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

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

Bachelor of Science (BS), Ohio University, 2014, Biological Sciences

Cancer is very prevalent in our society, and encompasses more than 100 different diseases. Additionally, each cancer can act distinctly, and current cancer therapies, such as chemotherapy and radiation, can lead to future complications. These facts implicate the need for new avenues of safer and more effective treatments. Oncogenic stresses are a hallmark characteristic of cancer, arising from the rapid growth and proliferation of cells. In response to these oncogenic stresses, the cells respond in a variety of ways, such as cell cycle arrest, DNA repair mechanisms, and cell death, in an attempt to overcome these stresses in and proliferate. Additionally, cancer is characterized by the activation of oncogenes and the loss of tumor-suppressor genes, which are involved in cell cycle arrest, as well as cell death. Therefore, in this project we investigated the link between cell death and cancer stresses by examining the interaction of p53, Clic, and the JAK/STAT pathway, all of which are known to be mutated in some cancers. The results presented in this thesis suggest that the activation of JAK/STAT pathway through the hopTum mutation creates a pro-apoptotic environment that likely stimulates p53 function. Furthermore, this stimulation is activating the canonical p53/apoptotic pathway, as well as a non-canonical p53 cell death pathway. Our results also implicate that Clic is acting in an anti-apoptotic manner.

Committee: Soichi Tanda (Advisor) Subjects: 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

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 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, 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: