Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 101-113).

Chez une bactérie, la régulation de l’expression génétique est essentielle afin de maintenir l’équilibre, s’assurer du bon fonctionnement des processus cellulaires et mieux s’adapter aux changements environnementaux. Elle peut s’effectuer à plusieurs niveaux (la transcription, la traduction et la synthèse ou la dégradation des protéines), et par le bais de différents mécanismes, dont les protéines, font le plus grand part de cette régulation. Cependant, au début des années 2000, une découverte fascinante a mis en évidence un nouveau mécanisme de régulation dont l’ARN est l’acteur principal. Ce sont les riborégulateurs (riboswitches). Ces derniers sont localisés dans la partie non traduite de certains ARNmessagers (ARNm) et capable de lier un ligand spécifique sans l’intervention des protéines, afin de réguler l’expression génique du gène d’intérêt. Aujourd’hui, plusieurs familles de riborégulateurs sont caractérisées, entre autres les riborégulateurs c-di-GMP. Ces derniers sont présents chez plusieurs espèces bactériennes notamment les bactéries pathogènes telles que Clostridium difficile, une bactérie nosocomiale opportuniste qui a causé des problèmes majeurs durant les dernières années, vu sa multirésistance aux antibiotiques. Le séquençage de son génome a révélé la présence de 66 riborégulateurs dont 16 sont des riborégulateurs c-di-GMP. Il a été proposé que parmi ces derniers, certains régulent l’expression des gènes impliqués dans deux phénotypes essentiels chez C. difficile : la motilité et la formation du biofilm. La présente étude porte sur la caractérisation structurale et fonctionnelle de deux riborégulateurs c-di-GMP chez le C. difficile, le Cdi1-1 et Cdi1-12, qui se trouvent en amont du gène CD1990 et le gène CD2830 (ZmpI) respectivement. Au début, nous avons prédit les deux structures liées (en présence du ligand) et non liées (en absence du ligand). Nous avons ainsi démontré qu’un des deux riborégulateurs (Cdi1-12) est fonctionnel et capable de lier le c-di-GMP in vitro. Ensuite, nous avons caractérisé les changements structuraux potentiels lors de l’interaction riborégulateur Cdi1-12/ligand. Nous avons également caractérisé le mécanisme de régulation en cis du riborégulateur Cdi1-12 in vitro et nous avons constaté que c’est un riborégulateur transcriptionnel Rho-indépendant. À la fin de notre étude, nous avons confirmé le mode de régulation de ce riborégulateur in vivo dans la bactérie modèle Bacillus subtilis.

Blutplättchen spielen unter physiologischen Bedingungen eine wichtige Rolle bei der Erhaltung der Hämostase. So verhindern sie ein andauerndes Bluten von Wunden, indem sie in Blutgefässen zwischen normalen Zellen des Endothels und beschädigten Bereichen unterscheiden und sich dort gezielt anheften können. Das Zusammenspiel der Plättchenagonisten und den dazugehörigen Rezeptoren wird durch intrazelluläre Signalmoleküle kontrolliert, die die Aktivierung der Blutplättchen regulieren. Äusserst wichtige intrazellulare Signalmoleküle stellen dabei die zyklischen Nukleotide cGMP und cAMP dar, die bei der Hemmung der Plättchen beteiligt sind. Die Bildung von cGMP und cAMP in den Blutplättchen wird durch die aus dem Endothel freigesetzten Moleküle NO und Prostacyclin (PGI2) stimuliert, die ihrerseits Blutplättchen hemmen, indem sie Proteinkinase G (PKG) und Proteinkinase A (PKA) aktivieren. Neuerdings wird vorgeschlagen, dass es sich bei ROS („reactive oxygen species“) um einen neuen Modulator bei der Signaltransduktion zwischen verschiedenen Zelltypen handelt. Die hier zusammengefasste Arbeit beschreibt die Rolle der ROS-Produktion bei der Aktivierung von Blutplättchen, die Beziehung zwischen dem NO/cGMP/PKG I Signalweg und der ROS bzw. MAP-Kinase Signaltransduktion, und die Rolle von zyklischen Nukleotiden bei der Entwicklung von Megakaryozyten und Blutplättchen. Werden Blutplättchen durch unterschiedliche Einflüsse aktiviert, so produzieren sie über die Aktivierung von NAD(P)H-Oxidase nur intrazelluläres aber nicht extrazelluläres ROS. Dabei beinflusst das in den Blutplättchen produzierte ROS signifikant die Aktivierung von αIIbβ3 Integrin, nicht jedoch die Sekretion von alpha- bzw. dichten Granula oder die Gestalt der Blutplättchen. Die Thrombin-induzierte Integrin αIIbβ3-Aktivierung ist nach Behandlung der Blutplättchen mit Hemmstoffen der NAD(P)H-Oxidase oder Superoxid-Fängern signifikant reduziert. Diese Inhibitoren reduzieren auch die Aggregation der Blutplättchen bzw. die Thrombusbildung auf Kollagen, wobei diese Effekte unabhängig vom NO/cGMP Signalweg vermittelt werden. Sowohl ADP, das von dichten Granula der Blutplättchen sezerniert wird und zur Aktivierung von P2Y12-Rezeptoren führt, als auch die Freigabe von Thromboxan A2 stellen wichtige, vorgeschaltete Vermittler bei der p38 MAP Kinase-Aktivierung durch Thrombin dar. Jedoch spielt die p38 MAP-Kinase-Aktivierung keine signifikante Rolle bei der Thrombin-induzierten Kalzium-Mobilisierung, P-Selektin Exprimierung, αIIbβ3 Integrin Aktivierung oder Aggregation der Blutplättchen. Abschliessend kann festgestellt werden, dass sich die Aktivierung der PKG insgesamt klar hemmend auf die p38 and ERK MAP-Kinasen in menschlichen Blutplättchen auswirkt. Desweiteren zeigt diese Studie, dass zyklische Nukleotide nicht nur die Blutplättchen hemmen, sondern auch einen Einfluss auf die Entwicklung der Megakaryozyten und Blutplättchen haben, aber auf unterschiedliche Weise. cAMP ist an der Differenzierung von embryonalen hämatopoietischen Zellen zu Megakaryozyten beteiligt, wobei cGMP keine Rolle bei diesem Prozess spielt. Während PKA in embryonalen Zellen schon vertreten ist, steigt beim Reifungsprozess der Megakaryozyten die Expression von Proteinen, die bei der cGMP Signalverbreitung („soluble guanylyl cyclase“, sGC; PKG) mitwirken, stetig an. In der letzten Phase der Reifung von Megakaryozyten, die durch die Freisetzung der Blutplättchen charakterisiert ist, zeigen cGMP und cAMP leicht divergierende Effekte: cGMP verstärkt die Bildung von Blutplättchen, während cAMP dieselbe reduziert. Dies deutet auf einen fein abgestimmten Prozess hin, abhängig von einem Stimulus, der von den benachbarten Zellen des Sinusoid-Endothels stammen könnte. Die Ergebnisse dieser Dissertation tragen zu einen besseren Verständnis der Regulation von Blutplättchen sowie der möglichen molekularen Mechanismen bei, die eine Rolle bei der Reifung von Megakaryozyten im vaskularen Mikroumfeld des Knochenmarks innehaben. / In physiological conditions platelets have a major role in maintaining haemostasis. Platelets prevent bleeding from wounds by distinguishing normal endothelial cells in vasculature from areas with lesions to which they adhere. Interaction of platelet agonists and their receptors is controlled by intracellular signaling molecules that regulate the activation state of platelets. Very important intracellular signaling molecules are cyclic nucleotides (cGMP and cAMP), both involved in inhibition of platelet activation. Formation of cGMP and cAMP in platelets is stimulated by endothelial-derived NO and prostacyclin (PGI2), which then mediate inhibition of platelets by activating protein kinase G (PKG) and protein kinase A (PKA). Recently, it has been suggested that reactive oxygen species (ROS) represent new modulators of cell signaling within different cell types. The work summarized here describes the involvement of platelet ROS production in platelet activation, the relation of NO/cGMP/PKG I pathway to ROS and to mitogen-activated protein kinases (MAP kinase) signaling, and the involvement of cyclic nucleotides in megakaryocyte and platelet development. Platelets activated with different agonists produce intracellular but not extracellular ROS by activation of NAD(P)H oxidase. In addition, ROS produced in platelets significantly affects αIIbβ3 integrin activation but not alpha/dense granule secretion and platelet shape change. Thrombin induced integrin αIIbβ3 activation is significantly decreased after pretreatment of platelets with NAD(P)H oxidase inhibitors and superoxide scavengers. These inhibitors also reduce platelet aggregation and thrombus formation on collagen under high shear and achieve their effects independently of the NO/cGMP pathway. ADP secreted from platelet dense granules with subsequent activation of P2Y12 receptors as well as thromboxane A2 release are found to be important upstream mediators of p38 MAP kinase activation by thrombin. However, p38 MAP kinase activation does not significantly contribute to calcium mobilization, P-selectin expression, αIIbβ3 integrin activation and aggregation of human platelets in response to thrombin. Finally, PKG activation does not stimulate, but rather inhibit, p38 and ERK MAP kinases in human platelets. Further study revealed that cyclic nucleotides not only inhibit platelet activation, but are also involved, albeit differentially, in megakaryocyte and platelet development. cAMP is engaged in haematopoietic stem cell differentiation to megakaryocytes, and cGMP has no impact on this process. While PKA is already present in stem cells, expression of proteins involved in cGMP signaling (soluble guanylyl cyclase, sGC; PKG) increases with maturation of megakaryocytes. In the final step of megakaryocyte maturation that includes release of platelets, cGMP and cAMP have mild but opposing effects: cGMP increases platelet production while cAMP decreases it indicating a finely regulated process that could depend on stimulus coming from adjacent endothelial cells of sinusoids in bone marrow. The results of this thesis contribute to a better understanding of platelet regulation and of the possible molecular mechanisms involved in megakaryocyte maturation in bone marrow vascular microenvironment.

In der vorliegenden Arbeit ging es darum, die inhibierende Wirkung von extrazellulär zugefügten GMP-Derivaten auf die Thrombozytenaktivierung nachzuweisen. Anhand verschiedener thrombozytärer Aktivierungsmarker wie ERK, der Expression von P-Selektin und intrazellulärem Kalziumeinstrom zeigte sich eine signifikante Inhibition der Thrombozytenaktivierung durch zyklische GMP-Analoga. Neu ist, dass auch nicht-zyklische GMP-Derivate eindeutig einen hemmenden Effekt aufweisen. Guanosin-Derivate alleine führen dagegen weder zu einer Hemmung noch zu einer vermehrten Stimulation der Plättchenaktivierung. Die Wirkung der GMP-Analoga scheint von der negativen Phosphatgruppe abhängig zu sein. Der frühe Zeitpunkt der Inhibition und die Tatsache, dass auch Hemmer der PKG einen inhibierenden Effekt auf die Thrombozytenaktierung aufweisen, führen zu der Hypothese, dass es sich um PGK-unabhängige Effekte handelt. Der Thrombinrezeptor als Wirkort der GMP-Analoga konnte in Biacore-Messungen ausgeschlossen werden. Es gilt nun, speziell den Thromboxanrezeptor auf mögliche Interaktionen mit GMP-Derivaten hin zu untersuchen. / Platelets play a key role in hemostasis through their ability to rapidly adhere to activated or injured endothelium, subendothelial matrix proteins and other activated platelets. A strong equilibrium between activating and inhibiting processes is essential for normal platelet and vascular function, impairment of this equilibrium being associated with either thrombophilic or bleeding disorders. Cyclic guanosine monophosphate is a crucial and synergistic intracellular messenger that mediates the effects of platelet inhibitors such as nitric oxide (NO) and prostacyclin (PG-I2). This work investigated the effects of extracellular-cyclic-nucleotides on platelet-activation. Different platelet-activation markers like ERK, P-Selectin and intracellular Calcium were tested to be effected by extracellular added cyclic nucleotides. We could show, that cyclic nucleotide derivatives have the power to inhibit platelet activation (stimulated by Thrombin or TRAP6). New is a similar effect with non-cyclic nucleotides like 8-pCPT-5´-GMP and 8-Br-5´-GMP. Guanosine derivatives alone have no effect on platelet activation, so we conclude that the effect might depend on the negative phosphate group of GMP. Furthermore we think that it might be a PGK-independet mechanism because of an early effect even after 15-30 seconds. We could exclude the thrombin receptor as direct interactor by Biacore measurement and suppose that there is some interaction between the guanosine monophosphates and the thromboxane-receptor on the platelet surface.

Myokardhypertrophie stellt einen Adaptationsmechanismus des Herzens an erhöhte Belastungen wie chronische arterielle Hypertonie, Myokardinfarkt, Koronare Herzkrankheit, Myokarditis und Kardiomyopathien dar. Sowohl biomechanische als auch neurohumorale Stimuli führen auf einer Reihe von Signalwegen zur Myokardhypertrophie, die als Größenzunahme postmitotischer Kardiomyozyten definiert ist. Zyklisches 3´5´-Guanosinmonophosphat ist ein ubiquitärer intrazellulärer Signalträger, der im kardiovaskulären System auf mindestens drei bekannten Wegen aus Guanosintriphosphat durch Abspaltung von Pyrophosphat synthetisiert wird. Die Synthese erfolgt unter anderem durch Aktivierung der partikulären, membranständigen Guanylylzyklase A durch Atriales Natriuretisches Peptid oder „B-Typ“ Natriuretisches Peptid und der Guanylyzyklase B, durch „C-Typ“ Natriuretisches Peptid. Des Weiteren wird über neuronale, endotheliale, oder induzierbare Stickstoffmonoxid-Synthase Stickstoffmonoxid synthetisiert, welches die lösliche, vorwiegend zytosolisch lokalisierte Guanylyzyklase aktiviert, die wiederum aus Guanosintriphosphat zyklisches 3´5´-Guanosinmonophosphat synthetisiert. Das intrazelluläre zyklische 3´5´-Guanosinmonophosphat lässt sich durch Behandlung mit Sildenafilcitrat, einem unter dem Markennamen Viagra® zur Therapie der Erektilen Dysfunktion und Revatio® zur Therapie der pulmonalen arteriellen Hypertonie zugelassenem Pharmakon, erhöhen. Sildenafil hemmt die Phosphodiesterase 5A, ein unter anderem in Lunge, Kardiomyozyten, Kleinhirn und glatter Muskulatur vorkommendes Isoenzym. In der vorliegenden Dissertation wurden die kardialen Effekte des Phosphodiesterase 5-Inhibitors Sildenafil an zwei Mausmodellen mit funktionell gut kompensierter Herzhypertrophie getestet. Die mittlere Tagesdosis betrug 150 (Studie 1) bzw. 240 (Studie 2) mg/kg Körpergewicht Sildenafil, in Anlehnung an publizierte Studien mit Nagern (Takimoto et al., 2005b). Diese Dosis wurde oral über vier (Studie 1) bzw. neun Wochen (Studie 2) zugeführt. Die mittlere Plasmakonzentration von Sildenafil lag mit 60 nM weit über dessen IC 50 (3,5 nM) zur Hemmung der PDE 5 in vitro. Studie 1 In der ersten Studie wurde die Nachlast des Herzens mittels operativer transverser Aortenkonstriktion um ca. 25 - 35 mmHg über vier Wochen erhöht. Vergleichbare Anstiege des systolischen Blutdrucks werden in Patienten mit ausgeprägter essenzieller arterieller Hypertonie gemessen. Nekropsie, echokardiographische, histologische und morphometrische Untersuchungen zeigten übereinstimmend die Entwicklung einer konzentrischen Herzhypertrophie ohne interstitielle Fibrose. Echokardiographische Bestimmungen der linksventrikulären Funktion zeigten einen leichten Anstieg der Ejektionsfraktion bei unveränderter fraktioneller Verkürzung der Wand des linken Ventrikels. Proteinchemische Analysen an linksventrikulären Gewebeproben zeigen die Aktivierung von Signalwegen der Herzypertrophie, insbesondere der Mitogen-Aktivierten Protein Kinase ERK 1 / 2. Zusammengenommen belegen diese morphologischen, funktionellen und biochemischen Analysen, dass durch die moderate operative Aortenstenose die Entwicklung einer ausgeprägten, aber funktionell gut kompensierten Linksherzhypertrophie induziert wurde. Die Entwicklung dieser Herzhypertrophie wurde durch Sildenafil nicht verhindert. Im Gegenteil, es zeigte sich unter der Gabe des Pharmakons sogar eine leichte Tendenz zur funktionellen Verschlechterung des linken Ventrikels, mit leicht reduzierter Ejektionsfraktion und gesteigertem Lungenfeuchtgewicht. Auch die kardiale Aktivierung des Hypertrophiesignalweges MAPK / ERK wurde durch Sildenafil nicht beeinflusst. Studie 2 In der zweiten Studie wurden die Effekte von Sildenafil an einem monogenetischen Mausmodell mit globaler Deletion der Guanylylzyklase-A (GC-A -/-), dem Rezeptor für Atriales natriuretisches Peptid, getestet. Wie zuvor in publizierten Studien gezeigt wurde, haben GC-A -/- Mäuse eine chronische arterielle Hypertonie, mit Anstiegen des systolischen Blutdrucks um 20 - 25 mmHg. Nekropsie, Echokardiographie, Histologie und Morphometrie ergaben übereinstimmend, dass diese arterielle Hypertonie von einer ausgeprägten globalen, funktionell gut kompensierten Herzhypertrophie mit leichter interstitieller Fibrose begleitet wurde. Unter der neunwöchigen Behandlung mit Sildenafil kam es zu einem signifikanten aber inkomplettem Rückgang der systemischen arteriellen Hypertonie (um ca 8 - 10 mmHg). Trotz dieser hypotensiven Effekte wurden die Rechts- und Linksherzhypertrophie und die kardiale Fibrose der GC-A -/- Tiere durch Sildenafil nicht beeinflusst. Dagegen zeigte sich auch hier mittels Echokardiographie eine leichte Verschlechterung der linksventrikulären Funktion unter Sildenafil, mit Abnahmen der Ejektionsfraktion und der fraktionellen Verkürzung des linken Ventrikels sowie einer Zunahme des endsystolischen Kammerdurchmessers. Zusammenfassend wurden die in der Literatur beschriebenen protektiven, kardialen antihypertrophen Effekte von Sildenafil in der vorliegenden experimentellen Dissertation nicht bestätigt. / Synthesis of cGMP in cardiomyocytes is stimulated by natriuretic peptides via GC-A signalling as well as nitric oxide (NO) via soluble guanylyl cyclase (sGC). Different studies in vitro / in vivo have demonstrated that cGMP can inhibit myocardial hypertrophic responses. In particular, genetic ablation of the ANP/GC-A or NO/sGC systems in mice led to exacerbated cardiac hypertrophy in response to pressure overload. Conversely, inhibition of cGMP catabolism with the PDE5 inhibitor sildenafil prevented and even reversed pathological cardiac hypertrophy after aortic banding [1]. The present ongoing study was originally designed to elucidate whether the antihypertrophic effects of PDE5 inhibition are linked to the formation of cGMP by either the NP/GC-A or the NO/sGC system. We subjected male C57Bl/6 mice (8 weeks of age, Charles River) to constriction of the transverse aorta (TAC) for 4 weeks. They were concurrently treated with the PDE5 inhibitor sildenafil (100 mg/kg/d) or vehicle mixed in solid food (n=7 per group).Furthermore, mice with genetic ablation of the GC-A receptor were treated with Sildenafil or Placebo. The studies demonstrate that Sildenafil does not exhibit direct antihypertrophic effects, but does exhibit indirect effects on left ventricular myocardium. Furthermore, Sildenafil is supposed to amplify the cGMP that is generated via ANP/GC-A system.

Stroke is one of the leading causes of human death in the United States. The debilitating effects of an ischemic stroke are due to the fact that mammalian neurons are highly susceptible to hypoxia and subsequent oxygen reperfusion. From studies in Drosophila melanogaster, cGMP-dependent Protein Kinase (PKG) enzyme is thought to affect anoxia tolerance by modifying the electrical current through potassium ion channels. In this research, two animal models were employed: Drosophila melanogaster and mammalian neurons exposed to stroke-like conditions. First, in vivo studies using Drosophila were performed to further our knowledge about the differences between the naturally occurring variants of the Drosophila foraging gene, which shows different protein levels of PKG. Mitochondrial density and metabolic activity between two fly genotypes exposed to anoxia and reoxygenation were compared. It was found that flies with less enzyme potentially showed mitochondrial biogenesis and higher metabolic rates upon reoxygenation. Next, in vivo studies where PKG enzyme was activated pharmacologically were performed; it was found that the activation of the cGMP/PKG pathway led to neuroprotection upon anoxia and reoxygenation. Furthermore, this model was translated into the in vitro model using Drosophila cells. Instead of anoxia and reoxygenation, hypoxia mimetics and hydrogen peroxide were used to induce cellular injury. After showing the cGMP/PKG pathway activation-induced cell protection, the potential downstream targets of the molecular signaling as well as underlying biochemical changes were assessed. It was found that mitochondrial potassium ion channels were involved in the protective signaling and the signaling modulated metabolic function. Furthermore, it was found that acidosis protected Drosophila cells from cell death, metabolic disruption, and oxidative stress. Finally, this research was translated to a mammalian in vitro model of neuronal damage upon stroke-like conditions; there, it was demonstrated that the cGMP/PKG pathway activation in rat primary cortical neurons and human cortical neurons was protective from low oxygen and acute oxidative stress. The results of this study lead to a better understanding of molecular mechanisms taking place during low oxygen and oxidative stresses. Consequently, this knowledge may be used to identify potential therapeutic targets and treatments that may prevent detrimental neurological effects of an ischemic stroke in humans. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

by Tung Sin Yi, Cindy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 117-132). / Acknowledgements --- p.I / List of Abbreviations --- p.II / Abstract --- p.IV / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Astrocytes in the Central Nervous System (CNS) l / Chapter 1.1.1 --- Characteristics of astrocytes / Chapter 1.1.2 --- Functions of astrocytes / Chapter 1.1.2.1 --- General functions of astrocytes / Chapter 1.1.2.2 --- Effects of neuroactive peptides on astrocytes / Chapter 1.1.3 --- Gliomas and the rat C6 glioma cells / Chapter 1.2 --- C-Type natriuretic peptide (CNP) in the CNS --- p.9 / Chapter 1.2.1 --- Structure and distribution of natriuretic peptides in the CNS / Chapter 1.2.2 --- Actions of CNP / Chapter 1.2.3 --- Natriuretic peptide receptors and signal transduction in astrocytes / Chapter 