Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2000. / Includes bibliographical references (leaves 174-192). Also available online through Digital Dissertations.

The connexins are a family of 21 proteins that represent the structural units of intercellular gap junctions and single membrane hemichannels. These channels provide a means for cells to exchange small metabolites and signaling molecules with adjacent cells and the extracellular space, respectively. Compelling evidence implicates connexins, and the more recently discovered pannexins, in the control of neural progenitor cell proliferation, survival and migration. Moreover, connexin and pannexin dysregulation following central nervous system injuries such as cerebral ischemia, spinal cord injury, and epilepsy contributes to the secondary expansion of lesions days and weeks after the initial insult. While these data suggest that connexins and pannexins represent novel therapeutic targets to both reduce the extent of neural injury and promote neural repair and regeneration, we currently lack the necessary repertoire of therapeutically useful connexin- and pannexin-specific compounds to test these hypotheses. In this thesis, I conducted targeted screening of a large, ethnobotanically-derived library to address my overarching objective of identifying compounds that selectively alter connexin and/or pannexin channel function. To accomplish this, I characterized the repertoire of connexins and pannexins expressed by neural progenitor cell-like NT2/D1 cells, quantified the intercellular flux of calcein through connexin gap junctions, and measured the uptake of lucifer yellow and propidium iodide through pannexin hemichannels. Collectively, these screens identified several promising lead compounds and ethanolic plant extracts that selectively alter connexin and pannexin channel activity.

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.

Rapid and well-orchestrated action potential propagation through the myocardium is essential to each heartbeat. Gap junctions comprising primarily Cx43 reside within the intercalated discs connecting cardiomyocytes, effecting not only direct intercellular electrical coupling, but the localization of other junctional structures and ion channels. Alterations in Cx43 expression occur in essentially all forms of heart disease and is therefore a topic of intense study. Posttranslational modification of Cx43 is understood to impact trafficking, conduction, and stability. Altered Cx43 phosphorylation is well described during pathological remodeling of gap junctions in response to cellular stress. Research has revealed how phosphorylation of specific residues elicit specific effects on Cx43, but the complexity of this process has left much unknown. In particular, the role phosphorylation of a triplet of double serine residues, Ser365, Ser368, and Ser373, plays in GJ function and Cx43/14-3-3 interaction has been called into question. Using an ex vivo whole heart ischemia model we find a decrease in pS368 in mice lacking the ability to phosphorylate S365 and S373 while under stress. In vitro transfection of human induced pluripotent stem cell-derived cardiomyocytes when stressed with PMA were also carried out. These data allow us to piece together the exquisite interplay of gatekeeper phosphorylation events upstream of channel closure, altered protein-protein interactions, and gap junction internalization and degradation. It is hoped that our increasing understanding of this important area of gap junction biology will facilitate better understanding of arrhythmogenesis, and potential therapeutic strategies to restore or preserve normal electrical coupling in diseased hearts. / Master of Science / The heart, an electrically active organ, relies on the propagation of an electrical signal throughout its entirety in order to produce a healthy heartbeat. In order to do so, the heart uses specialized muscle cells known as cardiomyocytes which can not only contract but pass along chemical signals to the cardiomyocyte next in line to signal it to contract as well. The passage of signals occurs through protein units called gap junctions and are made predominantly of Cx43 proteins in the heart. Gap junctions look and function like tubes that travel from the inside space of one cell to the other and allow a flow of small molecules to occur; these small molecules, namely ions, are part of the signal needed to initiate contraction in the adjacent cell. Cx43, like many proteins in our bodies, is slightly altered after it is produced through a process known as posttranslational modification. This allows the cell to alter the localization and function of the protein and tailor it for the needs of the cell. Rather than changing the backbone composition of the protein, small chemical groups are attached, and this imparts a change to how the protein interacts with other proteins or its environment. In particular, one form of modification is known as phosphorylation where a phosphate group is attached to the protein at specific locations along its chain. Cx43 too can be phosphorylated, and while under pathological stress, such as a lack of oxygen or infection, cardiomyocytes increase the amount of phosphorylated Cx43 at a site known to cause pathological changes to the function of Cx43. These changes include how well the gap junctions can transmit signals or associate with other proteins and, in the heart, can predispose the development of arrhythmias or unhealthy heartbeats. However, not all phosphorylation is bad and phosphorylation at other locations also occurs during normal healthy functions of the cardiomyocyte can affect how other sites along Cx43 are phosphorylated. The process of one phosphorylated site affecting another is known as the gatekeeper effect and add a new layer to our understanding of how cells use phosphorylated Cx43 to fine tune its effects. Using cells that do not produce their own Cx43 and subsequently giving them the instructions to produce specific forms of mutant Cx43 that can and cannot be phosphorylated at specific sites, we can understand with greater detail of how cardiomyocytes respond to stress and how some of those responses can be pathological. This will allow future research into the creation of therapies that prevent negative Cx43 phosphorylation after illness, potentially avoiding the development of dangerous arrhythmias.

No description available.

Gap Junctions als wichtigste Elemente der Zelle zur Ermöglichung einer interzellulären Kommunikation erlauben eine koordinierte Antwort auf externe und interne Stimuli und somit ein Zusammenspiel von Zellgruppen und Organen im Gesamtorganismus. In der vorliegenden Arbeit wurde der Einfluss einer mittelfristigen und chronischen Stimulation neonataler Rattenkardiomyozyten durch Insulin und den Wachstumsfaktoren Insulin-like Growth Factor-1 (IGF-1), Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor-β (TGF-β) und Fibroblast Growth Factor-2 (FGF-2) auf die Expression der Connexine 40, 43 und 45 untersucht. Dabei zeigte sich unter der Insulin-Stimulation eine konzentrationsabhängige Regulation der Connexin 43 (Cx43) Expression. Die Exposition gegenüber IGF-1 hatte einen signifikanten Anstieg der Cx43 Proteinmenge zur Folge. Unter 24stündiger VEGF- oder FGF-2-Stimulation fand sich dagegen diesbezüglich kein relevanter Unterschied. Die Analysen nach langfristiger Exposition gegebenüber TGF-β zeigten eine signifikante Abnahme der Cx43 Proteinmenge bei unveränderter Cx43 mRNA. Zur Erfassung mittelfristiger Veränderungen wurden die Kardiomyozyten jeweils 3 Stunden mit den Wachstumsfaktoren VEGF und TGF-β inkubiert. Dabei zeigte sich jeweils eine signifikante Zunahme der Cx43 Proteinmenge und –mRNA. Die Connexine 40 und 45 waren in den ventrikulären Kardiomyozyten nur spärlich nachweisbar und durch keinen der untersuchten Faktoren signifikant induzierbar.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Thu Annelise Nguyen / Colorectal cancer is one of the most common cancers in the United States with an early detection rate of only 39%. Colorectal cancer cells along with other cancer cells exhibit many deficiencies in cell-to-cell communication, particularly gap junctional intercellular communication (GJIC). GJIC has been reported to diminish as cancer cells progress. Gap junctions are intercellular channels composed of connexin proteins, which mediate the direct passage of small molecules from one cell to the next. They are involved in the regulation of the cell cycle, cell differentiation, and cell signaling. Since the regulation of gap junctions is lost in colorectal cancer cells, the goal of this study is to determine the effect of GJIC restoration in colorectal cancer cells. Overexpression of connexin 43 (Cx43) in SW480 colorectal cancer cells causes a 6-fold increase of gap junction activity compared to control un-transfected cells. This suggests that overexpressing Cx43 can restore GJIC. Furthermore, small molecule directly targeting gap junction channel was used to increase GJIC. Gap junction enhancers, PQs, at 200 nM showed a 4-fold increase of gap junction activity in SW480 cells. Using Western blot analysis, Cx43 isoform expression was seen to shift from P0 to P1 and P2 isoforms after treatment with PQ1 200 nM for 1 hour. Overall, the results show that overexpression of connexin and small molecules such as gap junction enhancers, PQs, can directly increase gap junction activity. The findings provide an important implication in which restoration of gap junction activity can be targeted for drug development.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Thu Annelise Nguyen / Gap junctional intercellular channels allow the cells to communicate with each other. A breach in gap junctional intercellular communication (GJIC) affects cell growth and proliferation. In addition, many neoplastic cells exhibit a decrease in GJIC. Many factors that decrease GJIC have been shown to potentiate cancer formation. 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), an environmental pollutant, is a carcinogen; however, its mechanism of carcinogenicity is unclear. Therefore, we examined the effect of TCDD on GJIC in MCF-7, a human breast cell line and normal mammary epithelial cells (HMEC). TCDD showed a decrease in GJIC in MCF-7 cells caused by increased phosphorylation of gap junctional protein, Cx43. PKCα-mediated phosphorylation of Cx43 was confirmed by inhibitor studies using calphostin C. Interestingly, TCDD affected GJIC in HMEC through a novel pathway involving redistribution of Cx43 to the perinuclear membrane. Our studies suggest that TCDD causes decrease in GJIC which could potentially lead to cancer. This also indicates that if GJIC is restored it could decrease cell growth and proliferation. Therefore, we investigated the role of substituted quinolines (PQ1), shown to bind with gap junctional proteins by computational docking. The results showed that indeed PQ1 significantly increases GJIC and exerts anti-tumor effect in human breast cancer cells compared to control without treatment or HMEC. We found an increase in GJIC, growth attenutation and increased apoptosis in T47D human breast cancer cell line. Our studies suggest that PQ1 is a novel gap junctional activator causing a decrease in tumor growth. Since PQ1 alone is effective in decreasing tumor growth in breast tumors, we proposed to test its efficacy with the current drug of choice for breast cancer, tamoxifen. The combinational treatment of tamoxifen and PQ1 showed a significant decrease in cell viability, increase in BAX (Bcl2-associated X), and, increase in caspase 3 activation compared to individual treatments. Hence, combinational treatment of PQ1 and tamoxifen can potentiate decrease in tumor growth. In conclusion, downregulation of gap junctions can potentiate tumor growth while restoration of GJIC can induce apoptosis and decrease tumor growth.

In Drosophila melanogaster, the GFS is synaptically coupled to the Tergotrochanteral motoneurons; these neurons form a signaling pathway from the brain to the jump muscles (Thomas and Wyman, 1983). Part of this signaling is done through gap junctions, and placement of these gap junctions was partially shown to be regulated by the binding of Netrin, a class of guidance molecule (Orr et al., 2014). In the present study we investigate the role of Netrin's receptor Frazzled in the placement of gap junctions in Drosophila at: 1) Presynaptic neurons (Giant Fibers [GF]), 2) Postsynaptic neurons (Tergotrochanteral motoneurons [TTMn]), and 3) Presynaptic + Postsynaptic neurons simultaneously. Effects of Frazzled were tested using Frazzled RNAi and a combination of electrophysiological recordings and imaging of the GF-TTMn synapse. The results from this study show that presynaptic and postsynaptic knockdown of Frazzled delayed muscular responses and altered the anatomy of both the GF's and TTMn's. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection