Thesis (Ph. D.)--University of Nevada, Reno, 2008. / "December, 2008." Includes bibliographical references. Online version available on the World Wide Web.

Glycinergic neurotransmission is a major inhibitory influence in the CNS and defects are associated with paroxysmal neuromotor disorder, hyperekplexia with mutations in subunits of the inhibitory glycine receptor which facilitates postsynaptic ligand-binding, ion-channels. This study investigates the human glycinergic system by; 1) Mutation analysis of glycinergic candidate genes in hyperekplexia: the DNA sequencing of GLRAl in 88 hyperekplexia patients revealed 30 sequence variants; 21 were inherited in recessive mode or part of compound heterozygosity, indicating that recessive hyperekplexia is more common than previously expected. Further screening of the glycine transporter-2 gene (SLC6A5) as a candidate gene, 12 SLC6A5 mutations were found in 7 human hyperekplexia cases inherited predominantly by compound heterozygosity. 2) Biophysical analysis and molecular modelling of GLRAl mutations: which demonstrated that subcellular localisation defects were the major mechanism underlying recessive mutations. Other mutants typically show alterations in the dose-response curve for glycine suggestive of disrupted signal transduction. This study reports the first hyperekplexia mutation associated with leaky current suggesting tonic channel opening as a new receptor mechanism and fully-supported by molecular modelling. 3) Molecular and immunoreactive analysis of gephyrin heterogeneity in human brain: gephyrin encodes a multifunctional cytoplasmic protein important for organizing glycine and GABAa receptors at the postsynaptic membrane. Gephyrin has many different transcript isoforms and the study describes the population / distribution of gephyrin isoforms in neuronal tissues using molecular and immunohistochemical techniques. The heterogeneity of gephyrin cassettes indicates that each cassette is temporally and spatially regulated with unique patterns of glycine receptors co-localisation and we hypothesise that different gephyrin isoforms exhibit differential binding specificity affecting protein-protein interactions. This thesis describes that hyperekplexia is definitively a glycinergic disorder with several mechanism of molecular pathogenicity. Moreover, the underlying complexity of proteins, such as gephyrin, reveals further challenges in interpretating the functional significance of the neuronal heterogeneity.

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Orientador: Lília Freira Rodrigues de Souza Li / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T19:22:58Z (GMT). No. of bitstreams: 1 Nascimento_PaulaHespanholo_M.pdf: 1475049 bytes, checksum: f1ec759ca189c1e3404667fb1ce01abc (MD5) Previous issue date: 2012 / Resumo: A esclerose hipocampal está associada à epilepsia de lobo temporal medial (ELT) e causa expressão alterada de receptores tais como o Receptor Metabotrópico de Glutamato (mGluR1). Contudo, ainda há controvérsias se sua expressão está aumentada ou diminuída em ELT. O Receptor Sensor de Cálcio (CASR), outro receptor da mesma família do mGluR1, é expresso em hipocampo, mas seu papel no cérebro ainda é desconhecido. VILIP-1 é uma proteína sensora de cálcio neuronal (NCS) expressa predominantemente no cérebro e em humanos e sua expressão foi mapeada por imunoistoquímica na subpopulação de neurônios piramidais em CA1 e CA4 de hipocampo. Sugere-se também que a ativação de mGluR possa regular a expressão de VILIP-1 durante a plasticidade hipocampal. No entanto, não há estudos associando VILIP-1 e esclerose hipocampal. Nós hipotetizamos que além do mGluR1, o CASR e o VILIP-1 estão associados a esclerose hipocampal em ELT. O objetivo deste trabalho foi analisar o padrão de expressão de VILIP-1, CASR e mGluR1, em hipocampo de pacientes com ELT submetidos a amigdalohipocampectomia. Nossos resultados demonstraram a presença de EH nos tecidos hipocampais de pacientes com ELT com redução no número de neurônios em CA1 e presença de intensa gliose. Pela análise da expressão dos transcritos VILIP-1, CASR e mGluR1 em hipocampo total utilizando PCR em tempo real não encontramos diferença na expressão dos RNAs mensageiros dos pacientes quando comparado com os controles. Entretanto, quando comparamos a expressão protéica em hipocampo de pacientes e controles, utilizando o método de imunoistoquímica, encontramos não somente redução significativa no número de neurônios presentes em CA1 de pacientes, mas também redução importante nos neurônios positivamente marcados para VILIP-1, CASR e mGluR1. Estes achados sugerem que não apenas mGluR1, mas também CASR e VILIP-1, estão associados à EH em pacientes com ELT / Abstract: Hippocampal sclerosis (HS) is associated to temporal lobe epilepsy (TLE) and cause altered expression of neurotransmitter receptors such as metabotropic glutamate receptor 1 (mGluR1). However, whether its expression level is increased or decreased in temporal lobe epilepsy is still controversial. Calcium-sensing receptor (CASR), another receptor from the same family of mGluR1, is expressed in hippocampus, but its role in brain is unknown. VILIP-1, a neuronal calcium sensing protein (NCS) is expressed predominantly in brain and in humans its expression was identified by immunohistochemistry in subpopulations of pyramidal neurons in CA1 and CA4 in hippocampus. Activation of mGluR1 is suggested that may regulates VILIP-1 expression during hippocampal plasticity. However, there are no studies associating VILIP-1 and hippocampal sclerosis. We hypothesized that not only mGluR1 but also VILIP and CASR is involved in hippocampal sclerosis in TLE patients. The objective of this study was to analyze the pattern of expression of VILIP-1, CASR and mGluR1 in hippocampal tissues from patients with TLE who underwent amygdalohippocampectomy. Our results demonstrated the presence of hippocampal sclerosis in hippocampal tissues in patients with TLE with reduction in the number of neurons in CA1 and gliosis. By the expression analysis of the transcripts of VILIP-1, CASR and mGluR1 in total hippocampus using real time PCR, we did not find differences on mRNAS expression of patients compared with controls. However, when we compared the protein expression from hippocampi from patients with controls, by immunohistochemistry, we not only found an important reduction on neuron cell number in patients, but also an important reduction on positively stained neurons for VILIP-1, CASR and mGluR1, suggesting that not only mGluR1, but also CASR and VILIP1 are associated to HS in patients with TLE / Mestrado / Saude da Criança e do Adolescente / Mestre em Saude da Criança e do Adolescente

Fathead minnows (Pimephales promelas) were exposed to mercury (1.69, 6.79, and 13. 57 µg/l HgCl2; 10 d exposure) and afterwards tested using various metrics of foraging ability while feeding in a vegetated habitat. Among the foraging metrics were foraging efficiency, capture speed, and the ability to learn and retain information regarding habitat characteristics. Comparisons with control fish and fish from the two highest exposure groups revealed consistent performance deficits in foraging efficiency and capture speed. However, no treatment effects on learning were detected. In determining the underlying proximate cause of the foraging deficits, it is believed that the greater pause time exhibited by treatment fish while foraging was the main cause of treatment differences. In the future, behavioral studies will continue to allow toxicity testing of environmentally relevant variables such as those used by behavioral ecologists. Such tests, when combined with tests of field collected specimens, could prove powerful in linking laboratory toxicity to toxicity in wild populations. / Master of Science

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This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

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An adequate supply of oxygen (02) is essential to the survival of all higher organisms. The mammalian carotid body, located at the common carotid artery senses blood levels of 0 2, carbon dioxide (C02) and acidity. Glomus cells, or type I cells in the carotid body are the main 0 2-sensors which regulate blood p02 via reflex control of ventilation. The carotid body secretes multiple neurotransmitters including dopamine (DA), which is potentiated during low p02 levels and is thought to modulate sensory signaling by apposing afferent nerve fibers. Catecholamine (CA) release is also critical for the animal's ability to survive hypoxic stress associated with the birthing process and the transition to extrauterine life. However, the source for this CA release (primarily epinephrine; EPI) is from adrenal chromaffin cells. The primary 02-sensor in both adrenal chromaffin cells and carotid body type I cells is unknown. One potential candidate is the cytochrome b55s/NADPH oxidase complex that generates the respiratory burst in phagocytes. To test this hypothesis, cultured adrenal medulla chromaffin cells and intact carotid bodies from wild type (WT) and oxidase deficient (OD) mice (knockout gp91 phox, the glycoprotein subunits in the NADPH oxidase complex) were investigated. High performance liquid chromatography and immunocytochemistry were used to quantify amine release in these two chemoreceptors following exposure to hypoxia. Both WT and OD chromaffin cells and carotid bodies responded to the hypoxic challenge with increased monoamine secretion. Norepinephrine and epinephrine were the principal amines released from chromaffin cells, compared to dopamine and serotonin from carotid bodies. These findings suggest that NADPH oxidase is not the primary 02- sensor in either chemosensory system. Quantification of monoamine secretion in intact carotid body from mouse and rat was also compared under basal conditions and after exposure to hypoxia and acid/hypercapnia (pH 7.10). Significantly larger amounts of basal serotonin was secreted from mouse carotid body as compared to the rat. Interestingly, serotonin release was potentiated by hypoxia in mouse carotid body, but this was not observed in the rat. Additionally, ratio of basal level serotonin-to-dopamine secretion was significantly higher in mouse than rat CB. Surprisingly, acid/hypercapnic (pH 7.1 0) had no detectable effect on amine secretion from either mouse or rat carotid body. / Thesis / Master of Science (MSc)

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