There is no abstract available for this dissertation. / School of Physical Education

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available via the Internet as viewed 10 July 2008.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 169-208) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 169-208) Also available online.

Les canaux calciques dépendants du voltage sont impliqués dans de nombreux processus cellulaires tels que la contraction musculaire, la libération des neurotransmetteurs et la régulation de l’expression génique. Ces canaux sont constitués d’une sous-unité canalaire α1, qui permet l’entrée des ions calciques dans le milieu intracellulaire, généralement associée à différentes sous-unités auxiliaires (α2δ, γ et β) qui régulent les fonctions du canal. La sous-unité auxiliaire β (Cavβ) joue un rôle capital dans l’adressage membranaire du canal et dans la régulation de ses propriétés biophysiques. Des études récentes décrivent cette sous-unité comme une protéine multifonctionnelle capable d’accomplir des fonctions indépendantes du canal. Quatre différentes isoformes de Cavβ sont codées par 4 gènes différents et caractérisées par des similarités structurales mais une distribution tissulaire différente. L’isoforme Cavβ4 est principalement exprimée dans le cerveau et le cervelet jouant ainsi un rôle important dans la régulation des courants calciques neuronaux. L’importance des fonctions neuronales de Cavβ4 a été soulignée par le fait que la mutation R482X de Cavβ4 a été associée à une forme d’épilepsie humaine.Mon travail de thèse a porté sur l’étude du rôle de Cavβ4 dans le contrôle de la division cellulaire et son implication dans la voie de signalisation Wnt. J’ai également étudié l’influence de la mutation R482X sur cette nouvelle fonction de Cavβ4.Dans ce but, des cellules CHO exprimant de manière stable Cavβ4 ou son mutant épileptique (Cavβ1-481) ont été générées et la localisation subcellulaire des deux protéines ainsi que leur implication dans la prolifération et la progression du cycle cellulaire ont été étudiées. Dans ces cellules, Cavβ4 subit une translocation nucléaire et se retrouve préférentiellement dans les nucléoles. Néanmoins, la délétion de 38 acides aminés de l’extrémité C-terminale de Cavβ4, correspondante à la mutation R482X, empêche sa translocation nucléolaire. L’expression de Cavβ4 réduit considérablement la capacité proliférative des cellules. Cette réduction semble être dépendante de la localisation nucléaire, voir nucléolaire de Cavβ4, parce que le mutant Cavβ1-481 n’induit aucune inhibition de la prolifération. D’un autre côté, l’expression de chacune de ces protéines entraine une modification du cycle cellulaire et l’altération de l’expression de certains gènes liés au cycle.Étant donné que la voie de signalisation Wnt/β-caténine est connue comme l'une des voies les plus importantes contrôlant la prolifération cellulaire, j’ai étudié l'effet de l’expression de Cavβ4 sur cette voie. J’ai ainsi pu montrer que Cavβ4, mais pas Cavβ1-481, réduit considérablement la transcription des gènes cibles de la β-caténine et ralenti la prolifération cellulaire. Cette inhibition est due à une interaction directe entre Cavβ4 et TCF4 qui empêche l’interaction de TCF4 avec la β-caténine et prévient la transcription des gènes cibles.L’ensemble de ces résultats suggèrent que Cavβ4 peut jouer un rôle dans le contrôle de la prolifération au cours du développement, en particulier dans les cellules neuronales. / The voltage gated calcium channels are involved in many cellular processes such as muscle contraction, neurotransmitter release and regulation of gene expression. These channels consist of the pore-forming subunit α1 usually associated with different regulatory subunits: α2δ, β and γ. The auxiliary subunit β (Cavβ) plays a key role in regulating membrane trafficking of the channel and its biophysical properties. Recent studies describe this subunit as a multifunctional protein that can also perform calcium channel-independent functions such as gene regulation. Four different isoforms of Cavβ are encoded by 4 different genes and are characterized by structural similarities but different tissue distribution. Cavβ4 isoform is mainly expressed in the brain and cerebellum, thus, playing an important role in the regulation of neuronal calcium currents. The importance of Cavβ4 neuronal functions has been highlighted by its R482X mutation that was associated to a form of human epilepsy.The aim of my thesis was to study the role of Cavβ4 in the control of cell division and its involvement in the Wnt signaling pathway. I also studied the influence of the R482X mutation on this new function of Cavβ4.To this end, CHO cells stably expressing Cavβ4, or its epileptic mutant (Cavβ1-481), were generated and the subcellular localization of the two proteins and their implication in the proliferation and cell cycle progression were studied. In these cells, Cavβ4 undergoes nuclear translocation and is found preferentially in the nucleoli. However, the deletion of 38 amino acids in the C-terminus domain of Cavβ4, corresponding to the R482X mutation, prevents its nucleolar translocation. In addition, the expression of Cavβ4 significantly reduces the proliferative rate of the cells. This reduction seems to be linked to Cavβ4 nuclear localization because the epileptic mutant is unable to slow down cell proliferation. On the other hand, the expression of each of these proteins is able to deregulate cell cycle progression and to alter the expression of many genes linked to the cycle.Since the Wnt/β-catenin pathway is known as one of the most important pathways controlling cell proliferation, I studied the effect of Cavβ4 expression on this signaling pathway. Indeed, Cavβ4, but not Cavβ1-481, substantially reduces the transcription of β-catenin-dependant genes and therefore slows down cell proliferation. This inhibition is due to a direct interaction between Cavβ4 and TCF4 that prevents the interaction of TCF4 with β-catenin, and thereafter negatively regulates the transcription of targeted genes.These findings suggest that Cavβ4 can play a role in the control of proliferation during development, particularly in neuronal cells.

Voltage-gated Cav2.1 (P/Q-type) and Cav2.2 (N-type) channels are two closely related calcium channels that play indispensable roles in signal transduction pathways by regulating neurotransmitter release. Despite having highly conserved amino acid sequences, they are differentially modulated by calmodulin, which mediate two important feedback mechanisms known as Ca2+-dependent inactivation (CDI) and Ca2+-dependent facilitation (CDF). These dual regulatory mechanisms contribute to synaptic plasticity, but only CDI is observed in Cav2.2 channel, while both CDI and CDF are present in Cav2.1 channel. Previously, it was hypothesized that the lack of CDF in Cav2.2 channel is due to the pre-IQ-IQ domain of the channel’s lower binding affinity for calmodulin compared to that of Cav2.1 channel. Now that the EF-hand domain of calcium channels is identified as one of the two minimally required molecular determinants that are responsible for supporting CDF in Cav2.1 channel and preventing CDF in Cav2.2 channel, it was necessary to determine the role of EF-hand domain in calmodulin binding of Cav2.1 and Cav2.2 channels. Using pull-down binding assays, this study finds that the EF-hand domain enhances calmodulin binding to the proximal C-terminal domain of Cav2.2 channel, which suggests that the lack of CDF in Cav2.2 does not result from the channel’s weak interaction with CaM, but from the EF-pre-IQ-IQ domain of the channel’s inability to allow calmodulin from fully exerting its effects.

A clear definition of the mechanisms involved in synaptic transmission is of paramount importance for the understanding of the processes governing synaptic efficacy. Despite decades of intense study, these mechanisms remain poorly understood. The work contained in this thesis examines several such mechanisms using the hypothalamic-neurohypophysial system (HNS), a classical preparation for the study of Ca2+-dependent neuropeptide release. The first portion of this thesis is comprised of my efforts to define the cellular machinery essential for the exocytosis of secretory granules isolated from peptidergic neurohypophysial terminals of the HNS. Here, using the planar lipid bilayer model system, I have been able to show that syntaxin alone in the target membrane is sufficient to elicit fusion of modified neurohypophysial secretory granules. Surprisingly, SNAP-25 does not appear to be necessary for this process. This suggests that syntaxin may be able to substitute for SNAP-25 to form functional non-cognate fusion complexes. Additionally, the coupling of amperometric detection with the planar lipid bilayer system has allowed me to confirm these results using native, unmodified secretory granules, and also provides some insight into the kinetics of release in this reconstituted system. This model system should provide a convenient means for the study of additional regulatory factors believed to be involved in secretory vesicle exocytosis. The second and third sections of this thesis involve my examination of the role of presynaptic Ca2+ stores in neuropeptide secretion from isolated peptidergic neurohypophysial terminals (NHT). I initially examined the source of recently discovered ryanodine-sensitive Ca2+ stores in this system. Using Immuno-electron microscopy I have found that ryanodine receptor (RyR) labeling appears to co-localize with large dense core granules. Additionally, I have shown that a large conductance cation channel, with similarities to the RyR, found in the membrane of these granules has the same characteristic response to pharmacological agents specific for the RyR. Further, application of RyR agonists modulates basal neuropeptide release from NHT. These results suggest that the large dense core granules of NHT serve as the source of a functional ryanodine-sensitive Ca2+store. Recent work has revealed that spark-like Ca2+ transients, termed syntillas, can be observed in NHT. These syntillas arise from ryanodine-sensitive intracellular stores. In other neuronal preparations, similar Ca2+ transients have been suggested to affect spontaneous transmitter release. However, such a role for syntillas had yet to be examined. To assess if syntillas could directly trigger spontaneous release from NHT, I used simultaneous Ca2+imaging along with amperometric detection of release. Amperometry was adapted to this system via a novel method of false-transmitter loading. Using this approach I have found no apparent correlation between these two events, indicating that syntillas are unable to directly elicit spontaneous transmitter release. As this finding did not rule out an indirect modulatory role of syntillas on release, I additionally present some preliminary studies examining the ability of ryanodine-sensitive Ca2+ release to modulate vesicular priming. Using immunocytochemistry, I have shown that RyR agonist treatment shifts the distribution of neuropeptides toward the plasma membrane in oxytocinergic NHT, but not in vasopressinergic NHT. RyR antagonists have the opposite affect, again only in oxytocinergic NHT. Further, I have found that application of RyR agonists result in a facilitation of elicited release in NHT using membrane capacitance recording. This facilitation appears to be due primarily to an increase in recruitment of vesicles to the readily-releasable pool. These findings suggest that ryanodine-sensitive Ca2+stores may be involved in vesicular priming in NHTs. Taken together, the work presented in this thesis provides some new and interesting insights into the underlying mechanisms and modulation of transmitter release in both the HNS and other CNS terminals.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The monoterpene citral is the major constituent of the essential oil of Cymbopogon citratus, medicinal plant popularly known as capim-limão or capim-santo , widely used to treat hypertension. This study evaluated the cardiovascular effects induced by the citral in normotensive rats by using in vivo and in vitro approaches. In non-anaesthetized rats, citral (1, 5, 10, and 20 mg/kg, i.v.) induced transient hypotension and bradycardia. Both effects were significantly attenuated by the pre-treatment with atropine (2 mg/kg, i.v.), hexamethonium (20 mg/kg, i.v.), sodium thiopental (45 mg/kg; i.p.) or indomethacin (5 mg/kg, i.v.) after dose of 5 mg/kg of the citral. After pre-treatment with L-NAME (20 mg/kg, i.v.), hypotension was significantly attenuated, while bradycardia was not altered. Furthermore, electrocardiogram records demonstrated that citral (10 and 20 mg/kg) was also able to induce sinoatrial block, which was reverted by atropine (2 mg/kg). In rings of rat mesenteric artery pre-contracted with phenylephrine (10 μM), citral (10-5 - 10-2 M) was able to induce relaxations (pD2 = 2.52 ± 0.10; Emax = 103.4 ± 10.2%) that was not affected after removal of the endothelium (pD2 = 2.34 ± 0.15; Emax = 107.2 ± 4.3%) or in rings without endothelium pre-contacted with KCl 80 mM (pD2 = 2.04 ± 0.12; Emax = 101.3 ± 7.1%) or in rings without endothelium after tetraethylammonium (pD2 = 3.25 ± 0.05; Emax = 109.3 ± 9.8%). At concentrations of 3 x 10-4 and 10-3 M, citral was able significantly to inhibit the contractions induced by CaCl2 (from 10- 5 to 10-2 M) or sodium orthovanadate (from 3 x 10-4 to 3 x 10-2 M) up to 88.6 ± 3.1% and 93.3 ± 3.8%, respectively. These results demonstrate that citral induces hypotension, which appears to be caused by activation of muscarinic receptors, NO release and, in part, by PGI2 release, associated to bradycardia, which seems to be due to an activation of muscarinic and nicotinic receptors, involving compounds of central nervous system, and sinoatrial block. Furthermore, citral induces vasorelaxation of mesenteric artery possibly caused by the inhibition of the Ca2+ influx through voltage-operated Ca2+ channels associated to a decrease of calcium sensitivity. / O monoterpeno citral é o composto majoritário do óleo essencial de Cymbopogon citratus, planta medicinal conhecida popularmente como capim-santo ou capim-limão , e é utilizada na medicina popular brasileira para o tratamento da hipertensão. Este estudo buscou investigar os efeitos cardiovasculares do citral em ratos normotensos através de experimentos in vivo e in vitro. Em animais não-anestesiados, a administração i.v. do citral (1, 5, 10 e 20 mg/kg) induziu uma resposta transiente caracterizada por hipotensão associada à bradicardia. Estes efeitos foram significativamente atenuados em animais pré-tratados com atropina (2 mg/kg, i.v.), hexametônio (20 mg/kg, i.v.), tiopental (45 mg/kg; i.p.) ou indometacina (5 mg/kg, i.v.) após dose de 5 mg/kg do citral. Em animais pré-tratados com L-NAME (20 mg/kg, i.v.), o efeito hipotensor foi significativamente atenuado, enquanto que a bradicardia não foi alterada. Além disso, registros de ECG mostraram que o citral (10 e 20 mg/kg) foi capaz de induzir bloqueio sinoatrial e que este efeito foi inibido totalmente com a administração de atropina (2 mg/kg; i.v.). Em anéis de artéria mesentérica de rato précontraídas com FEN (10 μM), o citral (10-5 - 10-2 M) induziu relaxamento (pD2 = 2,52 ± 0,10; Emáx = 103,4 ± 10,2%) que não foi alterado após a remoção do endotélio (pD2 = 2,34 ± 0,15; Emáx = 107,2 ± 4,3%), nem em preparações, sem endotélio, pré-contraídas com KCl 80 mM (pD2 = 2,04 ± 0,12; Emáx = 101,3 ± 7.1%) e nem em anéis, sem endotélio, após incubação com 100 μM de TEA (pD2 = 3,25 ± 0,05; Emáx= 109,3 ± 9,8%). Nas concentrações de 3 x 10-4 e 10-3 M, o citral foi capaz de inibir significativamente as contrações induzidas por CaCl2 (10-5 - 10-2 M) e por ortovanadato de sódio (3 x 10-4 - 3 x 10-2 M) em 88,6 ± 3,1% e 93,3 ± 3,8%, respectivamente. Estes resultados demonstram que o citral induz hipotensão, que parece ser causada por ativação de receptores muscarínicos, liberação de NO e em parte, por liberação de PGI2, associada à bradicardia, que parece ser causada pela ativação de receptores muscarínicos cardíacos, nicotínicos ganglionares, envolvendo componentes do sistema nervoso central, e bloqueio sinoatrial. Além disso, o citral induz vasorelaxamento que parece ser causado por bloqueio do influxo de Ca2+ através dos canais de Ca2+ operados por voltagem associado à diminuição da sensibilidade ao Ca2+.