Doctor of Philosophy, The Ohio State University, 2021, Comparative and Veterinary Medicine

As one of the top causes of cancer in the United States and the leading cause of skin cancer related death, melanoma has been the focus of extensive research in recent years. Such efforts have resulted in remarkable progress in identifying mediators of melanoma oncogenesis and resistance to therapy, prognostication, diagnosis, and management of melanoma patients. However, the translation of these innovations to less common presentations of melanoma, such as ulcerated cutaneous melanoma and vulvovaginal melanoma, which characteristically have a significantly reduced prognosis, has been limited. Thus, additional studies to identify the molecular mediators contributing to aggressive biologic behavior observed with ulcerated cutaneous melanoma and melanomas originating from gynecologic sites are needed. microRNAs are small, non-coding RNA molecules that function to inhibit the expression of specific target genes. Through this action microRNAs can function as either tumor suppressors or oncogenes in cancer. Patterns of microRNA expression in ulcerated cutaneous melanoma are not well defined, and have not been examined in melanomas originating from gynecologic sites. We hypothesized unique patterns of microRNA expression occur with each of these presentations of melanoma that inversely correlate with the expression of microRNA target genes. Using the Nanostring platform the microRNA and mRNA expression patterns in ulcerated melanomas, vaginal melanomas, and vulvar melanomas were characterized. This resulted in identification of 24 differentially expressed microRNAs (p<0.05) and 21 differentially expressed mRNA transcripts (p < 0.01) in ulcerated melanoma, 21 microRNAs and 17 mRNA transcripts with differential expression in vaginal melanoma (p < 0.01), and 47 microRNAs and 89 mRNA transcripts with differential expression in vulvar melanoma (p < 0.01). Using mean expression patterns and correlation analysis across individual samples between microRNAs a (open full item for complete abstract)

Committee: William Carson MD (Advisor); Naduparambil Jacob (Committee Member); Krista La Perle PhD, Dipl. ACVP (Committee Member); Xue-Feng Bai MD, PhD (Committee Member) Subjects: Biology; Molecular Biology; Oncology

Bachelor of Science, Marietta College, 2022, Chemistry

After finding that heptyl paraben, a novel paraben, causes melanoma cells to die with a LC50 value of 0.195 millimolar when treated for an hour, the next step in this project was to determine how the cells die; they may die through apoptosis or necrosis. Parabens are esters of p-hydroxybenzoic acid and are used as preservatives. Melanoma is a type of cancer that starts in skin cells. Apoptosis was proven to be induced through the caspase assay procedure. During apoptosis, caspases are activated, so by showing caspase activity is increased with increased concentration of heptyl paraben, apoptosis is proven to be induced. After this, the cytochrome c concentrations in the mitochondria and in the cytoplasm of the cell were standardized using total protein concentration and compared. There was no change in the amount of cytochrome c in the cytoplasm or in the amount of cytochrome c in the mitochondria as the concentration of heptyl paraben was increased, which indicates that intrinsic apoptosis did not occur. These results lead to the conclusion that extrinsic apoptosis was induced when the melanoma cells were treated with heptyl paraben.

Committee: David Brown (Committee Member); Suzanne Parsons (Advisor) Subjects: Biochemistry; Cellular 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

Master of Sciences, Case Western Reserve University, 2019, Clinical Research

Primary site is considered an important prognostic factor for cutaneous malignant melanoma (CMM); however, opinions vary regarding its influence on survival. This study compares overall survival between Head and Neck Melanoma (HNM) and Melanoma of Other Regions (MOR), and between Melanoma of the Scalp and Neck (MSN) and Melanoma of other Head Regions (MOHR). We identified patients with HNM (MSN and MOHR) and MOR, stages I-IV (n=39,965), and their linked survival data using the National Cancer Database. After adjustment using propensity score models, subjects with HNM had an increased mortality (HR=1.25, [95% CI, 1.17-1.33]; p<0.05) compared to MOR. Among those with HNM, subjects with MSN and ≤ 3.5 years follow up (75% of subjects) had an increased mortality (HR 1.14, [95% CI, 1.04-1.26]; p<0.05) compared to MOHR; however, after 3.5 years no difference was noted (HR=0.91, [95% CI, 0.67-1.23]; p=0.53). These results suggest that a lower threshold of suspicion for recurrence/metastasis may be warranted for patients with HNM so as to improve survival and prevent non-resectable disease.

Committee: Douglas Einstadter M.D., M.P.H. (Committee Chair); Thomas Love Ph.D. (Committee Member); Mark Schluchter Ph.D. (Committee Member) Subjects: Health; Health Sciences; Medicine; Oncology; Surgery

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

Growth hormone (GH) is known to play a crucial role in melanoma progression and the development of drug resistance. Recent studies have implicated melanoma-derived exosomes in these processes, although the direct effects of GH on melanoma-derived exosomes remain unexplored. This study aims to investigate the impact of GH in modulating cargoes in melanoma-derived exosomes. Characterization of melanoma-derived exosomes revealed that GH caused changes in exosomal cargo without affecting the morphological features or secretion characteristics of exosomes. We observe that GH elevated multiple mRNAs within exosomes, including stearoyl-CoA desaturase (SCD), potassium channel tetramerization domain 20 (KCTD20), and zinc-finger CCCH-type containing antiviral 1 (ZC3HAV1), which are associated with cancer progression. Concurrently, GH suppressed non-coding RNAs such as mir-98 and mr-185, which are linked to tumor suppression. Inhibition of GH action by pegvisomant reversed these expression patterns. Strikingly, pegvisomant alone elevated various cargoes such as tumor suppressing exosomal lncRNA Growth Arrest Specific transcript (GAS5), indicating independent action of pegvisomant in modulating exosomal cargoes. Previous studies from our lab and others have demonstrated that GH expression exacerbates chemotherapy resistance and cancer invasion in melanoma via upregulating the levels of ATP-binding cassette transporters (ABC) transporters, epithelial to mesenchymal transition (EMT) markers, and matrix metalloproteinases (MMPs). We assessed exosomal ABC transporters, EMT markers, MMPs associated with GH. GH treatment elevated exosomal ABC transporters, specifically ABCC1 and ABCB1, with levels further increasing in the presence of chemotherapy, in particular, doxorubicin. Furthermore, assessing the exosomal transfer of these ABC transporters revealed that GH-induced exosomes elevated the levels of these ABC transporters in recipient cells and resulted in decreased drug retentio (open full item for complete abstract)

Committee: John Kopchick (Advisor) Subjects: Cellular Biology; Molecular Biology

PhD, University of Cincinnati, 2024, Pharmacy: Pharmaceutical Sciences

A hallmark of metastatic melanoma is the ability of tumor cells to escape primary sites, invade underlying dermal tissues, and metastasize to distant organs. During this process, melanoma cells develop a dynamic and lopsided relationship with neighboring stromal cells in the host microenvironment, which consists of a heterogeneous mix of non-cancer cells embedded in a tumor-specific extracellular matrix (ECM). Genetically stable fibroblasts that surround and infiltrate melanoma stroma, often termed cancer-associated fibroblasts (CAFs), are known to substantially contribute to melanoma progression through reciprocal tumor-stromal interactions although the exact mechanisms remain to be elucidated. Moreover, CAFs are a potential target for optimizing therapeutic strategies against melanoma. However, a major unknown still exists in CAFs, which is how CAFs maintain and even reinforce their functions in drug therapy and influence melanoma cell drug sensitivity. We discovered that Yes-associated oncoprotein 1 (YAP1) function is required for CAFs to proliferate, migrate, remodel the cytoskeletal machinery and matrix, and promote cancer cell invasion. To identify YAP1-regulated genes that are involved in a CAF-elicited melanoma program we applied RNA-Seq. By RNA-seq N-cadherin was identified as a major downstream effector of YAP1 signaling in CAFs. N-cadherin is a homophilic transmembrane protein that is known to function in cell-cell adhesion and is essential in tumor metastasis. YAP1 silencing leads to Ncadherin downregulation in CAFs, which subsequently induces the downregulation of N-cadherin in co-cultured BRAF-mutant melanoma cells. Furthermore, N-cadherin downregulation inhibits the PI3K-AKT signaling pathway in melanoma cells, contributing to suppressed melanoma cell proliferation. Mechanistically, YAP1 depletion in CAFs induces the downregulation of pAKT signaling in BRAF-mutant melanoma cells through homotypic N-cadherin-mediated interaction between melanoma (open full item for complete abstract)

Committee: Yuhang Zhang Ph.D. (Committee Chair); Joan Garrett Ph.D. (Committee Member); Georg Weber M.D. Ph.D. (Committee Member); Bingfang Yan D.V.M. Ph.D. (Committee Member); Chenran Wang Ph.D. (Committee Member) Subjects: Biology

Master of Science (MS), Wright State University, 2024, Biochemistry and Molecular Biology

