PhD, University of Cincinnati, 2024, Medicine: Molecular, Cellular and Biochemical Pharmacology

Cardiovascular disease is the leading cause of death in the United States. Members of the low-density lipoprotein receptor (LDLR) family play integral roles in maintaining cardiovascular and metabolic homeostasis, and defects in these receptors are known to precede vascular occlusive diseases through their various roles. Apolipoprotein E Receptor-2 (ApoER2) is a transmembrane receptor in the LDLR family with a unique tissue expression limited to the brain, testis, and vascular and circulatory cells such as endothelial cells, smooth muscle cells, monocytes/macrophages, and platelets. Studies have revealed roles for ApoER2 in regulating atherosclerotic disease development, smooth muscle cell growth, tissue inflammation, and diet-induced obesity and diabetes. A single nucleotide polymorphism that encodes the R952Q sequence variant of ApoER2 has been associated with elevated plasma cholesterol levels and increased myocardial infarction risk in humans. The objective of this study was to delineate the mechanism underlying the association between the ApoER2-R952Q variant and increased atherosclerosis risk. Using a hyperlipidemic ApoER2-R952Q mouse model generated via a CRISPR/Cas9 strategy and intercrossing with LDLR knockout mice, this study investigated the development of atherosclerotic cardiovascular disease by feeding a Western-type, high-fat, high-cholesterol diet. Immunohistological analysis of atherosclerotic lesions revealed an acceleration of disease progression in ApoER2-R952Q mice. Plasma lipids and lipid distributions among the various lipoprotein classes were analyzed by colorimetric assay and revealed that hypercholesterolemia is exacerbated in ApoER2-R952Q mice. Tissue-specific effects of the R952Q sequence variant on atherosclerosis were analyzed by bone marrow transplant studies and showed that the ApoER2-R952Q mutation in bone marrow-derived cells instead of non-bone marrow-derived cells is responsible for the increase in hypercholesterolemia and atheros (open full item for complete abstract)

Committee: David Hui Ph.D. (Committee Chair); Guo-Chang Fan Ph.D. (Committee Member); Konstantinos Drosatos Ph.D. (Committee Member); Evangelia Kranias Ph.D. (Committee Member); Moises Huaman (Committee Member) Subjects: Pathology

Doctor of Philosophy, University of Akron, 2024, Chemistry

Cell communication is crucial for regulating cell growth, proliferation, survival, and function. The process of cell communication and cell signaling involves the recognition of exogenous or endogenous signaling molecules by receptors expressed on the surface of the plasma membrane. The activation of surface receptors such as enzyme-linked receptors results in the activation of the receptor's intracellular domain, which might have an intrinsic kinase activity like receptor tyrosine kinase (RTK). The activation of RTK triggers multiple downstream signaling pathways that regulate cell proliferation, angiogenesis, apoptosis, motility, adhesion, and metastasis. Therefore, a thorough understanding of RTK activation mechanisms and subsequent signaling events is crucial to provide insights into the control of RTK activity. The work presented in this dissertation is studying two different receptors from two subfamilies of RTKs, c-KIT, and EGFR/HER receptors family. The first project in this dissertation is focused on studying the SCF-mediated c-KIT signaling in mast cells (MCs) and the role of the underlying mechanisms in modulating mast cell viability and proliferation. The number of MCs in tissues usually remains constant, inflammation and asthma disturb this homeostasis, leading to proliferation of MCs. Understanding the signaling events behind this proliferative response could lead to the development of novel strategies for better management of allergic diseases. MC survival, proliferation, differentiation, and migration are all maintained by a MC growth factor, stem cell factor (SCF) via its receptor, KIT. Here, we explored how protein kinase C (PKC) redundancy influences MC proliferation in bone marrow-derived MC (BMMC). We found that SCF activates PKCα and PKCβ isoforms, which in turn modulates KIT phosphorylation and internalization. Further, PKCα and PKCβ activate p38 mitogen-activated protein kinase (MAPK), and this axis subsequently regulates SCF-induced MC cel (open full item for complete abstract)

Committee: Yilin Liu (Advisor); Christopher J. Ziegler (Committee Chair); Jordan Renna (Committee Member); Aliaksei Boika (Committee Member); Chrys Wesdemiotis (Committee Member) Subjects: Biochemistry; Chemistry; Physical Chemistry

PhD, University of Cincinnati, 2024, Medicine: Neuroscience/Medical Science Scholars Interdisciplinary

Epilepsy affects over 50 million patients worldwide, and one-third of those patients are resistant to current therapeutic options. The development of acquired epilepsy typically begins with a brain insult such as status epilepticus (SE), traumatic brain injury, genetic mutation, or infection. Following such insults, patients enter what is known as the “latent period” of the disease. During the latent period, the brain is changing but patients do not exhibit spontaneous recurrent seizures (SRSs). The latent period ends when a patient first experiences an SRS, at which point the disease has progressed to epilepsy. Epileptogenesis is the process by which a healthy brain becomes prone to SRSs, and it occurs during initial insult as well as throughout the latent period and chronic disease. While the majority of available treatment options aim to reduce SRSs in patients with chronic epilepsy, research has increasingly sought to define and inhibit epileptogenic changes. Successful anti-epileptogenic intervention has the potential to prevent or delay the onset of chronic epilepsy following brain insult and/or prevent continued brain alterations in patients who have already developed chronic epilepsy. The work in this thesis aimed to clarify the molecular mechanisms underlying epileptogenesis. Chapters 2 and 3 discuss the role of microRNAs (miRNAs) in epileptogenesis, and Chapter 4 discusses the role of Ras-MAPK signaling in epileptogenesis. MiRNAs are short, non-coding RNA sequences that regulate post-transcriptional gene expression via translational suppression or degradation of target messenger RNAs (mRNAs). Although many miRNAs have been implicated in epilepsy development, this thesis primarily discusses the role of miR-324-5p. Previous work in our lab showed an anti-convulsant and anti-epileptic effect of miR-324-5p inhibition when administered before brain insult or in chronic epilepsy. Using the intrahippocampal kainic acid model in mice, we tested (open full item for complete abstract)

Committee: Mark Baccei Ph.D. (Committee Chair); Christina Gross Ph.D. (Committee Member); Katrina Peariso M.D. Ph (Committee Member); Anil Jegga DVM MRe (Committee Member); Steve Danzer Ph.D. (Committee Member) Subjects: Neurology

Doctor of Philosophy, The Ohio State University, 2023, Comparative and Veterinary Medicine

Metastasis is the primary killer of patients with osteosarcoma. To effectively treat metastasis, we must first understand how tumor cells colonize and survive within the lungs. It is well established that only a small number of cells from the primary tumor go on to metastasize, but whether this is a pre-existing capability of metastatic cells or a phenotype they develop in response to a new microenvironment has yet to be established. Furthermore, osteosarcoma metastases are often chemoresistant, such that surgical resection is the only treatment option that has been shown to provide a reasonable chance of a longer term “cure”. Identifying the survival requirements of metastatic cells in the lung may allow us to target those dependencies therapeutically. Because osteosarcoma is a rare disease, there is a critical need for models that recapitulate what is seen in human patients as faithfully as possible so that clinical trials focus on therapeutics with the greatest potential for success. Interrogating disease biology and screening therapeutics in dogs holds great potential in this regard. While investigating tumor heterogeneity, we found that osteosarcoma cells undergo clonal selection when crossing the so-called metastatic bottleneck, although the degree of transcriptional heterogeneity among tumor cells remains similar to that seen in primary tumors. We also identified heterogeneous expression of glycolysis markers among tumor cells in tissue culture, primary tumors, and metastases. These findings indicated that transcriptional heterogeneity may be an intrinsic aspect of osteosarcoma, and implied that metastasis may be a cooperative event. Building on our findings of transcriptional heterogeneity and previous work evaluating subpopulations in metastases, we sought to identify factors that allow metastatic cells to survive in the lungs. We pharmacologically targeted an identified factor, MCL1, in vitro, and found that it readily killed osteosarcoma cell spheroids. (open full item for complete abstract)

Committee: Ryan Roberts (Advisor); Cheryl London (Committee Member); Estelle Cormet-Boyaka (Committee Member); Maxey Wellman (Committee Member) Subjects: Biology; Cellular Biology; Comparative; Oncology

PhD, University of Cincinnati, 2022, Engineering and Applied Science: Biomedical Engineering

Cancer is the second leading cause of death in the world after heart disease. The current treatment modalities to fight this disease are limited and there is a critical need for better treatment strategies. Electric field (EF)-based modalities have been used clinically to treat cancer for many years, but current available treatment options include invasive electroporation and non-invasive tumor treating fields (TTField) with limited therapeutic efficiency and significant side effects. Recently, cancer biomarker-targeted therapy has been a growing interest in the cancer field. Phosphatidylserine (PS) is a cancer cell surface exposed biomarker. The number of preclinical and clinical studies demonstrate that PS-selective targeting is a promising therapeutic approach for cancers. Importantly, multiple research investigations have shown that increasing cell surface PS exposure induced by chemodrugs and radiation can sensitize cancer cells to the PS-targeting agents. However, chemo and radiation treatments have the drawback of potentially harming healthy cells because of their high toxicity. In this dissertation, we used a potent non-contact EF inducer device to investigate the regulation effects of a particular EF modality on cancer-specific surface PS biomarkers. Our results indicate that this innovative EF technology modulate cancer surface PS levels. To our knowledge, this is the first study demonstrating the regulation effect of EF to modulate the cancer cell surface biomarker. The long-term goal of this research is to develop an EF-based therapy technique which can sensitize cancer cells to PS-targeting drugs. Our research contributes to this goal by showing the EF effect on surface PS exposure modulation in cancer cells. Further we elucidate the molecular mechanism of this cancer-selective EF-regulated PS exposure, showing that EF treatments can differentially regulate the intracellular calcium (Ca2+) levels, actin polymerization, p38 mitogen-activated protein k (open full item for complete abstract)

Committee: Xiaoyang Qi Ph.D. (Committee Member); Daria Narmoneva Ph.D. (Committee Member); Jing-Huei Lee Ph.D. (Committee Member); Andrei Kogan Ph.D. (Committee Member) Subjects: Biomedical Research

PhD, University of Cincinnati, 2022, Medicine: Toxicology (Environmental Health)

This dissertation focused on the role of the Stathmin (STMN1) oncoprotein in regulating epithelial mesenchyme transition (EMT). EMT is implicated in cancer progression and understanding the mechanisms of EMT is necessary in developing approaches for preventing or treating metastasis. STMN1 levels and STMN1 serine 25 (S25) phosphorylation are upregulated in many cancers, correlating with cancer progression and poor patient survival; yet the role(s) of STMN1 phosphorylation in driving metastasis remains unclear. As p38/mitogen-activated protein kinase (MAPK) signaling up-regulates metastasis, and p38 phosphorylates STMN1 S25, we hypothesized that p38-mediated phosphorylation of S25 promoted EMT. Utilizing the prostate cancer (PCa) DU-145 cell line, treatment with anisomycin induced p38 and S25 phosphorylation which was inhibited by the p38 inhibitor SB203580 (SB), suggesting S25 was a target of p38 signaling. Unexpectedly, both anisomycin and AEBSF, and the p38 inhibitor SB, inhibited DU-145 cell migration. Consequently, we were unable to address our hypothesis with these chemicals. Therefore, we utilized hepatocyte growth factor (HGF) to determine whether HGF-induced p38 signaling regulated STMN1 S25 phosphorylation and promoted EMT. We provide the first evidence that treatment with HGF promotes STMN1 S25 phosphorylation in PCa cells. Furthermore, both extracellular regulated kinase (ERK) and p38 were activated by HGF and contributed to S25 phosphorylation. With HGF treatment, inhibition of ERK alone reduced pS25, while inhibition of both ERK and p38 abolished pS25, suggesting these kinases converge to phosphorylate STMN1 S25. HGF-treated DU-145 cells also exhibited increased migration and invasion and presented an EMT phenotype in vitro. A novel finding showed HGF-mediated EMT involved both ERK and p38 pathways, where p38 promoted cell migration while ERK promoted cytoskeletal rearrangements and loss of cell-cell junctions. We also demonstrated that cotreatment of H (open full item for complete abstract)

Committee: Susan Kasper Ph.D. (Committee Member); Ying Xia Ph.D. (Committee Member); Saulius Sumanas Ph.D. (Committee Member); Katherine Burns Ph.D. (Committee Member) Subjects: Organismal Biology

Doctor of Philosophy, The Ohio State University, 2021, Biomedical Sciences

Few therapeutic options exist for the 15-25% of melanoma patients whose disease is driven by oncogenic NRAS. NRAS is a member of the RAS family of proto-oncogenic GTPase proteins which trigger signal transduction pathways involved in cellular motility, survival, proliferation, and metabolism. Therapeutic targeting of NRAS is a decades-old challenge, hindered by the inability to develop small molecule inhibitors specific for the mutant protein. Furthermore, oncogenic NRAS can circumvent treatments targeting post-translational RAS modifications, interacting partners, and downstream signaling pathways. Current first-line therapies for NRAS-driven melanoma are immune-based. While such drugs are effective in 40-50% of individuals, many patients suffer from high-grade adverse events and only a subset of responders experience durable remissions. With NRAS-driven melanomas being the most aggressive subtype of this disease, new and effective therapeutic options are needed. Oncogenic NRAS mutations primarily affect codons 12, 13, and 61, resulting in constitutive GTP-binding, activation, and downstream signal transduction. However, each NRAS-driven malignancy shows selection bias for a different subset of NRAS mutants. For example, NRAS-driven melanomas are enriched for genetic mutations in codon 61 (>80%) while mutations in acute myeloid leukemia primarily occur in codons 12 and 13. This mutational bias remains poorly understood, especially in melanoma where the codon 61 alterations are not directly attributed to ultraviolet light. I developed a suite of conditional, Nras knock-in mouse models (LSL-Nras Q61R, -K, -L, -H, -P, -Q; G12D and G13D, -R) to test the hypothesis that NRAS mutants commonly observed in melanoma possess functional properties required for efficient melanocyte transformation. Expression of these alleles in melanocytes revealed that the melanomagenic potential of each NRAS variant parallels the frequency of that mutation in human melanoma. Specifical (open full item for complete abstract)

Committee: Christin Burd E (Advisor); Joanna Groden (Committee Chair); Michael Freitas A (Committee Member); Terence Williams M (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology; Oncology

PhD, University of Cincinnati, 2020, Medicine: Pathobiology and Molecular Medicine

Tuberous Sclerosis Complex (TSC) is an autosomal dominant genetic disease with multi-system manifestations including benign neoplastic growth in the brain, heart, kidneys, and lung. Inactivating gene mutations in either TSC1 or TSC2 result in hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1), which leads to the uncontrolled cell growth and proliferation in TSC. Lymphangioleiomyomatosis (LAM) is the lung manifestation of TSC and can also present as sporadic form. It occurs predominantly in women during their reproductive age and is exacerbated by female hormones including estrogen, progesterone and prolactin. To explore the underlying molecular mechanism of this hormonal predominance, we utilized TSC2-deficient models and showed that estrogen promotes glycolytic metabolism in tumor cells through activation of pyruvate kinase muscle isozyme M2 (PKM2). Accumulation of phosphorylated PKM2 was evident in pulmonary nodule and renal angiomyolipoma cells from LAM patients. These data suggest that the female predominance of LAM might partially be attributed to estrogen stimulation of PKM2-mediated cellular metabolic alterations. The clinical application of mTORC1 specific inhibitors (mTORi), including sirolimus (rapamycin) and everolimus, have been shown to promote tumor regression and stabilize lung function in TSC and LAM patients. However, sustained improvement requires continuous exposure, as refractory growth occurs upon drug cessation, suggesting a cytostatic rather than a cytocidal nature of the medicine. We recapitulated rapamycin- induced refractory response in mouse xenograft tumors. RNA sequencing analysis of xenograft tumors from different growth stages revealed upregulation of pro-survival mitogen activated protein kinase (MAPK) pathway under rapamycin induction. This studyrevealed a MAPK-evoked positive feedback loop that dampens the efficacy of mTOR inhibition in TSC models. Pharmacological inhibition of MAPK abrogated this feedback loop (open full item for complete abstract)

Committee: Phillip Owens Ph.D. (Committee Chair); Tanya Kalin M.D. Ph.D. (Committee Member); Mario Medvedovic Ph.D. (Committee Member); Manoocher Soleimani M.D. (Committee Member); Yan Xu Ph.D. (Committee Member) Subjects: Surgery

Doctor of Philosophy, The Ohio State University, 2021, Molecular, Cellular and Developmental Biology

The centrosome is the microtubule organizing center of the cell and is highly conserved in structure and function. The centrosome cycle mirrors the cell cycle and is tightly controlled to maintain genetic fidelity. Centrosome duplication is limited to once per cell cycle to form a normal bipolar mitotic spindle and potentiate a functional spindle assembly checkpoint (SAC) that ensures proper kinetochore attachment before the cell proceeds through anaphase. However, the presence of extra centrosomes, through a process known as centrosome amplification, threatens the fidelity of chromosome segregation and can lead to chromosomal instability (CIN). Both centrosome amplification and CIN are hallmarks of most human cancers including those characterized by constitutive activation of the mitogen activated protein kinase (MAPK) pathway. The MAPK pathway primarily controls cell proliferation and has also been implicated in control of centrosome duplication and SAC function. MPS1 is a dual-specificity protein kinase first discovered for its role in centrosome duplication and later also shown to play a critical role in the SAC. It has been shown that MAPK signaling is necessary for targeting MPS1 to kinetochores and proper SAC function, but less is known about how MPS1 and MAPK proteins interact at centrosomes and whether that relationship contributes to centrosome amplification in cancer. The present study aimed to further characterize the interaction between MPS1 and the MAPK pathway to better understand how supernumerary centrosomes form in cancer cells. To address these questions, we assessed the localization of MAPK proteins, identified binding partners, and generated constitutively active mutants used in fluorescent microscopy to identify their impact on centrosome amplification. We have shown that ERK localizes to centrosomes where it accumulates under prolonged S-phase arrest. ERK, but not its upstream activators MEK or BRAF, interacts with MPS1 in S-phase when cen (open full item for complete abstract)

Committee: Harold Fisk (Advisor); Jane Jackman (Committee Member); Matthew Ringel (Committee Member); Amanda Toland (Committee Member) Subjects: Cellular Biology; Molecular Biology

PhD, University of Cincinnati, 2020, Medicine: Pathobiology and Molecular Medicine

Pancreatic cancer is projected to be second deadliest malignancy in the US by 2020. Pancreatic ductal adenocarcinoma (PDAC) makes up 90% of pancreatic cancers. Current treatments for PDAC are ineffective and new treatments are urgent. Gemcitabine (GEM) used in combination with Abraxane (Abr) is one of the first line treatments for advanced PDAC. Unfortunately, GEM/Abr affords only marginal benefits to patients compared to no treatment. Phosphatidylserine (PS) is exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy and is overexpressed on the surface of pancreatic cancer cells compared to healthy cells. PS is normally expressed in the inner leaflet of healthy cells. However, oxidative stress (OS) in the tumor microenvironment is thought to induce PS externalization in cancer cells. In cancer cell lines, variations of surface PS expression is associated with increased intracellular calcium, reduced flippase activity and increased expression of PS synthase genes PTDSS1 and PTDSS2. Studies have shown that OS affects intracellular calcium levels through the regulation of calcium channels and pumps, actin rearrangement and p38 MAPK activation. However, the mechanisms regulating PS externalization in PDAC cells are not clear. Additionally, the effects of OS on PS externalization has not been studied in the context of PDAC and the function for PS externalization in pancreatic cancer progression remains unknown. PS is targetable by several therapeutic agents including SapC-DOPS. Furthermore, existing cancer treatments are known to induce PS externalization, sensitizing cells to PS-targeting drugs. GEM, a first line treatment for metastatic PDAC, has been shown to increase the expression of PS on cancer cells. Although GEM treatment increases PS externalization in cancer cells, the therapeutic effect of combining GEM with a PS-targeting drug for PDAC treatment have not been investigated. The aims of our study were to: 1)

Committee: Xiaoyang Qi Ph.D. (Committee Chair); Vladimir Bogdanov Ph.D. (Committee Member); William Miller Ph.D. (Committee Member); Susan Waltz Ph.D. (Committee Member); Trisha Wise-Draper Ph.D. (Committee Member) Subjects: Dance; Demographics; Molecular Biology; Polymer Chemistry

Doctor of Philosophy (PhD), University of Toledo, 2018, Biomedical Sciences (Molecular Medicine)

Hypertension is a complex disease caused by a combination of genetic and environmental factors. Renin-angiotensin system (RAS) dysfunction leads to essential hypertension. Polymorphisms in the AGT gene increase RAS activity and cause blood pressure elevation. We and others have shown that SNPs in the promoter of hAGT gene can be arranged mainly in two haplotypes. Variants -1670A, -1562C, -1561T and1164A always occur with -6A and form the haplotype-I (Hap-I), while variants -1670G, -1562G, -1561G and 1164G always occur with -6G and constitute haplotype-II (Hap-II). We have made transgenic animals containing these two haplotype blocks and shown that Haplotype-I is the risk haplotype and increases hAGT expression in TG animals. We hypothesized that these SNPs, when present together as Hap-I or Hap-II in transgenic mice may lead to haplotype-dependent DNA methylation of CpG sites in the promoter of the hAGT gene. Methylation patterns may modulate gene transcription at an epigenetic level and this, in turn, could be dependent on individual polymorphisms. Hypomethylation is associated with higher gene expression and hypermethylation is associated with lower gene expression. We have found that the number of methylated CpG sites in the hAGT promoter is decreased after high salt (HS) treatment in the liver and kidney of Hap-I TG animals as compared to the liver of Hap-II animals. In the liver of Hap-II animals, the hAGT promoter CpG sites are methylated at -460,-434,-386,-261,+42, whereas after high salt treatment, the promoter DNA methylation is observed at -460,-386,+65. In the kidney of Hap-II animals, the hAGT promoter CpG sites are methylated at -460,-434,-386,-346,-261,-153,+65, whereas after high salt treatment, the promoter DNA methylation is observed at -460,-386,-153,-18,+65. On the other hand methylation of hAGT promoter CpG sites in the liver and kidney of Hap-II are increased as compared to the liver of Hap-I after high salt treatment. In the liver of Hap-I anim (open full item for complete abstract)

Committee: Ashok Kumar PhD (Committee Chair); Guillermo Vazquez PhD (Committee Chair); Andrew Beavis PhD (Committee Member); Nikolai Modyanov PhD (Committee Member); Zahoor Shah PhD (Committee Member); Jiang Tian PhD (Committee Member) Subjects: Biology; Biomedical Research; Genetics

Master of Science (MS), Wright State University, 2018, Biochemistry and Molecular Biology

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) involved in cell growth and differentiation. Recent studies have revealed important roles for ERK3 in promoting cancer cell migration and invasion. In addition, ERK3 was shown to be highly upregulated in human lung cancers and to be associated with tumor metastasis. Altogether, these findings suggest an important role for ERK3 in lung tumor formation and progression. However, no in vivo study of ERK3 in lung tumorigenesis has been reported. For this purpose, a transgenic mouse model conditionally overexpressing ERK3 in lung bronchial epithelial cells was generated. First, a transgenic mouse line harboring ERK3 transgene under the control of a ubiquitous promoter and a STOP sequence flanked by two Lox P sites (LSL-ERK3) was generated. The obtained LSL-ERK3 mice were then crossed with a mouse line harboring the Cre recombinase transgene driven by a Clara cell secretory protein gene promoter (CCSP-Cre), resulting in CCSP-Cre/LSL-ERK3 transgenic mice that show ERK3 overexpression in Clara cells, the non-ciliated epithelial cells lining the bronchioles of lung. No clear phenotype, however, was observed in CCSP-Cre/LSL-ERK3 transgenic mice. As lung tumorigenesis usually requires multiple genetic alterations, the CCSP-Cre/LSL-ERK3 transgenic mice were then crossed with a PTENFlox/Flox mouse line in which the exon 5 of the tumor suppressor gene PTEN (Phosphatase and Tensin homolog) is flanked by two Lox P sites. These crossings led to the generation of CCSP-Cre/LSL-ERK3/PTENFlox/Flox mice which display PTEN deletion and ERK3 overexpression in the Clara cells of lung. Thus, four transgenic mouse groups were included in the study and monitored on daily basis: 1) LSL-ERK3; 2) CCSP-Cre/LSL-ERK3; 3) CCSP-Cre/PTENFlox/Flox; 4) CCSP-Cre/LSL-ERK3/PTENFlox/Flox. Tumor growth or other abnormalities in the lungs were analyzed by examining the whole lungs for surface tumors and by his (open full item for complete abstract)

Committee: Weiwen Long Ph.D. (Advisor); Steven Berberich Ph.D. (Committee Member); Michael Markey Ph.D. (Committee Member) Subjects: Biochemistry; Biomedical Research

Doctor of Philosophy, The Ohio State University, 2018, Pharmaceutical Sciences

Circadian rhythms are endogenous and entrainable 24-hour oscillations in biological processes that influence nearly every aspect of physiology. Located in the brain, the suprachiasmatic nucleus (SCN) is the `master clock' that facilitates the maintenance of circadian rhythms and orchestrates the adaptation of these rhythms to exogenous light cues. Within the SCN, complex signaling mechanisms are essential in maintaining the fidelity of the transcriptional translational feedback loop (TTFL) by ensuring robust and timed oscillations of genes within the TTFL known as `clock' genes. In line with this, the p44/42 mitogen activated protein kinase (MAPK) pathway is a critical signaling motif within the SCN that's activity and induction is gated by time-of-day. Here, with the use of transgenic animal models, molecular techniques and behavioral paradigms, I expand upon the current details of how the MAPK pathway is involved in cellular signaling within the SCN. First, I show that phosphoprotein enhanced in astrocytes (PEA-15), a scaffold for the main MAPK effector kinase ERK, is highly expressed in the SCN, light-regulated at the level of phosphorylation, and it's physical interaction with ERK is disrupted by light during the early subjective night within the mouse SCN. Next, I demonstrate that the phosphorylation of cAMP response element binding protein (CREB) at serine-133 is a critical signaling event mediating transcriptional regulation within the SCN that impacts gene expression and behavioral rhythms. Finally, I examine the role of CREB-regulated transcription coactivator (CRTC1) as a potential phosphorylation-independent compensatory activator of CREB that is regulated by the expression of a micro-RNA, MiR-132, to maintain the functional integrity of SCN timing. I also pursue a clinical angle to circadian alignment by assessing sleep, which is a clock-gated clinical parameter, in Huntington's disease (HD) patients. HD is a devastating neurodegenerative disease an (open full item for complete abstract)

Committee: Kari Hoyt Ph.D. (Advisor); Karl Obrietan Ph.D. (Committee Member); John Oberdick Ph.D. (Committee Member); James McAuley Ph.D. (Committee Member) Subjects: Neurosciences; Pharmacy Sciences

Doctor of Philosophy (PhD), Wright State University, 2017, Biomedical Sciences PhD

Mitogen-activated protein kinases (MAPKs) are Ser/Thr kinases that regulate diverse cellular processes. Extracellular signal-regulated kinase 3 (ERK3) is an atypical MAPK that has recently gained interest for its roles in cancer. ERK3 promotes cancer cell migration, invasion and drug resistance, and its expression is upregulated in multiple cancers. However, little is known about the molecular mechanisms that regulate ERK3's expression and kinase activity. Here, we have identified the oncogenic polycomb group protein BMI1 as a positive regulator of ERK3 level in cancer cells. Mechanistically, BMI1 upregulates ERK3 expression by inhibiting the expression of the tumor suppressor microRNA (miRNA) let-7i, which directly targets ERK3 mRNA. ERK3 then acts as an important downstream mediator of BMI1 in promoting cancer cell migration. Importantly, ERK3 protein level is positively correlated with BMI1 level in head and neck tumor specimens of human patients. In addition, we have investigated the roles of the activation loop phosphorylation and the unique elongated C-terminus extension in regulating ERK3's kinase activity and functions in cancer cells. Our study revealed that mutating the activation loop phosphorylation site S189 or truncating the C-terminus extension greatly decreases ERK3's in vitro kinase activity towards the oncogenic protein steroid receptor co-activator 3 (SRC-3), an important downstream target for ERK3 signaling in cancer. While the reduced activity of ERK3 S189A mutant can be partly attributed to its decreased interaction with the substrate SRC-3, the importance of the C-terminus extension in kinase activity possibly stems from its intramolecular interaction with the kinase domain of ERK3. In agreement with their decreased kinase activity towards SRC-3, the S189A mutant and the C-terminus deletion mutants have greatly diminished ability to stimulate migration and invasion of lung cancer cells as compared to wild type ERK3. Furthermore, we have identi (open full item for complete abstract)

Committee: Weiwen Long Ph.D. (Advisor); Madhavi Kadakia Ph.D. (Committee Member); Steven Berberich Ph.D. (Committee Chair); Christopher Wyatt Ph.D. (Committee Member); Shulin Ju Ph.D. (Committee Member) Subjects: Biomedical Research; Molecular Biology

PHD, Kent State University, 2017, College of Arts and Sciences / School of Biomedical Sciences

Glaucoma is a group of neurodegenerative disorders characterized by axonal transport deficits and progressive retinal ganglion cell (RGC) loss, which comprise the leading cause of irreversible blindness worldwide. Due to the complexity of the disease, little is known about the stressors, mechanisms, or progression of pathology in glaucoma. This lack of knowledge hinders our ability to prevent and treat glaucoma. The purpose of the studies in this thesis was to investigate links between two major components of neurodegeneration: axon dysfunction and neuroinflammation. First, I determined the progression of cytoskeletal protein modifications in relation to transport deficits in a common murine model of glaucoma. Second, I identified the timeline of changes in neuroinflammatory protein expression during the onset and progression of functional and structural changes in the retina and its projection. Finally, I explored signaling mechanisms that may link cytokine and cytoskeletal changes within the glaucomatous projection. This work sets the stage for the development of new therapies for this and other degenerative conditions. My work used the DBA/2J inbred mouse, one of the most common animal models of glaucoma. These mice show elevated intraocular pressure (IOP), transport deficits, axonopathy, and altered cytokine expression with age. Anterograde tracing, along with protein quantification techniques including enzyme-linked immunosorbent assays (ELISA), bead-based multiplexing, automated western blot, and immunofluorescence staining were employed. Major findings included the first reports of cytoskeletal protein hyperphosphorylation and increased pro-inflammatory cytokine expression in the retinal projection of the DBA/2J model. Importantly, these often occurred prior to IOP elevation or RGC death. Data also indicated upregulation of likely candidates producing these changes leading to neurodegeneration, including mitogen-activated protein kinases (MAPKs) and relate (open full item for complete abstract)

Committee: Samuel Crish (Committee Member); Brett Schofield (Committee Member); Denise Inman (Committee Member); Christine Dengler-Crish (Committee Member); Jennifer McDonough (Committee Member) Subjects: Neurosciences

PhD, University of Cincinnati, 2017, Medicine: Cancer and Cell Biology

Angiosarcoma is a rare malignancy of the endothelium, making up about 2% of sarcoma diagnoses. The causes of this disease are relatively unknown, especially in the pediatric population. Currently, the best treatment for angiosarcoma is complete surgical resection. If complete resection is unachievable due to anatomic location, chemotherapeutics are used, although these are not very effective. Five year median survival remains poor around 31-43%. As angiosarcoma is so rare, there has been little research done regarding the pathogenesis of the disease. There is a lack of model systems in which to study angiosarcoma in order to develop novel therapeutic strategies.

Committee: Lionel Chow M.D. Ph.D. (Committee Chair); Elisa Boscolo Ph.D. (Committee Member); Biplab Dasgupta Ph.D. (Committee Member); James Driscoll M.D. Ph.D. (Committee Member); Nicolas Nassar Ph.D. (Committee Member) Subjects: Biology

Master of Sciences, Case Western Reserve University, 2017, Systems Biology and Bioinformatics

Motivation: The prognosis of low-grade-glioma (LGG) patients is very poor. Identifying subnetworks related to LGG can better describe the genetic make-up of the tumor. Methods: n-Node Subnetwork Enumeration Algorithm (nSEA) was developed to identify significantly dysregulated subnetworks. We utilized a filtered protein network to enumerate n-node subnetworks exhaustively and score each subnetwork to carry out feature selection. These subnetwork seeds were expanded to identify tumor-specific subnetworks. Clustering these subnetworks provided patient groups with different subnetwork states. Results: We identified 92 subnetwork features, 8 subnetwork groups and 5 patient groups. A new patient group was identified with favorable outcomes. By decision tree modeling, the new group were characterized as down-regulated MAPK/B-Raf pathway and up-regulated Notch pathway. It had fewer mutations of candidate genes, hypomethylation of NIPBL and hypermethylation of KALRN. Conclusions: These results could provide opportunities for improved treatment options and personalized interventions of LGG.

Committee: Gurkan Bebek (Committee Chair); Mark Cameron (Committee Member); Jean-Eudes Dazard (Committee Member) Subjects: Bioinformatics; Oncology

PhD, University of Cincinnati, 2016, Medicine: Molecular and Developmental Biology

The mammalian intestine is a rapidly self-renewing organ with epithelial cell turn over occurring every 4-6 days. Along the length of the small and large intestine are crypt invaginations that contain rapidly proliferating progenitors and stem cells that continuously divide and give rise to all cell types. Regulation of this activity requires an intricate network of signaling and gene regulation to maintain homeostasis. KLF5 is a zinc-finger transcription factor that is found to be highly expressed in the proliferative crypts. It has been shown to mediate the onset and progression of diseases in the intestine; however its function in normal intestinal homeostasis is not well defined. To examine this role, we first utilized inducible knockout mouse models to delete KLF5 from the adult intestine, and found it to be required for normal proliferation. We discovered that loss of KLF5 correlated with decreased expression of active stem cell transcripts, Lgr5, Ascl2, and Olfm4. Restoration of protein expression in the crypt occurred within 14 days; however this had no impact on the loss of stem cell expression and did not restore progenitor proliferative to wildtype levels. We found a transient decrease in the enteroendocrine cells, but no other differentiated cell types were affected. These studies indicated that KLF5 is required for stem cell renewal and proliferation. We then specifically overexpressed KLF5 in the intestine discern its effects on proliferation, cytodifferentiation, and stem cell dynamics. After 2.5 days of overexpression villus blunting and crypt hyperplasia were associated with decreased villus cytodifferentiation and increased proliferative progenitor cell numbers in mice overexpressing KLF5 in the intestinal epithelium. While no effect was seen on stem cells, we were only able to examine these mice 2.5 days after induction as ectopic KLF5 expression in the intestine proved to be fatal by day 4. Results from these experiments indicated (open full item for complete abstract)

Committee: Noah Shroyer Ph.D. (Committee Chair); Simon Hogan Ph.D. (Committee Member); Tatiana Kalin M.D.Ph.D. (Committee Member); James Wells Ph.D. (Committee Member); Jeffrey Whitsett M.D. (Committee Member) Subjects: Biology; Developmental Biology

PHD, Kent State University, 2015, College of Arts and Sciences / School of Biomedical Sciences

Growth arrest and DNA damage inducible protein 45 alpha (GADD45a) is a nuclear protein involved in genomic stability, epigenetic modulation, DNA repair, cell cycle arrest, cell apoptosis and senescence. The overall purpose of our study is to establish a novel treatment paradigm targeting cellular GADD45a level for non-small cell lung carcinoma (NSCLC). We first reported the sequential activation of JNK/ERK, Elk-1, Egr-1 and GADD45a by arsenite in BEAS-2B normal human bronchial epithelial cells as a protective mechanism to suppress aberrant cell proliferation, and the direct binding of Egr-1 protein to GADD45a promoter under arsenite stress to initiate the transcription of GADD45a. The CArG elements are the chemo/radio-responsive regions in the Egr-1 promoter, and resveratrol, a nontoxic natural compound, is also an effective inducer of CArG elements. We revealed that the activation of Egr-1 by resveratrol in A549 NSCLC cells is through JNK/ERK-Elk-1 signaling pathway, which is also upstream of arsenite-induced Egr-1 in BEAS-2B cells. However, as opposed to arsenite, resveratrol only exhibited negligible effects on GADD45a expression in A549 cells. We then designed synthetic CArG promoters containing different repeats and core A/T sequences of isolated CArG elements and a deletion construct of native Egr-1 promoter, but no discrepancy was observed in resveratrol-responsiveness among these promoters. The suicide gene therapy vector was constructed by inserting GADD45a opening reading frame (ORF) downstream to the inducible CArG promoter. Further investigations confirmed the activation of synthetic CArG promoter by standard NSCLC regimen cisplatin and radiation as well as resveratrol, and evaluated the therapeutic effects of each suicide gene therapy combination in three NSCLC cell lines, H1299 (deleted p53), A549 (wild type p53) and H23 (mutated p53). The results demonstrate that the spatial and temporal-controlled GADD45a overexpression achieved by our suicide gene t (open full item for complete abstract)

Committee: Werner Geldenhuys (Advisor); Deepak Bhatia (Committee Member); Prabodh Sadana (Committee Member); Christine Crish (Committee Member); Gail Fraizer (Committee Member) Subjects: Biomedical Research; Oncology; Pharmacology

Doctor of Philosophy, Case Western Reserve University, 2015, Neurosciences

The ERK/MAPK pathway is an intracellular signaling pathway that mediates many cellular functions. Mutations in the different elements of this pathway lead to a variety of congenital disorders termed neuro-cardio-facial-cutaneous syndromes (NCFC). These disorders result in many overlapping features including various levels of intellectual disability and cognitive function. In addition, dysregulation of the ERK/MAPK pathway has been implicated in other cognitive and behavioral disorders such as autism, schizophrenia and major depression. Understanding the role of this pathway during development and maturation of the brain will provide critical insight into the pathophysiology of these disorders. The ERK/MAPK pathway has previously been shown to regulate proliferation of neural progenitors during cortical development. Furthermore, pharmacologic studies inhibiting the ERK/MAPK pathway resulted in impaired synaptic plasticity and spatial memory. These studies have identified potential roles for ERK signaling during development and neurophysiologic functions in the hippocampus. The hippocampus is a critical mediator of memory formation and hippocampal volume is altered in various cognitive and behavioral disorders. An important question that remains to be answered is identifying the relative contributions from developmental and post-natal dysregulation of the MAPK pathway in the hippocampus as it relates to the pathophysiology of cognitive deficits observed in NCFC and related syndromes. In examining the development of the hippocampus, we observe that loss of both ERK1 and ERK2 isoforms impairs morphogenesis of the hippocampus resulting in a dentate gyrus that is 1/3 the size of a normal dentate gyrus by post-natal day 10. Furthermore, this phenotype is due to the precocious maturation of radial glial cells and depletion of progenitor populations in the germinal zones that generate the granule cells of the dentate gyrus. Interestingly, the presence of ERK1 is su (open full item for complete abstract)

Committee: Evan Deneris PhD (Committee Chair); Gary Landreth PhD (Advisor); Narla Goutham MD.PhD (Committee Member); Friel David PhD (Committee Member); Dubyak George PhD (Committee Member) Subjects: Biomedical Research; Molecular Biology