Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2007, College of Science

Background: This project examined JAK-STAT pathway activation in two mouse models of cardiac hypertrophy: autoimmune myocarditis and pressure overload (PO). Methods: Myocarditis was induced with cardiac myosin; PO was induced by transverse aortic constriction. STAT1, 3, and 5 binding was assessed by gel shift. STATs, JAKs, SERCA2A, and calsequestrin (CSQ) were quantified. In myocarditis, P-STAT3 localization to cardiac myocyte nuclei was ascertained, and plasma IL-6 and ventricular ANF mRNA were analyzed. Results: In myocarditis, STAT3/3 and STAT1/1 activation, inflammation, increased ventricle weight (P < 0.0001), and ANF mRNA (P = 0.005) occurred on days 14, 21, and 28. In PO, activation appeared on day 7 and persisted to failure. P-STAT3 increased (myocarditis, P < 0.0001; pressure overload, P < 0.05). P-JAK1 increased in myocarditis on days 21 and 28 (P < 0.007). In PO, CSQ and SERCA2A levels did not differ, but in myocarditis CSQ decreased (P = 0.02). In myocarditis, a biphasic elevation in plasma IL-6 occurred (P = 0.003). Conclusions: Although JAK-STAT signaling is activated in both models, it occurs earlier in PO and persists to heart failure, whereas in myocarditis it declines to basal levels as inflammation and plasma IL-6 return to baseline.

Committee: Christine Moravec (Advisor) Subjects:

Bachelor of Sciences, Ohio University, 2013, Biological Sciences

Cancer is a leading cause of death in the United States. Cancer arises due to the accumulation of multiple mutations. These mutations can occur in signaling transduction pathways that then impact other pathways through crosstalk. Crosstalk between pathways is an expanding area of research as it demonstrates how one mutation can affect other systems downstream. The identification of proteins that mediate crosstalk also allows for the potential development of therapeutic drugs to interrupt the crosstalk, potentially nullifying the initial mutation. Two pathways that are involved in many types of human cancers are the Jak-Stat and Wnt (Wingless, Wg) pathways. These two pathways are also involved in the development of a leukemia-like disease seen in Hop[Tum-l] mutants in Drosophila. Here I show that these two pathways crosstalk in vivo, mediated by the protein Mad. I show that this crosstalk is important to the proliferation of larval hemocytes and their differentiation into a specialized cell type, the lamellocyte. I propose and test a model whereby the Jak-Stat pathway activates proliferation and differentiation while the Wnt pathway activates proliferation and inhibits differentiation. I examine if differentiation in an overactive Jak-Stat mutant, Hop[Tum-l], requires activity of Mad. Additionally, I examine if Mad inhibits Wg pathway activity in hematopoiesis. From my results, I propose a system where the protein Mad mediates the crosstalk between the Jak-Stat and Wnt pathways by inhibiting the Wnt pathway.

Committee: Soichi Tanda (Advisor) Subjects: Biology; Cellular Biology; Genetics; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2017, Biomedical Sciences

Chronic pancreatitis (CP) is a devastating disease characterized by persistent inflammation and fibrosis of the pancreas leading to exocrine and endocrine insufficiency and increased risk of malignancy. Unfortunately, diagnosis of CP remains difficult and no curative therapeutic options exist, largely due to a limited understanding of the cellular and molecular mediators of CP pathology. Recent findings implicate pancreatic stellate cells (PSC) as prominent mediators of the inflammatory and fibrotic phenotype observed during CP. We hypothesized that the pro-survival Jak2/STAT3 and MAPK pathways play a role in PSC activation and proliferation and that these pathways would serve as therapeutic targets to reduce the pathogenic activity of these cells during disease. In vitro, cultured PSC demonstrated activation of both the Jak2/STAT3 and MAPK pathways and robust secretion of several soluble immunomodulatory factors including monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), and vascular endothelial growth factor (VEGF). Treatment of PSC with the small molecule Jak1/2 inhibitor Ruxolitinib reduced STAT3 phosphorylation and decreased cell growth. Treated cells remained adherent and did not display poly (ADP-ribose) polymerase (PARP) cleavage by western blot, suggesting the effects of Ruxolitinib on cell growth are not pro-apoptotic. Instead, western blot and fluorescent microscopy demonstrate a dose-dependent decrease in alpha-smooth muscle actin (aSMA), a marker of PSC activation. Treatment with a MAPK pathway inhibitor (MEK162) had no effect on growth or activation. These data suggest that the Jak/STAT pathway, and not the MAPK pathway, functions to regulate PSC growth and activation, thereby representing a viable therapeutic target. To examine this hypothesis in vivo, we utilized the caerulein-induced murine model of chronic pancreatitis. Ruxolitinib was administered at 90mg/kg twice daily by oral gavage during the final week of cae (open full item for complete abstract)

Committee: Gregory Lesinski PhD, MPH (Advisor); Aharon Freud MD, PhD (Advisor); William Carson III, MD (Committee Member); Traci Wilgus PhD (Committee Member) Subjects: Biomedical Research; Immunology

Master of Sciences (Engineering), Case Western Reserve University, 0, Biomedical Engineering

pH-stat is a pH control system that is used for studying biochemical reactions such as enzyme assays. Electrochemical micro-pH-stat based on the rotating sample system as well as microfabrication techniques have been developed in our laboratory previously, with an unmet need of reliable micro pH electrode. In this work we evaluate the Pd electrode using a novel voltammetric pH sensing technique. Pd disk microelectrodes were fabricated and the electrodes showed linear pH reseponse and super-Nerstian sensitivity (~160 mV/pH) at pH 6-7.15 in bulk solution. A micro electrochemical cell combining voltammetric pH sensing and coulometric titration was designed and constructed, together with the analog circuits and control software. The Pd electrodes, however, showed elusive pH response upon further testing in the micro electrochemical cell. In the future a different type of material and/or method may need to be considered for pH sensing in the micro-pH-stat.

Committee: Miklos Gratzl (Advisor); Christian Zorman (Committee Member); Steven Eppell (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy (PhD), Ohio University, 2016, Molecular and Cellular Biology (Arts and Sciences)

The Jak/Stat signaling pathway is one of the most conserved signaling pathways regulating cellular processes such as cell proliferation and cellular differentiation. Mutations in Jak that make it constitutively active are implicated in the development of leukemia and myeloproliferative disorders in humans. A dominant mutation in the Drosophila Janus Kinase (or hopscotch) gene called hopTum-l causes an increase in Jak/Stat pathway activity levels and significantly increases the hemocyte count. Removal of one copy of the Phosphatase 61F gene, a negative regulator of the Jak/Stat pathway, in the hopTum-l background further increases pathway activity at the molecular level, but surprisingly causes a reduction in the hemocyte count. Our results suggest that the Drosophila Signal Transducer and Activator of Transcription (Stat92E), can regulate the expression of two different sets of target genes. Low threshold genes (LTG) are expressed at moderate levels of Jak/Stat pathway activity and high threshold genes (HTG) are activated at much higher levels of Jak/Stat pathway activity. Based on our hypothesis we propose a model that predicts certain transcriptional repressors negatively regulate the expression of the HTGs at moderate levels of Jak/Stat pathway activity. Loss of function (LOF) screening helped us identify C-terminal binding protein (CtBP) and Suppressor of Hairless [Su(H)] as potential transcriptional repressors of HTGs. Two independent in silico approaches were used to discover possible regions in the Drosophila melanogaster genome that have Stat92E and repressor binding sites within 1000 bp of each other. These scans identified thirty-three potential Jak/Stat pathway target genes. RNAi analysis of thirty of these candidates was performed in the hopTum-l background to examine their effect on hematopoiesis and classify them as either LTGs or HTGs. Eleven of the thirty genes showed a genetic interaction with the Jak/Stat pathway and these eleven genes were then ex (open full item for complete abstract)

Committee: Soichi Tanda (Advisor); Mark Berryman (Committee Member); Donald Holzschu (Committee Member); Sarah Wyatt (Committee Member) Subjects: Bioinformatics; Genetics; Molecular Biology

Master of Science (MS), Ohio University, 2014, Biological Sciences (Arts and Sciences)

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease and renal failure in humans. The molecular pathways that lead to DN are not well known. This research investigates possible roles of several signal transducers and activators of transcription (STAT) proteins in this disease using a STAT5A/B knockout (SKO) mouse model. Based on previous observations of increased inflammation-related gene expression in the kidneys of diabetic SKO mice, the hypothesis of the current project was that the combination of the loss of STAT5 repression and increase of STAT3 activity escalates inflammation-related gene expression in the kidneys of diabetic SKO mice. In support of this hypothesis, an increase of IRF-1 RNA expression, reflective of the loss of STAT5 repression, was observed in the kidneys of diabetic SKO mice. Levels of phosphorylated STAT3 were also increased in the kidneys of diabetic SKO mice. These results suggest that STAT5 acts as a repressor of inflammation-related genes in DN and, in its absence, expression of these genes is no longer repressed, either due to direct loss of the STAT5 repression or due to increased STAT3 activity which could potentially increase their expression.

Committee: Karen Coschigano PhD (Advisor); Calvin James PhD (Committee Member); Ramiro Malgor MD (Committee Member) Subjects: Animals; Biology; Biomedical Research; Immunology; Molecular Biology

PhD, University of Cincinnati, 2013, Medicine: Molecular and Developmental Biology

Heparan sulfate proteoglycans (HSPGs) are macromolecules that stay on the cell surface and within the extracellular matrix (ECM). HSPG is composed of a core protein and some heparan sulfate (HS) glycosaminoglycan (GAG) chains. It is already known that HSPGs interact with a number of ligand proteins to regulate signaling activities in various developmental processes. In this thesis work, we revealed some important functions of HSPGs in the regulation of cell signaling and stem cell development. First we demonstrated that Sulf1, which removes sulfation groups on the HS chains, is a negative feedback regulator of Wingless distribution and signaling activity. Sulf1 probably modifies Dally to help the formation of a precise Wg gradient. Second, we demonstrated that JAK/STAT signaling is regulated by HSPGs. Dally and Dally-like (Dlp) are essential for the Upd distribution and thus positively regulate the JAK/STAT signaling activity. Third, we revealed the critical roles of Perlecan (Pcan) in the regulation of stem cell activity and stem cell-ECM attachment in drosophila adult posterior midgut. Our results showed that Pcan is required for the normal proliferation of intestinal stem cells (ISCs), and ISC attachment to the basement membrane. Importantly, our results allow us to propose a novel model to explain how ISC niches are formed. Our data suggested that Pcan may help ISCs to create an "activated ECM", which restricts a niche around each ISC. Altogether, this dissertation work will provide some novel insights into the functions of HSPGs in the regulation of cell signaling and stem cell development.

Committee: Xinhua Lin Ph.D. (Committee Chair); Iain Cartwright Ph.D. (Committee Member); Tiffany Cook Ph.D. (Committee Member); Brian Gebelein Ph.D. (Committee Member); James Wells Ph.D. (Committee Member) Subjects: Biology

PhD, University of Cincinnati, 2012, Medicine: Immunology

The inflammatory bowel diseases (IBD) are chronic, relapsing and remitting gastrointestinal diseases of unknown etiology. It is postulated that in genetically susceptible hosts, epithelial barrier function is disrupted and intestinal microbes activate microbial sensing systems in macrophages (MΦs)/dendritic cells, which initiate an adaptive immune response, characterized by both CD4+ T helper (TH)1 and TH17 cells in Crohn's disease (CD) and by a TH17/TH2 response in ulcerative colitis (UC). These activated T cells release an array of pro-inflammatory cytokines, which promotes the recruitment of other immune cells, including monocytes/MΦs, neutrophils and eosinophils, all of which cause extensive damage to the local tissues. The involvement of cell-cell interactions between eosinophils and MΦs and the importance of these interactions to the pathophysiology of IBD remain poorly defined. In this dissertation, we employ a mouse model of experimental colitis to further understand the interactions between eosinophils and MΦs in the pathogenesis of the chronic inflammatory response in IBD. We demonstrate increased eosinophil numbers and degranulation in rectosigmoid pediatric UC colonic biopsies compared to healthy control biopsies and that eosinophil levels correlate with disease severity. In preclinical studies employing eosinophil-deficient mice, we demonstrate that eosinophils play a pathogenic role in the histopathology of DSS-induced colitis. Furthermore, we demonstrate increased levels of the eosinophil-specific chemokines, eotaxin-1/CCL11 and eotaxin-2/CCL24 in the colon during DSS-induced colitis, and we identify that CCL11 and not CCL24 is required for DSS-induced eosinophilia. Furthermore, we identify F4/80+ MΦs as a cellular source of CCL11 and demonstrate a role for a subset of pro-inflammatory colonic MΦs (F4/80+CD11b+Ly6Chi) in CCL11 expression and eosinophil recruitment in DSS-induced colitis. Consistent with this preclinical observation, we show that CCL11 (open full item for complete abstract)

Committee: Simon Hogan PhD (Committee Chair); Lee Denson MD (Committee Member); Kris Steinbrecher PhD (Committee Member); Marsha Wills-Karp PhD (Committee Member); Nives Zimmermann PhD (Committee Member) Subjects: Immunology

Doctor of Philosophy, The Ohio State University, 2003, Veterinary Clinical Sciences

The objective of this dissertation was to determine if the pH-stat modified therapy is more effective than the pH-stat therapy in treating hemorrhagic shock and vascular hyporeactivity in dogs. The specific aims were to compare the effectiveness of these two treatments on systemic hemodynamics and acid-base balance and to evaluate their efficacy in preventing vascular hyporeactivity in dogs with hemorrhagic shock. Twenty-three anesthetized dogs were randomly assigned to four groups: I: One (1) dog included as control to provide baseline and control data; II: Seven (7) hemorrhaged, hypothermic [HT] (32° C) dogs received lactated Ringers solution (LRS); III: Seven (7) hemorrhaged, HT dogs received LRS and were subjected to the pH-stat therapy; IV: Eight (8) hemorrhaged, HT dogs received LRS and underwent the pH-stat modified therapy. Dogs were intubated and mechanically ventilated on room air and end tidal CO2 of 35-40 mm Hg. Approximately 40% of each dogs blood volume was removed until mean arterial pressure (MAP) reached 50 mm Hg. MAP was held constant for 60 minutes. After bleeding, all dogs were treated with LRS. Dogs in group II received LRS. Dogs in group III and IV received LRS and either pH-stat or pH-stat modified therapies. pH-stat keeps arterial pH at 7.40 and PCO2 at 40 mm Hg regardless of body temperature. PaO2, PCO2 and pH were corrected for body temperature. pH-stat modified maintains a fixed PCO2 of 60 mm Hg and a pH of 7.30. Hemodynamics and acid-base data were measured at baseline, through hemorrhage, end of treatment, and at 60, 120 and 180 minutes post-resuscitation. Vascular reactivity to norepinephrine infusion and blood flow to the hind limb and small intestine were determined. Through bleeding, heart rate increased insignificantly in all groups and returned to values not different from baseline. None of the treatments was superior in sustaining left ventricular systolic pressure and preload. The 3 methods produced transient improvements in phys (open full item for complete abstract)

Committee: William Muir (Advisor) Subjects: Biology, Veterinary Science

Doctor of Philosophy (PhD), Ohio University, 2010, Chemistry and Biochemistry (Arts and Sciences)

Melanoma is an aggressive form of skin cancer with high occurrence in the United States. Interferon α2b (IFNα2b/IFNα2) has been used in high doses to treat melanoma. However, problems associated with small therapeutic proteins, such as with interferon treatment, include degradation by serum proteases and rapid kidney clearance because of small molecular size. Pegylation increases the size of the molecule but creates a host of other issues, such as decrease receptor binding, non-specific chemical derivatization, low overall yields and additional purification steps. In this study we used an alternative approach of IFN produced as an arabinogalactan fusion protein in plant cells. These IFN analogues bind to IFN receptors and follow the IFN induced JAK-STAT signaling pathway in melanoma cells. Experiments also demonstrate that these fusion proteins of higher molecular weight cause similar growth inhibition and affect cell cycle distribution. Further, the fusion proteins increased translation of 2'5' OAS1 and PKR, known IFN induced proteins, showing similar downstream signaling as native recombinant IFNα2. The tumor suppressor p53 gets activated in response to DNA damage and has interferon stimulated response elements (ISREs) in its promoter region and hence can be induced by IFN. Additionally, it has a significant role in mediating apoptosis by activating several intracellular pathways as well as up regulating proteins involved in cell cycle arrest. In this study we show that the fusion analogue IFNα2-(SO)20,as well as recombinant IFNα2b, were able to stabilize p53 protein levels and its pro-apoptotic target Bax. Also, there was a decrease in HDM2 levels, the negative regulator of p53. These results suggest that p53 is a downstream signaling target of IFNs and has a possible role in IFN mediated effects in these melanoma cells.

Committee: Susan Evans PhD (Advisor); Xiaozhuo Chen PhD (Committee Member); Marcia Kieliszewski PhD (Committee Member); Glen Jackson PhD (Committee Member) Subjects: Biochemistry

Doctor of Philosophy (PhD), Ohio University, 2007, Biological Sciences (Arts and Sciences)

The evolutionarily conserved JAK/STAT pathway plays a critical role in Drosophila hematopoiesis. A gain-of-function mutation of the Janus kinase (JAK), hopscotchTumorous-lethal (hopTum-l), leads to hemocyte overproliferation and abnormal lamellocyte differentiation. In this study, I demonstrated that effects of hopTum-l were manifested in different types of hemocytes and its activity was regulated in various ways. Firstly, hopTum-l affects proliferation and differentiation of hemocytes in a temperature-dependent manner. Male larvae raised at a lower temperature exhibited the most pronounced effect in hemocyte proliferation and were used for further study. Activation of the JAK/STAT pathway in specific compartments of the lymph gland resulted in different proportions of terminally differentiated hemocytes. Furthermore, to understand the regulatory roles of the Drosophila SUMO conjugase Lesswright (Lwr) and the putative Drosophila SUMO ligase protein inhibitor of activated STAT (dPIAS) in the JAK/STAT pathway, lwr and dpias were expressed in the hopTum-l background. Expression of lwr and dpias both suppressed hopTum-l-induced hemocyte overproduction and the effects were additive, which raises the possibility that lwr and dpias egatively regulate the JAK/STAT pathway through SUMOylation. Finally, the JAK/STAT pathway interacts with the Toll (Tl) pathway, another important pathway involved in larval hematopoiesis. Removal of the Tl pathway transcription factors, Dl and Dif, did not diminish hopTum-l-induced hemocyte overproduction. Moreover, activation of the Tl pathway led to nuclear accumulation of activated Stat mainly in lamellocytes, which suggests that the Tl pathway may be functionally upstream of the JAK/STAT pathway in lamellocyte differentiation. On the other hand, activation of the Tl pathway suppressed hopTum-l-induced hemocyte overproduction, possibly through downstream component Dl. This suppression was observed primarily in plasmatocytes, the professional (open full item for complete abstract)

Committee: Soichi Tanda (Advisor) Subjects:

Doctor of Philosophy, Miami University, 2004, Microbiology

Interferon (IFN)-gamma-induced indoleamine 2,3-dioxygenase (IDO) activity inhibits the growth of some intracellular pathogens by catalyzing the oxidative cleavage of the indole ring of L-tryptophan and depleting pools of this essential amino acid. Tumor necrosis factor (TNF)-alpha synergistically enhances the IDO activity induced by IFN at the level of transcription in human epithelial cells. The purpose of this study was to characterize the molecular mechanisms responsible for synergistic gene expression in response to IFN and TNF. It was found that IFN-induced binding of STAT-1 to gamma activation sequences (GAS) and IFN responsive factor (IRF)-1 to IFN-stimulated response elements (ISRE), is more highly activated following treatment with interferon and TNF. This enhanced signal transduction is due to the increase in IFN receptor expression following combined cytokine stimulation. CCAAT enhancer binding protein (C/EBP)-beta binds to one of three consensus C/EBP sites in the IDO regulatory region in response to TNF alone or in combination with IFN. A transcriptional reporter construct consisting of green fluorescent protein expressed from the IDO regulatory region was utilized to understand which enhancer regions are responsible for synergistic IDO gene expression in response to IFN and TNF. Mutation of other individual enhancers and large deletions within the regulatory region showed that increased binding of IFN-specific factors to GAS and ISRE sites is alone responsible for synergistic transcriptional activation. An indirect requirement for NF-KB in synergistic IDO expression by regulating IRF-1 expression was also explored. A gamma activated sequence and a KB site, respectively, reside in the IRF-1 regulatory region. It was important to identify whether increased translocation of NF-KB to the nucleus and binding to the kB site upstream of the IRF-1 gene in response to IFN and TNF, is rate-limiting in enhanced IRF-1 expression. Limiting NF-KB translocation to th (open full item for complete abstract)

Committee: Joseph Carlin (Advisor) Subjects:

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2009, College of Science

Cytokines are intracellular messengers that activate multiple signaling pathways and regulate cell survival, differentiation, migration, apoptosis and immune responses. We are investigating the homeostatic control of cytokine-mediated cell signaling using interleukin-4 (IL-4) as a model cytokine. Protein tyrosine phosphorylation is essential for cytokine-dependent signal transduction through the Janus Kinase-Signal Transducer and Activator of Transcription (Jak-STAT) pathway. Therefore, protein tyrosine dephosphorylation is a key mechanism providing the homeostatic control of the pathway. Here, we have shown for the first time that immediately following ligand-dependent activation, IL-4 receptor induces an intracellular calcium flux via insulin receptor substrate (IRS)-phosphoinositide 3-kinase (PI3K)-phospholipase C (PLC)-gamma pathway which, in turn, induces protein kinase C (PKC)-dependent activation of NAD(P)H oxidase (NOX)5 that generates reactive oxygen species (ROS). IL-4 also induces NOX1-mediated ROS production via IRS-PI3K-Ras-related C3 botulinum toxin substrate (RAC) 1 pathway. Moreover, we have also demonstrated that IL-4-generated ROS, in turn, promote IL-4 receptor activation by oxidatively inactivating protein tyrosine phosphatase 1B (PTP1B) that physically associates with and deactivates IL-4 receptor. In addition, we have shown that antioxidant enzymes peroxiredoxin II, IV and VI (Prx II, IV, VI) physically interact with the IL-4 receptor and become oxidized by IL-4 generated ROS, thereby neutralizing IL-4 generated ROS and compromising ROS-mediated amplification of IL-4 signaling. Cytokine-activated Jak-STAT pathway is believed to operate from cell surface to the nucleus via DNA-protein and protein-protein interactions without involving any second messengers. Here, we demonstrate, for the first time to our knowledge, a role for second messengers (ROS) in the amplification of Jak-STAT signal transduction in IL-4-stimulated cells. Further, our data (open full item for complete abstract)

Committee: Saikh J. Haque Ph.D. (Advisor); Anton Komar Ph.D. (Committee Member); Barsanjit Mazumder Ph.D. (Committee Member); Dennis J Stuehr Ph.D. (Committee Member); Xiaoxia Li Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Cellular Biology; Immunology; Molecular Biology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2008, College of Science

FUNCTIONAL CHARACTERIZATION OF SCN5A, THE CARDIAC SODIUMCHANNEL GENE ASSOCIATED WITH CARDIAC ARRHYTHMIAS AND SUDDEN DEATH LING WU ABSTRACT The cardiac sodium channel α subunit Nav1.5 (encoded by the SCN5A gene) plays an important role in the generation and propagation of electrical signals in the heart, and can cause cardiac arrhythmias, heart failure and sudden death when mutated or dysregulated. However, the precise composition of the multi-protein complex for the sodium channel has not been completely defined. The molecular mechanisms by which Nav1.5 mutations cause cardiac arrhythmias have not yet been well-studied in vivo. It remains to be explored whether Nav1.5 is expressed in other tissues and plays novel roles in other tissues or organs. This dissertation addresses these aspects of Nav1.5 regulation. I found that MOG1, a small protein that is highly conserved from yeast to humans, is a central component of the channel complex and distinctly modulates the physiological function of Nav1.5. A yeast two-hybrid screen identified MOG1 as a new protein that interacts with the cytoplasmic loop II (between transmembrane domain DII and III) of Nav1.5. The interaction was further demonstrated by both in vitro GST pull-down and in vivo co-immunoprecipitation assays. Co-expression of MOG1 with Nav1.5 in HEK293 cells increased sodium current densities, whereas two siRNAs that knocked down expression of MOG1 decreased current densities. In neonatal myocytes, over-expression of MOG1 increased current densities nearly two-fold, and MOG1 siRNAs down-regulated the sodium currents. Immunostaining revealed that in the heart, MOG1 was expressed in both atrial and ventricular tissues and was highly localized in the intercalated discs. These results suggest that MOG1 may be a critical regulator of sodium channel function in the heart and reveal a new function for MOG1. Furthermore, I showed that MOG1 increased sodium current density by increasing cell membrane localization of Nav (open full item for complete abstract)

Committee: Dr. Qing Wang (Advisor) Subjects: Biochemistry; Biology; Biomedical Research; Biophysics; Biostatistics

Master of Sciences (Engineering), Case Western Reserve University, 2010, EECS - Electrical Engineering

Conventional pH-stats use titration to add acid or base solution to control sample pH. This technique is impractical for small volume samples because fluidic addition leads to dilution. The goal of this work is to produce a microfluidic electrochemical pH-stat, which utilizes water electrolysis for hydrogen/hydroxyl ion generation. This technique does not induce volume change and can be precisely controlled and measured to determine reaction rates. The device consists of a PDMS-based microchannel that is sandwiched between two glass substrates. The glass substrates support microfabricated electrodes for current injection and pH sensing. Fabrication of the pH-stat utilizes a novel vacuum-assisted micromolding process capable of producing microfluidic channels while maintaining substrate and electrode surface integrity. The pH-stat analyzes two microliters of fluid, uses capillary flow for fluid transport, and ion diffusion as the mixing mechanism. Preliminary evaluation with a sample containing pH-dye shows successful fluid transport, pH modification, and ion diffusion.

Committee: Christian Zorman (Advisor); Miklos Gratzl (Committee Member); Frank Merat (Committee Member) Subjects: Electrical Engineering

Doctor of Philosophy, Case Western Reserve University, 2009, Biomedical Engineering

Point-of-care testing (POCT) generally refers to the implementation of laboratory tests near the patient with the goal to minimize turnaround time, reduce medical cost, and improve medical outcome. The aim of this research is to develop, validate, and optimize a preliminary POCT system for body fluid diagnostics using our existing analytical platform, Rotating Sample System (RSS). The RSS is a convective platform for different optical and electrochemical analyses in microliter-sized samples. In this work we have developed a reagent-free electrochemical micro pH-stat for in vitro enzyme assays where a titrant of acid or base is produced by water electrolysis on the RSS platform. As water electrolysis induces no volume change and the current that generates the reagent can be precisely measured even at low levels, very small samples in the 1-20 μL volume range become accessible for pH-stating: a reduction of more than an order of magnitude in specimen size relative to the most conventional methods. More importantly, analysis using untreated biological sample is feasible as turbidity would not influence pH-stat measurement. The RSS micro pH-stat effectively operates as a galvanostat with an output range of ±0.1-100 μA and can withstand sample impedance up to 100 kΩ. The cathodic current efficiency is virtually 100% in both buffer and serum samples. Results of cholinesterase activities in both buffer and serum using the proposed reagent-free pH-stat have been validated with standard optical techniques (r2 ≥ 0.97). This novel technique hence has great potential to become a miniaturized analyzer for point-of-care diagnostics.

Committee: Miklos Gratzl PhD (Advisor); Clive Hamlin PhD (Committee Member); Uziel Landau PhD (Committee Member); Andrew Rollins PhD (Committee Member); Christian Zorman PhD (Committee Member) Subjects: Biomedical Research; Engineering