Master of Sciences, Case Western Reserve University, 2018, Physiology and Biophysics

Erythropoietin-producing human hepatocellular (Eph) receptors account for the majority of the membrane-bound receptor tyrosine kinase (RTK) family. Eph receptors have significant roles during embryonic development, cell maturation, and adulthood. Furthermore, studies have shown that drastic increase in the expression of Eph receptors is detected in the malignant proliferation of tumor cells in numerous cancers, such as melanoma, breast cancer and glioblastoma. Indeed, recently, oncogenic activities have been observed in non-canonical unliganded Ephrin type-A receptor 2 (EphA2). This project aims to probe the effects of phosphorylation on the intracellular domain interactions of EphA2 in solution. Results from this study indicate that deletion of the sterile a motif (SAM) domain leads to a strong binding affinity between kinase domains in solution. Upon oligomerization, less kinase activity is observed, compared to that of the monomeric state of the intracellular domain (ICD) of EphA2. Moreover, in this study, microscale thermophoresis (MST) is utilized to assess the binding interactions of EphA2 in complex with liposomes, a membrane-like environment. Upon the truncation of the SAM domain, binding of the intracellular domains of EphA2 to the membrane is abrogated. Data from this study will help to elucidate and further our understanding of the signaling mechanism of receptor tyrosine kinases and the regulatory role of phosphorylation.

Committee: Matthias Buck (Advisor); Sudha Chakrapani (Committee Member); Rajesh Ramachandran (Committee Member); Bingcheng Wang (Committee Member); William Schilling (Committee Chair) Subjects: Biophysics; Physiology

Doctor of Philosophy, The Ohio State University, 2018, Biomedical Engineering

Collagen type I is the most abundant extracellular matrix (ECM) protein found in vertebrates. A number of cell surface receptors and as well as glycoproteins and proteoglycans found in the ECM are known to interact with collagen. The clustering or oligomeric state of these proteins is emerging as an important factor in modulating their interaction with collagen. This thesis attempts to better understand the role of oligomeric status of the collagen receptors, Discoidin domain receptors 1 and 2 (DDR1 and DDR2), in their interaction with collagen. DDRs are widely expressed receptor tyrosine kinases which modulate cells signaling as well as ECM remodeling. The first aim of this thesis (Chapter 2) was to understand how ligand binding impacts the clustering or oligomeric state of DDRs interaction. AFM imaging provided insight on how the extracellular domain (ECD) of DDR1 but not DDR2 was able to cluster upon collagen binding. Live cell assays where the murine osteoblastic cell line, MC3T3-E1, was transfected to express full length fluorescent DDR1 and DDR2 proteins showed that DDR1 clusters upon collagen stimulation whereas DDR2 does not. Further, receptor phosphorylation after collagen binding occurred in DDR1 clusters. On the other hand, DDR2 phosphorylation was found in unique filamentous organizations of the protein. Receptor phosphorylation for both DDR1 and DDR2 was observed several hours post collagen binding, consistent with earlier studies. The second aim of this work (Chapter 3) was to investigate how the oligomeric state of DDR ECD affects their binding to collagen and the structural and mechanical properties of the collagen network. Solid phase binding was used to observe how DDR oligomerization increased their binding affinity towards collagen. AFM and Confocal fluorescence microscopy imaging of collagen gels revealed that DDR2-Fc dimers, and to an even greater extent, DDR2-Fc oligomers reduced the fibril diameter and disrupted the network architecture (open full item for complete abstract)

Committee: Gunjan Agarwal (Advisor); Heather Powell (Committee Member); Gregory Lafyatis (Committee Member); Do-Gyoon Kim (Committee Member) Subjects: Biomedical Engineering

MS, University of Cincinnati, 2016, Medicine: Cancer and Cell Biology

Prostate cancer (PCa) is one of the leading causes of death among men in the United States, particularly given that many patients will present with or develop castration-resistant prostate cancer. Identifying novel biomarkers is therefore essential to develop new therapeutics for the treatment of this disease. While current therapeutics have targeted the androgen receptor, a major driver in the development and progression of PCa, this has been met with limited success. Understanding the role of other potent drivers of PCa is paramount to successful patient outcomes. One potential driver may be the Ron receptor tyrosine kinase. Chronic inflammation has been suggested to be a precursor for the development of PCa and interestingly, Ron has been shown previously to be a regulator of macrophage inflammatory responses. In addition, new studies have suggested its role in oncogenesis. While there are Ron inhibitors in clinical trials for the treatment of several malignancies, there have been no trials looking at Ron inhibition in PCa. Therefore, understanding how Ron functions to mediate inflammatory responses and drive oncogenic events are crucial to potentially utilizing Ron inhibitors for the treatment of prostate cancer.

Committee: Tatiana Kalin M.D.Ph.D. (Committee Chair); Rafeeq Habeebahmed Ph.D. (Committee Member); Atsuo Sasaki Ph.D. (Committee Member) Subjects: Cellular Biology

PhD, University of Cincinnati, 2022, Medicine: Cancer and Cell Biology

Despite a 40% decline in the breast cancer mortality rate over the past 40+ years, breast cancer continues to claim the lives of more than 44,000 individuals each year. Imaging advances have increased the proportion of breast cancers diagnosed in early stages, and treatment advances have allowed for high survival rates of breast cancer patients, however 5 year survival drops to <30% when locoregional recurrence is detected, and <10% when recurrence is distant metastasis. Critically, breast cancer recurrence remains a significant barrier in the treatment of this disease. In this dissertation, we demonstrate the relationship between RON expression and clinical outcomes of breast cancer patients wherein RON expression predicts early death and metastatic progression. In pre-clinical models, RON drives breast cancer recurrence, metastatic progression, and breast cancer stem cell self-renewal. RON sustains activation through co-overexpression with Hepatocyte Growth Factor-Like (HGFL) that cooperates with physiologic sources of HGFL. Tumor cell produced HGFL acts in an autocrine manner to alter the tumor cell secretome to attract macrophages and dampen T cell responses. It also acts in a paracrine fashion to activate RON on macrophages within the tumor microenvironment to alter the macrophage secretome, further dampening T cell responses and promoting tumor growth. Within tumor cells, RON signaling alters metabolic pathways including enhancement of glycolysis and cholesterol biosynthesis. Under sustained RON activation, glycolysis-provided substrate for cholesterol production is required for breast cancer stem cell self-renewal, recurrence, and metastatic progression. Upregulated glycolysis occurs by RON-dependent ERK1/2 activation that upregulates c-Myc, which upregulates glycolysis genes such as hexokinase-2 and lactate dehydrogenase a. Upregulated cholesterol biosynthesis occurs by RON-dependent non-canonical ß-catenin activation that upregulates SREBP2, which upregula (open full item for complete abstract)

Committee: Susan Waltz Ph.D. (Committee Member); John Cunningham Ph.D. (Committee Member); Lisa Vinnedge Ph.D. (Committee Member); Vinita Takiar M.D. Ph.D. (Committee Member); Jun-Lin Guan Ph.D. (Committee Member) Subjects: Cellular Biology

PhD, University of Cincinnati, 2020, Medicine: Cancer and Cell Biology

Prostate cancer is the second most common cancer in men and the second leading cause of cancer-related deaths in men in the United States, with 191,930 men estimated to be diagnosed with prostate cancer and 33,330 patients expected to succumb to this disease in 2020 alone. Standard-of-care treatments, including active surveillance, surgery, radiation, and androgen deprivation therapy, provide positive clinical outcomes in patients with localized and regional disease. However, these strategies fail to effectively treat advanced prostate cancer, leading to a 5-year survival rate of 31% in patients with distant stage disease. Cancer immunotherapy has emerged as a powerful therapeutic strategy by enhancing the host immune response, particularly cytotoxic CD8+ T cells, to attack tumors. These strategies have proven successful in numerous cancers, such as melanoma, non-small cell lung cancer, and bladder cancer, however prostate cancer remains poorly responsive to these agents. The work presented in this dissertation demonstrates that the RON receptor tyrosine kinase, an oncogene implicated in the tumorigenesis of several epithelial cancers, is a critical regulator of the antitumor immune response in prostate cancer. Our studies herein identify novel RON-mediated communication networks between prostate tumor cells and immune cells as well as between different immune cell populations in the tumor microenvironment. We show that RON signaling in prostate epithelial cells and macrophages supports M2 macrophage activation and modulates the infiltration of multiple immune cell types with known roles in regulating prostate tumor growth and progression, such as CD4+ and CD8+ T cells. Importantly, our studies reveal that loss of RON signaling in macrophages promotes CD8+ T cell activation. Our deeper analyses show that the regulation of M2 macrophage activation by RON is in part through upregulation of RON expression in macrophages. Further, our preliminary data suggest that RON s (open full item for complete abstract)

Committee: Susan Waltz Ph.D. (Committee Chair); George Deepe M.D. (Committee Member); Susan Kasper Ph.D. (Committee Member); David Plas Ph.D. (Committee Member); Kathryn Wikenheiser-Brokamp M.D. (Committee Member) Subjects: Cellular Biology

Master of Science (MS), Wright State University, 2019, Microbiology and Immunology

Macrophages are phagocytic cells located in tissues, organs and even circulated within our body as white blood cells. They are critical in detecting tissue damage and infection. Resident tissue macrophages initiate the signals for inflammation recruiting neutrophils and blood monocytes which mature into macrophages at sites of infection and in the resolution of inflammation. Based on the local cytokine milieu in tissue sites, macrophages may be polarized into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. Receptor tyrosine kinase Mer (MERTK) helps in clearing dead neutrophils and other apoptotic cells from damaged tissue sites preventing chronic inflammation and autoimmune disorders. MERTK aids in the maintenance of tissue homeostasis and wound healing. Phosphatidylserine (PtdSer) present on the surface of apoptotic cells release “eat me” signals which are recognized by the two “bridging ligands” of MERTK receptor, Gas6 and ProS. The binding of the ligands to PtdSer initiates intracellular signals leading to phagocytosis of the cell. MERTK receptor is expressed mostly on M2c macrophages. The current study explores the expression rate of the phagocytic receptor MERTK, on macrophages polarized with either IL-10 (M2c ells) or IL-4 or IL-13 (M2a macrophages) following treatment with the suppressor of cytokine signaling SOCS3 in comparison with macrophage polarization with only IL-10 or IL-4 or IL-13 . The current study exhibits an enhancement in the expression of the phagocytic MERTK receptor on the surface of IL-10 polarized M2c macrophage when treated with SOCS3 in comparison to IL-10 polarized M2c macrophage, IL-4 polarized M2a macrophage and IL-13 treated M2a macrophage. IL-13 polarized M2a macrophage also shows an increase in the expression of MERTK receptor which is similar to a previous study where a similar receptor to MERTK termed “Axl receptor” is enhanced by IL-13 treatment on bone- marrow derived macrophage. SOCS3 when treated with IL-13 polar (open full item for complete abstract)

Committee: Nancy J. Bigley Ph.D. (Advisor); Dawn P. Wooley Ph.D. (Committee Member); Marjorie Markopoulos Ph.D. (Committee Member) Subjects: Immunology; Microbiology

Doctor of Philosophy, The Ohio State University, 2019, Vision Science

Purpose. Half of the nearly 500 million diabetic patients globally will develop degenerative corneal changes, termed diabetic keratopathy, during the course of their disease, leading to sight-threatening scarring. Dogs are a naturally occurring model of insulin-dependent diabetes and develop ocular surface changes with disease; however, corneal fragility and delayed wound healing are not observed clinically in the dog. Due to the consistently increasing prevalence and estimated costs to treat diabetic keratopathy, there has been a growing effort to identify novel therapies to minimize discomfort and sight-threatening scarring. Topical insulin has been shown to reduce wound area and restore corneal sensitivity in diabetic rats and promote cellular proliferation. Both the insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1-R) stimulate cell signaling events through activation of a receptor tyrosine kinase (RTK), although different cellular pathways can be initiated due to the different properties of each receptor. Additionally, the two RTKs can each contribute two subunits to yield a hybrid receptor (Hybrid-R). RTK expression within ocular tissue has been evaluated in humans and rodents. The purpose of this study was to assess in vitro cytotoxicity with insulin treatment and suggest a mechanism by which improved wound healing occurs by characterizing RTK and induced kinase expression within the PI3K-Akt pathway in both human and canine corneal cells. Methods. Lactate dehydrogenase (LDH) assays were performed to assess for cytotoxicity. In vitro scratch tests were used to evaluate wound healing outcomes under variable glucose conditions in the presence or absence of human recombinant insulin. Fluorescent cellular glucose uptake and protein and DNA synthesis assays were performed to characterize the effect of insulin on glucose transport dynamics and protein and DNA synthesis, respectively. Protein co-immunoprecipitation, RT-PCR, and immunohistoche (open full item for complete abstract)

Committee: Heather Chandler PhD (Advisor); Andrew Hartwick OD, PhD (Committee Member); Timothy Plageman PhD (Committee Member); Amit Sharma PhD (Committee Member) Subjects: Biomedical Research; Cellular Biology; Molecular Biology; Ophthalmology

PhD, University of Cincinnati, 2018, Medicine: Cancer and Cell Biology

Granulocyte-colony stimulating factor receptor (G-CSFR) controls myeloid progenitor proliferation and differentiation to neutrophils. Mutations in CSF3R (encoding G-CSFR) have been reported in patients with chronic neutrophilic leukemia (CNL) and acute myeloid leukemia (AML); however, despite years of research, the malignant downstream signaling of the mutated G-CSFRs is not well understood. To have a system level understanding of the normal and malignant G-CSFR signaling, a global quantitative phosphoproteomics approach was utilized. BaF3 murine cell line based in vitro model system was developed expressing wild type (WT), proximal mutation (T618I), and distal truncation mutation (Q741x). SILAC-based quantitative phosphoproteomics studies were performed with WT and mutant receptors by sequential enrichment for phosphotyrosine (pTyr) followed by nano-LC-MS/MS analyses. The flow-through of pTyr enrichment was further processed for phospho-serine/threonine using titanium dioxide columns. Based on computational, bioinformatics and kinase enrichment analysis of the pTyr data, Bruton's tyrosine kinase (Btk) was found to be activated (increase in phosphorylation) in the mutated receptors but not in WT. The aberrant activation of Btk was further validated using immunoblots in the BaF3 and 32D cell line lysates (murine myeloid 32D cell line based in vitro model system was generated expressing WT, and mutant receptors similar to BaF3 system). Furthermore, ibrutinib based chemical inhibition of Btk was performed in the 32D as well as primary bone marrow cells from WT and truncation G-CSFR knock-in mouse and the readout was measured by cell death (for IC50 analysis) and colony forming unit (CFU) assays. Chemical inhibition of Btk led to increased sensitivity (lower IC50) in both 32D and primary cells and significantly lower clonogenicity in the mutant cells compared to WT primary cells. To further validate the findings of the in vitro and mouse in vivo studies in human sam (open full item for complete abstract)

Committee: Kenneth Greis Ph.D. (Committee Chair); Mohammad Azam Ph.D. (Committee Member); H. Grimes Ph.D. (Committee Member); Patrick Limbach Ph.D. (Committee Member); Daniel Starczynowski Ph.D. (Committee Member); Xiaoting Zhang Ph.D. (Committee Member) Subjects: Health Sciences

PhD, University of Cincinnati, 2010, Medicine : Cell and Molecular Biology

Prostate cancer is a leading cause of cancer related death among men in the United States, and despite current therapies, once the cancer progresses and metastasizes, the mortality rate is dramatically increased. Therefore, advances in our understanding of prostate cancer signaling pathways involved in prostate tumor progression are critical for the development of new therapeutic options. Prostate tumor angiogenesis has been shown to be critical for prostate tumor growth and metastasis, and the production of angiogenic chemokines is important for tumor vascularization. Angiogenic chemokines are a group of cytokines that act as chemoattractants for endothelial cells, and have been implicated in the pathogenesis of prostate cancer. A receptor tyrosine kinase termed Ron has also been implicated in several human cancers, including prostate cancer. In our studies, we sought to determine the impact of Ron receptor signaling in prostate cancer cells on the production of angiogenic chemokines. Our data show that Ron is highly expressed in human prostate cancer specimens compared to normal prostate tissue, and is also highly expressed in PC-3 and DU145 prostate cancer cell lines. Interestingly, Ron expression correlated with angiogenic chemokine production in prostate cancer cells. Inhibition of Ron in PC-3 or DU145 cells resulted in decreased angiogenic chemokine production, with no effect on VEGF or on the angiostatic chemokine CXCL10. The impact of Ron on the production of angiogenic chemokines is at least partially dependent on the activation of the NF-kappaB transcription factor, which has previously been shown to regulate angiogenic chemokine production in prostate cancer cells. Additionally, overexpression of Ron in LNCaP prostate cancer cells is sufficient to induce production of the angiogenic chemokine CXCL8, and these levels can be abrogated using an inhibitor of NF-kappaB. Furthermore, compared with control PC-3 cells, Ron-knockdown PC-3 cells orthotopically tran (open full item for complete abstract)

Committee: Susan Waltz PhD (Committee Chair); Arthur Buckley PhD (Committee Member); Susanne Wells PhD (Committee Member); Alex Lentsch PhD (Committee Member); Karen Knudsen PhD (Committee Member) Subjects: Molecular Biology

PhD, University of Cincinnati, 2009, Medicine : Cell and Molecular Biology

The Ron receptor tyrosine kinase is overexpressed in many human cancers including colorectal and breast, and studies have established Ron as a predictor of disease outcome and as a therapeutic target. Ron overexpression and constitutive activation contributes to the tumorigenic properties of human colon cancer cells. Moreover, metastatic dissemination of colon cancer cells from primary orthotopic tumors in mice can be reduced upon Ron knockdown. The majority of hereditary and sporadic colorectal cancers harbor aberrant Apc/β-catenin signaling, however, the relationship between Ron, Apc, and β-catenin signaling in intestinal tumorigenesis is not well understood. We sought to test the requirement of Ron tyrosine kinase signaling for initiation of intestinal tumors in vivo using a well-characterized mouse model of mutant Apc-driven intestinal tumorigenesis. By generating (ApcMin/+ mice with a targeted deletion of the tyrosine kinase domain of Ron (RonTK-/-), we found that Ron is not required for intestinal adenoma formation, and that Ron loss increases tumor burden in a large fraction of mice. Unexpectedly, the loss of Ron in non-transformed intestinal epithelium significantly increases crypt cell proliferation, which may lead to an increased susceptibility to tumor initiation in this model. β-catenin localization and target gene expression were not significantly altered in ApcMin/+;RonTK-/- mouse tumors or normal intestine compared to controls, suggesting that Ron is not required for β-catenin signaling in this model. Like in colon cancer, Ron overexpression has also been observed in approximately half of human breast cancers. Mammary-specific overexpression of Ron in mice results in mammary carcinomas in 100% of mice that metastasize to the lungs and liver, supporting the conclusion that Ron overexpression is a causal oncogenic factor in breast cancer. Interestingly, mammary glands from virgin mice with aberrant Ron expression have dilated mammary ducts and sparse du (open full item for complete abstract)

Committee: Susan Waltz PhD (Committee Chair); Kathleen Goss PhD (Committee Member); Christopher Wylie PhD (Committee Member); Nelson Horseman PhD (Committee Member); Erik Knudsen PhD (Committee Member); Sohaib Khan PhD (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy, The Ohio State University, 2011, Comparative and Veterinary Medicine

Osteosarcoma (OSA) is the most common malignant bone tumor in children and dogs. Currently, 30-40% of children and greater than 90% of dogs succumb to the disease following treatment. The receptor tyrosine kinase (RTK) Met has emerged as a potential target for therapeutic intervention for OSA. Met overexpression in human OSA is associated with a more aggressive phenotype and poor survival, and aberrant expression in normal human osteoblasts induces malignant transformation. The purpose of this work was to evaluate Met as a target for therapeutic intervention in OSA. The first objective was to determine if Met interacts with heat shock protein 90 (Hsp90) in OSA and evaluate a novel Hsp90 inhibitor, STA-1474, for OSA treatment. Hsp90 associated with co-chaperones in canine OSA cells but not normal canine osteoblasts, indicating formation of an active superchaperone complex in malignant versus normal tissue. STA-1474 promoted loss of cell proliferation, apoptosis, and induction of Hsp70. STA-1474 treatment also resulted in the down-regulation of HGF (hepatocyte growth factor) induced p-Met/Met, p-STAT3, and p-Akt/Akt. These data suggest that STA-1474 may be useful for the treatment of OSA. The second objective was to determine whether RTKs Met, epidermal growth factor receptor (EGFR), and Recepteur d'origine nantais (Ron) interact in OSA and explore the functional consequences of such interactions. EGFR and Ron phosphorylation was present in canine OSA tumor tissues, and Met was associated with EGFR and Ron in canine OSA cell lines. High Ron expression was prognostic for survival. Gefitinib (small molecule EGFR inhibitor) and crizotinib (small molecule Met inhibitor) inhibited OSA cell proliferation in an additive manner. Prolonged TGF alpha exposure promoted Met phosphorylation. Co-activation of EGFR and Met with their ligands resulted in amplified ERK1/2 and STAT3 phosphorylation. These data indicate that Met, Ron, and EGFR functionally interact in canine OSA, alter (open full item for complete abstract)

Committee: Cheryl London (Advisor); William Kisseberth (Committee Member); Carlos Alvarez (Committee Member); Ramiro Toribio (Committee Member) Subjects: Oncology

Doctor of Philosophy, The Ohio State University, 2004, Molecular, Cellular, and Developmental Biology

The vertebrate lens provides an excellent model to study mechanisms of cell cycle regulation and differentiation. Lens is composed of proliferative epithelium covering the anterior hemisphere and a core of postmitotic fiber cells that are differentiated from the epithelial cells at the lens equator. However, relatively little is known about the identity of molecules in the ocular environment that promote fiber differentiation in vivo. Despite circumstantial evidence suggesting the importance of Fibroblast Growth Factor (FGF) signaling in lens development, its role during lens development in vivo is still unclear and controversial. FGFs act through binding and activation of FGF receptors (FGFRs). The mouse lens expresses at least 3 different FGFR genes (Fgfr1-3) in a dynamic spatio-temporal fashion. In this study, we examined the role of FGF receptor signaling in lens fiber differentiation by targeting Fgfrs individually or in different combinations. To address the role of Fgfr1 and Fgfr2, null mutations of which cause early embryonic lethality prior to ocular induction, in lens development, we performed chimeric analysis and tissue-specific gene inactivation. To achieve lens-specific gene ablation, a transgenic mouse line, MLR10, was generated using this modified alphaA-crystallin promoter that drives Cre recombinase expression in a lens-specific manner from embryonic days 10.5 (E10.5). Our analyses showed that lens fiber differentiation was not prevented in the absence of any single Fgfr. Given the possibility of redundancy among different Fgfrs during lens fiber differentiation, we proceeded to inactivate Fgfrs in different combinations. Histological and gene expression analyses indicated that lens fiber differentiation was intact in all double Fgfr mutants. However, profound lens defects were observed in mice lacking all three Fgfrs. Lens-specific inactivation of Fgfrs causes abnormal proliferation, reduced expression of cell cycle inhibitors p27kip1 and p57kip2 (open full item for complete abstract)

Committee: Michael Rbinson (Advisor) Subjects: Biology, Genetics