Doctor of Philosophy, The Ohio State University, 2016, Evolution, Ecology and Organismal Biology

Venom has independently evolved several times across diverse animal lineages, resulting in toxins targeting a variety of functionally important protein complexes and macromolecules involved in cellular homeostasis. Sea anemones (Actiniaria) are members of the oldest venomous animal lineage (Cnidaria) and use a diverse array of toxic peptides to incapacitate and immobilize prey, deter potential predators, and fight with conspecifics. When compared to other venomous lineages outside of Cnidaria, sea anemones are atypical venomous animals, as they have venom being expressed throughout their body, engage in conspecific aggression, and host ectosymbionts that are members of lineages that are typical food sources. For these reasons, sea anemones present an opportune lineage to ask questions about venom evolution in a comparative framework. For my dissertation, I use a combination of next-generation sequencing, bioinformatics, and gene tree reconstructions to a) characterize the toxin assemblage in an anatomical structure used exclusively in intraspecific aggressive encounters, b) contrast the toxin assemblage and differential gene expression across the tentacles, mesenterial filaments, and column in three species of sea anemones, c) investigate evolutionary processes and selection events shaping a neurotoxin gene family found exclusively in sea anemones, and d) characterize evolutionary history and functionally important regions in a pore forming toxin. In chapter 1, a tissue-specific RNA-seq approach is used to investigate the venom composition and gene ontology of acrorhagi, specialized structures used in intraspecific competition, in aggressive and non-aggressive polyps of the aggregating sea anemone Anthopleura elegantissima. The resulting assemblage of expressed genes may represent synergistic proteins associated with toxins or proteins related to the morphology and behavior exhibited by the aggressive polyp. In chapter 2, a tissue specific RNA-seq approach is used t (open full item for complete abstract)

Committee: Marymegan Daly (Advisor); John Freudenstein (Committee Member); Lisle Gibbs (Committee Member); Zakee Sabree (Committee Member) Subjects: Animals; Bioinformatics; Biology; Evolution and Development; Toxicology; Zoology

Doctor of Philosophy (PhD), Wright State University, 2024, Biomedical Sciences PhD

Myotonia Congenita (MC) is a rare, inherited ion channelopathy caused by a loss-of-function mutation in the CLCN1 gene. The resulting downregulation of the skeletal muscle chloride channel (ClC-1) results in hyperexcitable skeletal muscle fibers that fire action potentials involuntarily. Patients with MC suffer from debilitating stiffness due to myotonia, described clinically as delayed muscle relaxation following voluntary contraction. Skeletal muscle is a unique system we use in this study to advance our understanding of the pathophysiology underlying MC, and other channelopathies characterized by hyperexcitable cells (i.e., forms of epilepsy and cardiac arrhythmia). Furthermore, re-assessing what makes anti-myotonic drugs such as mexiletine efficacious, yet imperfect, may help us improve the quality of life of affected patients through optimizing treatment outcomes. The current therapeutic approach to treating myotonia is to block voltage-gated Na+ channels in skeletal muscle (Nav1.4) to reduce the fast-transient Na+ current responsible for action potential generation (NaT) via use-dependent block (Statland et al., 2012),(Lo Monaco et al., 2015). Two assumptions are made by the field when taking this approach. First, open channel blockers, those that increase their block of Na channels during repetitive firing, are the preferred type of drug. Second, the block of NaT is the mechanism underlying efficacy. We tested both hypotheses utilizing both transgenic and pharmacologic models of MC in mice. Techniques used included current clamp of the extensor digitorum longus (EDL) muscle and voltage clamp of the flexor digitorum brevis (FDB) muscle. We compared the efficacy of two open-state NaCh blockers; mexiletine and ranolazine, to a closed-state blocker, µ-conotoxin GIIIA (uCTX). We quantitated efficacy against myotonia and determined the optimal concentration of each drug using intracellular recordings from EDL fibers. We were surprised to find that open-channel b (open full item for complete abstract)

Committee: Mark M. Rich M.D., Ph.D. (Advisor); David Ladle Ph.D. (Committee Member); Eric Bennett Ph.D. (Committee Member); David Cool Ph.D. (Committee Member); Andrew Voss Ph.D. (Committee Member) Subjects: Anatomy and Physiology; Biomedical Research; Physiology

Master of Science (MS), Wright State University, 2023, Physiology and Neuroscience

Abnormal glycosylation can impact cardiac proteins, affecting signaling, contraction, and metabolism, which leads to compromised cardiac function. O-GlcNAcylation, the only form of intracellular glycosylation, involves adding a single GlcNAc molecule to proteins via enzymes: O-GlcNAc Transferase (OGT) for addition and O-GlcNAcase (OGA) for removal. The Ednie/Bennett lab has previously demonstrated that in both chronic (OGT KO) and acute (OGT inhibition) models of reduced cardiomyocyte O-GlcNAcylation, a disruption of Ca2+ homeostasis occurs. This study delved into the cardiac voltage-gated sodium channel (NaV 1.5) in these models. NaV 1.5 expression was analyzed through western blotting and immunocytochemistry, while its functional activity was gauged using whole cell voltage-clamping. We also investigated how the trafficking proteins LITAF and NEDD4-2 might influence NaV 1.5 expression in the OGT KO. Initial results highlight distinct regulatory mechanisms, warranting additional studies

Committee: Andrew Ednie Ph.D. (Advisor); Mark Rich M.D., Ph.D. (Committee Member); Eric Bennett Ph.D. (Committee Member) Subjects: Physiology

Master of Science, The Ohio State University, 2020, Anatomy

The sinoatrial node (SAN) is the primary pacemaker of the human heart, which is responsible for initiating and conducting normal cardiac rhythm. When pacemaker cells of the SAN are unable to initiate or conduct heart rhythm, abnormalities such as bradycardia, tachycardia, and/or sinus arrest can occur. These abnormalities constitute just part of a much larger and complex pathological phenomenon known as sinus node dysfunction (SND). In the United States, SND is projected to increase among the elderly with an increasing aging population. However, the only current available treatment for SND is permanent pacemaker (PPM) implantation. PPM implantation is not without its own limitations. By itself, PPM implantation is a costly invasive procedure, which involves a long waiting period between its indication and implantation, not to mention the possibility of adverse events such as infections, and even death. Hence, the work described herein represents an effort to uncover the molecular characteristics of SND induced by heart failure (HF) to aid in the discovery and development of less invasive treatment options for a growing population of SND patients. HF is known to alter important microRNAs (miRs) and mRNAs across the human heart. However, the expression profiles of these miRs and mRNAs in the human HF SAN has not been studied. Our goal was to identify important genes and their modifying miRs in the SAN from HF human hearts, which could be used to target and treat SND. We first compared the mRNA and miR expression profiles between HF and non-heart failing (non-HF) human hearts by next generation sequencing (NGS) in human SAN samples. By NGS, we saw that out of >15,000 mRNAs and >2500 human microRNAs examined, HF significantly altered 831 mRNAs and 44 miRs in the human SAN. After this, we proceeded to use the ingenuity pathway analysis (IPA) software to predict miRs that target mRNAs involved in either SAN pacemaking or conduction. To further confirm the interactio (open full item for complete abstract)

Committee: Melissa Quinn PhD (Advisor); Vadim Fedorov PhD (Committee Member); James Cray PhD (Committee Chair) Subjects: Anatomy and Physiology

Doctor of Philosophy, The Ohio State University, 2016, Biomedical Sciences

Cardiovascular disease is the leading cause of death in the United States, claiming nearly 800,000 lives each year. Regardless of the underlying cardiovascular dysfunction, nearly 50% of these patients die of sudden cardiac arrest caused by arrhythmia. Development and sustainment of cardiac arrhythmia begins with dysfunction of excitability and structure at the cellular level. Therefore, in order to improve therapeutic options for these patients, a basic understanding of the molecular mechanisms regulating cardiac cellular excitability and structure is required. Decades of research have demonstrated that intracellular scaffolding polypeptides known as ankyrins are critical for the regulation of cellular excitability and structure in multiple cell types. Ankyrin-G (ANK3) is critical for regulation of action potentials in neurons and lateral membrane development in epithelial cells. Given its central importance for cellular physiology in excitable and non-excitable cell types, we hypothesized that functional ankyrin-G expression is critical for proper cardiac function. To test this hypothesis in vivo, we generated cardiac-specific ankyrin-G knockout (cKO) mice. In the absence of ankyrin-G, mice display significant reductions in membrane targeting of the voltage-gated sodium channel Nav1.5. This disruption in turn causes severely reduced whole cell sodium current, leading to significant conduction abnormalities, bradycardia, and ventricular arrhythmia and atrioventricular nodal block following infusion of NaV channel antagonists. In addition to regulating cardiac excitability, we also demonstrate a critical role for ankyrin-G in the regulation of the cardiomyocyte cytoarchitecture. Specifically, ankyrin-G cKO mice show disrupted cellular distribution of the desmosomal protein plakophilin-2 (PKP2) at baseline. In a setting of pressure overload-induced heart failure we observed severe disruptions to the cellular localization of PKP2. Further, as desmosomes mediate the in (open full item for complete abstract)

Committee: Peter Mohler (Advisor); Noah Weisleder (Committee Chair); Thomas Hund (Committee Member); Philip Binkley (Committee Member) Subjects: Cellular Biology; Physiology

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2012, College of Sciences and Health Professions

Type-3 long QT syndrome, which is related to type 5 voltage-gated sodium channel alpha subunit (SCN5A) mutation, has been identified since 1995. LQTS mutation in SCN5A is a gain-of-function mutation producing late sodium current, INa,L. Brugada mutation in SCN5A is a loss-of-function causing INa decrease. Whereas, the mechanism for Dilated Cardiomyopathy mutations in SCN5A is still not fully understood. N1325S is one of the first series of mutations identified for type-3 LQTS. Our lab created a mouse model for LQTS by expressing SCN5A mutation N1325S in the mouse hearts (TG-NS) and a matched experimental control line with overexpression of wild- type SCN5A (TG-WT). There are some interesting findings in TG-NS mice: (i) Intracellular sodium (Na+) level is higher in TG-NS myocytes compared with TG-WT myocytes. (ii) Ca2+ handling is abnormal in TG-NS myocytes, but not in TG-WT myocytes. (iii) Apoptosis was also found in TG-NS mouse heart tissue, but not in TG-WT hearts. These results provoke the hypothesis that gain-of-function mutation N1325S in SCN5A leads to LQTS through abnormal cytosolic Ca2+ homeostasis. Another LQTS mutation in SCN5A R1193Q was identified in 2004 and the electrophysiological property is similar to other gain-of-function SCN5A mutations. The transgenic mouse model for this mutation was also established and the surface Electrocardiogram (ECG) results indicate longer corrected QT interval also present in transgenic mice carrying R1193Q mutation. Besides, quinidine, an anti-arrhythmic medication, can cause arrhythmic symptoms such as premature ventricular contraction (PVC), premature atrial contraction (PAC) and atrioventricular (AV) block in R1193Q transgenic mice. In order to further study the relationship between abnormal Ca2+ handling and the type of SCN5A mutation, either gain-of-function or loss-of-function, we have chosen HL-1 cells, a cell line with indefinite passages in culture with all the adult cardiac phenotypes. The similar abnormal (open full item for complete abstract)

Committee: Qing Wang PhD, MBA (Advisor); Yan Xu PhD (Advisor); Xue-long Sun PhD (Committee Member); Robert Wei PhD (Committee Member); Wu Yuping PhD (Other) Subjects: Biology; Chemistry; Genetics; Health Sciences

Doctor of Philosophy, Case Western Reserve University, 2012, Physiology and Biophysics

Monogenic disorders caused by mutations in ion channels produce alterations of the cardiac action potential that lead to life-threatening arrhythmias. However, although Long QT (LQT) and Brugada Syndrome (BrS) are autosomal dominant diseases, they also display incomplete penetrance, a phenomenon where individuals carrying a disease-causing mutation are asymptomatic. There are several prevailing hypotheses that have attempted to explain why a particular gene expression pattern might produce variable phenotypic expression. This dissertation focuses on one possible mechanism involving disease modifying genes. I focused on this since emerging evidence indicates that disease modifying genes, such as ion channel polymorphisms, modulate and ultimately affect the function of ion channels, and thus cause genotype-phenotype discordance in both common forms of arrhythmias such as atrial fibrillation and in more rare forms such as LQT or BrS. Thus, the overall hypothesis of my dissertation is that the common H558R sodium channel polymorphism contributes to the genotype-phenotype discordance observed in heritable arrhythmias. In my first project, I propose a novel mechanism that explains how the sodium channel polymorphism H558R can modulate the function of a gain-of-function sodium channel mutation. Surprisingly, this gain-of-function mutation was identified in an asymptomatic family and is also found in control population. However based on its biophysical phenotype, this mutation would be expected to lead to life-threatening arrhythmias. I demonstrated that the H558R polymorphism acted as a disease-modifying gene by restoring normal gating kinetics of the mutant channel which explains the absence of a clinical phenotype in patients carrying the polymorphism along with the mutation. Furthermore, we reported that this H558R polymorphism could restore trafficking of a BrS mutation and there again explain the incomplete penetrance phenomenon seen in a BrS family. Therefore in my s (open full item for complete abstract)

Committee: Isabelle Deschenes PhD (Advisor); Thomas M. Nosek PhD (Committee Chair); George R. Dubyak PhD (Committee Member); Kevin J. Donahue MD (Committee Member); Kenneth R. Laurita PhD (Committee Member); Stephen W. Jones PhD (Committee Member) Subjects: Biomedical Research

Master of Sciences (Engineering), Case Western Reserve University, 2010, Biomedical Engineering

Abundant clinical evidence suggests an association between mesial temporal lobe epilepsy (MTLE) and sleep, but studies of sleep are lacking in animal models. We investigated the reciprocal seizure-sleep relationship using the Scn2aQ54 mice, a sodium channel transgenic MTLE animal model. Chronic 24-hour continuous video EEG/EMG (electroencephalograph /electromyograph) was utilized to monitor seizure and sleep activities in free-running animals. Spontaneous secondarily generalized tonic seizures and inter-ictal HFOs were characterized in the Scn2aQ54 transgenic mice (tg/+). The seizures were highly related to different sleep states. Predominantly more frequent seizure occurred after NREM sleep compared to after REM sleep and wakefulness. The transgenic animal also had changed sleep structures compared to controls, as seizures fragmented all sleep states, and resulted in longer wake time. High frequency oscillations (HFOs) were also detected from hippocampal electrodes during pre-ictal periods, and were under the influences of different sleep states.

Committee: Dominique Durand PhD (Advisor); Kingman Strohl MD (Committee Co-Chair); Kenneth Loparo PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, Case Western Reserve University, 2008, Biomedical Engineering

Individuals with epilepsy experience recurrent and unprovoked seizures characterized by uncontrolled, excessive neurological activity. Seizures can be debilitating and often resistant to available drug therapies. Deep brain stimulation is an excellent new therapy in the treatment of Parkinsons, nevertheless its mechanisms of action are not clearly understood. Ongoing research is essential for a complete understanding of the mechanisms of action of DBS in order to determine the most effective stimulation parameters for epilepsy therapy including target location, frequency, amplitude, and duration. Genetic models of disease can provide invaluable insight into disease development, progression, and treatment. The Q54 model, is a mouse model of epilepsy resulting from a single sodium channel mutation that was utilized in this study to examine inherited seizure characteristics in vitro and in vivo, and evaluate the application of novel DBS parameters. The data presented here suggest that Q54 mice experience increased excitability and spontaneous activity in the hippocampal slice in vitro, as well as chronic recurrent hippocampal seizures in vivo. Furthermore, low frequency stimulation applied to the ventral hippocampal commissural fibers (VHC) in Q54 mice is successful in the reduction of seizure frequency and is therefore a potential new therapy for the treatment of epilepsy syndromes. In addition, an electrode model was developed for application of low frequency deep brain stimulation to white matter tracts, such as the VHC, in the treatment of epilepsy.

Committee: Dominique Durand PhD (Advisor); Dawn Taylor PhD (Committee Member); David Friel PhD (Committee Member); Joseph Nadeau PhD (Committee Member); Mary Ann Werz MD, PhD (Committee Member) Subjects: Biomedical Research; Engineering