The changes in splicing of MDM4 in human melanomagenesis are critical to p53 activity and represent potential therapeutic targets. Genomic characteristics of melanoma and precursor lesions, such as copy number variation and gene expression, have been investigated in detail. However, there has never been a detailed survey of splicing changes that occur during melanomagenesis. Splicing changes can be a very early event in melanoma tumor progression, with characteristic changes in the p53 pathway already in place in early nevi. MDM4 is upregulated in a strong majority of melanoma cases and has been described as a “key therapeutic target in cutaneous melanoma”. Identifying splicing changes in human tissue specimens will provide therapeutic targets during initiation, dysplasia, and progression toward metastatic melanoma. Splicing of MDM4 has been characterized in melanomas with increased MDM4 isoform expression over canonical MDM4-FL. However, these studies have utilized new or existing data obtained by next-generation sequencing (NGS) methods, typically Illumina sequencing, relying on short reads. A difficulty associated with NGS analysis is the loss of “connectivity” data in which full transcripts are inferred from the presence of splice junction reads. To address this problem, long-read, third generation sequencing, was utilized to read the entire length of transcripts, intact. Oxford Nanopore flow cell sequencing allowed for quantification and identification of the MDM4 transcripts, both alternative and canonical, present in melanoma specimens. RT-PCR and Nanopore sequencing provided a direct view into the isoform landscape of melanoma specimens while RT-qPCR quantified the expression of major MDM4 isoforms represented in the samples. This study identifies and quantifies MDM4 isoforms present in malignant melanoma tumor samples and provides evidence of a novel hybrid MDM4-A & MDM4-S isoform (MDM4-A/S).

Committee: Michael Markey Ph.D. (Advisor); Courtney Sulentic Ph.D. (Committee Member); Weiwen Long Ph.D. (Committee Member) Subjects: Biochemistry; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2024, Biomedical Engineering

Objective : Breast and skin cancers are one of the most lethal cancers with triple negative breast cancer (TNBC) and melanoma as most deadlier subtypes, respectively. It is well known that cancers are innervated with sensory, sympathetic and parasympathetic nerves and these nerves interact with stromal and tumoral cells in order to control and modify every significant aspect of tumor microenvironment. Despite the crucial roles of nerves, the neuroelectric phenomena in malignancy initiation, the branch specific origin of tumoral nerve activity and its implications on secondary metastasis are vastly unexplored. Principle Global Hypothesis: Solid primary tumors with malignant nature are characterized by presence of neural impulses while the primary tumors with lower metastatic potential, are devoid of any significant neural activity. The neural activity originates in the autonomic nervous system with both its branches i.e. sympathetic and parasympathetic, exerting antagonistic effects on secondary distant metastasis. Methods : Triple negative- 4T1, D2A1; HER2+ - MMTV/NeuT and B16-F10, B16-F1 cell lines were used for in-vivo modeling of solid breast and melanoma tumors. Neural recordings were obtained using a differential pair of electrodes, comprising drawn filled tubing (DFT) wires directly implanted in breast tumors while human grade microneurography needles implanted in melanoma tumors. Bioluminescence imaging was used to monitor the growth of the primary tumors as well as the secondary metastases to prevalent sites. Immunohistochemical (IHC) staining was used to confirm the presence of neurofilaments as well as sympathetic and parasympathetic fibers. Results : Experiments show that the presence of neural activity is a unique and distinct feature of primary highly metastasizing tumors of breast and skin cancers while their counterparts with low metastatic potential have significantly reduced neural action potentials. Noradrenergic sympathetic nerves are (open full item for complete abstract)

Committee: Efstathios Karathanasis (Committee Chair); Susann Brady-Kalnay (Committee Member); Jennifer Yu (Committee Member); Dominique Durand (Committee Member); Ruth Keri (Committee Member); Mark Cohen (Committee Member) Subjects: Biomedical Engineering

Master of Engineering, Case Western Reserve University, 2023, Biomedical Engineering

Solid tumors were previously believed to lack innervation, but now tumor innervation is being explored as a method to target for therapy and diagnosis. This study intends to understand characteristics of melanoma innervation to provide insight to important neural markers of cancer progression. These patterns may be exploited as a predictive biomarker of cancer aggression. This study quantifies neural activity within murine melanoma B16 cell line tumors with microneurography and analyzes activity patterns to its growth and metastasis monitored by bioluminescent imaging (BLI). Two variants of the B16 cell line were utilized in this study, the B16-F10 which is recognized for its high metastatic potential, and B16-F1 which exhibits a low metastatic potential. Analysis of neural activity finds statistically significant differences, such that the B16- F10 tumors have greater activity than the B16-F1 tumors (p< 0.05). We have also found statistically significant differences in neural activity between B16-F10 tumor bearing mice and B16-F10 sympathectomized tumor bearing mice (p< 0.05). From histology and imaging, we find statistically significant differences between B16-F10 and B16-F1 tumors in nerve density (p< 0.01), but not of nerve diameter (p>0.05).

Committee: Dominique Durand (Committee Chair); Grant McCallum (Committee Member); Andrew Shoffstall (Committee Member); Jennifer Yu (Committee Member); Shuyang Chen (Committee Member) Subjects: Biomedical Research; Engineering

Bachelor of Science, Marietta College, 2023, Chemistry

Parabens are commonly used as preservatives in regularly used topical products, but their safety is under discussion since small amounts of paraben have been found in tumor tissue. Mononitroparaben causes cell death in melanoma cells with an LC50 value of 7.02mM after twelve hours of treatment. This study focused on how mononitroparaben affects the cellular lipid content during induced cell death. The experiment was conducted by growing the M624 melanoma cells, dissolving the paraben in methanol, and then leaving 0 mM, 5 mM, and 10 mM concentrations of paraben on the melanoma cancer cells for twelve hours. The paraben was then removed from the cells, and the cells were lysed. Colorimetric cholesterol assays and ceramide assays were completed to determine the changes in cellular cholesterol and ceramide content and the role of cellular lipids in cell death signaling. The results showed that cholesterol concentrations did not significantly change among cells treated with mononitroparaben. However, ceramide significantly increased for cells treated with 10 mM mononitroparaben, which indicated that apoptosis occurred.

Committee: Suzanne Parsons Ph.D. (Advisor); Adam Jacoby Ph.D. (Committee Member); Rakibul Sarker Ph.D. (Committee Member) Subjects: Biochemistry

PhD, University of Cincinnati, 2022, Pharmacy: Pharmaceutical Sciences

The use of BRAF and MEK inhibitors for patients with BRAF-mutant melanoma is limited as patients relapse on treatment as quickly as 6 months due to resistance. We used two approaches to treat resistant melanomas based on known and novel mechanisms of resistance. For the first approach, we generated trametinib and dabrafenib resistant (TDR) melanoma cell lines to trametinib, a MEK and dabrafenib, a BRAF inhibitor. TDR cells exhibited increased viability and maintenance of downstream p-ERK and p-Akt as compared to parental cells. Receptor tyrosine kinase arrays revealed an increase in phosphorylated insulin like growth factor 1 receptor (IGF1R) and phosphorylated insulin receptor (IR) in the drug resistant cells versus drug sensitive cells. RNA-sequencing analysis identified IGF1R and INSR upregulated in resistant cell lines compared to parental cells. Analysis of TCGA PanCancer Atlas (skin cutaneous melanoma) showed that patients with a BRAF mutation and high levels of IGF1R and INSR had a worse overall survival. BMS-754807, an IGF1R/IR inhibitor, suppressed cell proliferation along with inhibition of intracellular p-Akt in TDR cells. Dual inhibition of IGF1R and INSR using siRNA reduced cell proliferation. The combination of dabrafenib (BRAF inhibitor), trametinib (MEK inhibitor), and BMS-754807 (IGF1R/IR inhibitor) treatment reduced in vivo xenograft tumor growth. Examining the role of IGF1R and IR in mediating resistance to BRAF and MEK inhibitors will expand possible treatment options to aid in long-term success for BRAF-mutant melanoma patients. Reactive oxygen species (ROS) levels are elevated after acquisition of resistance to BRAF and MEK inhibitors. For the second approach we utilized a ROS-induced drug release (RIDR)-PI-103, a novel prodrug with a self-cyclizing moiety linked to PI-103, a PI3K inhibitor. Under high ROS conditions, RIDR-PI-103 releases PI-103, which inhibits conversion of PIP2 to PIP3. Studies in our lab demonstrate that TDR cells ma (open full item for complete abstract)

Committee: Joan Garrett Ph.D. (Committee Member); Zalfa Abdel-Malek Ph.D. (Committee Member); Yuhang Zhang Ph.D. (Committee Member); Lisa Vinnedge Ph.D. (Committee Member); Timothy Phoenix Ph.D. (Committee Member) Subjects: Oncology

PhD, University of Cincinnati, 2021, Pharmacy: Pharmaceutical Sciences

Although the groundbreaking discovery of RAF and MEK inhibitors has revolutionized targeted therapy for the treatment of advanced melanoma, a high percentage of patients still die from metastasis despite the initial positive responses to targeted therapy drugs. Both tumor progression and the development of therapeutic resistance are strongly modulated by stromal cells that reside in the tumor microenvironment (TME). The TME is composed of noncancer stromal cells, including endothelial cells, immune cells, and cancer-associated fibroblasts (CAFs), and the noncellular extracellular matrix (ECM). During the past decade, there is a great expansion in research on CAFs, which is one of the most abundant and active stromal cell types to aid the creation of a desmoplastic tumor niche. CAFs have multifaceted roles in regulating tumor progression, including synthesis and remodeling of the ECM and production of growth factors, modulating anti-tumor immune response, and influencing angiogenesis, tumor mechanics, drug access and therapy responses. Although TME-mediated drug resistance mechanism has been extensively studied in the past, it is still not clear how therapy-induced pressure would act on the genetically stable stromal cells and how these cells could add in the occurrence of drug resistance. Here, we show that the number of intratumoral CAFs increases in BRAF inhibitors (BRAFi)/MEK inhibitors (MEKi)-treated melanoma stroma. CAFs also exhibit increased amounts of nuclear ß-catenin under BRAFi/MEKi therapy. ß-catenin has critical roles in both the Wnt/ ß-catenin signaling pathway and cell-cell adhesion. Wnt/ß-catenin signaling is one of the key signaling pathways that are deregulated not only in a variety of human cancer cells, and also in fibroblasts. Aberrant activation of ß-catenin signaling in fibroblasts has been demonstrated to lead to diseases, such as skin fibrosis. Our results suggest that CAFs, which possess the wild-type BRAF gene, could be paradoxical (open full item for complete abstract)

Committee: Yuhang Zhang Ph.D. (Committee Chair); Zalfa Abdel-Malek Ph.D. (Committee Member); Joan Garrett Ph.D. (Committee Member); Ana Luisa Kadekaro Ph.D. (Committee Member); Georg Weber M.D. (Committee Member) Subjects: Pharmaceuticals

Master of Science (MS), Wright State University, 2020, Biochemistry and Molecular Biology

Melanoma is a potentially lethal type of skin cancer and regarded to be the third most common type of skin cancer. Although melanoma is not as common as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), it is more likely to metastasize than BCC and SCC. Interestingly, the incidence of melanoma continues to go up (expected 2% in 2020), but the deaths continue to decrease (-5.3% in 2020) due to improvements in detection and treatment. The treatment of melanoma depends on several aspects but most importantly the tumor's stage and the location. In the early stages, melanoma can be removed via surgical operation, but in the late stages, melanoma usually treated with radiation or targeted therapy. Many factors predict the development of melanoma like skin pigmentation, age, sun exposure (UV light), and genetic mutations. The p53 protein initiates apoptosis and cell cycle arrest in response to stresses that cause DNA damage like UV light. However, mutant p53 is frequently detected in numerous cancers, including melanoma. MDM2 and MDM4 negatively regulate p53. Notably, a study indicates that MDM4 is overexpressed in 65% of melanoma. Therefore, there is a strong rationale to target MDM4 therapeutically, but MDM4 has several spliced variants, and some variants were detected in human and murine cancers. Yet, no studies have assessed the relative levels of MDM4 splice variants in melanoma. Therefore, in this study, we aimed to identify which variants are expressed in melanoma. We collected clinical specimens and designed specific primers for each isoform by RT-PCR. We observed no expression of MDM4-211 in either malignant and nevi samples. MDM4-Alt1 was present only once in nevi samples. Other variants were similarly present in nevi and malignant samples, but MDM4-A was the most commonly expressed variant in melanoma samples. Data were also collected and analyzed from publicly available databases to examine MDM4 expression in normal skin and melanoma and the surviva (open full item for complete abstract)

Committee: Michael Markey Ph.D. (Advisor); Weiwen Long Ph.D. (Committee Member); Michael Craig Ph.D. (Committee Member) Subjects: Biochemistry; Molecular Biology

Doctor of Philosophy (PhD), Wright State University, 2020, Biomedical Sciences PhD

ΔNp63α is a p53 transcription factor family member that promotes cellular proliferation and survival. In squamous cell carcinoma (SCC), ΔNp63α overexpression is associated with poor prognosis and survival, implicating it as a proto-oncogene. Despite its importance in SCC, the mechanisms regulating ΔNp63α expression are poorly understood. We have identified the acetyltransferase TIP60 as a novel upstream regulator of ΔNp63α levels. We discovered that TIP60 regulates ΔNp63α via two mechanisms. First, TIP60 upregulates ΔNp63α mRNA levels. Moreover, pharmacological inhibition of TIP60 activity reduces ΔNp63α transcript levels, indicating that TIP60-cataltyic activity is vital to amplified transcription of ΔNp63α. Second, we have demonstrated that TIP60 promotes ΔNp63α protein stability by preventing its ubiquitination and proteasomal degradation. This study further shows that TIP60 co-localizes with, interacts with, and directly acetylates ΔNp63α. Utilizing mass spectrometry and site directed mutagenesis, we found that TIP60 acetylates ΔNp63α residues K138, K139, and K494, respectively. We additionally revealed that preventing acetylation of these sites inhibit TIP60-mediated stabilization of ΔNp63α, providing evidence that acetylation by TIP60 enhances ΔNp63α protein stability. We further investigated the functions of the TIP60-ΔNp63α axis in the regulation of SCC proliferation. We discovered that regulation of ΔNp63α by TIP60 increases cellular proliferation. In accordance with the functional role of ΔNp63α, pharmacological inhibition of TIP60 reduced SCC cell proliferation suggesting TIP60 may have therapeutic potential in cancers exhibiting ΔNp63α overexpression. Furthermore, we investigated the mechanisms by which TIP60 regulation of ΔNp63α enhances cancer cell proliferation. We discovered that TIP60 upregulation of ΔNp63α represses p21Cip1/Waf1 levels, resulting in increased G2/M progression. In conclusion, this study uncovers a novel mechanism promoting ΔNp (open full item for complete abstract)

Committee: Madhavi Kadakia Ph.D. (Advisor); Thomas L. Brown Ph.D. (Committee Chair); Paula A. Bubulya Ph.D. (Committee Member); Weiwen Long Ph.D. (Committee Member); Michael P. Markey Ph.D. (Committee Member) Subjects: Biochemistry; Biomedical Research; Cellular Biology; Molecular Biology; Oncology

PhD, University of Cincinnati, 2020, Arts and Sciences: Chemistry

Reactive oxygen species(ROS) are a family of radical and non-radical byproducts of aerobic metabolism. It plays essential roles as secondary signaling molecules in cell proliferation, differentiation, sentence, and apoptosis. Ultraviolet radiation (UVR) overexposure can upregulate ROS in skin cells and results in further damage to deoxyribonucleic acid (DNA), protein, and lipid. As UVR is an essential risk factor for the development of premalignant skin lesions as well as of melanoma and nonmelanoma skin cancer, sunscreen agent was developed to prevent it. The chemoprevention strategy is found and developed since 1976, which is being developed to present. It includes two different types: one is applying the chemical which can absorb or reflect UVR to prevent UVR radiation on the skin surface, the other type affects the metabolism of skin cells to stop the cell damage and malignant initiation. The second category is developing fast in the most recent 20 years to meet the need of human beings. A series of antioxidants and natural products prove to effectively prevent UVR by reducing the ROS level by ROS scavenging or as an inhibitor of the ROS generator. And a ROS-activated prodrug strategy is developed to enhance the selectivity of chemoprevention. The first project was finished by designing a novel ROS-activated moiety attached with apocynin, which is not only an antioxidant but a nicotinamide adenine dinucleotide phosphate oxidase(NOX) inhibitor. Releasing manner about it was studied by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). The skin protection function of it was further proved by gel electrophoresis, dichlorofluorescein diacetate(DCFDA) assay, MTT assay, western blot, and cyclobutane pyrimidine dimers (CPD) quantification. To sum, this prodrug was proved to release the NOX1 inhibitor and protect the DNA from UVR radiation by lowing the ROS level. Considering the drug attached has a controversial mechanism to inhibit (open full item for complete abstract)

Committee: Edward Merino Ph.D. (Committee Chair); Ana Luisa Kadekaro Ph.D. (Committee Member); David Smithrud Ph.D. (Committee Member); Peng Zhang Ph.D. (Committee Member) Subjects: Pharmaceuticals

Bachelor of Science, Marietta College, 2020, Chemistry

Melanoma is the deadliest form of skin cancer that affects the melanin-producing cells in the human body and it accounts for 60 - 80% of all skin cancer-related deaths. The prevalence of this disease has been increasing during the past several decades and the desired type of cell death in the elimination of these cells is apoptosis. Parabens are a class of organic, antimicrobial compounds widely used in industrial, food, and cosmetic products and have also displayed cytotoxic effects towards cancerous human cells. Cyclohexylmethylparaben is a newly synthesized paraben that has been shown to be cytotoxic to human M624 melanoma cells. Human M624 melanoma cells were treated with 0.35, 0.45, 0.55, and 0.65 mM cyclohexylmethylparaben and then cell viability was analyzed with clonogenic assays using crystal violet dye. An IC50 value of 0.4768 mM was calculated for cyclohexylmethylparaben in human M624 melanoma cells. Caspase-3 activity assays were performed to analyze the activation of the caspase-3 pathway to determine if apoptosis occurred in treated human M624 melanoma cells. Cyclohexylmethylparaben was found to induce its highest levels of caspase-3 activation at 0.35 mM when compared to the other treatment groups and the control group. This data suggests that cyclohexylmethylparaben could be a useful compound in the activation of apoptosis and treatment of human melanoma.

Committee: Suzanne Parsons Ph.D. (Advisor); David Brown Ph.D. (Committee Member) Subjects: Biochemistry

Master of Science (MS), Wright State University, 2019, Biochemistry and Molecular Biology

Mitogen activated protein kinases (MAPKs) are Ser/Thr kinases that relay the extracellular signal into intracellular responses and regulate several biological responses. They are classified into conventional MAPKs and atypical MAPKs. Extracellular signal regulated kinase 3 (ERK3) is an atypical MAPK that has a single phospho-acceptor site (Ser 189) in its activation motif instead of the canonical Thr-Xaa-Tyr (TXY) motif of conventional MAPK like ERK1/2. ERK3 comprises of a unique C terminal tail and a central C34 domain that further distinguishes it from ERK1/2. Moreover, compared to ERK1/2, much less is known about the upstream activators and the downstream targets of ERK3. Here, our study identifies IL-6 signaling to be negatively regulated by ERK3. We show that ERK3 downregulates the IL-6 target genes and STAT3 phosphorylation. IL-6 is a multifunctional pleiotropic cytokine that signals predominantly via JAK/STAT signaling pathway. In response to IL-6, STAT3 is phosphorylated at Tyrosine 705 (Y705) residue by JAK2, which successively activates STAT3 as a transcription factor. The IL-6/STAT3 pathway is negatively regulated by a feedback inhibitor SOCS3 (Suppressor of Cytokine Signaling 3) which binds to JAK2, hence, preventing the phosphorylation and activation of STAT3. Interestingly, this study reveals that ERK3 interacts with SOCS3 via its C34 domain. Until now, there has been no information about the physiological or the biochemical function of the C34 domain of ERK3. Hence this study has identified a novel role of ERK3's C34 domain. Furthermore, we show that ERK3 facilitates the interaction between SOCS3 and JAK2, promoting the inhibition of STAT3 Y705 phosphorylation and activation. Taken together, the findings of our study provide important and novel insights into the negative regulation of IL-6/STAT3 signaling pathway by ERK3.

Committee: Weiwen Long Ph.D. (Advisor); Madhavi Kadakia Ph.D. (Committee Member); Hongmei Ren Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research

Master of Science, University of Toledo, 2019, Electrical Engineering

Over the past few years, Deep learning has been widely used in medical imaging for classification and segmentation and has been successful in providing better diagnostic accuracy. The state-of- the-art deep learning algorithms are built using neural networks arranged in layers where the first layer extracts basic information of images like edges, colors etc so that the output of one layer is fed as input to the next consecutive layers. Thus, increasing the complexity of learning with increase of layers. In comparison with the traditional machine learning algorithms, deep learning has many advantages and is an automatic process. However, it requires large scale annotated data for better performance and is thus constrained by limited size of available public datasets. To overcome data constraints, this thesis proposes a more efficient and novel scheme comprising techniques that use an ensemble of convolution neural networks (CNN) with generative adversarial network (GAN) based augmentation to improve the diagnostic accuracy. Also, the implementation of the proposed technique on skin lesion dataset of limited size to classify melanoma is presented. In this work , images are augmented to increase the dataset size using two different augmentation techniques: traditional augmentation and GAN based augmentation. GAN based augmentation is based on neural networks, where, two neural networks compete against each other to produce visually realistic synthetic images. For the classification, CNN and an ensemble of CNNs are trained on the final enlarged training dataset comprising original images and synthetic images. Here, the ensemble of CNN techniques combines five trained CNNs into a single meta-classifier. One hundred and ninety-three skin lesion test images were used to validate our proposed methods. The first method proposed in this research incorporated traditional to enlarge the training dataset and trained a CNN classifier to classify skin cancer into malignant (open full item for complete abstract)

Committee: Ezzatollah Salari PhD (Advisor) Subjects: Artificial Intelligence; Computer Science; Electrical Engineering

Master of Science (MS), Wright State University, 2019, Biochemistry and Molecular Biology

DNp63a, a member of the p53 transcription factor family, is overexpressed in non-melanoma skin cancer and regulates cell survival, migration and invasion. TIP60 is histone acetyltransferase (HAT) which mediates cellular processes such as transcription and the DNA damage response (DDR). Previous studies in our lab have shown that overexpression of TIP60 induces DNp63a protein stabilization in a catalytic-dependent manner. Since DNp63a is known to transcriptionally regulate several DDR genes and promote cisplatin resistance, its stabilization by TIP60 may contribute to the failure of platinum-based drugs in squamous cell carcinoma (SCC). We hypothesize that TIP60 regulates the transcriptional activity of DNp63a thereby modulating chemoresistance. In this study, we showed that overexpression of TIP60 in both H1299 and A431 cells led to increase in the levels of DNp63a, while TIP60 silencing in A431 cell lines led to a decrease in endogenous DNp63a transcript and protein levels, thus confirming that TIP60 positively regulates DNp63a in these cell lines. Increased levels of DNp63a and TIP60 were observed in a cisplatin resistant A431 SCC line. Further, stable expression of TIP60 or DNp63a individually promoted resistance to cisplatin, whereas loss of DNp63a and TIP60 sensitized cells to cisplatin. Higher acetylation of DNp63a and TIP60 were seen in cisplatin resistant cells. High-throughput transcriptome sequencing was performed using the Ion ProtonTM AmpliSeq panel to identify downstream mRNA targets of TIP60. An NGS data set generated from Lenti viral transduced A431 cells resulting from overexpression of TIP60 and eGFP as a control identified 228 mRNA showing differential gene expression. Taken together, our data suggest that TIP60-mediated regulation of DNp63a increases cisplatin resistance and has potential implications for cancer treatment and drug design. Additionally, since DNp63a confers cisplatin resistance through regulation of genes involved in DNA damage (open full item for complete abstract)

Committee: Madhavi Kadakia Ph.D. (Advisor); Michael P. Markey Ph.D. (Committee Member); Hongmei Ren Ph.D. (Committee Member) Subjects: Biochemistry; Bioinformatics; Molecular Biology

Master of Science (MS), Wright State University, 2019, Biochemistry and Molecular Biology

Non-melanoma skin cancer (NMSC) is a group of skin cancer that includes basal cell carcinoma of the skin (BCC), squamous cell carcinoma of the skin (SCC), actinic keratoses, a precursor to SCC, and other rare cutaneous carcinomas. p63, a member of the p53 gene family, is an important regulator for epithelial tissue growth and development. ∆Np63α, a main isoform of p63, is highly expressed in NMSC and plays essential roles in NMSCs development. Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the MAPK family. It possesses a single phosphorylation site (serine 189) in its activation loop, which makes it different from the conventional MAPKs. Similar to ∆Np63α, the expression level of ERK3 is upregulated in cutaneous SCC and actinic keratoses. While ERK3 has been shown to promote invasiveness of squamous cell lung cancer, little is known about ERK3's role in NMSCs. In addition, how ERK3 expression is upregulated in NMSCs remains largely unknown. Given that the expression levels of both ∆Np63α and ERK3 are upregulated in NMSCs, we wanted to test whether ∆Np63α, as a transcriptional factor, regulates ERK3 expression in SCCs and if ERK3 acts as a downstream mediator of ∆Np63α in controlling NMSCs cell growth and invasiveness. We showed that ∆Np63α and ERK3 are co-overexpressed and there was a positive correlation between their expression in the skin from normal humans, and patients with actinic keratosis, squamous cell carcinomas, and basal cell carcinoma. We further showed that while silencing ∆Np63α reduced ERK3 expression level in HaCaT keratinocytes and A431 squamous cell carcinoma cells, overexpression of ∆Np63α increased ERK3 expression level in H1299. Therefore, ∆Np63α positively regulates ERK3 expression. Moreover, silencing either ∆Np63α or ERK3 greatly enhanced A431 cell migration. Importantly, restoration of ERK3 expression rescued the increased cell migration observed upon silencing ∆Np63α. Knockdown of ERK3 in squamous cells does not sh (open full item for complete abstract)

Committee: Weiwen Long Ph.D. (Advisor); Madhavi Kadakia Ph.D. (Committee Member); Hongmei Ren Ph.D. (Committee Member) Subjects: Biochemistry; Biomedical Research; Molecular Biology