1.3 --- Endothelin-3 (ET-3) in the CNS --- p.18 / Chapter 1.3.1 --- Structure and distribution of endothelins (ETs) in the CNS / Chapter 1.3.2 --- Actions of ET-3 / Chapter 1.3.3 --- Endothelin receptors and signal transductionin astrocytes / Chapter 1.4 --- cGMP second messenger system in astrocytes --- p.28 / Chapter 1.4.1 --- Second messenger systems in astrocytes / Chapter 1.4.2 --- cGMP as second messenger in astrocytes / Chapter 1.4.3 --- Post cGMP cascade effects / Chapter 1.5 --- The aims of this project --- p.33 / Chapter Chapter 2 --- Methods / Chapter 2.1 --- In vitro culture of rat C6 glioma cells --- p.36 / Chapter 2.1.1 --- Preparation of reagents / Chapter 2.1.2 --- Culture of C6 glioma cells / Chapter 2.1.3 --- "Cell plating in 6-well, 24-well and 96-well plastic trays" / Chapter 2.2 --- Determination of cGMP --- p.40 / Chapter 2.2.1 --- Measurement of cGMP / Chapter 2.2.2 --- Data analysis / Chapter 2.3 --- Determination of the effect of CNP on cGMP productionin C6 cells --- p.41 / Chapter 2.4 --- Determination of the effect of ET-3 on the action of CNPin C6cells --- p.44 / Chapter 2.4.1 --- Measurement of intracellular cGMP levels affected by ET-3 / Chapter 2.4.2 --- Measurement of intracellular cGMP levels affected by CNP with ET-3 pretreatment / Chapter 2.5 --- Determination of the effects of PKC activator and inhibitor on CNP-treated C6 cells --- p.46 / Chapter 2.5.1 --- Measurement of intracellular cGMP levels affected by PKC activator or inhibitor / Chapter 2.5.2 --- Measurement of intracellular cGMP levels affected by CNP with PKC activator or inhibitor pretreatment / Chapter 2.5.3 --- Measurement of intracellular cGMP levels affected by CNP with PKC inhibitor antagonized PMA or ET-3 pretreatment / Chapter 2.6 --- Determination of the effect of arachidonic acid on the action of CNP in C6 cells --- p.49 / Chapter 2.7 --- Determination of the effects of ET-3 and CNP on calcium uptake in C6 cells --- p.50 / Chapter 2.8 --- Determination of the effects of CNP and ET-3 on cell volume change in C6 cells --- p.51 / Chapter 2.9 --- Determination of the effects of CNP and ET-3 on glucose and amino acids uptake in C6 cells --- p.53 / Chapter 2.9.1 --- Measurement of glucose uptake in CNP - and/or ET- 3-treated C6 cells / Chapter 2.9.2 --- Measurement of amino acids uptake in CNP - and/or ET-3-treated C6 cells / Chapter 2.10 --- "Determination of thymidine, uridine and leucine incorporation in CNP - and/or ET-3- treated C6 cells" --- p.55 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effects of CNP and ET-3 on cGMP production in cultured rat C6 glioma cells --- p.56 / Chapter 3.1.1 --- Effect of CNP on cGMP production in cultured C6 glioma cells --- p.57 / Chapter 3.1.1.1 --- The time course of CNP on cGMP production / Chapter 3.1.1.2 --- Dosage-response of CNP on cGMP production / Chapter 3.1.2 --- Effect of ET-3 on cGMP production in C6 glioma cells --- p.61 / Chapter 3.1.2.1 --- Effect of ET-3 on basal cGMP production / Chapter 3.1.2.2 --- Effect of pre-exposure duration to ET-3 on CNP-induced cGMP formation / Chapter 3.1.2.3 --- Dosage-response of ET-3 on CNP-induced cGMP production / Chapter 3.1.3 --- Effect of PMA on cGMP production in C6 glioma cells --- p.65 / Chapter 3.1.3.1 --- Effect of PMA on basal cGMP production / Chapter 3.1.3.2 --- Effect of pre-exposure duration to PMA on CNP-induced cGMP formation / Chapter 3.1.3.3 --- Dosage-response of PMA on CNP-induced cGMP production / Chapter 3.1.4 --- Effects of PKC inhibitors on cGMP production in C6 glioma cells --- p.73 / Chapter 3.1.4.1 --- Effects of PKC inhibitors on basal cGMP production / Chapter 3.1.4.2 --- Effects of PKC inhibitors on CNP-induced cGMP formation / Chapter 3.1.4.3 --- Antagonism of PKC inhibitors on the action of PMA on CNP-induced cGMP formation / Chapter 3.1.4.4 --- Antagonism of PKC inhibitors on the action of ET-3 on CNP-induced cGMP formation / Chapter 3.1.5 --- Effect of arachidonic acid on CNP-induced cGMP production in C6 glioma cells --- p.82 / Chapter 3.2 --- Effects of CNP and ET-3 on cellular metabolism in cultured rat C6 glioma cells --- p.83 / Chapter 3.2.1 --- Effects of CNP and ET-3 on calcium uptake in C6 glioma cells --- p.86 / Chapter 3.2.2 --- Effects of CNP and ET-3 on cell volume changes in C6 glioma cells --- p.89 / Chapter 3.2.3 --- Effects of CNP and ET-3 on glucose and amino acids uptake in C6 glioma cells --- p.91 / Chapter 3.2.4 --- Effects of CNP and ET-3 on C6 cell proliferation --- p.98 / Chapter 3.2.5 --- Effects of CNP and ET-3 on RNA synthesis --- p.101 / Chapter 3.2.6 --- Effects of CNP and ET-3 on protein synthesis --- p.103 / Chapter Chapter 4 --- Discussion and Conclusion --- p.105 / References --- p.117

Chan Ho Yu, Richard. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 115-122). / Abstracts in English and Chinese. / Declaration --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Table of Contents --- p.vii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Potassium Channels --- p.1 / Chapter 1.1.1 --- Potassium Ions --- p.1 / Chapter 1.1.2 --- Potassium Channels --- p.1 / Chapter 1.1.3 --- Structure of K Channels --- p.2 / Chapter 1.1.4 --- Classification ofK Channels --- p.3 / Chapter 1.1.5 --- Mechanisms Contributed to K Channel Functions and Diversity --- p.5 / Chapter 1.1.5.1 --- RNA Editing --- p.5 / Chapter 1.1.5.2 --- Alternative Splicing --- p.6 / Chapter 1.1.5.3 --- Heteromultimeric Assembly of Principal Subunits --- p.6 / Chapter 1.1.5.4 --- Auxiliary Subunits --- p.7 / Chapter 1.1.5.5 --- Posttranslational Modifications --- p.7 / Chapter 1.2 --- Voltage-gated Potassium (Kv) Channels --- p.9 / Chapter 1.2.1 --- Diversity of Kv Channel Structure --- p.9 / Chapter 1.2.2 --- Early Origin of the Kv Family --- p.10 / Chapter 1.2.3 --- Structural Diversity of Kv Channels in Drosophila --- p.11 / Chapter 1.2.4 --- Structural Diversity of Kv Channels in Mammals --- p.11 / Chapter 1.2.5 --- Phylogenetic Tree of Kv Family --- p.13 / Chapter 1.2.6 --- Tissue Expression of Kv Channels --- p.13 / Chapter 1.2.7 --- "Three Main Functions of Kv Channels as Signaling Proteins: Ion Permeation, Gating and Sensing" --- p.16 / Chapter 1.2.7.1 --- Ion Permeation --- p.16 / Chapter 1.2.7.2 --- Gating --- p.18 / Chapter 1.2.7.2.1 --- Gating at the S6 Bundle Crossing --- p.18 / Chapter 1.2.7.2.2 --- Ball-and-Chain Gating --- p.19 / Chapter 1.2.7.2.3 --- Gating at the Selectivity Filter --- p.19 / Chapter 1.2.7.3 --- Sensing Mechanisms --- p.20 / Chapter 1.2.7.3.l --- Voltage Sensor --- p.20 / Chapter 1.2.7.3.2 --- Gating Sensors for Ligands --- p.21 / Chapter 1.3 --- KCNA10 --- p.22 / Chapter 1.3.1 --- "Rabbit Homologue of KCNA10, Kcnl" --- p.22 / Chapter 1.3.2 --- Genomic Localization of Human KCNA10 --- p.23 / Chapter 1.3.3 --- Human Gene for KCNA10 --- p.23 / Chapter 1.3.4 --- Basic Kinetic and Pharmacological Properties of KCNA10 --- p.25 / Chapter 1.3.5 --- "Regulation of KCNAlO by KCNA4B, a β -subunit" --- p.27 / Chapter 1.4 --- Aim of the Present Study --- p.30 / Chapter Chapter2: --- Materials and Methods --- p.31 / Chapter 2.1 --- Molecular Sub-Cloning ofKCNAlO --- p.31 / Chapter 2.1.1 --- Polymerase Chain Reaction (PCR) ofKCNA10 Fragment from KCNA Clone --- p.10 / Chapter 2.1.2 --- Separation and Purification of PCR Products --- p.32 / Chapter 2.1.2.1 --- Separation --- p.32 / Chapter 2.1.2.2 --- Purification --- p.33 / Chapter 2.1.3 --- Polishing the Purified PCR Products --- p.33 / Chapter 2.1.4 --- Ligation of PCR Products and pPCR-Script Amp SK(+) Cloning Vector --- p.34 / Chapter 2.1.5 --- Transformation --- p.34 / Chapter 2.1.6 --- Preparing Glycerol Stocks Containing the Bacterial Clones --- p.35 / Chapter 2.1.7 --- Plasmid DNA Preparation --- p.35 / Chapter 2.1.8 --- Clones Confirmation --- p.36 / Chapter 2.1.8.1 --- Restriction Enzyme Digestion --- p.36 / Chapter 2.1.8.2 --- Automatic Sequencing --- p.37 / Chapter 2.2 --- In situ Hybridization --- p.39 / Chapter 2.2.1 --- Probe Preparation --- p.39 / Chapter 2.2.1.1 --- Antisense KCNA10 RNA Probe --- p.39 / Chapter 2.2.1.2 --- Sense KCNA10 RNA Probe (Control Probe) --- p.40 / Chapter 2.2.2 --- Testing of DIG-Labeled RNA Probes --- p.43 / Chapter 2.2.3 --- Paraffin Sections Preparation --- p.43 / Chapter 2.2.4 --- In situ Hybridization: Pretreatment --- p.44 / Chapter 2.2.5 --- "Pre-hybridization, Hybridization and Post-hybridization" --- p.45 / Chapter 2.2.5.1 --- Pre-hybridization --- p.45 / Chapter 2.2.5.2 --- Hybridization --- p.45 / Chapter 2.2.5.3 --- Post-hybridization --- p.46 / Chapter 2.2.6 --- Colourimetnc Detection of Human KCNA10 --- p.46 / Chapter 2.3 --- Cell Culture --- p.47 / Chapter 2.3.1 --- Human Kidney Proximal Epithelial Cell Line (OK) --- p.47 / Chapter 2.3.2 --- Mouse Micro-vessel Endothelial Cell Line (H5V) --- p.48 / Chapter 2.3.3 --- Mouse Neuroblastoma Cell Line (NG108-15) --- p.48 / Chapter 2.3.4 --- Human Bladder Epithelial Cell Line (ECV304) --- p.48 / Chapter 2.3.5 --- Human T Cell Leukemia Cell Line (Jurkat) --- p.49 / Chapter 2.4 --- Total RNA Extraction --- p.49 / Chapter 2.5 --- Reverse Transcription from Cell Line --- p.51 / Chapter 2.6 --- Polymerase Chain Reaction (PCR) ofKCNAl 0 Fragment from Frist Strand cDNA --- p.51 / Chapter 2.7 --- Northern Hybridization --- p.52 / Chapter 2.7.1 --- Probe Preparation --- p.52 / Chapter 2.7.2 --- Separating RNA on an Agarose Gel --- p.52 / Chapter 2.7.3 --- RNA Transfer and Fixation --- p.52 / Chapter 2.7.4 --- Hybridization --- p.54 / Chapter 2.7.5 --- Post-hybridization --- p.54 / Chapter 2.7.6 --- Chemiluminescent Detection --- p.55 / Chapter 2.8 --- Intracellular Free Calcium Ion ([Ca2+］i) Measurement by Confocal Imaging System --- p.56 / Chapter 2.8.1 --- Bathing Solutions --- p.56 / Chapter 2.8.2 --- Preparation of Cells for [Ca2+]i Measurement --- p.56 / Chapter 2.8.3 --- Confocal Imaging System --- p.57 / Chapter 2.8.3.1 --- Fluo-3/AM Dye Loading --- p.57 / Chapter 2.8.3.2 --- [Ca2+]i Measurement --- p.57 / Chapter Chapter3: --- Results --- p.59 / Chapter 3.1 --- Phylogenetic Tree Reconstruction ofKCNAl0 --- p.59 / Chapter 3.2 --- Hydropathy Analysis ofKCNAl0 --- p.60 / Chapter 3.3 --- Molecular Sub-Cloning ofKCNAl0 --- p.61 / Chapter 3.3.1 --- Polymerase Chain Reaction (PCR) ofKCNAl0 Fragment from KCNA10 Clone --- p.61 / Chapter 3.3.2 --- Clones Confirmation --- p.63 / Chapter 3.4 --- In situ Hybridization Analysis ofKCNAl0 mRNAExpression --- p.65 / Chapter 3.4.1 --- Expression ofKCNAl0 in Human Kidney (Nephron) --- p.66 / Chapter 3.4.2 --- Expression ofKCNAl0 in Human Cerebral Artery --- p.69 / Chapter 3.4.3 --- Expression ofKCNAl0 in Human Cerebellum --- p.71 / Chapter 3.4.4 --- Expression ofKCNAl0 in Human Hippocampus --- p.73 / Chapter 3.4.5 --- Expression ofKCNAl0 in Human Occipital Cortex --- p.75 / Chapter 3.4.6 --- Expression ofKCNAl0 in Human Esophagus --- p.77 / Chapter 3.4.7 --- Expression ofKCNAl0 in Human Lung --- p.79 / Chapter 3.4.8 --- Expression ofKCNAl0 in Human Thyroid Glands --- p.81 / Chapter 3.4.9 --- Expression ofKCNAl0 in Human Adrenal Glands --- p.83 / Chapter 3.4.10 --- Expression ofKCNAl0 in Human Spleen --- p.86 / Chapter 3.5 --- RT-PCR ofKCNAl0 Fragment from Different Tissues --- p.88 / Chapter 3.6 --- Northern Blot Analysis of KCNA10 in Different Tissues --- p.90 / Chapter 3.7 --- Effects of Blocking KCNA10 on Ca2+ influx in Human Renal Proximal Tubule Epithelial Cells --- p.91 / Chapter Chapter4: --- Discussion --- p.97 / Chapter 4.1 --- Phylogency ofKCNAlO --- p.97 / Chapter 4.2 --- Hydropathy Plot for KCNA10 --- p.97 / Chapter 4.3 --- Expression ofKCNAl0 --- p.98 / Chapter 4.3.1 --- In situ Hybridization --- p.98 / Chapter 4.3.2 --- RT-PCR & Northern Blot Analysis --- p.99 / Chapter 4.4 --- Functional Implication of KCNA10 Expression in Different Human Tissues --- p.100 / Chapter 4.4.1 --- Unique Functional Properties ofKCNAlO --- p.100 / Chapter 4.4.2 --- Role ofKCNAlO in Renal Proximal Tubule --- p.101 / Chapter 4.4.2.1 --- Functions ofK+ Channels in Kidney --- p.101 / Chapter 4.4.2.2 --- The Function ofKCNAlO --- p.104 / Chapter 4.4.3 --- Role ofKCNAl0 in Blood Vessels --- p.106 / Chapter 4.4.3.1 --- Endothelial Cells --- p.106 / Chapter 4.4.3.2 --- Smooth Muscle Cells --- p.108 / Chapter 4.4.4 --- Role ofKCNA10 in CNS --- p.109 / Chapter 4.4.5 --- Role ofKCNAl0 in Secretory Cells --- p.111 / Chapter 4.4.6 --- Role ofKCNAl0 in Lung --- p.112 / Chapter 4.5 --- Conclusion --- p.114 / Chapter Chapter5： --- Reference --- p.115

Lau Kin Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 97-103). / Abstracts in English and Chinese. / Contents --- p.1 / Chapter Chapter 1 --- Introduction --- p.5 / Chapter 1.1 --- Calcium Signaling in Endothelial Cells --- p.5 / Chapter 1.1.1 --- Calcium and its functions --- p.5 / Chapter 1.1.2 --- "Second Messengers: Inositol-1,4,5-Triphosphate and Diacylglycerol" --- p.6 / Chapter 1.1.3 --- Propagation of Ca2+ Signals --- p.8 / Chapter 1.1.4 --- Ca2+-ATPases --- p.9 / Chapter 1.1.5 --- Regulation of Sarcoplasmic Reticulum --- p.10 / Chapter 1.1.6 --- Agonist-induced Ca2+ Entry --- p.11 / Chapter 1.2 --- Mechanism of Store-Operated Ca2+ Entry --- p.14 / Chapter 1.2.1 --- Signaling Mechanisms of SOC --- p.14 / Chapter 1.2.1.1 --- A Diffusible Messenger --- p.14 / Chapter 1.2.1.2 --- Conformational Coupling --- p.15 / Chapter 1.2.1.3 --- Vesicle Secretion --- p.16 / Chapter 1.3 --- Regulation of Ca2+ Entry by cGMP --- p.20 / Chapter 1.4 --- Molecular Structres of Store-operated Channels --- p.22 / Chapter 1.4.1 --- Drosophila Transient Receptor Potential (trp) Gene --- p.22 / Chapter 1.4.2 --- Trpl Gene --- p.23 / Chapter Chapter 2 --- Methods and Materials --- p.27 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Phosphate-buffered saline --- p.27 / Chapter 2.1.2 --- Culture Media and Materials --- p.27 / Chapter 2.2 --- Preparations and Culture of Cells --- p.28 / Chapter 2.2.1 --- Culture of Rat Aortic Endothelial Cells --- p.28 / Chapter 2.2.2 --- Culture of Human Bladder Epithelial Cell Line --- p.29 / Chapter 2.2.3 --- Culture of Human Embryonic Kidney Epithelial Cell Line --- p.29 / Chapter 2.3 --- Cell. Subculture and Marvest --- p.29 / Chapter 2.4 --- Intracellular Free Calcium Ions ([Ca2+]i) measurment --- p.30 / Chapter 2.4.1 --- Chemicals --- p.30 / Chapter 2.4.2 --- Bathing solutions --- p.31 / Chapter 2.4.3 --- Preparations of Cells for [Ca2+]i Measurement --- p.31 / Chapter 2.4.3.1 --- Plating cells on Glass Cover Slips for [Ca2+]i Measurement with PTI RatioMaster Fluorescence System --- p.31 / Chapter 2.4.3.2 --- Plating cells on Glass Cover Slips for [Ca2+]i Measurement with Confocal Imaging System and Confocal Laser Scanning Microscopy --- p.32 / Chapter 2.4.4 --- PTI RatioMaster Fluorescence System --- p.35 / Chapter 2.4.4.1 --- Experimental Setup --- p.35 / Chapter 2.4.4.2 --- Fura-2/AM Dye loading --- p.35 / Chapter 2.4.4.3 --- Background Fluorescence and [Ca ]i Measurement --- p.37 / Chapter 2.4.5 --- Confocal Imaging System --- p.37 / Chapter 2.4.5.1 --- Experimental Setup --- p.37 / Chapter 2.4.5.2 --- Fluo-3/AM Dye Loading --- p.39 / Chapter 2.4.5.3 --- [Ca2+]i Measurement --- p.39 / Chapter 2.4.6 --- Confocal Laser Scanning Microscopy --- p.40 / Chapter 2.4.6.1 --- Principles --- p.40 / Chapter 2.5 --- Cloning and expression of Trpl in HEK293 cell line --- p.43 / Chapter 2.5.1 --- Cloning of Htrpl Gene into pcDNA3 Vector --- p.43 / Chapter 2.5.1.1 --- Enzyme Digestion --- p.43 / Chapter 2.5.1.2 --- Gel electrophoresis and Isolation of Htrpl by GeneCIean II Kit --- p.44 / Chapter 2.5.1.3 --- Ligation of Trpl and pcDNA3 Vector --- p.44 / Chapter 2.5.1.4 --- Transformation --- p.47 / Chapter 2.5.1.5 --- Purification of cloned Trpl-pcDNA3 by QIAprep Spin Miniprep Kit --- p.47 / Chapter 2.5.2 --- Transfection of HEK293 Cells with Htrpl and pEGFP-Nl Vector --- p.48 / Chapter 2.5.2.1 --- Cell Preparation for Transfection --- p.48 / Chapter 2.5.2.2 --- Transfection --- p.48 / Chapter 2.5.3 --- Fluorescence Labeling of Expressed Htrpl Channel in HEK293 Cells --- p.49 / Chapter 2.5.3.1 --- Immunostaining with Anti-TRPCl Antibody --- p.49 / Chapter 2.5.3.2 --- Labeling with FITC2° Antibody --- p.50 / Chapter Chapter 3 --- Results --- p.51 / Chapter 3.1 --- Propagation of Ca2+ Signaling --- p.51 / Chapter 3.2. --- Effect of cGMP on SERCA --- p.55 / Chapter 3.2.1 --- ATP stimulated Ca2+ release from internal stores --- p.55 / Chapter 3.2.2 --- Effect of cGMP on the falling phase of [Ca2+]i --- p.55 / Chapter 3.2.3 --- Effect of CPA on the falling phase of [Ca2+]i --- p.58 / Chapter 3.2.4 --- Effect of KT5823 on cGMP --- p.63 / Chapter 3.3. --- Effect of cGMP on bradykinin-activated capacitative Ca2+ entry --- p.65 / Chapter 3.3.1 --- Bradykinin induced capacitative Ca2+ entry --- p.65 / Chapter 3.3.2 --- Effect of cGMP on Ca2+ entry activated by bradykinin --- p.67 / Chapter 3.3.3 --- Effect of KT5823 on the inhibitory effect of cGMP on Ca2+ entry activated by bradykinin --- p.67 / Chapter 3.3.4. --- Effect of cGMP and KT5823 on capacitative Ca2+ entry activated by a combination of different agonists. --- p.71 / Chapter 3.4 --- Cloning and expression of htrpl in HEK 293 cell line --- p.75 / Chapter 3.4.1 --- Optimizing transfection conditions using pEGFP-Nl --- p.78 / Chapter 3.4.2 --- Transient transfection of htrpl channel in HEK293 cells --- p.81 / Chapter 3.4.3 --- Channel properties of expressed htrpl channel --- p.84 / Chapter Chapter 4 --- Discussion --- p.88 / Chapter 4.1 --- Ptopagation of Ca2+ Signaling --- p.88 / Chapter 4.2 --- Effect of cGMP on[Ca2+]i of Vascular Endothelial Cells --- p.89 / Chapter 4.2.1 --- Effect of cGMP on SERCA --- p.89 / Chapter 4.2.2 --- Effect of cGMP on Regulation of Agonist-Activated Capacitative Ca2+ Entry --- p.92 / Chapter 4.2.3 --- Physiological Property of Expressed Htrpl in HEK293 cells --- p.95 / References --- p.97

Diseases such as epilepsy, pain, and neurodegenerative disorders are associated with changes in neuronal dysfunction due to an imbalance of excitation and inhibition. This work details a novel electroconvulsive seizure assay for C. elegans using the well characterized cholinergic and GABAergic excitation and inhibition of the body wall muscles and the resulting locomotion patterns to better understand neuronal excitability. The time to recover normal locomotion from an electroconvulsive seizure could be modulated by increasing and decreasing inhibition. GABAergic deficits and a chemical proconvulsant resulted in an increased recovery time while anti-epileptic drugs decreased seizure duration. Successful modulation of excitation and inhibition in the new assay led to the investigation of a cGMP-dependent protein kinase (PKG) which modulates potassium (K+) channels, affecting neuronal excitability, and determined that increasing PKG activity decreases the time to recovery from an electroconvulsive seizure. The new assay was used as a forward genetic screening tool using C. elegans and several potential genes that affect seizure susceptibility were found to take longer to recover from a seizure. A naturally occurring polymorphism for PKG in D. melanogaster confirmed that both genetic and pharmacological manipulation of PKG influences seizure duration. PKG has been implicated in stress tolerance, which can be affected by changes in neuronal excitability associated with aging, so stress tolerance and locomotor behavior in senescent flies was investigated. For the first time, PKG has been implicated in aging phenotypes with high levels of PKG resulting in reduced locomotion and lifespan in senescent flies. The results suggest a potential new role for PKG in seizure susceptibility and aging. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection