specificity determinant for binding to UAS[subscript GABA]. The data available from the Saccharomyces genome database indicates that there are multiple repeats of -CCG-N4-CGG- regions within the genome. These transcription factors are involved in the activation of specific pathways and the question arises as to how their specificity of binding is determined. The aim of this study was to understand the binding characteristics of Uga3p to UAS[subscript GABA] and to determine the affinity and specificity of this interaction. In this study, full-length (tagged and untagged) and truncated (1-124 a.a.) Uga3p was produced in a heterologous expression system (E. coli). The interaction of Uga3p with UAS[subscript GABA] in Saccharomyces cerevisiae was characterized in terms of binding in vitro and the transcriptional activation of lacZ reporter genes in vivo. The Uga3p was capable of binding to these sites in vitro independent of exogenous GABA. Electrophoretic mobility shift assays (EMSA) of the full-length Uga3p with the wild type UAS[subscript GABA] sequences produced two distinct mobility complexes. The complexes formed in the EMSA of the full-length Uga3p were those specific to the interaction of the Uga3p to UAS[subscript GABA]. The truncated Uga3p(1-124 a.a.), which has the DNA-binding zinc cluster domain, the linker region and the putative coiled-coil domain was not functionally equivalent to the full-length protein with respect to binding in vitro because the EMSAs of the UAS[subscript GABA] with the truncated Uga3p produced indistinct complexes. EMSAs using mutant UAS[subscript GABA] sequences and heterologously-produced full-length Uga3p, demonstrated that UAS[subscript GABA] consists of two, independent Uga3p-binding sites. This work presents evidence that the two Uga3p molecules bound to UAS[subscript GABA] most likely interact with each other. Unlike other zinc cluster binding sites the Uga3p-binding site is an asymmetric site of 5’-SGCGGNWWT-3’ (S= G or C, W = A or T and N = no nucleotide or G or C). UAS[subscript GABA] is a palindrome containing the two asymmetric Uga3p-binding sites. The two-site consensus sequence required for the binding of Uga3p to the UAS[subscript GABA] is present upstream of UGA1 (region -387 to -370) and UGA4 (region -403 to -387). Furthermore, a single Uga3p-binding site was identified in the 5’ untranslated regions of UGA2 (region -219 to -211). GABA-dependent transcriptional activation by UAS[subscript GABA] in vivo could be directly correlated to a high affinity, specific interaction of two Uga3p molecules to this UAS. Binding with high affinity required the conserved sequences flanking the everted repeat. This study provided evidence that the binding pattern of Uga3p is novel compared to other zinc cluster motifs investigated, as the sequences flanking the everted repeat are important regions for recognition by Uga3p. The studies with the truncated Uga3p (1 –124 a.a.), also suggested that the regions C-terminal to the DNA-binding motif and putative coiled-coil area of this protein are important for Uga3p-specific interactions with UAS[subscript GABA]. Investigation of regions C-terminal to the zinc cluster, linker and putative coiledcoil revealed an eight-motif regulatory region similar to that in other zinc cluster proteins. This indicated that the regions C-terminal to these domains are important for the regulation and activity of these proteins. A putative seven repeat WD40-like motif was identified within this region. This putative domain has been speculated to be important for protein-protein interactions. Phosphorylation and dephosphorylation in other proteins of this class have been indicated to be important for the regulation of the activity of these proteins. The bioinformatic analysis of Uga3p revealed two possible cAMP/cGMP-dependent protein kinase phosphorylation sites, four putative protein kinase C phosphorylation motifs and four putative casein kinase II phosphorylation motifs. This study has contributed to the understanding of the nature of interactions between Uga3p and its specific UAS [subscript GABA] and how the regions flanking the everted repeat determine its specificity. The comparison of the nature of the binding of truncated and full-length Uga3p in vitro provided evidence for the role played by the full-length protein in determining this specific interaction. This evidence suggested that the in vitro binding evidence for other proteins of this family, using truncated peptides that carry the DNA-binding domain, might not reflect the true nature of interactions between the proteins of this class and their specific UASs in vivo.

Members of the zinc binuclear cluster family are important fungal transcriptional regulators sharing a common DNA binding domain. Da181p is a pleotropic zinc binuclear cluster protein involved in the induction of the UGA genes required for the γ-aminobutyrate nitrogen catabolic pathway in Saccharomyces cerevisiae. The zinc binuclear cluster domain is indispensable for function in Da181p and little is known about other domains in this protein. The aim of the study was to explore the zinc binuclear cluster protein family using comparative bioinformatics as a complement to biochemical and structural approaches. A database of all zinc binuclear cluster proteins was composed. A total of 118 zinc binuclear proteins are reported in this work. Thirty nine previously unidentified zinc binuclear cluster proteins were found. Four homologues of Da181p were identified by homology searching. Important sequence motifs were identified in the aligned sequences of Da181p and its homologues. The coiled coil motif found in the Ga14p zinc binuclear cluster protein could not be identified in Da181p and its homologues. This suggested that Da181p did not dimerise through this structural motif as other zinc binuclear cluster proteins. Solvent accessible site that could be phosphorylated by protein kinase C or casein kinase II and the role of such sites in the possible regulation of Da181p function were discussed.

Le cerveau immature est le siège de processus développementaux qui permettent de passer d'une structure primitive à un réseau mature et fonctionnel. L'activité synaptique spontanée générée dans le système nerveux en développement joue un rôle fondamental dans ces processus. Un des principaux moyens par lesquels cette activité peut être traduite en changement phénotypique au niveau neuronal est la sécrétion de neurotrophines. Les neurotrophines sont sécrétées par les neurones et contrôlent toutes les étapes du développement neuronal. Dans l'hippocampe en développement, la neurotrophine principale est le BDNF (brain derived neurotrophic factor). Cette protéine est synthétisée sous forme immature, le proBDNF, dont le rôle est encore méconnu. Durant ma thèse, j'ai montré que le BDNF exerce un contrôle bidirectionnel sur l'efficacité des synapses GABAergiques en développement. La polarité de la plasticité est déterminée par le type d'activité endurée par les neurones et la forme sous laquelle le BDNF est présenté à ces derniers. J'ai ainsi décrit une séquence développementale qui pourrait s'inscrire dans les processus développementaux permettant la maturation du réseau GABAergique dans l'hippocampe de rat. / The immature brain is the place of developmental processes that allow the switch from a primitive structure to a mature and functional network. Spontaneous synaptic activity generated in the developing nervous system plays a fundamental role in these processes. One of the principal ways this activity is translated into phenotypical changes at the neuronal level is the secretion of neurotrophins. Neurotrophins are secreted by neurons and control each step of neuronal development. In the developing hippocampus, the major neurotrophin is BDNF (brain derived neurotrophic factor). This protein is synthetized under an immature form, proBDNF, which role is still poorly known. During my thesis, I showed that BDNF exerts a bidirectional control on the efficacy of developing GABAergic synapses, which polarity is set by the type of activity endured by neurons and the form of BDNF that is presented to them. I described a developmental sequence which could be a part of the developmental processes allowing the maturation of the GABAergic network in the developing rat hippocampus.

O fipronil é um inseticida fenilpirazol de amplo espectro, empregado na Medicina Veterinária e na Agricultura para o controle de pragas; é um potente inibidor do canal de cloreto ligado ao ácido gama aminobutírico (GABA), um dos neurotransmissores responsáveis por efeitos inibitórios no sistema nervoso central (SNC) de mamíferos. Embora vários estudos procurem compreender os mecanismos da toxicidade neuronal dos praguicidas em mamíferos, há poucos relacionados aos efeitos neurocomportamentais. Neste trabalho estudou-se os efeitos da exposição aguda a ao fipronil, utilizando-se modelos comportamentais ligados ao sistema GABAérgico: campo-aberto (CA), labirinto em cruz elevado (LCE), dose convulsivante mínima (DCM) com picrotoxina e pentilenotetrazol, e avaliação dos níveis cerebrais de alguns neurotransmissores e metabólitos. Ratos Wistar machos receberam, por via intragástrica (gavage), dose única de fipronil (1,0; 10,0; 30,0 ou 100,0 mg/Kg) ou água destilada 1 mL/Kg. No CA, uma hora após o tratamento, houve redução significante da distância percorrida, da velocidade média, do tempo em movimento e do número de levantamentos nos animais tratados. No LCE, observou-se diminuição da distância percorrida e número de entradas nos braços fechados e diminuição do número de movimentos iniciados na arena, nos animais tratados. Quanto às convulsões, foi observada redução significante apenas na DCM de pentilenotetrazol em animais expostos a maior dose de fipronil. Uma hora após o tratamento não foram observadas alterações relevantes nos níveis dos neurotransmissores (noradrenalina; dopamina, serotonina e GABA) e seus metabólitos no córtex, no hipotálamo e no striatum. Finalizando, os resultados do presente estudo mostraram que ratos expostos a dose única de fipronil apresentaram alterações comportamentais, caracterizadas por redução da atividade motora no campo aberto, no labirinto em cruz elevado e na dose convulsivante mínima do pentilenotetrazol. Além disso, a maior dose de fipronil (100,0 mg/Kg) causou a morte de 60% dos ratos, num período entre 24 horas a 7 dias após a exposição oral. Esses efeitos comportamentais do fipronil não puderam ser atribuídos a um único sistema de neurotransmissão central. / The Fipronil is a phenylpyrazole insecticide with broad spectrum, both in Veterinary Medicine, and in Agriculture it has been used for the control of nuisances, is a potent inhibitor of chloride channel connected to the gamma aminobutyric acid (GABA), one of the neurotransmitters responsible for inhibitory effects on the central nervous system (CNS) of mammals. 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 acute exposure, being observed some behaviors models connected to GABAergic system, as open field, elevated plus maze (EPM), as well as in the seizures induced by picrotoxinin and pentylenotetrazole and determinate the brain levels of some neurotransmitter and metabolic levels. Male Wistar rats received by intragastric (gavage), single dose of fipronil (1.0; 10.0; 30.0 or 100.0 mg/Kg) or distilled water. In open field, one hour after treatment, there was a significant reduction of distance moved, average speed, time moving and the number of surveys in treated animals. In the EPM, it was observed decrease in the distance moved, in the number of entries in the closed arms and decrease the number of started movements in the arena, in treated animals. As for seizures, a significant decrease was observed only in minimum convulsant dose of pentylenotetrazole in animals exposed to higher dose of fipronil. One hour after treatment were not observed any relevant changes in the levels of neurotransmitters (norepinephrine, dopamine, serotonin and GABA) and its metabolites in the cortex, the hypothalamus and the striatum. Finally, the results of this study showed that rats exposed to single dose of fipronil showed behavioral changes, characterized by reduction of motor activity in the open field, in the elevated plus-maze and the minimum convulsant dose of pentyletetrazole. Moreover, the higher dose of fipronil (100.0 mg/Kg) caused the death of 60% of rats, over a period from 24 hours to 7 days after oral exposure. These behavioral effects of fipronil could not be attributed to a single system of central neurotransmission.

A Doença de Parkinson, uma condição neurodegenerativa e progressiva, está relacionada à morte de neurônios localizados na Substância Negra compacta, um dos componentes dos Núcleos da Base. Quando há a morte de neurônios dopaminérgicos nigrais, esta via modulatória é perdida, levando ao desequilíbrio entre as vias direta e indireta, esta última tendo sua atividade aumentada em detrimento da outra. O estriado tem um papel importante no recebimento e filtração de sinais motores corticais e talâmicos e suas maiores populações neuronais são GABAérgicas, demonstrando a importância do neurotransmissor GABA nesta modulação. O estriado recebe projeções dopaminérgicas vindas da Substância Negra compacta e, na falta desta aferentação, surgem os sintomas e sinais da Doença de Parkinson. Nosso objetivo é caracterizar a liberação de GABA nesta estrutura, avaliando os efeitos de outros transmissores e também o papel de alguns sinalizadores intracelulares neste processo. Para isto, empregamos o método de superfusão e liberação de GABA radiomarcado, previamente carregado, em tecido picado in vitro. A lesão nigral é produzida por cirurgia estereotáxica e microinjeção de 6-OHDA no feixe medial prosencefálico (mfb). Diversas drogas foram utilizadas para avaliarmos diferentes passos na liberação do transmissor. Concluímos que a liberação é fortemente dependente de cálcio e segue o modelo de exocitose vesicular, além de a subpopulação neuronal GABAérgica estrital estudada sofrer pouca influência de aferências glutamatérgicas e colinérgicas. No entanto, drogas dopaminérgicas regulam complexamente a liberação de GABA no estriado e ela também é bastante dependente de calmodulina. Conjecturamos se algumas drogas antipsicóticas que agem sobre calmodulina devem seu efeito terapêutico, ou parte dele, a esta ação e se, no modelo de DP de lesão unilateral por 6-OHDA, há comunicação entre os hemisférios lesado e não lesado após o estabelecimento da lesão e processo de rearranjo neuronal / Parkinsons disease, a progressive and neurodegenerative condition, is related to the death of neurons located in Substantia Nigra compacta, a component of Basal Ganglia. When nigral dopaminergic neurons die, this modulatory pathway is lost leading to imbalance between direct and indirect pathways, the latter having its activity increased over the former. Striatum has an essential role in receiving and filtering motor signals from cortex and thalamus and its major neuronal populations are composed by GABAergic neurons, showing how important is GABA in this modulation. Striatum receives dopaminergic projections from Substantia Nigra compacta and in its absence the typical signals and symptoms of the disease arise. We aimed to characterize GABA relase at this structure, assessing the effect of other transmitters as well the role of some intracellular signaling molecules in this process. For that, we employed the superfusion method and release of preloaded radiolabeled GABA from chopped striatal tissue. Nigral injury was produced by stereotaxic surgery and 6-OHDA microinjection at medial forebrain bundle (mfb). Several drugs were used to evaluate different steps in transmitter release. We concluded that the release is strongly calcium-dependent and follows vesicular exocytosis model; in addition the striatal GABAergic subpopulation of neurons studied here undergo little influence of glutamatergic and cholinergic afferents. However, dopaminergic drugs complexly regulate striatal GABA release and it also shows high involvement of calmodulin. We wonder if some antipsychotic drugs that act over calmodulin owe their therapeutical effects, or at least part of it, to this activity and if in 6-OHDA unilateral lesion parkinsonism model there is communication between injuried and healthy hemispheres after the establishment of the injury and neuronal rearrangement process

O fipronil é um inseticida fenilpirazol de amplo espectro, empregado na Medicina Veterinária e na Agricultura para o controle de pragas; é um potente inibidor do canal de cloreto ligado ao ácido gama aminobutírico (GABA), um dos neurotransmissores responsáveis por efeitos inibitórios no sistema nervoso central (SNC) de mamíferos. Embora vários estudos procurem compreender os mecanismos da toxicidade neuronal dos praguicidas em mamíferos, há poucos relacionados aos efeitos neurocomportamentais. Neste trabalho estudou-se os efeitos da exposição aguda a ao fipronil, utilizando-se modelos comportamentais ligados ao sistema GABAérgico: campo-aberto (CA), labirinto em cruz elevado (LCE), dose convulsivante mínima (DCM) com picrotoxina e pentilenotetrazol, e avaliação dos níveis cerebrais de alguns neurotransmissores e metabólitos. Ratos Wistar machos receberam, por via intragástrica (gavage), dose única de fipronil (1,0; 10,0; 30,0 ou 100,0 mg/Kg) ou água destilada 1 mL/Kg. No CA, uma hora após o tratamento, houve redução significante da distância percorrida, da velocidade média, do tempo em movimento e do número de levantamentos nos animais tratados. No LCE, observou-se diminuição da distância percorrida e número de entradas nos braços fechados e diminuição do número de movimentos iniciados na arena, nos animais tratados. Quanto às convulsões, foi observada redução significante apenas na DCM de pentilenotetrazol em animais expostos a maior dose de fipronil. Uma hora após o tratamento não foram observadas alterações relevantes nos níveis dos neurotransmissores (noradrenalina; dopamina, serotonina e GABA) e seus metabólitos no córtex, no hipotálamo e no striatum. Finalizando, os resultados do presente estudo mostraram que ratos expostos a dose única de fipronil apresentaram alterações comportamentais, caracterizadas por redução da atividade motora no campo aberto, no labirinto em cruz elevado e na dose convulsivante mínima do pentilenotetrazol. Além disso, a maior dose de fipronil (100,0 mg/Kg) causou a morte de 60% dos ratos, num período entre 24 horas a 7 dias após a exposição oral. Esses efeitos comportamentais do fipronil não puderam ser atribuídos a um único sistema de neurotransmissão central. / The Fipronil is a phenylpyrazole insecticide with broad spectrum, both in Veterinary Medicine, and in Agriculture it has been used for the control of nuisances, is a potent inhibitor of chloride channel connected to the gamma aminobutyric acid (GABA), one of the neurotransmitters responsible for inhibitory effects on the central nervous system (CNS) of mammals. 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 acute exposure, being observed some behaviors models connected to GABAergic system, as open field, elevated plus maze (EPM), as well as in the seizures induced by picrotoxinin and pentylenotetrazole and determinate the brain levels of some neurotransmitter and metabolic levels. Male Wistar rats received by intragastric (gavage), single dose of fipronil (1.0; 10.0; 30.0 or 100.0 mg/Kg) or distilled water. In open field, one hour after treatment, there was a significant reduction of distance moved, average speed, time moving and the number of surveys in treated animals. In the EPM, it was observed decrease in the distance moved, in the number of entries in the closed arms and decrease the number of started movements in the arena, in treated animals. As for seizures, a significant decrease was observed only in minimum convulsant dose of pentylenotetrazole in animals exposed to higher dose of fipronil. One hour after treatment were not observed any relevant changes in the levels of neurotransmitters (norepinephrine, dopamine, serotonin and GABA) and its metabolites in the cortex, the hypothalamus and the striatum. Finally, the results of this study showed that rats exposed to single dose of fipronil showed behavioral changes, characterized by reduction of motor activity in the open field, in the elevated plus-maze and the minimum convulsant dose of pentyletetrazole. Moreover, the higher dose of fipronil (100.0 mg/Kg) caused the death of 60% of rats, over a period from 24 hours to 7 days after oral exposure. These behavioral effects of fipronil could not be attributed to a single system of central neurotransmission.