Hyperkalemic periodic paralysis, HyperKPP, is an inherited progressive disorder of the muscles caused by mutations in the voltage gated sodium channel (NaV1.4). The objectives of this thesis were to develop a technique for measurement symptoms in vivo using electromyography (EMG) and to determine the mechanism by which Ca2+ alleviates HyperKPP symptoms, since this is unknown. Increasing extracellular [Ca2+] ([Ca2+]e) from 1.3 to 4 mM did not result in any increases in45Ca2+ influx suggesting no increase in intracellular [Ca2+] ([Ca2+]i) acting on an intracellular signaling pathway or on an ion channel such as the Ca2+sensitive K+ channels. HyperKPP muscles have larger TTX-sensitive22Na+ influx than wild type muscles because of the defective NaV1.4 channels. When [Ca2+] was increased from 1.3 to 4 mM, the abnormal 22Na+ influx was completely abolished. Thus, one mechanism by which Ca2+alleviates HyperKPP symptoms is by reducing the abnormal Na+ influx caused by the mutation in the NaV1.4 channel.

Synaptic transmission occurs as a result of either a spontaneous release of presynaptic vesicles or a batch release of presynaptic vesicles driven by action potentials. The physiological consequence of synaptic transmission driven by different patterns and frequencies of presynaptic stimulation has been extensively investigated. However, the physiological nature, mechanism as well as relevance of prolonged presynaptic stimulation have been poorly characterized. In this dissertation, I present three projects in which prolonged stimulation of synaptic transmission in different forms and different brain regions was studied for its effect on synaptic transmission, mechanisms and physiological relevance. In the first project, prolonged electrical stimulation (100 sec) at high frequency induced a deep synaptic depression in acute hippocampal slices, followed by a recovery of synaptic transmission after ~15 min. The deep synaptic depression was attributed to a complete depletion of presynaptic vesicle pools. In the second project, attempts were made to characterize the mechanism of nuclear activation of gene transcription induced by prolonged electrical stimulation (100 sec). Our results demonstrated that reduced inactivation of non-L-type calcium channels failed to provide calcium required for gene transcription, leaving the activation of gene transcription a selective function for L-type calcium channels. In the third project, we sought to study the physiological relevance of enhanced miniature events of inhibitory synapses induced by prolonged chemical stimulation. We showed that prolonged application (2 min) of nicotine to the striatal slice enhanced the frequency of miniature inhibitory currents that was accompanied with a reduction in the amplitude of evoked response. This reduction in the amplitude of evoked responses was ascribed to a compromised action potential invasion of presynaptic terminals possibly due to inactivation of sodium channels resulting from nicotine-induced depolarization. To summarize, prolonged stimulation of presynaptic vesicle release imposes significant influence upon neuron-to-neuron communication, with distinct mechanisms in different brain regions.

The sialyltransferase family is a group of enzymes that transfer sialic acid from donor CMP-Neu5Ac onto suitable carbohydrate chains of glycoproteins and glycolipids. In vertebrates, sialylation is implicated in many physiological and pathobiological processes, including nervous and immune system development and functioning, pathogen-host interaction, cancer cell proliferation and apoptosis. However, the complexity of the sialylation pathway and limitation of genetic and in vivo approaches interferes with functional analyses in mammalian organisms. We use Drosophila because of its simplified physiology and reduced genetic redundancy to characterize the evolutionarily conserved function of sialylation and to reveal its relationship to the role of sialic acids in humans. This dissertation focuses primarily on Drosophila sialyltransferase, DSIAT, so far the only sialyltransferase described in protostomes. Gene targeting of the DSIAT endogenous locus with a DSIAT-HA tagged version uncovered its remarkably dynamic stage- and cell-specific expression. I found that the expression of DSIAT is developmentally regulated and is restricted to motor neurons and cholinergic interneurons within the central nervous system of Drosophila. To reveal the role of DSIAT in development and functioning of fly nervous system I performed characterization of neurological phenotypes of DSIAT knockout flies, also generated by gene targeting approach. I observed that DSIAT mutant larvae are sluggish and have abnormal neuromuscular junction (NMJ) morphology. Electrophysiological analysis of mutant larval NMJ showed altered evoked NMJ activity. It was also observed that DSIAT knockout adult flies are paralyzed when are exposed to higher temperatures. Longevity assays showed that DSIAT adult mutants have significantly reduced life span. I used genetic interaction analysis to identify possible sialylated targets in Drosophila and found that ?-subunit of voltage gated sodium channel is a potential sialylated protein in the fly nervous system. All these data strongly supports the hypothesis that DSIAT plays an important role for neural transmission and development in Drosophila. This research work establishes Drosophila as a useful model system to study sialylation which may shed light on related biological functions in higher organisms including humans.

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Synaptic transmission occurs as a result of either a spontaneous release of presynaptic vesicles or a batch release of presynaptic vesicles driven by action potentials. The physiological consequence of synaptic transmission driven by different patterns and frequencies of presynaptic stimulation has been extensively investigated. However, the physiological nature, mechanism as well as relevance of prolonged presynaptic stimulation have been poorly characterized. In this dissertation, I present three projects in which prolonged stimulation of synaptic transmission in different forms and different brain regions was studied for its effect on synaptic transmission, mechanisms and physiological relevance. In the first project, prolonged electrical stimulation (100 sec) at high frequency induced a deep synaptic depression in acute hippocampal slices, followed by a recovery of synaptic transmission after ~15 min. The deep synaptic depression was attributed to a complete depletion of presynaptic vesicle pools. In the second project, attempts were made to characterize the mechanism of nuclear activation of gene transcription induced by prolonged electrical stimulation (100 sec). Our results demonstrated that reduced inactivation of non-L-type calcium channels failed to provide calcium required for gene transcription, leaving the activation of gene transcription a selective function for L-type calcium channels. In the third project, we sought to study the physiological relevance of enhanced miniature events of inhibitory synapses induced by prolonged chemical stimulation. We showed that prolonged application (2 min) of nicotine to the striatal slice enhanced the frequency of miniature inhibitory currents that was accompanied with a reduction in the amplitude of evoked response. This reduction in the amplitude of evoked responses was ascribed to a compromised action potential invasion of presynaptic terminals possibly due to inactivation of sodium channels resulting from nicotine-induced depolarization. To summarize, prolonged stimulation of presynaptic vesicle release imposes significant influence upon neuron-to-neuron communication, with distinct mechanisms in different brain regions.

Τα πενταμερή ιοντικά κανάλια που ενεργοποιούνται από τη δέσμευση ενός προσδέτη (pLGICs-pentameric Ligand Gated Ion Channels) της υπεροικογένειας των υποδοχέων Cys-θηλιάς είναι διαμεμβρανικές γλυκοπρωτεΐνες που εμπλέκονται σε ποικιλία βιολογικών λειτουργιών. Στην παρούσα εργασία παρουσιάζεται η NMR μελέτη της εξωκυττάριας περιοχής ενός προκαρυωτικού pLGIC, της GLIC (Gloeobacter ligand-gated ion channel), που προέρχεται από το κυανοβακτήριο Gloeobacter violaceus και εμφανίζεται ως μονομερές σε διάλυμα. / Pentameric ligand-gated ion channels (pLGICs) of the Cys loop family are transmembrane glycoproteins implicated in a variety of biological functions. Here, we present a solution NMR study of the extracellular domain of a prokaryotic pLGIC homologue from the bacterium Gloeobacter violaceus that is found to be a monomer in solution.

Made available in DSpace on 2014-06-11T19:28:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-11-17Bitstream added on 2014-06-13T19:57:51Z : No. of bitstreams: 1 zandonadi_jc_me_prud.pdf: 8814232 bytes, checksum: 639b792fb6ddcb95c07af901afcff862 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Nesta dissertação, trata-se de mudanças ocorridas no processo de urbanização, principalmente após a década de 1970, na cidade de Marília, dando-se ênfase ao seu setor leste. Tais alterações consistem em mudanças na estrutura da cidade, a qual passa de um padrão concentrado e contínuo para extenso e descontinuo. Essas mudanças vêm acompanhadas de maior homogeneização dos subespaços da cidade, ou seja, acentuação da segregação socioespacial, bem como do surgimento de novas formas de habitat urbano, os condomínios horizontais e loteamentos fechados, situados, sobretudo em áreas periféricas, assim como o aparecimento de novos espaços de consumo, marcados pela especialização e segmentação, mudando a estrutura da cidade, passando de monocêntrica para a cidade multi(poli)cêntrica. Tais mudanças são observadas no setor leste de Marília, onde se nota forte homogeneidade socioeconômica da população, inúmeros loteamentos fechados e condomínios horizontais, bem como a consolidação de três novos espaços de consumo, marcados por níveis... / This dissertation is about changes in the urbanization process, specially after the 1970’s decade, in the city of Marília, with emphasis in its eastern sector. These changes are structural changes in the city, which becomes from a concentrated and continuous pattern to an extense and discontinuous one. These changes happen with greater homogenization of the city subspaces, ie, accentuation of sociospace segregation, and the emergence of new forms of urban habitat, the horizontal condominiuns and closed lots located especially in remote areas, as well as the emergence of new areas of consumption, marked by specialization and segmentation, changing the structure of the city, from a monocentric to a multi(poli)centric city. Such chances are observed in the eastern sector of Marilia, where high socioeconomic homogeneity of the population, the existence of many closed lots and horizontal condominiuns are noted, as well as the consolidation of three new areas of consuption, marked by different levels of centrality... (Complete abstract click electronic access below)

Orientador: Maria Encarnação Beltrão Sposito / Banca: Arthur Magon Whitacker / Banca: Luis Renato Bezerra Pequeno / Resumo: Nesta dissertação, trata-se de mudanças ocorridas no processo de urbanização, principalmente após a década de 1970, na cidade de Marília, dando-se ênfase ao seu setor leste. Tais alterações consistem em mudanças na estrutura da cidade, a qual passa de um padrão concentrado e contínuo para extenso e descontinuo. Essas mudanças vêm acompanhadas de maior homogeneização dos subespaços da cidade, ou seja, acentuação da segregação socioespacial, bem como do surgimento de novas formas de habitat urbano, os condomínios horizontais e loteamentos fechados, situados, sobretudo em áreas periféricas, assim como o aparecimento de novos espaços de consumo, marcados pela especialização e segmentação, mudando a estrutura da cidade, passando de monocêntrica para a cidade multi(poli)cêntrica. Tais mudanças são observadas no setor leste de Marília, onde se nota forte homogeneidade socioeconômica da população, inúmeros loteamentos fechados e condomínios horizontais, bem como a consolidação de três novos espaços de consumo, marcados por níveis... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This dissertation is about changes in the urbanization process, specially after the 1970's decade, in the city of Marília, with emphasis in its eastern sector. These changes are structural changes in the city, which becomes from a concentrated and continuous pattern to an extense and discontinuous one. These changes happen with greater homogenization of the city subspaces, ie, accentuation of sociospace segregation, and the emergence of new forms of urban habitat, the horizontal condominiuns and closed lots located especially in remote areas, as well as the emergence of new areas of consumption, marked by specialization and segmentation, changing the structure of the city, from a monocentric to a multi(poli)centric city. Such chances are observed in the eastern sector of Marilia, where high socioeconomic homogeneity of the population, the existence of many closed lots and horizontal condominiuns are noted, as well as the consolidation of three new areas of consuption, marked by different levels of centrality... (Complete abstract click electronic access below) / Mestre

Synaptic transmission occurs as a result of either a spontaneous release of presynaptic vesicles or a batch release of presynaptic vesicles driven by action potentials. The physiological consequence of synaptic transmission driven by different patterns and frequencies of presynaptic stimulation has been extensively investigated. However, the physiological nature, mechanism as well as relevance of prolonged presynaptic stimulation have been poorly characterized. In this dissertation, I present three projects in which prolonged stimulation of synaptic transmission in different forms and different brain regions was studied for its effect on synaptic transmission, mechanisms and physiological relevance. In the first project, prolonged electrical stimulation (100 sec) at high frequency induced a deep synaptic depression in acute hippocampal slices, followed by a recovery of synaptic transmission after ~15 min. The deep synaptic depression was attributed to a complete depletion of presynaptic vesicle pools. In the second project, attempts were made to characterize the mechanism of nuclear activation of gene transcription induced by prolonged electrical stimulation (100 sec). Our results demonstrated that reduced inactivation of non-L-type calcium channels failed to provide calcium required for gene transcription, leaving the activation of gene transcription a selective function for L-type calcium channels. In the third project, we sought to study the physiological relevance of enhanced miniature events of inhibitory synapses induced by prolonged chemical stimulation. We showed that prolonged application (2 min) of nicotine to the striatal slice enhanced the frequency of miniature inhibitory currents that was accompanied with a reduction in the amplitude of evoked response. This reduction in the amplitude of evoked responses was ascribed to a compromised action potential invasion of presynaptic terminals possibly due to inactivation of sodium channels resulting from nicotine-induced depolarization. To summarize, prolonged stimulation of presynaptic vesicle release imposes significant influence upon neuron-to-neuron communication, with distinct mechanisms in different brain regions. / Medicine, Faculty of / Graduate

Hyperkalemic periodic paralysis, HyperKPP, is an inherited progressive disorder of the muscles caused by mutations in the voltage gated sodium channel (NaV1.4). The objectives of this thesis were to develop a technique for measurement symptoms in vivo using electromyography (EMG) and to determine the mechanism by which Ca2+ alleviates HyperKPP symptoms, since this is unknown. Increasing extracellular [Ca2+] ([Ca2+]e) from 1.3 to 4 mM did not result in any increases in45Ca2+ influx suggesting no increase in intracellular [Ca2+] ([Ca2+]i) acting on an intracellular signaling pathway or on an ion channel such as the Ca2+sensitive K+ channels. HyperKPP muscles have larger TTX-sensitive22Na+ influx than wild type muscles because of the defective NaV1.4 channels. When [Ca2+] was increased from 1.3 to 4 mM, the abnormal 22Na+ influx was completely abolished. Thus, one mechanism by which Ca2+alleviates HyperKPP symptoms is by reducing the abnormal Na+ influx caused by the mutation in the NaV1.4 channel.