Master of Science, The Ohio State University, 2017, Horticulture and Crop Science

Autophagy (self-eating) is an intracellular process by which macromolecules (i.e. proteins and lipids) or entire organelles are degraded in the vacuole. Micro- and macro-autophagy have been identified in plants. The hallmark of macroautophagy (hereafter autophagy) is the autophagosome. Autophagy is regulated by the autophagy-related (ATG) genes, and it is involved in cellular homeostasis, nutrient recycling during senescence, and environmental stress responses. Autophagy deficient plants senesce prematurely, and they are hypersensitive to abiotic stresses. Premature senescence (leaf or flower) and abiotic stress damage reduce postharvest quality of floriculture crops. The goal of this project was to describe the role of autophagy in senescence and abiotic stress responses of Petunia × hybrida 'Mitchell Diploid'. Better understanding of the processes involved in senescence and abiotic stress responses may lead to the development of floriculture crops with extended leaf/flower longevity or higher abiotic stress tolerance. Petunia is an important floriculture commodity, and it is a good plant model for both senescence and abiotic stress research. Quantitative PCR was used to study how gene expression was regulated during certain developmental stages or by external factors. We analyzed how PhATG4, PhATG5, PhATG6, PhATG7, PhATG8a, and PhATG13 were regulated during age-induced flower senescence, nutrient starvation, and salt stress. All six ATG genes were expressed at basal levels in recently opened flowers and leaves from non-stressed plants. Petunia ATG genes were upregulated upon the onset of flower senescence (corolla wilting). Low fertility stress and individual nutrient (-N or -P) deficiencies increased the expression of the ATG genes. Changes in expression of the ATG genes happened within the first 24 h after salt treatment. Thus, expression of the ATG genes is regulated by flower senescence and abiotic stress responses of petunia. RNA interference (RNAi) is a co (open full item for complete abstract)

Committee: Michelle Jones (Advisor); John Finer (Committee Member); Joshua Blakeslee (Committee Member) Subjects: Horticulture

Doctor of Philosophy, Case Western Reserve University, 2025, Pathology

The COVID-19 pandemic claimed the lives of millions of people and affected communities worldwide. SARS-CoV-2, the virus that causes COVID-19, continues to be a global health concern, as does the inevitable threat of new viral outbreaks. We must therefore learn from this virus in the hope of better preparing for future pandemics. We investigated the SARS-CoV-2 accessory protein ORF3a and its roles in modulating host antiviral strategies, namely inflammatory signaling and autophagy regulation. ORF3a activates NF-κB signaling, which induces an inflammatory response in infected cells and can also prime certain cells for inflammasome assembly and subsequent cell death. We found that, unlike the homologous protein SARS-CoV ORF3a, SARS-CoV-2 ORF3a does not depend on its N-terminal TRAF-binding sequence to activate NF-κB. The ORF3a homologs thus affect NF-κB signaling through different mechanisms. Second, SARS-CoV- 2 ORF3a blocks autophagy by binding to the human protein VPS39, a member of the complex that facilitates membrane fusion between autophagic compartments. We discovered that the predicted β-propeller domain of VPS39 is critical to its interaction with ORF3a. Regulating autophagy is important for productive SARS-CoV-2 infection; disrupting the ORF3a:VPS39 interaction could therefore be a future strategy to hinder SARS-CoV-2 propagation.

Committee: Tsan Sam Xiao (Advisor); George Dubyak (Committee Chair); Clive Hamlin (Committee Member); Allison Kraus (Committee Member); Focco van den Akker (Committee Member) Subjects: Biochemistry; Biomedical Research; Biophysics; Immunology; Molecular Biology; Pathology; Virology

Doctor of Philosophy, The Ohio State University, 2024, Biomedical Sciences

Plasma membrane repair is an evolutionary conserved cellular mechanism observed in multiple tissue types including striated muscle, skin, gastrointestinal tract, and peripheral nervous system. Plasma membrane repair is critical following damage to restore the barrier function of the membrane and avoid cell death, and many diseases have a defect in the membrane repair mechanism that contributes to disease progression. Some of these diseases have an intrinsic gene mutation which cause the defect in cell membrane repair, and others have an extrinsic factor causing the defect in cell membrane repair. Amyloid beta (A) is a hallmark protein implicated in Alzheimer's Disease (AD) and A has an intimate relationship with the neuronal plasma membrane by which it alters the integrity of the membrane and has been shown to damage and penetrate the membrane, which would require an effective repair response to compensate for the damage to remain viable. We hypothesized neuronal death in AD is due at least in part to a defect in neuronal plasma membrane repair in due to A deposition. We discovered a neuronal membrane repair defect in cell types treated with A and AD patient CSF, and in ex vivo APP/PS1 mice. We determined A was the cause of the induced repair defect in CSF treated cells because when it was depleted the repair defect was rescued. Furthermore, we identified a decrease in dysferlin protein expression, a membrane repair protein, which was due to overactive autophagy. Lastly, we tested a novel therapeutic, recombinant human MG53, as a viable therapeutic to increase membrane repair and reduces neurotoxicity in vitro. Limb Girdle Muscular Dystrophy (LGMD) is a genetic muscle disease caused by a mutation in dysferlin which leads to dysferlin deficiency in cells. LGMD is known to have an intrinsic membrane repair defect due to the loss of dysferlin protein expression. We hypothesized there was an extrinsic factor that exacerbated the membrane repair defect in LGMD mus (open full item for complete abstract)

Committee: Noah Weisleder (Advisor); Christoph Lepper (Advisor); Olga Kokiko-Cochran (Committee Member); Nuo Sun (Committee Member); Loren Wold (Committee Member) Subjects: Biomedical Research; Cellular Biology; Molecular Biology; Neurobiology

PhD, University of Cincinnati, 2024, Medicine: Cancer and Cell Biology

Clear cell renal cell carcinoma (ccRCC) is the most common cancer of the kidneys. Understanding the mechanisms that govern tumor suppression and tumor progression is critical to facilitate the discovery of novel therapeutic avenues for treatment of ccRCC and to develop biomarkers for predicting relapse and response to treatment. Presented here are three studies investigating several of these mechanisms that we have recently identified. The first part elucidates the contribution of copper in driving tumor progression through metabolic rewiring and the pathways involved in maintaining cellular homeostasis in this condition. Namely, copper accumulation and allocation to cytochrome c oxidase is associated with higher stage tumors. Furthermore, copper augments tumor growth in xenograft models. Copper drives oxidative phosphorylation (oxphos) supported by mitochondrial remodeling which contributes to energy and biosynthesis. This induction of ETC/oxphos is coordinated with synthesis of a pool of glutathione derived from glucose through the activity of glutamate pyruvate transaminase 2 (GPT2) which couples the two pathways. Synthesis of glutathione is required to maintain redox homeostasis in these cells and represents an area of possible therapeutic vulnerability. The second part focuses first on the regulation of the non-canonical tumor-suppressing autophagic program requiring Microtubule Associated Protein 1 Light Chain 3 Gamma (MAP1LC3C/LC3C). LC3C is regulated by non-canonical upstream regulatory complex components whose assembly is coordinated by the unique C-terminal peptide that is present on the LC3C. The post-division midbody, associated with cancer cell stemness, is a LC3C specific target. Finally, we show that high lysosomal exocytosis is induced by loss of LC3C and leads to a decrease in intracellular zinc and transcriptomic reprogramming. This drives aggressive ccRCC tumor formation in mice. Together these studies deepen our understanding of the pathogenesis (open full item for complete abstract)

Committee: Maria Czyzyk-Krzeska M.D. Ph.D. (Committee Chair); David Plas Ph.D. (Committee Member); Atsuo Sasaki Ph.D. (Committee Member); Krushna Patra Ph.D. (Committee Member); Nicolas Nassar Ph.D M.A B.A. (Committee Member); John Cunningham Ph.D. (Committee Member) Subjects: Cellular Biology

MS, University of Cincinnati, 2024, Medicine: Cancer and Cell Biology

Healthy adults must generate approximately 2 million red blood cells (RBCs) per second to maintain normal hematocrit through an orderly process called erythropoiesis. Any disruption in this process often results in pathological outcomes. As erythroblasts mature, they undergo significant morphological transformations, including cell size reduction, chromatin condensation, and an increase in hemoglobinization. This intricate process concludes with the expulsion of the nucleus and organelles like mitochondria, accompanied by membrane restructuring to form highly specialized cells. Autophagy, a “self-eating” mechanism that breaks down cellular components, plays a vital role in maintaining the balance and quality of hematopoietic stem cells (HSCs) and in reshaping terminal erythroblasts. Multiple studies have investigated the impacts of various autophagy-related proteins on erythropoiesis through targeted gene deletion in mouse models or silencing in CD34+ HSCs. These investigations indicate that disturbances in autophagy contribute to anemia by impairing erythrocyte maturation and mitophagy. Canonical macroautophagy relies on the autophagy-related gene (ATG) family, with ATG5 and ATG7 serving as crucial rate-determining participants. While extensive research examining the consequences of Atg7 loss in mouse models has documented severe anemia as a result, patients with deficiency or absence of ATG7 have been described to suffer from complex neurodevelopmental disorders with brain, muscle, and endocrine involvement, but no anemia. Conversely, the role of ATG5 in erythropoiesis has not received as much scrutiny. An infant enrolled in the Congenital Dyserythropoietic Anemia Registry (CDAR) of North America after parental consent (clinicaltrials.gov: NCT02964494; P.I. Theodosia Kalfa, MD, PhD) had presented with a multi-system congenital syndrome, including transfusion-dependent anemia with evidence of dyserythropoiesis. Genetic evaluation including whole genome sequencing ( (open full item for complete abstract)

Committee: Theodosia Kalfa M.D. Ph.D. (Committee Chair); Andrew Volk Ph.D. (Committee Member); Amanda Wasylishen Ph.D. (Committee Member); Nicolas Nassar Ph.D. (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy (PhD), Ohio University, 2024, Molecular and Cellular Biology (Arts and Sciences)

Functional studies of the ubiquitin (Ub)-26S proteasome system (UPS) have demonstrated that virtually all aspects of the plant's life involve UPS-mediated turnover of abnormal or short-lived proteins. However, developmental characterization of the UPS, including in seeds and fruits, remains scarce. Unfortunately, early termination of embryogenesis limits the scope for characterizing the UPS activities in reproductive organs. In this dissertation, I utilized a biochemical approach to tackle the developmental role of UPS in plants, using Arabidopsis thaliana (Arabidopsis hereafter) as a model. The overarching goal of my research is to unravel the molecular mechanisms of UPS underpinning seed development so that new molecular breeding technologies could be developed to promote seed production. First, I systematically compared expression changes of multiple 26S proteasome subunits along with the dynamics of proteasome activity and total protein ubiquitylation in seedlings and developing siliques of Arabidopsis. Because autophagy plays the second largest role in maintaining proteome stability, I parallelly studied three late-limiting enzymes that are involved in autophagy influx. My experiments unexpectedly discovered that, in opposite to the activities in seedlings, both protein and transcript levels of six selected 26S proteasome subunits gradually decline in immature siliques toward maturation while the autophagy influx rises, albeit in a nutrient-rich condition. I also discovered a reciprocal turnover pathway between the proteasome and autophagy. While the autophagy influx is suppressed in seedlings by UPS-mediated degradation of its three key enzymes, transcriptional reprogramming dampens this process in siliques that in turn stimulates a bulk autophagy degradation of proteasomes. Collectively, my discovery about the developmental changes of the UPS and autophagy activities suggests that they relay the proteome homeostasis regulation in early seed development, w (open full item for complete abstract)

Committee: Zhihua Hua (Advisor) Subjects: Biology; Plant Biology; Plant Sciences

Doctor of Philosophy (PhD), University of Toledo, 2023, Experimental Therapeutics

Triple-negative breast cancer (TNBC), the most lethal and aggressive subtype of breast cancer, lacks estrogen receptors, progesterone receptors, and human epidermal receptors, rendering it unsuitable with targeted-based treatment. TNBC has higher relapse rate, worst prognosis and higher metastasis rate compared to non-TNBC because of their tendency to resist to apoptosis, a form of programmed cell death, induced by chemotherapy. Hence, non-apoptotic cell death inducers could be a potential alternative to circumvent the apoptotic drug resistance. In this study, we discovered two novel compounds, TPH104c and TPH104m, which induce non-apoptotic cell death in TNBC cells. These lead compounds were 15 to 30-fold more selective in TNBC cell lines and significantly decreased the proliferation of TNBC cells compared to normal mammary epithelial cell lines. TPH104c and TPH104m induced a unique type of non-apoptotic cell death characterized by no cellular shrinkage, absence of nuclear fragmentation and f apoptotic blebs. Although TPH104c and TPH104m produced the loss of the mitochondrial membrane potential, TPH104c- and TPH104m-induced cell death did not increase total cytochrome c and intracellular ROS, lacked caspases activation, and was not rescued by pan-caspase inhibitor, zVAD-FMK. Moreover, TPH104c and TPH104m significantly downregulated mitochondrial fission protein, Drp1 and its levels determined their cytotoxic efficacy. Studies have shown that protein, Bcl-2 interacting protein 3 (BNIP3), mediates a non-apoptotic, necrosis-like cell death similar to that produced by TPH104c and TPH104m that lacked activation of caspases and reduced mitochondrial transmembrane potential. Therefore, we determined the effect of TPH104c and TPH104m on various mitochondrial functions, in triple negative breast cancer (TNBC) cells, BT-20 and MDA-MB-231. TPH104c and TPH104m (2 and 5 μM), compared to vehicle, significantly increased the levels of reactive oxygen species (ROS) BNIP3 and c-Jun (open full item for complete abstract)

Committee: Amit K. Tiwari (Committee Chair); Aniruddha Ray (Committee Member); Ana Maria Oyarce (Committee Member); Frederick E. Williams (Committee Member) Subjects: Pharmacology

Master of Science (M.S.), University of Dayton, 2023, Interdisciplinary Studies

The SARS-CoV2 virus was responsible for the COVID-19 Pandemic, one of the most fatal international public health emergencies experienced in the past century. SARS-CoV2 induces symptoms like increased inflammatory response, severe acute respiratory syndrome (SARS), cognitive dysfunction like brain fog, and cardiovascular defects. Prolonged or long-term infection led to the emergence of Post-COVID-19 Syndrome, or PCS. PCS is characterized by chronic cardiovascular, autoimmune, and neurological manifestations and remains understudied. Individuals with pre-existing neurological insult like those with neuroinflammatory or neurodegenerative diseases are likely more vulnerable to such PCS effects. Furthermore, individuals with pre-existing neurological conditions often have comorbidities like obesity, hypertension, hyperlipidemia, and low activity levels. However, little is understood about the molecular effects of SARS-CoV2 on neuron in both healthy and neuro-compromised individuals. Currently, many individuals experiencing PCS-related neurological symptoms require management of their symptoms even though our knowledge in this area is still limited. Therefore, this study utilized an interdisciplinary approach to better understand how SARS-CoV2 impacts both neurons at a cellular level and clinically in neurologically compromised populations such as Multiple Sclerosis (MS). This interdisciplinary approach sheds light on how translational work is being done where basic science efforts complement efforts made clinically to make connections and identify relationships between observed effects and known science. To do so, SARS-CoV2 proteins were misexpressed in the Drosophila eye and through a forward genetic screen evaluated for changes to cellular structure or function. To corroborate these findings, SARS-CoV2 proteins were also transfected into Neuro-2a cells to assess how these proteins affected cellular functioning. Furthermore, SARS-CoV2 protein structure-function analys (open full item for complete abstract)

Committee: Kurt Jackson (Advisor); Mrigendra Rajput (Advisor) Subjects: Biomedical Research; Neurology; Neurosciences; Physical Therapy; Virology

Doctor of Philosophy, The Ohio State University, 2022, Biomedical Sciences

The projects described within address a knowledge gap regarding specific epigenetic regulatory mechanisms underlying the dysfunction of microglia in Alzheimer's disease. AD is the 6th leading cause of death in the US. Of the top 10 causes of death, AD is the only one which currently cannot be prevented or cured. AD brain pathology is characterized by build-up of amyloid-β (Aβ) plaques and neurofibrillary tangles of tau protein. Previous research has established that neuroinflammation also contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the central nervous system, in AD pathogenesis. For instance, microglia are unable to effectively phagocytose and degrade Aβ aggregates, and they instead respond by releasing pro-inflammatory cytokines which are implicated in AD neurodegeneration. How microglia initiate these inflammatory responses without simultaneous clearance of Aβ is unknown. Autophagy is a normal, degradative process that discards non-functional cellular organelles, internalized microbes, protein aggregates, and regulates release of inflammatory proteins. Extensive research in AD established that diseased neurons exhibit dysfunctional autophagy, which contributes to neuronal death. However, autophagy function in other brain cells in AD, such as microglia, remains overlooked. This work uncovered the first-described mechanism underlying dysfunctional autophagy in AD microglia. The MicroRNA (miR) Mirc1/Mir17-92 cluster, known to diminish autophagy effectors, is elevated in AD microglia from human patient samples. Microglia from a mouse model of AD fail to clear Aβ due to the elevation of the Mirc1/Mir17-92 cluster. However, autophagy function and clearance of Aβ can be recovered by inhibiting miR-17 in diseased mouse microglia. Moreover, this work established a nanoparticle-based therapeutic (open full item for complete abstract)

Committee: Amal Amer (Advisor); Amy Lovett-Racke (Committee Chair); Douglas Scharre (Committee Member); Stephanie Seveau (Committee Member); Kathryn Lenz (Committee Member); Ruth Barrientos (Committee Member) Subjects: Immunology; Neurosciences

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

Cystic fibrosis (CF) is an autosomal recessive disease that mainly affects the Caucasian population with no definitive available cure. CF is caused by specific mutations in the Cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes an ion channel which has been mostly studied in epithelial cells. CFTR F508del mutation is the most common mutation which, in epithelial cells, prevents the CFTR protein from reaching the plasma membrane due to misfolding. People with CF (pwCF) are more prone to infections by opportunistic pathogens such as Burkholderia cenocepacia (B. cenocepacia) resulting in fatal respiratory infections. Macrophages play critical roles in the process of clearance of lung pathogens, the locations and functions of CFTR in macrophages are unclear. In CF, human and mouse macrophages expressing F508del CFTR are defective in their ability to kill bacteria. Furthermore, the autophagy process in CF macrophages is halted, and the underlying mechanism remains to be elucidated. Using 3D reconstruction and confocal microscopy, we demonstrate that CFTR protein is expressed in the macrophage and on the autophagosome. We identified that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia, but not to vacuoles enclosing Escherichia coli (E. coli). Using several complementary approaches, we found that F508del CFTR macrophages display defective lysosomal acidification as well as impaired degradative function for cargos destined to autophagosomes such as B. cenocepacia, whereas non-autophagosomal cargos such as E. coli are effectively degraded. CFTR modulators are used for treating CF patients, via correction of misfolded CFTR, and improving its function in epithelial cells. Yet, their effect on macrophages is still unclear. Here, we show that treatment of CF macrophages with CFTR modulators increased F508del CFTR localization to B. cenocepacia containing vacuoles, improved the autophagy flux, and lysosomal function. Additionall (open full item for complete abstract)

Committee: Amal Amer (Advisor); Mark Wewers (Committee Member); Stephen Kirkby (Committee Member); Estelle Cormet-Boyaka (Committee Co-Chair); Stephanie Seveau (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy (PhD), University of Toledo, 2021, Experimental Therapeutics

The development of resistance to apoptosis-inducing chemotherapeutic compounds is a major challenge associated with the treatment of solid malignant tumors. An example of such cancer is colorectal cancer (CRC). CRC cancer cells often contain mutations in genes that regulate programmed apoptotic cell death (e.g., Bax, KRAS, and p53), which produces resistance to conventional chemotherapy. Our lab has designed and characterized a novel, structurally constrained compound 4-(((1H-Indol-3-yl) methylene) imino)-5-(pyridin-4-yl)-4H-1, 2, 4-triazole-3-thiol termed BAPT-27. BAPT-27 causes a form of non- apoptotic cell death in CRC cells characterized by the simultaneous induction of methuosis (i.e., self-drinking) and macroautophagy (i.e., self-eating). We defined this distinctive type of caspase-independent cell death as `methuophagy' (from the Greek' methuo,' to drink to intoxication, and `phagy,' self-eating). The specific sequence of subcellular events or the molecular pathways producing this novel form of cell death remains to be elucidated. The synthesis of BAPT-27 - related compounds, including 6-(2-methyl-1H-indol-3-yl)-3-(pyridin-4-yl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (BAPT-38), 2-methyl-3-(4-(pyridin-4-yl)-2H-1,2,3-triazole-2-yl)-1H-indole (BAPT-54), and 4-(2-methyl-1H-indol-3-yl)-6-(pyridin-4-yl)pyrimidine-2(1H)-thione (BAPT-42), allowed us to study the structure-activity relationships (SAR) of a directed library of BAPT-27. The new series of compounds will be useful in delineating the cellular and molecular mechanisms underlying this novel type of cancer cell death. This dissertation reports the characterization of the anti-cancer efficacy and mechanisms by which the novel BAPT-analogs produce anti-cancer effect in CRC cells. We evaluated whether structural differences of novel BAPT-analogs could produce mechanistic differences in CRC cell lines. BAPT-38 and BAPT-54, at submicromolar concentrations, induced non-apoptotic cell death associated with (open full item for complete abstract)

Committee: Amit K. Tiwari (Committee Chair); William S. Messer (Committee Member); Ana Maria Oyarce (Committee Member); F. Scott Hall (Committee Member); Yuan Tang (Committee Member) Subjects: Pharmacology

PHD, Kent State University, 2021, College of Arts and Sciences / School of Biomedical Sciences

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by the accumulation of beta-amyloid plaques and neurofibrillary hyperphosphorylated tau tangles. According to the most recent report from the U.S. Centers for Disease Control and Prevention, Alzheimer's disease is the 6th leading cause of death in the United States and the number of cases as increased almost 150% in the last 20 years in the United States. The disease starts as a subtle change in memory that progresses into noticeable cognitive decline, often referred to as mild cognitive impairment, and progresses into severe and pervasive memory loss. One of the hallmarks of Alzheimer's disease is the accumulation of the misfolded protein product of the amyloid precursor protein, beta-amyloid. Beta-amyloid has been long considered and widely supported by genetics and biochemical observation to promote Alzheimer's disease pathology. One of the earliest and long-supported theories, the amyloid hypothesis, posits beta-amyloid accumulation as having a central role in the pathogenesis of Alzheimer's disease as a promoter of tau seeding and inflammatory signaling which causes immune dysfunction. Beta-amyloid therapeutics represent the majority of the clinical trial candidates for Alzheimer's disease therapeutics. Recent clinical failures of anti-beta-amyloid trials, including Aducanumab and Gantenerumab, have raised concerns for the ability to manage beta-amyloid accumulation. The current pipeline of drugs targeting both beta-amyloid and tau are ineffective; therefore warranting alternative therapeutic options. A possible alternative non-pharmacological option for targeting beta-amyloid plaque aggregation is using energy to disrupt large beta-amyloid plaques into smaller fragments that may be cleared by microglia, the innate immune cells of the brain. One manner in which we can generate sufficient energy in a minimal to non-invasive safe manner is to use an alternating magnetic (open full item for complete abstract)

Committee: Min-Ho Kim Ph.D. (Advisor); Fayez Safadi Ph.D. (Advisor); Colleen Novak Ph.D. (Committee Chair); Gary Koski Ph.D. (Committee Member); Songping Huang Ph.D. (Committee Member) Subjects: Biomedical Engineering; Biomedical Research; Nanoscience; Nanotechnology; Neurobiology; Neurology; Neurosciences

Doctor of Philosophy (PhD), Ohio University, 2021, Chemistry and Biochemistry (Arts and Sciences)

Ultraviolet B (UVB) radiation has several detrimental effects in the skin, such as promoting aging and carcinogenesis. Previous studies from our group demonstrated that immediately after UVB exposition, the constitutive nitric oxide synthase (cNOS) enzyme produces elevated levels of nitric oxide (NO•) and peroxynitrite (ONOO−) in keratinocytes. These strong oxidants induced DNA damage and the activation of NF-κB-dependent on the phosphorylation of the eukaryotic initiation factor 2 (eIF2). This work focuses on study the role of cNOS after UVB on the regulation of two mechanisms that are tightly related to skin cancer development: DNA damage and autophagy. Our results indicate that cNOS increases the DNA damage by the induction of cyclobutane pyrimidine dimers (CPDs) at early times after UVB irradiation; and that UVB induces the autophagy response, which dynamic balance is regulated by cNOS.

Committee: Shiyong Wu (Advisor) Subjects: Biochemistry

Doctor of Philosophy, Case Western Reserve University, 2020, Pharmacology

Breast cancer is the most commonly diagnosed cancer in women, claiming the lives of more than 40,000 women each year. The majority of these cancer associated deaths are due to metastasis, a clinically incurable disease with few treatment options. The multifactorial cytokine, TGF-Β, functions to mediate epithelial mesenchymal transition (EMT), a critical process by which breast cancer cells metastasize. Following dissemination from the primary tumor, breast cancer stem cells (BCSCs), cells capable of tumor initiation, can lie dormant and non-proliferative for decades before emerging as distant metastases. The studies presented herein have identified a glycolytic gene, Pfkfb3, as a key mediator of TGF-Β induced EMT and as a driver of emergence from metastatic dormancy. We show that aberrant Pfkfb3 expression can mediate escape from metastatic dormancy, doing so through the upregulation of BCSC frequency. Furthermore, we show that inhibition of autophagy, a key cytoprotective mechanism employed by BCSCs, can promote aberrant Pfkfb3 expression and drive metastatic outgrowth following a period of metastatic dormancy. We have identified Pfkfb3 as an autophagy substrate, interacting with the UBA domain of p62. During periods of high autophagy, p62 bound Pfkfb3 is degraded. However, upon autophagy inhibition, p62 accumulation promotes the stabilization of Pfkfb3 which ultimately drives emergence from dormancy and overt metastasis. Dissemination of BCSCs to distant metastatic sites is mediated in large part through TGF-Β-stimulated EMT. We demonstrate that Pfkfb3 expression is critical for breast cancer invasion and metastasis both in vitro and in vivo. In addition to roles in mediating survival through EMT, we also show that Pfkfb3 can modulate autophagy activation, a key regulatory mechanism of TGF-Β stimulated EMT. Importantly, we demonstrate that autophagy inhibition promotes the stabilization of the critical EMT mediator, Twist, suggesting that autophagy can se (open full item for complete abstract)

Committee: William Schiemann (Advisor); Marvin Nieman (Committee Chair); Ruth Keri (Committee Member); Alex Almasan (Committee Member); Mark Jackson (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology; Oncology; Pharmacology

Doctor of Philosophy, The Ohio State University, 2019, Pharmacy

Natural products were once the only source of medicine and, since the dawn of modern drug discovery, have continued to serve as inspiration for the development of novel pharmaceuticals. The use of purified plant constituents greatly advanced the treatment of cancer towards the end of the 20th century. However, there remain many shortcomings with current treatment options with regards to toxicity, resistance, and overall effectiveness. The phyllanthusmin class of natural products, initially identified in 2006 to consist primarily of arylnaphthalene lignan arabinosyl glycosides, possess potent antiproliferative activity as well as a versatile pharmacophore whose exploration has recently been well studied, but not yet exploited into a known drug. The primary work described herein involves utilizing the phyllanthusmin scaffold to identify a novel anticancer lead compound, its mechanism of action, and develop novel synthetic routes in order to further streamline drug property optimization.

Committee: James Fuchs PhD (Advisor); Liva Rakotondraibe PhD (Committee Member); Mark Mitton-Fry PhD (Committee Member) Subjects: Organic Chemistry; Pharmacy Sciences

Doctor of Philosophy (PhD), Wright State University, 2019, Biomedical Sciences PhD

Human adenoviruses (AdV) are double-stranded DNA viruses that can cause a range of diseases. While AdV respiratory infections are often mild and self-limited, severe manifestations such as fulminant pneumonia and acute respiratory distress syndrome (ARDS) are not uncommon. In order to identify potential modalities to treat AdV infections and potentially enhance the effectiveness of AdV vector delivery, a thorough mechanistic understanding of how AdV enters host cells is needed. Previous studies have revealed polarized epithelia, one of the primary targets for AdV respiratory infections, are highly resistant to AdV entry from the apical (luminal) surface. However, upon exposure to interleukin 8 (IL-8), the 8-exon encoded isoform of the Coxsackie and Adenovirus receptor (CAREx8) localizes to the apical surface of airway epithelial cells where it is able to mediate AdV entry. Furthermore, neutrophils, or polymorphonuclear leukocytes (PMN), that migrate to the epithelium as a result of the IL-8 stimulus have been shown to further enhance AdV infection of the epithelium through an uncharacterized mechanism. I hypothesized that neutrophilic factors alter epithelial physiology in such a way that renders them more susceptible to AdV infection and that a combination of enhanced apical CAREx8 expression and PMN factor signaling drastically enhances epithelial susceptibility to AdV5 infection. In order to address this hypothesis, a variety of pharmacological inhibitors were tested for their ability to influence AdV entry in epithelial cells that had been exposed to PMN or various PMN factors. Furthermore, CRISPR/Cas9 techniques were used to develop a CAREx8-knockdown epithelial model system to address the importance of CAREx8 in AdV entry in the presence and absence of neutrophilic factors. Using these methods, I demonstrate that neutrophil elastase (NE), a PMN serine protease with diverse functions, is a major neutrophilic factor that drives PMN-mediated enhancement of epit (open full item for complete abstract)

Committee: Katherine J.D.A. Excoffon Ph.D. (Advisor); Jeffrey B. Travers M.D., Ph.D. (Committee Member); Dawn P. Wooley Ph.D. (Committee Member); Dan R. Halm Ph.D. (Committee Member); Hongmei Ren Ph.D. (Committee Member) Subjects: Biomedical Research; Cellular Biology; Virology

Doctor of Philosophy, Case Western Reserve University, 2019, Pathology

Toxoplasma gondii is an obligate intracellular protozoan parasite that can cause disease in the brain and the eye. It is estimated that 1/3 of the population worldwide is chronically infected. After host cell invasion, T. gondii resides in a specialized compartment known as the parasitophorous vacuole (PV) that protects the parasite from being targeted for lysosomal degradation. T. gondii, additionally, manages to avoid autophagy by inducing EGFR signaling in host cells early, during host cell invasion. The brain and eye are the most clinically relevant organs in infection with T. gondii. The parasite reaches these organs hematogeneously after overcoming the blood-brain and blood-retinal barriers. Therefore, we set out to determine the role of EGFR expression in modulating T. gondii invasion to the brain and the eye. Transgenic mice with conditional expression of a dominant negative form of EGFR (DN EGFR) were infected with T. gondii. Deficiency in EGFR activation resulted in diminished parasite load and histopathology in the brain and eye. This was accompanied by reduction of parasite foci in brain endothelial cells, accumulation of LC3 structures around the parasite and parasite killing that was dependent on autophagy proteins and lysosomal enzymes. Thus, expression of EGFR in endothelial cells is an important regulator of T. gondii invasion to the brain and eye that promotes survival of the parasite. The previously described mechanisms by which T. gondii engages EGFR signaling occur early during invasion. Therefore, we hypothesized that the parasite must have an alternative mechanism to maintain EGFR signaling at later timepoints. We found that there is phosphorylation of EGFR that persists for up to 18h and is dependent on PKC/-Src signaling. Since EGFR signaling is relevant to parasite survival throughout the cycle of infection, we set out to determine whether in vivo inhibition of EGFR after pre-established infection with T. gondii led to control (open full item for complete abstract)

Committee: Carlos Subauste MD (Advisor); Donald Anthony (Committee Chair); David Canaday (Committee Member); W Henry Boom (Committee Member); Cathleen Carlin (Committee Member); Clive Hamlin (Committee Member) Subjects: Biomedical Research; Immunology; Microbiology; Parasitology; Pathology

Doctor of Philosophy, The Ohio State University, 2019, Molecular, Cellular and Developmental Biology

Cancer is the second leading cause of death in the United States with over 1.6 million people diagnosed annually. Standard cancer treatments include surgery, radiation, and biological or chemical therapies. Although treatments result in primary tumor removal or reduction, there are often disseminated tumor cells (DTCs) that have left the primary tumor, sometimes before it has even been detected, and lodge at distant sites in the body. Here DTCs can remain dormant for long periods during which patients have no evidence of residual disease. After a period of time, sometimes lasting years, external or internal changes may trigger a break in dormancy and the growth of DTCs into active tumors. Hence, targeting DTCs, while dormant, could prevent metastatic relapse. Here I describe a Drosophila tumor cell model in which oncogenic RasV12 expression can be regulated to induce a reversible arrest in G0/G1. When RasV12 is expressed, the cells proliferate. When RasV12 is switched off, the cells stop proliferating but remain viable in a quiescent state. The stability of the quiescent state, which can last for weeks, allowed an analysis of the biological processes activated in the dormant cells. RNA-sequencing was performed to identify genes involved in tumor dormancy. These dormant cells exhibited altered metabolism and conserved features of mammalian DTCs including stress signaling (p38) and increased autophagy. Upregulated Foxo signaling was also identified. Both autophagy and Foxo signaling were essential because blocking these processes, genetically or with drugs, specifically killed the dormant cells. The conserved nature of dormancy in the fly model suggests the cells will be useful for gene and drug discovery.

Committee: Amanda Simcox (Advisor); Sharon Amacher (Committee Member); Mark Seeger (Committee Member); Anne Strohecker (Committee Member) Subjects: Biology; Cellular Biology; Genetics; Molecular Biology

Master of Science (MS), Wright State University, 2018, Biological Sciences

Optineurin, an autophagy adaptor protein genetically linked to Normal Tension Glaucoma and Amyotrophic Lateral Sclerosis, has been found in the pathological inclusions of various other neurodegenerative disorders, supporting an important role of optineurin in neurodegeneration. Using yeast as a model, we found that overexpression of optineurin drastically enhances the toxicity of the Parkinson's disease-causing protein, alpha-synuclein. Considering the conserved protein-protein interaction between optineurin and Ypt1, a yeast suppressor of the toxicity of both optineurin and alpha-synuclein, we hypothesize that cellular targets of optineurin underlie in the cytotoxicity and the enhancer effect. Using genome-wide yeast two-hybrid screens, we identified 97 yeast interacting proteins of optineurin and systematically tested their modifier effect on the toxicity of optineurin and alpha-synuclein. Given the neuroprotective effect of the mammalian homologs of Ypt1, the convergent pathways of the identified modifiers may represent conserved cellular perturbations induced by optineurin overexpression in higher eukaryotes.

Committee: Quan Zhong Ph.D. (Advisor); Mill Miller Ph.D. (Committee Member); Paula Bubulya Ph.D. (Committee Member) Subjects: Cellular Biology; Genetics; Neurosciences

Doctor of Philosophy, Case Western Reserve University, 2018, Pathology

Aging is a progressive, global degeneration in cellular functions which occurs as homeostatic mechanisms become insufficient, leading to an age-related exponential increase in risk of mortality and age-associated disease. Aging has characteristic features shared across phylogeny and is subject to biologic control. Here I describe a regulatory pathway which modulates lifespan and healthspan of the nematode Caenorhabditis elegans via enhancement of cellular proteostasis. Specifically, direct transcriptional control of macroautophagy by the family of Kruppel like factors (KLFs) alters nematode lifespan and age-associated phenotypes. Further, the KLFs are broadly required in at least four mechanistically distinct longevity pathways, suggesting that diverse upstream signaling converges on a KLF regulatory node to effect a common pro-longevity response. The KLF-autophagy pathway is conserved in mice vasculature to regulate endothelial autophagy and vascular aging. As the KLFs are well accepted as mechanotransducers of laminar shear stress, we also suggest that KLFs mediate the effects of shear stress on endothelial autophagy. Long-lived nematodes overexpressing a KLF also are protected from neurodegeneration in a model of Parkinson's Disease, therefore experiencing an extension of time spent free of age-associated debility. Interestingly, only an intestinal KLF is required for this effect and these nematodes do not experience lifespan extension. A putative secreted c-type lectin (ortholog for mammalian complement protein COLEC11) mediates intestinal KLF neuroprotection, linking gut signals with neuronal proteostatic mechanisms. Together, these observations outline a novel longevity pathway, in addition to known biologic mechanisms regulating aging, and define a role for the KLFs in determining organismal lifespan.

Committee: Mukesh Jain (Advisor); George Dubyak (Committee Chair); Jeff Coller (Committee Member); Goutham Narla (Committee Member); Clive Hamlin (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology; Neurobiology; Physiology