Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, June 1999. / Includes bibliographical references. Also available on the Internet.

The ubiquitously expressed pannexin 1 (Panx1) ion- and metabolite-permeable channel is capable of mediating ATP release in a multitude of cells and tissues. This leads to activation of nearby purinergic (P2X/P2Y) receptors in an autocrine/paracrine manner. Stimulation of P2 receptors also triggers Panx1 activation, leading to the formation of a positive feedback loop. Although the focus of Panx1 research has primarily been on its expression at the cell surface, there is robust and stable expression of Panx1 on intracellular membranes. Whether intracellular Panx1 was the consequence of direct diversion from the secretory pathway or internalization from the cell surface was unknown at the onset of my studies. I postulated that Panx1 internalization to these membranes would require a ubiquitous constitutively or episodically released stimulus to allow stable intracellular expression. ATP, a potent signalling molecule released via exocytosis (constitutive or regulated) or large pore channels, fit this criterion. My hypothesis was that ATP triggered Panx1 internalization to intracellular compartments. Upon elevation of extracellular ATP, I observed P2X7R-mediated non-canonical internalization of Panx1 via macropinocytosis. This involved upstream cholesterol-dependent P2X7R-Panx1 clustering via a physical interaction between P2X7R-Panx1 ectodomains and possible contribution of phospholipid (PA, PIP, PIP2) interactions localized to the Panx1 C-terminus. Physical P2X7R-Panx1 interaction may promote Panx1 association with actively endocytosing regions of the membrane. Internalized Panx1 was targeted to slow recycling Rab14/Rab11-positive endosomes in an Arf6-dependent mechanism. The data I presented here provides an additional negative feedback layer to P2X7R-Panx1 crosstalk in the many cell types where they are co-expressed. Further, this is the first evidence demonstrating that Panx1 surface expression is labile to changes in the cellular environment, which contributes to the understanding of the regulation of Panx1 and associated behaviours through trafficking mechanisms. / Graduate / 2018-06-20

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Cardiovascular disease is the leading cause of death world-wide, with 42% of sudden cardiac death in young adults caused by myocarditis. Viruses represent the main cause of myocarditis, with adenovirus being a leading pathogen. However, it is not understood how adenoviruses cause sudden cardiac arrest. Myocarditis is defined by two phases, acute and chronic. The acute phase involves viral-mediated remodeling of subcellular structures in the myocardium, which is thought to contribute to arrhythmogenesis. The chronic phase is immune response-mediated, where the host immune system causes damage that induces gross remodeling of the heart, which can result in cardiac arrest or heart failure. Electrical impulses of the heart are propagated by cardiomyocytes, via gap junctions, ion channels, and intracellular junctions, creating the healthy heartbeat. Cx43, the primary gap junction protein in the myocardium, not only propagates electrical signals, but also anti-viral molecules. Viral targeting of gap junction function leads to reduced anti-viral responses in neighboring cells. However, reduced cellular communication would dangerously alter cardiac conduction. Using a cardiotropic adenovirus, MAdV-3, we find that viral genomes are significantly enriched in the heart, with a decrease of gap junction and ion channel mRNA in infected hearts, however, their protein levels were unchanged. Phosphorylation of Cx43 at serine 368, known to reduce gap junction open probability, was increased in infected hearts. Ex vivo optical mapping illustrated decreased conduction velocity in the infected heart and patch clamping of isolated cardiomyocytes revealed prolonged action potential duration, along with decreased potassium current density during infection. Pairing mouse work with human induced pluripotent stem cell-derived cardiomyocytes, we found that human adenovirus type-5 infection increased pCx43-Ser368 and perturbation of intercellular coupling, as we observed with in vivo MAdV-3 infection. Allowing adenovirus infection to progress in vivo, we find myocardium remodeling and immune cell infiltration. Together, these data demonstrate the complexity of cardiac infection from viral-infection induced subcellular alterations in electrophysiology to immune-mediated cardiomyopathy of cardiac adenoviral infection. Our data describe virally induced mechanisms of arrhythmogenesis, which could lead to the development of new diagnostic tools and therapies, to help protect patients from arrhythmia following infection. / Doctor of Philosophy / Viral infection has long thought to be a cause of unexplained sudden cardiac death, especially in young adults. Viruses have been identified to cause many cases of deleterious remodeling of the heart, which can result in heart failure. The heart relies on electrical signaling that moves in a coordinated fashion to contract and pump blood throughout the body. The cells within the heart that do this are called cardiomyocytes, and they join end-to-end to communicate with each other via gap junctions. Gap junctions are tunnels that allow for ions that create electrical impulses to pass, and molecules, such as ones that are important in immune responses to infection. In addition to gap junctions in the heart, ion channels, which are highly selective to allow only one ion flow, unlike gap junctions, create the healthy heartbeat. The most common gap junction in the heart comprises Cx43 proteins. If a virus were to alter how Cx43 connects to a neighboring cell, this would cause a better environment for the virus, as this would keep anti-viral surveillance low, however, this would change how the electrical signal moves throughout the heart, creating arrhythmias. Adenoviruses are a common cold virus, but have been found in the hearts of many cardiac arrest patients. However, little is known on how adenoviruses may cause cardiac arrest, because human adenoviruses are only successful in humans, and mouse adenoviruses are only successful in mice. This creates a challenge when studying the dynamic heart, which does not translate well to cells in a dish. A mouse adenovirus, called Mouse Adenovirus Type-3 (MAdV-3) was reported to favor infecting the heart in mice, but no research has been published on if this virus can answer how adenoviruses change the heart. Because of this virus, and our prior research that adenoviruses can decrease Cx43 within skin cells in a dish, we used MAdV-3 to understand if, how adenoviruses could cause sudden cardiac arrest, and if longer infection could change the overall structure of the heart. We find that MAdV-3 infection prefers the heart to other organs, and that early stages, reduce both the speed of the electrical signal moves through heart and, looking within a cardiomyocyte, how it creates that electrical signal. These changes are arrhythmogenic and accompany modification of Cx43 that would close the gap junction between two cells, changing how ions and molecules move between cells. Using a human adenovirus infection in human cardiomyocytes created from stem cells, this result is also observed. If infection is allowed to continue in the mouse to cause chronic infection, the heart itself changes shape and is diseased. Together, this work shows that adenoviruses create a diseased heart, first the virus changes how the electrical signal moves and then later, causes thinning of the heart muscle. These data illustrate the role viruses play in causing cardiac arrest and could lead to diagnostic or drug targets.

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Zheng Tingting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 98-124). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Table of Contents --- p.vi / List of Tables --- p.viii / List of Figures --- p.ix / Abbreviations --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Triamcinolone acetonide(TA) --- p.1 / Chapter 1.1.1 --- Application in ophthalmology --- p.1 / Chapter 1.1.2 --- Mechanism of anti-inflammatory effect --- p.2 / Chapter 1.1.3 --- Side effects of TA --- p.4 / Chapter 1.1.4 --- Toxicity of TA --- p.5 / Chapter 1.2 --- Retinal pigment epithelial (RPE) cell --- p.6 / Chapter 1.3 --- Mechanism of cell death --- p.8 / Chapter 1.3.1 --- Apoptosis --- p.9 / Chapter 1.3.2 --- caspase --- p.11 / Chapter 1.3.3 --- Mitogen-activated protein kinase (MAPK) --- p.13 / Chapter 1.3.4 --- Activator Protein-1 (AP-1) --- p.15 / Chapter 1.4 --- Potassium channel (K+）） --- p.15 / Chapter 1.4.1 --- Molecular structure of KAtp channel --- p.16 / Chapter 1.4.2 --- Regulation of Katp channel --- p.17 / Chapter 1.4.3 --- Pinacidil --- p.18 / Chapter 1.5 --- Calcium channel --- p.20 / Chapter 1.5.1 --- VDCCs and subtypes --- p.21 / Chapter 1.5.2 --- Calcium channel blocker --- p.22 / Chapter 1.5.3 --- Verapamil --- p.23 / Chapter 1.6 --- Study objectives --- p.25 / Chapter Chapter 2 --- Methodology --- p.37 / Chapter 2.1 --- Cell biology --- p.37 / Chapter 2.1.1 --- Materials --- p.37 / Chapter 2.1.1.1 --- Culture related material --- p.37 / Chapter 2.1.1.2 --- Drugs --- p.37 / Chapter 2.1.1.3 --- Cell line and instrument --- p.37 / Chapter 2.1.2 --- Preparations --- p.38 / Chapter 2.1.2.1 --- Working medium --- p.38 / Chapter 2.1.2.2 --- Drugs --- p.38 / Chapter 2.1.2.3 --- MTT solution --- p.39 / Chapter 2.1.3 --- Cell culture and treatment process --- p.40 / Chapter 2.1.3.1 --- Seed cell --- p.40 / Chapter 2.1.3.2 --- Treatment --- p.40 / Chapter 2.1.4 --- MTT-Cell Proliferation Assay --- p.41 / Chapter 2.2 --- Molecular biology --- p.42 / Chapter 2.2.1 --- Materials --- p.42 / Chapter 2.2.1.1 --- "Chemicals, reagents, and kits" --- p.42 / Chapter 2.2.1.2 --- Solutions and Buffers --- p.42 / Chapter 2.2.1.3 --- Primers and Enzymes --- p.43 / Chapter 2.2.1.4 --- Equipment --- p.43 / Chapter 2.2.1.5 --- Software --- p.43 / Chapter 2.2.2 --- Reverse transcription 226}0ؤ Polymerase Chain Reaction (RT-PCR) --- p.44 / Chapter 2.2.2.1 --- Cell collection and RNA Isolation --- p.44 / Chapter 2.2.2.2 --- Reverse Transcription (RT) --- p.45 / Chapter 2.2.2.3 --- PCR Reaction --- p.46 / Chapter 2.3 --- Immunocytochemistry --- p.48 / Chapter 2.3.1 --- Materials and instrumentation --- p.49 / Chapter 2.3.1.1 --- Antibodies and Equipment --- p.49 / Chapter 2.3.1.2 --- Chemicals and other useful items --- p.49 / Chapter 2.3.2 --- Preparations --- p.50 / Chapter 2.3.2.1 --- Preparation of coverslips --- p.50 / Chapter 2.3.2.2 --- Prepations of solutions --- p.50 / Chapter 2.3.3 --- Procedures --- p.51 / Chapter 2.4 --- Expression of results and statistics --- p.52 / Chapter Chapter 3 --- Results --- p.57 / Chapter 3.1 --- Effects of TA on RPE cell culture --- p.57 / Chapter 3.1.1 --- Cell morphology --- p.57 / Chapter 3.1.2 --- MTT assay --- p.57 / Chapter 3.1.3 --- Gene expressions --- p.57 / Chapter 3.2 --- Effects of PIN/VP on TA treated RPE cells --- p.58 / Chapter 3.2.1 --- MTT assay --- p.58 / Chapter 3.2.2 --- Gene expression --- p.59 / Chapter 3.2.2.1 --- Expression of housekeeping gene --- p.59 / Chapter 3.2.2.2 --- Expression of apoptosis-related gene --- p.59 / Chapter 3.2.2.3 --- Expression of early-response genes --- p.60 / Chapter 3.2.3 --- Immunofluorescence --- p.61 / Chapter Chapter 4 --- Discussion --- p.86 / Chapter Chapter 5 --- References --- p.98

Tsoi Sze Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 82-104). / Text in English; abstracts in English and Chinese. / Tsoi Sze Man. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview of study --- p.1 / Chapter 1.2 --- Cerebellum --- p.2 / Chapter 1.2.1 --- General Structure of cerebellum --- p.3 / Chapter 1.2.2 --- Cell types of cerebellar cortex --- p.4 / Chapter 1.2.2.1 --- Basket cells --- p.5 / Chapter 1.2.2.2 --- Stellate cells --- p.6 / Chapter 1.2.2.3 --- Purkinje cells --- p.6 / Chapter 1.2.2.4 --- Granule cells --- p.7 / Chapter 1.2.2.5 --- Golgi cells --- p.8 / Chapter 1.2.2.6 --- Unipolar brush cells --- p.9 / Chapter 1.2.2.7 --- Deep cerebellar nuclear neurons --- p.11 / Chapter 1.2.3 --- The neuronal circuitry of the cerebellum --- p.12 / Chapter 1.2.4 --- Cerebellar function --- p.14 / Chapter 1.3 --- Cyclic nucleotide-gated (CNG) channels --- p.16 / Chapter 1.3.1 --- Molecular characterization of CNG channels --- p.16 / Chapter 1.3.2 --- Functional properties of CNG channels --- p.19 / Chapter 1.3.3 --- Expression of CNG channels in brain --- p.21 / Chapter 1.3.4 --- CNG channel and neuronal plasticity --- p.23 / Chapter 1.4 --- Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels --- p.26 / Chapter 1.4.1 --- Molecular characterization of HCN channels --- p.27 / Chapter 1.4.2 --- Functional properties of HCN channels and Ih current --- p.29 / Chapter 1.4.3 --- Modulation by cyclic nucleotides --- p.31 / Chapter 1.4.4 --- Expression of HCN channels in brain --- p.33 / Chapter 1.4.5 --- Physiological roles of Ih current in central nervous system --- p.35 / Chapter 1.5 --- Aims of study --- p.38 / Chapter Chapter 2 --- Material and Methods --- p.39 / Chapter 2.1 --- Immunohistochemistry Experiments --- p.39 / Chapter 2.1.1 --- Animal preparation --- p.39 / Chapter 2.1.2 --- Tissue preparation --- p.39 / Chapter 2.1.3 --- Primary and secondary antibodies --- p.40 / Chapter 2.1.4 --- Immunofluroescence staining --- p.41 / Chapter 2.1.5 --- Confocal laser scanning microscopy and data processing --- p.41 / Chapter 2.2 --- Whole cell patch clamp recordings --- p.42 / Chapter 2.2.1 --- Brain slice preparation and identification of the cerebellar Purkinje neurons --- p.42 / Chapter 2.2.2 --- Whole cell voltage- and current-clamp recordings --- p.43 / Chapter 2.2.3 --- Drug solutions and delivery --- p.44 / Chapter 2.2.4 --- Statistical analysis --- p.45 / Chapter Chapter 3 --- Expression of Various Cyclic Nucleotide-Gated (CNG) Channel Subunits in Rat Cerebellum --- p.46 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Results --- p.46 / Chapter 3.2.1 --- Immunoreactivity of CNGA1 in cerebellum --- p.46 / Chapter 3.2.2 --- Immunoreactivity of CNGA2 in cerebellum --- p.47 / Chapter 3.2.3 --- Immunoreactivity of CNGA3 in cerebellum --- p.47 / Chapter 3.3 --- Discussion --- p.48 / Chapter Chapter 4 --- Expression of Various Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channel Subunits in Rat Cerebellum --- p.53 / Chapter 4.1 --- Introduction --- p.53 / Chapter 4.2 --- Results --- p.53 / Chapter 4.2.1 --- Immunoreactivity of HCN 1 in cerebellum --- p.53 / Chapter 4.2.2 --- Immunoreactivity of HCN2 in cerebellum --- p.55 / Chapter 4.2.3 --- Immunoreactivity of HCN3 in cerebellum --- p.55 / Chapter 4.2.4 --- Immunoreactivity of HCN4 in cerebellum --- p.55 / Chapter 4.3 --- Discussion --- p.55 / Chapter Chapter 5 --- Electrophysiological Recordings of Cyclic Nucleotide-Gated Ionic Conductance in Rat Cerebellar Purkinje Neurons --- p.59 / Chapter 5.1 --- Introduction --- p.59 / Chapter 5.2 --- Results --- p.59 / Chapter 5.2.1 --- Effect of cyclic nucleotides on the membrane potential of cerebellar Purkinje neurons --- p.59 / Chapter 5.2.2 --- Ionic conductance of the cyclic nucleotide-induced inward current --- p.61 / Chapter 5.2.3 --- The mechanism of the cyclic nucleotide-induced inward current --- p.61 / Chapter 5.2.3.1 --- Site of action --- p.62 / Chapter 5.2.3.2 --- Involvement of CNG channels and HCN channels --- p.63 / Chapter 5.2.3.3 --- Involvement of protein kinase A (PKA) and protein kinase G (PKG) --- p.65 / Chapter 5.2.3.4 --- Involvement of inwardly rectifying potassium (Kir) channels and transient receptor potential (TRP) channels --- p.65 / Chapter 5.2.4 --- Effect of cyclic nucleotides on Ih current in Purkinje neurons --- p.67 / Chapter 5.3 --- Discussion --- p.68 / Chapter Chapter 6 --- Concluding remarks References --- p.78 / References --- p.82

Cyclic nucleotide-gated (CNG) ion channels are Ca2+ permeable nonselective cation channels that are directly gated by binding of cAMP or cGMP, thus providing a linkage between two important signal molecules, cyclic nucleotides and calcium. They are known to play an important role in sensory transduction and in second-messenger modulation of synaptic neurotransmitter release. Previous studies showed that besides in neuronal cells, CNG were found also in non-neuronal tissues including heart, kidney, blood vessels and spleen, they are reported to be involved in a variety of cell function. / Ion channels play an indispensable role in endothelial cells, which is a unique signal-transducting surface in the vascular system that is responsible in altering vascular tone. The present study investigated the expression and functional roles of the cyclic nucleotide gated channels (CNG) in regulating the intracellular calcium level of vascular endothelial cells using molecular and calcium measurement techniques. / The present study provided evidence that the CNG channels, especially that of CNGA2, were expressed in vascular tissues. I used a variety of different methods, including RT-PCR, northern blot, in-situ hybridization, immunohistochemistry and western blot to study the localization of CNGA2 channels. RT-PCR amplify a CNGA2 fragment of 582bp from RNAs isolated from bovine vascular endothelial cell line, rat vascular smooth muscle cell line and rat aorta. Northern blot analysis detected a 2.3-kilobase (kb) CNGA2 transcript in rat aorta mRNA. The cellular distribution of CNGA2 was further studied by in-situ hybridization, which demonstrated expression of CNGA2 mRNA in human vascular endothelial and vascular smooth muscle cells. Immunohistochemistry data also agreed with those generated from in-situ hybridization. Western blot data also demonstrated proteins of CNG2 was expressed in both human vascular endothelial cells and vascular smooth cells layer. Subcellular localization of CNGA2 inside the vascular endothelial cells was also investigated with the use of a GFP linked CNGA2 channel gene. Taken together, the results showed that CNGA2 proteins were expressed on the plasma membrane of the vascular endothelial cells. (Abstract shortened by UMI.) / Cheng Kwong Tai Oscar. / "July 2005." / Adviser: Xiaoqiang Yao. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3531. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 216-243). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

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