PhD, University of Cincinnati, 2023, Medicine: Cancer and Cell Biology

Human papillomavirus is the most common sexually transmitted pathogen in the US and drives benign and malignant diseases, namely high-risk (e.g. HPV16) and low-risk (e.g. HPV6; HPV11) strains. High-risk HPV strains are responsible for 5% of cancers worldwide while low risk strains cause benign tumors including those in recurrent respiratory papillomatosis (RRP). HPV specifically infects the stratified squamous epithelium, where the virus can either remain latent, thus avoiding immune detection, or actively replicate and express viral genes in synchrony with epithelial differentiation. Despite decades of research, no antivirals exist to clear HPV infection and it remains unknown which specific epithelial cell subpopulation(s) support disease formation and/or persistence. Single-cell RNA sequencing technologies enable a granular understanding of distinct cell types and subpopulations within complex tissues. Thus, we applied scRNAseq to epithelial tissues to (1) identify the transcriptomes of keratinocyte subpopulations and (2) discover HPV reprogramming and viral-driven disease phenotypes in infected epithelium. In Part One, we utilized 3D organotypic epithelial rafts, a model of stratified squamous epithelium, to compare isogenic HPV16+ versus HPV16-naive tissue. We identified twelve transcriptomically distinct keratinocyte clusters and spatially mapped select subpopulations, including actively replicating cells and those that comprise basal and differentiated layers. HPV16-driven reprogramming was evidenced by changes in gene signatures of defined subpopulations (e.g. basal cells), in the localization of replicating cells, and in the overall composition of the epithelium wherein we discovered an HPV-induced differentiated subpopulation. This subpopulation, termed HPV-induced differentiation dissonant epithelial nonconventional (HIDDEN) cells, was further determined to be driven by the transcription factor ELF3 and to be enriched in stages of HPV+ car (open full item for complete abstract)

Committee: Susanne Wells Ph.D. (Committee Chair); S. Steven Potter Ph.D. (Committee Member); Joseph Qualls Ph.D. (Committee Member); Kathryn Wikenheiser-Brokamp M.D. (Committee Member); David Smith M.D. Ph.D. (Committee Member); Daniel Starczynowski Ph.D. (Committee Member); Marc Rothenberg M.D. Ph.D. (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy (PhD), Ohio University, 2023, Chemistry and Biochemistry (Arts and Sciences)

The regulation of various genes associated with cell cycle, proliferation, and apoptosis relies significantly on NF-κB (Nuclear Factor Kappa-light-chain-enhancer of activated B cells) transcription factors. These transcription factors are critical for the UV-induced production of ROS, which is linked to skin disease pathophysiology. Our previous study showed that UV radiation can activate NF-κB through the activation of constitutive nitric oxide synthase(s) (cNOS) in human keratinocyte HaCaT cells in the early phase (within 6 h) post-UVB. One of the upstream NF-κB proteins, I kappa B kinase alpha (IKKα), is also involved in the pathway post-UVB. However, the regulation of IKKα after UVB irradiation is not clear. In this study, I used HEK293 cells to study the role of cNOS as it is known for the cNOS-null cell line in comparison with the overexpression of cNOS in HEK293cNOS cells. My evidence showed that the expression of cNOS increases the IKKα promoter activities in the presence and absence of UVB. In addition, second p53 and ETS-1 binding sites that are elements in IKKα promoter are critical for maintaining the IKKα activity and UVB inducibility. In addition to the promoter, our data also demonstrated that UVB inhibits the elongation of IKKα mRNA transcription but not mRNA stability. These results indicated that while IKKα promoter is activated by UVB, the elongation inhibition of the mRNA synthesis limited the expression of IKKα. Later, we checked their protein levels in the presence and absences of cNOS both in vitro and in vivo. The protein level also confirmed the presence of cNOS can increase the basal level of IKKα in order to prevent skin tumor formation. In addition to IKKα, the effect of cNOS was also studied in skin aging and wrinkle using L-NAME, a selective inhibitor of NOS, with UV treatment. Our data indicated cNOS could play a protective role in skin aging post-UV irradiation through activation of PERK/GCNE and p-eIF2α. Meanwhile, cNOS KO (nNOS+/- (open full item for complete abstract)

Committee: Shiyong Wu (Advisor); Michael Held (Committee Member); Jixin Chen (Committee Member); Xiaozhuo Chen (Committee Chair) Subjects: Biochemistry; Molecular Biology

PhD, University of Cincinnati, 2020, Medicine: Toxicology (Environmental Health)

The focus of this dissertation is the characterization of Cr(VI)'s disruption of chromatin architecture and its effects on the regulation of transcription, particularly through disruption of the CCCTC-binding factor's (CTCF) ability to recognize its cognate binding sites, and how this may promote tumor initiation and progression. The class of compounds containing Cr(VI), commonly known as chromates, are well-established respiratory carcinogens in occupational industries that include stainless steel production and welding, electroplating, and pigment production, among others. However, heavy use of these compounds in these processes results in environmental contamination, with global populations potentially exposed over the course of a lifetime to low levels of Cr(VI), primarily through ingestion in the form of drinking water. While several studies provide evidence for the adverse health outcomes associated with exposure, the mechanisms that induce these endpoints are not well understood. Cr(VI) promotes the generation of reactive oxygen species as well as the formation of Cr-DNA and ligand-Cr-DNA adducts, resulting in increased DNA damage, silencing of tumor suppressor genes, and genomic instability. Together with epidemiological and animal studies, this evidence suggests that Cr(VI) has carcinogenic properties regardless of exposure route and highlights the need to better understand how long-term exposure to low levels of chromates affects the risk of developing cancer. By studying the molecular mechanisms of Cr(VI) that promote carcinogenesis, this work contributes to the body of evidence used to inform public health decisions and risk mitigation. Chapter 1 provides a summary of the current understanding of Cr(VI), its relevance to Public Health, and the mechanisms that contribute to its carcinogenic properties. Chapter 2 characterizes the disruption of chromatin organization that occurs in vitro following treatment with Cr(VI) and how that affects CTCF's acces (open full item for complete abstract)

Committee: Alvaro Puga Ph.D. (Committee Chair); Susan Kasper Ph.D. (Committee Member); Liang Niu Ph.D. (Committee Member); Peter Stambrook Ph.D. (Committee Member); Ying Xia Ph.D. (Committee Member) Subjects: Toxicology

Doctor of Philosophy, The Ohio State University, 2018, Public Health

The increasing frequency and severity of harmful algal blooms (HABs) has quickly become an environmental health concern worldwide. As rates of anthropogenic nutrient use rise with growing food demand, and as extreme precipitation events are expected to increase with the changing climate, rates of nutrient influx into watersheds are expected to increase. With increasing nutrient loads and temperatures in watersheds, many of the world's water bodies and reservoirs are becoming eutrophic, establishing optimal conditions for HAB formation. HABs may change the ecology of water bodies; produce hypoxic zones resulting in fish deaths; and produce cyanotoxin compounds toxic to the liver, nervous system, and reproductive system of most eukaryotic organisms. Human and animal exposure to the most commonly occurring cyanotoxin, microcystin (MC), has been linked with hepatotoxicity, nausea, vomiting, and death in extreme circumstances. Within the MC family of toxins, microcystin-LR (MC-LR) is the most common, possesses the highest toxicity, and is cited as a suspected carcinogen. While HAB mitigation and MC exposure prevention efforts have often focused on large lakes and bodies of water, small lakes and ponds (SLaPs) remain understudied and unmonitored. SLaPs are the most numerous lentic bodies of water worldwide, providing vital ecosystem services and biodiversity support. Due to their location, low volume, and seasonal water level changes, SLaPs are at an increased risk for eutrophication and HAB formation. SLaPs have many uses including recreation, aquaculture, irrigation, and even drinking water sources in economically stressed areas of the world. The utility of SLaPs presents a potential exposure pathway to HABs and MC compounds. While our knowledge base of MC-related health outcomes continues to grow, several gaps exist, including: low dose, chronic exposure outcomes; differences in toxicity between pure toxic compounds and crude cyanobacterial extracts; and the cancer (open full item for complete abstract)

Committee: Jiyoung Lee (Advisor); Christopher Weghorst (Committee Member); CK Shum (Committee Member); Qinghua Sun (Committee Member) Subjects: Aquatic Sciences; Biology; Ecology; Environmental Health; Environmental Science; Geology; Land Use Planning; Public Health; Remote Sensing; Water Resource Management

Doctor of Philosophy, The Ohio State University, 2000, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1986, Graduate School

Committee: Not Provided (Other) Subjects: Health Sciences

Doctor of Philosophy, The Ohio State University, 1985, Graduate School

Committee: Not Provided (Other) Subjects: Health Sciences

Doctor of Philosophy, The Ohio State University, 1984, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1983, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1982, Graduate School

Committee: Not Provided (Other) Subjects: Health Sciences

Doctor of Philosophy, The Ohio State University, 1978, Graduate School

Committee: Not Provided (Other) Subjects: Biophysics

Doctor of Philosophy, The Ohio State University, 1978, Graduate School

Committee: Not Provided (Other) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 1974, Graduate School

Committee: Not Provided (Other) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 2016, Molecular, Cellular and Developmental Biology

Vitamin D, the precursor to the potent steroid hormone calcitriol (1,25(OH)2D), is obtained through synthesis following sunlight exposure, diet and supplements. Vitamin D is first metabolized by CYP2R1 in the liver to produce calcifediol (25(OH)D), a circulating form of vitamin D, which is further converted to 1,25(OH)2D by CYP27B1 primarily in the kidney. 1,25(OH)2D can also be produced by extrarenal CYP27B1 in local tissues, including the breast, functioning in an intracrine, autocrine or paracrine manner. 1,25(OH)2D binds to the vitamin D receptor (VDR) that regulates multiple gene expression. However, most studies reported circulating 25(OH)D level but not the level of 1,25(OH)2D in the target tissue. Laboratory studies indicate that vitamin D should decrease the risk of breast cancer, although epidemiological studies are mixed. Thus, the potential anticancer mechanisms for women remain elusive. In the breast, little is known about the metabolic consequences of vitamin D at the cellular level and how this may potentially impact early breast carcinogenesis. In order to address these issues, blood and tissue samples from 153 healthy women undergoing reduction mammoplasty were used to examine 25(OH)D, 1,25(OH)2D levels and VDR protein expression. These women never had cancer, so findings from their breast tissue would represent biological processes before cancer develops, e.g. early carcinogenesis. I aimed to assess the correlation of blood to breast vitamin D levels, to assess the usefulness of blood levels of 25(OH)D in epidemiology studies as markers for breast levels of 1,25(OH)2D (aim 1), and if there were breast cancer risk factors that affected breast vitamin D levels (aim 2) (Fig. 1.4). I also aimed to determine if there were breast VDR (receptor) that positively correlated to 1,25(OH)2D and 25(OH)D (ligands) (aim 3). To investigate if breast vitamin D alone or with VDR affected breast pre-carcinogenesis, different markers were determined in normal breast (open full item for complete abstract)

Committee: Peter Shields (Advisor); Jeffrey Parvin (Committee Member); Gregory Lesinski (Committee Member); Michael Freitas (Committee Member) Subjects: Biomedical Research; Biostatistics; Endocrinology; Epidemiology; Molecular Biology; Oncology; Womens Studies

PhD, University of Cincinnati, 2004, Medicine : Environmental Health Sciences

Iron is the most abundant transition metal in the body and on earth. Its ability to cause or promote DNA damage is therefore important for environmental mutagenesis and carcinogenesis. Iron's complex biology makes this a challenging task, requiring a multidisciplinary approach that considers both internal targets and external sources. Since in vitro evidence suggests iron potentiates oxidative DNA damage by chromate, this study examined internal mechanisms by which iron could promote chromium DNA damage, and identified external sources of co-exposure to the two metals. Mice exposed by intratracheal instillation showed greater chromium deposition and lacI transgene mutations in lung, liver and kidney. The mutational spectrum resembled that of hydrogen peroxide, suggesting Fenton-related chemistry. Similarity of human tumor p53 mutational spectra to lacI chromate spectra for large cell lung tumors, renal cell carcinoma, and cholangiocarcinoma could not be ruled out. An in silico model using lacI and p53 mutation databases was developed and validated to determine whether mutations cluster potential iron binding (PIB) DNA motifs. There was a significant association between PIB motifs and mutational odds, not only for chromate, but also for other compounds, some not thought to directly cause oxidative DNA damage. Randomly simulated mutations had no association with PIB motifs. Chromate induces oxidative damage at motifs susceptible to chromosomal instability, which is the predominant type of damage observed in chromate-linked cancers. The known ability of iron-binding to alter DNA strand conformation, viscosity and electrostatic charge may explain how iron promotes carcinogenicity of so many agents, many of which do not cause direct oxidative DNA damage. Iron's ability to promote chromate damage raises concerns about workplace co-exposures to the two metals. Department of Labor data from 40,000 individuals revealed patterns of co-exposure to iron and chromium from 1990-2 (open full item for complete abstract)

Committee: Dr. Glenn Talaska (Advisor) Subjects: Health Sciences, General

Doctor of Philosophy, The Ohio State University, 2009, Integrated Biomedical Sciences

Non-melanoma skin cancers (NMSCs), especially squamous cell carcinoma (SCC), are a significant problem in the general population, and a deadly one among immunosuppressed populations. Nearly a third of transplant patients die from aggressive SCCs, and the risk for development increases as the length of time under immunosuppression increases. It is not yet fully understood how immunosuppression impacts UVB-induced inflammation and carcinogenesis. In Chapter 2, we determine the impact of different immunosuppressive drugs on UVB-induced inflammation and carcinogenesis. We found that cyclosporine (CsA) exacerbates all markers of inflammation and carcinogenesis, including angiogenesis. Mycophenolate mofetil (MMF), when co-administered with CsA reduced the markers of inflammation and carcinogenesis to near vehicle levels. Choice of immunosuppression, therefore, plays arole in the development of SCC in transplant patients. The changes wrought in keratinocytes by the change from normal cell to malignant cell are poorly understood. Understanding the changes in cytokine and chemokine expression, as well as the changes in programmed cell death pathways would give new avenues for treatment and prevention. To expand the knowledge of these changes, we used an in vitro system of normal (JB6) and pre-malignant (308) keratinocytes in the presence and absence of UVB (Chapter 3). We found that 308 cells produce more inflammatory cytokines than JB6 cells, and 308 cells have an altered expression of autophagy proteins. These changes may illustrate the steps necessary for malignant transformation. Preventing the transformation of normal cells would be a life saver to the general population. The use of a treatment that would not interfere with immunosuppressive medications would be a boon to transplant patients. To address both of these we developed a post-UVB-exposure treatment derived from black raspberries (BRE). In Chapter 4, we explore the efficacy of BRE in the prevention of UVB-indu (open full item for complete abstract)

Committee: Tatiana Oberyszyn PhD (Advisor); Donna Kusewitt DVM, PhD (Committee Member); Gregg Hadley PhD (Committee Member); Traci Wilgus PhD (Committee Member) Subjects: Cellular Biology; Immunology; Molecular Biology; Oncology

Doctor of Philosophy, The Ohio State University, 2007, Veterinary Biosciences

Slug is a member of the Snail family of zinc-finger transcription factors and has been implicated in epithelial-mesenchymal transformation (EMT). EMT occurs during embryonic development and wound healing, and contributes to the invasive behavior of neoplastic cells. To study the impact of Slug in normal skin, we compared patterns of gene expression in epidermis from Slug null and wild type mice. Functional classification of genes with altered expression was consistent with a role for Slug in keratinocyte development and differentiation, proliferation, apoptosis, adhesion, motility, angiogenesis and response to environmental stimuli. These categories have important implications for skin homeostasis, wound healing, and carcinogenesis. We then investigated the role of Slug in the response to ultraviolet radiation (UVR). Although the extent of direct UVR-induced DNA damage was similar in both genotypes, Slug knockout mice did not develop the sunburns that were observed in wild type mice. Additionally, Slug knockout mice had reduced dermal neutrophil infiltrates and epidermal hyperplasia post-UVR. The resistance of the knockout mice to the acute effects of UVR exposure could be explained by delayed and sometimes exaggerated cytokine expression in these mice following UVR exposure. These findings indicated an important role for Slug in the acute inflammatory and proliferative responses of the skin to UVR exposure. Since chronic inflammation is strongly linked to carcinogenesis, we evaluated the role of Slug in the chronic UVR exposure and UVR-induced tumor formation. As compared to the wild type mice, Slug null mice had less cutaneous inflammation, less epidermal proliferation, a lower tumor burden, and a tendency to develop fewer aggressive spindle cell tumors – which arise from EMT within squamous cell carcinomas. Furthermore, we identified decreased vimentin and increased E-cadherin expression in these tumors suggesting impaired EMT in the Slug knockout mice, and there (open full item for complete abstract)

Committee: Donna Kusewitt (Advisor) Subjects: Biology, Veterinary Science

Doctor of Philosophy, The Ohio State University, 2006, Molecular, Cellular, and Developmental Biology

The goal of the present studies was to localize two proteins known to be involved in regulation of cell proliferation and survival in specific cell populations in normal mouse skin, during multi-stage skin carcinogenesis, following skin injury, and during tumor angiogenesis. The proteins evaluated included activated Akt, as defined by phosphorylation of Akt at Serine-473 (pAkt) and mTOR, defined by phosphorylation of mTOR at Serine-2448 (pmTOR). Our laboratory previously identified a novel murine VEGF splice variant, VEGF205*, which was differentially expressed in mouse skin carcinomas, but not in normal skin. VEGF205* encodes for a truncated 145 amino acid polypeptide with a unique 7 amino acid carboxyl-terminal tail, YVGAAAV, that is significantly different from the carboxyl-terminal tail of mouse or human VEGF proteins previously identified. VEGF205* stimulated a PI3-K-dependent-increases in pAkt and pmTOR in human vascular endothelial cells, which were significantly higher than pAkt and pmTOR levels induced following incubation of endothelial cells with VEGF120. Immunochemical staining analysis as well as triple color immunofluorescence were used in combination with confocal microscopy to evaluate the presence of pAkt and pmTOR in keratinocyte stem cells (KSC) located within a specific niche in hair follicles defined as “the bulge”, as identified by expression of markers of mouse KSC, CD34 and K15. The location of CD34+/K15+ KSC remained restricted to the bulge niche within hair follicles during multi-stage skin carcinogenesis and cutaneous wound healing. Our results also provide the first evidence for the presence of pAkt and pmTOR in CD34+/K15+ KSC localized to the ORS niche of the bulge region in normal skin, during multi-stage skin carcinogenesis, as well as during wound healing. Using the highly invasive and metastatic human Hs578T breast tumor cell line and the slow growing and non-invasive human MCF-7 breast tumor cell line, the present studies also compa (open full item for complete abstract)

Committee: Fredika Robertson (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2005, Medical Science

Ultraviolet light B (UVB) is a ubiquitous complete carcinogen of the skin. Due to their immunosuppressive therapy, organ transplant recipients have a 60-250 fold increased likelihood to develop aggressive, highly metastatic UVB-induced squamous cell carcinomas (SCC) compared to the general population. This thesis examines both UVB-induced cutaneous inflammation and UVB-induced skin tumor models mimicking transplant immunosuppression by depleting systemic CD4+ or CD8+ T lymphocytes. Our hypothesis is that a systemic decrease of CD4+, but not CD8+ T lymphocytes will increase the amount of UVB-induced DNA damage in acute UVB exposure and result in increases in tumor number following chronic UVB exposure.In the UVB-induced inflammation model, a decrease in systemic CD4+ but not CD8+ T lymphocytes increased UVB-induced neutrophil number and activity in the skin. Both T lymphocyte depletion groups showed increased DNA damage through increased levels of p53+ epidermal cells. This increase in DNA damage was not due to an increase in direct UVB-induced damage, as seen by immunohistochemical analysis of cyclobutane pyrimidine dimers (CPDs). Increased UVB-induced DNA damage could increase the amount of UVB-induced skin cancer that develops. The anti-inflammatory, anti-angiogenic drug, celecoxib, was able to almost completely abate the exacerbated UVB-induced inflammatory reaction in the CD4-depleted animals.In the chonic UVB-induced skin tumor model, a decrease in systemic CD4+ but not CD8+ T lymphocytes significantly increased tumor numbers. Tumors isolated from CD4-depleted mice also showed increased invasive properties and increased numbers of p53 point mutations. Topical treatment with the anti-inflammatory drug celecoxib significantly decreased tumor numbers in control, CD4- and CD8-depleted mice. Based upon our findings, it appears that there are multiple mechanisms by which systemic immunosuppression can cause an increase in tumor number and aggressiveness in transplant (open full item for complete abstract)

Committee: Tatiana Oberyszyn (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2005, Veterinary Biosciences

Squamous cell carcinoma (SCC) is a malignant tumor of keratinocytes. Head and neck SCC (H/N SCC) is a common diagnosis in the cat. SCCs often secrete parathyroid hormone related-protein (PTHrP) which induces osteoclastic resorption and contributes to bone destruction. Bone resorption results in the release of transforming growth factor-Β (TGF-Β), which regulates PTHrP via Smad signaling proteins.We developed a PTHrP-secreting feline oropharyngeal SCC cell line (SCCF1). Treatment of cells with TGF-Β increased PTHrP production. A segment of feline PTHrP had a high degree of homology to human and canine PTHrP cDNA and predicted amino acid sequence. PTHrP mRNA was alternatively spliced to the 1-139 and 1-141 isoforms. A mouse xenograft model of feline oral SCC was developed using luciferase-expressing SCCF1 cells. This allowed serial in vivo monitoring of tumor growth using bioluminescent imaging. Using a human pulmonary SCC line (HARA) in an intratibial metastasis model, we investigated the role of tumor-produced PTHrP in the growth of HARA in bone. We disrupted normal osteoclast function by treatment with zoledronic acid or osteoprotegerin. Tumor burden in bone was decreased with either treatment. Osteoclast numbers along tumor-associated bone were not decreased. High local levels of PTHrP may have partially antagonized the anti-osteoclast effects of either drug. Chemical induction of skin tumors in mice heterozygous for Smad2 and Smad3 was used to investigate the role of disruption of the TGF-Β signaling pathway on the development of SCC. Smad3± mice developed fewer tumors than wild-type mice, suggesting an oncogene role. Smad2± mice formed less-differentiated tumors than wild-type mice, supporting a tumor-suppressor role. There was a significant difference in tumor type between the two groups, suggesting that Smad2 and Smad3 regulate different targets. In conclusion, we propose the use of the cat as a natural model for human H/N SCC. In vitro and in vivo studies usi (open full item for complete abstract)

Committee: Thomas Rosol (Advisor) Subjects: