Master of Science (MS), Bowling Green State University, 2023, Biological Sciences

Bacterial pathogens are evolving resistance to all available antibiotics and are a leading cause of death worldwide. Thus, the discovery of new antibiotics that inhibit multi-drug resistant (MDR) pathogens is urgent. We hypothesize that environmental Pseudomonas strains that can inhibit the growth of MDR pathogens may be source of novel antibiotics that may have new or multiple mechanisms of action. We isolated 288 environmental Pseudomonas strains from the Portage River in northwest Ohio which represents a polluted water source. Pseudomonas strains were isolated and phylogenetically characterized using the gyrB housekeeping gene. To test our hypothesis, the environmental strains were competed against a collection of human and animal MDR pathogens including nine Burkholderia species, seven Staphylococcus aureus, and nine Pseudomonas aeruginosa strains, all isolated from lungs of cystic fibrosis patients, and five Aeromonas fish pathogens. Results from the 8,640 individual antagonistic assays showed that 115 of the environmental Pseudomonas strains demonstrated the ability to inhibit at least one pathogen. This suggests that these environmental strains may produce novel compounds which effectively inhibit these MDR pathogens. We performed transposon mutagenesis and screened for loss of inhibition mutants with strains J4D8, JA1H2, J1A5 and J3E11 to determine biosynthetic gene clusters involved in antibiotics production. Whole genome sequencing and arbitrary PCR coupled with antiSMASH were used to identify specific gene clusters involved in antagonistic activity. The compounds were predicted to include pyochelin, pyoverdine, MA026, lankacin C and cepacin A which are all derived from non-ribosomal peptides. Further analysis of these compounds is required to determine their novelty.

Committee: Hans Wildschutte Ph.D. (Committee Chair); Raymond Larsen Ph.D. (Committee Member); Christopher Ward Ph.D. (Committee Member) Subjects: Biochemistry; Bioinformatics; Biology; Biomedical Research; Microbiology; Molecular Biology

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

The human DNA topoisomerase IIα (170 kDa, TOP2α/170) enzyme is essential in proliferating cells by functioning as a homodimer resolving DNA topological entanglements that form during chromosome condensation, replication, and segregation. The TOP2α/170 homodimer disentangles DNA by introducing transient double strand breaks in a DNA helix via a transesterification reaction between the active site Tyr805 from each TOP2α/170 subunit and the phosphodiester DNA backbone, creating the intermediate state known as the TOP2α/170-DNA cleavage complex. An intact DNA duplex passes through the cleaved DNA. Subsequently, the DNA strands are religated restoring the integrity of the DNA and preparing the TOP2α/170 dimer for another catalytic cycle. TOP2α/170 enzymatic activity is indispensable for the survival of highly proliferating cells including cancer cells. This has made TOP2α a prominent target for anticancer therapies. Some of the most widely used topoisomerase II targeting drugs such as etoposide, mitoxantrone, amsacrine and doxorubicin, stabilize the TOP2α/170-DNA cleavage complex preventing the religation of the DNA strands. As a result, these agents exert their cytotoxic effects by the accumulation of double strand DNA breaks which ultimately lead to the initiation of apoptotic pathways. Acquired chemoresistance to topoisomerase II targeting drugs continues to be a major obstacle in cancer treatment in the clinic. In order to characterize the mechanisms of resistance to etoposide, our laboratory developed etoposide resistant human leukemia K562 cells, designated K/VP.5 in which levels of TOP2α/170 were decreased along with identification of a novel C-terminal truncated isoform of TOP2α, TOP2α/90. This 90 kDa protein is present in both in K562 and etoposide resistant K/VP.5 cells with expression levels increased ~3-fold in K/VP.5 cells. TOP2α/90 is the translation product of novel alternatively spliced mRNA via intron 19 (I19) retention and processing, confirmed by 3 (open full item for complete abstract)

Committee: Christopher Coss (Committee Member); Jack Yalowich (Advisor); Dawn Chandler (Committee Member) Subjects: Pharmaceuticals; Pharmacology; Pharmacy Sciences

Doctor of Philosophy (PhD), Ohio University, 2016, Molecular and Cellular Biology (Arts and Sciences)

Human melanomas are one of the most therapy resistant types of cancer, expressing a repertoire of mechanisms for drug resistance. Intracellular signaling networks used by the melanoma cells for active proliferation, migration, invasion, therapy resistance and metastases happen to strongly overlap with those regulated by the human growth hormone (hGH). Indeed consistently high levels of GH receptor (GHR) expression have been observed in almost all melanoma cell lines in the NCI60 human cancer panel. There are no comprehensive studies investigating the effects of GH action or GHR antagonism on the GH-responsive intracellular signaling pathways and downstream effects in human melanoma. Here we report for the first time, a detailed analysis of the effect of siRNA mediated GHR knock-down (KD) and effect of hGH on the intracellular signaling and downstream phenotypes in human melanoma cells. We report the existence of an autocrine loop of hGH-GHR in the aforesaid human melanoma cell lines, changes observed in relevant intracellular signaling pathways, RNA transcript level changes observed in multiple key modulators of the GH/GHR axis. Phosphorylation states of the JAK2, SRC, STAT1, 3, and 5, p44/42-MAPK, AKT and mTOR increased in a dose-dependent manner with hGH stimulation and were significantly attenuated by GHR-KD. Differential yet significant changes were observed in the relative mRNA transcript levels of prolactin (PRL), insulin and related growth factors (IGF1, IGF2) and their receptors, following hGH treatment or GHR-KD. Thus, the GH/GHR interaction can influence the levels of the JAK2, SRC, STAT1, 3, and 5, p44/42-MAPK, AKT and mTOR in human melanoma cells and may provide leads as to potential targets for therapeutic intervention.

Committee: Shiyong Wu (Advisor); John Kopchick (Committee Member) Subjects: Biochemistry; Biology; Endocrinology; Molecular Biology; Oncology

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2007, Psychology/Clinical

Theories of adolescent experimental substance use note that proximal influences, including refusal skills and substance use decisions in response to drug offers, are more amenable to change than distal influences. However, research on adolescents' responses to offers of alcohol or drugs has not addressed whether resistance responses vary by type of drug, or whether emotional reactions to an offer of drugs or alcohol are associated with adolescents' intentions to use resistance strategies. Therefore, I assessed whether type of drug (alcohol vs. marijuana), relationship with the offerer (close friends vs. acquaintance), and drug refusal policy (uniform rejection, marijuana-only refusal, open-to-offers) affected anticipated emotional reactions and intended use of a broad range of resistance responses. I administered a questionnaire to high school students to assess resistance responses adolescents have used in the past, resistance responses adolescents expect to use in the future, and expected emotional reactions to drug offers. Although there was statistically significant variation in anticipated use of different types of strategies according to drug and offerer, two particular strategies (“say no,” “say don't want it”) were endorsed most often regardless of the drug/offerer condition. Participants who had made an a priori decision to refuse offers of alcohol or marijuana more often anticipated that an offer would be a negative experience, and more often anticipated that they would use various types of resistance strategies in response to the offer. Background characteristics such as age, gender, race, grade, locality, grade point average, and intentions of going to college had little association with endorsement of emotional reactions or resistance strategies. However, prior experience of receiving drug offers predicted anticipation of less negative emotional reactions and lower endorsement of resistance strategies. Emotional reactions were correlated with resistance (open full item for complete abstract)

Committee: Harold Rosenberg (Advisor) Subjects: Psychology, Clinical

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

Immune checkpoint inhibitors (ICIs), and immunotherapy as a whole, have been the at the forefront of cancer research for the past two decades. They have revolutionized the oncology field, and their efficacy has helped cure cancer patients, not just treat them. However, for reasons unknown, only 25-45% of patients respond to treatment. Mechanisms of ICI resistance are poorly understood and there are few biomarkers available to identify patients that will respond. Two factors associated with ICI resistance are cancer cachexia-associated increases in exogenous mAb clearance (CL) and the sex-dependent differences in nuclear hormone signaling. In this dissertation, I describe novel immune-intrinsic cancer-induced fluctuations in expression of the neonatal Fc receptor (FcRn). I also determined that in mice absent FcRn:ICI binding, cachexia-associated increased ICI CL disappears, suggesting the role of this receptor in cachexia-induced elevated ICI CL. In a separate tumor model of ICI resistance, female mice were treated with anti-PD1 in combination with two structurally distinct estrogen receptor beta (ERβ) agonists – OSU-ERβ-12 and LY500307. Tumor growth was limited in the combination treatment groups, and in a dose-dependent fashion in the OSU-ERβ-12-treated mice. Splenic immunophenotyping suggests the synergistic effects of the ERβ agonists and anti-PD1 in modulating tumor growth were due to immune activation, rather than direct action on the tumor. Together, these studies advance our knowledge of immune checkpoint inhibitor resistance mechanisms and how to overcome them.

Committee: Christopher Coss (Advisor); Emanuele Cocucci (Committee Member); Thomas Mace (Committee Member); Mitch Phelps (Advisor) Subjects: Pharmaceuticals; Pharmacology

Master of Science, The Ohio State University, 1971, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 2007, Graduate School

Committee: Not Provided (Other) Subjects:

Doctor of Philosophy, Case Western Reserve University, 2024, Nutrition

Drug resistance in both cancer and infectious disease is a major driver of mortality across the globe. In infectious disease, the emergence of antimicrobial resistance (AMR) outpaces our ability to develop novel drugs, and within-host evolution confounds the use of previously effective drugs during the course of treatment. In cancer, while targeted therapies have improved outcomes for some, many patients continue to face metastatic, drug-resistant disease, with limited therapeutic options available. As both disease types are driven by clonal evolution, a complementary approach to treatment that leverages tools and ideas from evolutionary biology has been beneficial. However, this evolutionary-inspired therapy has thus far been limited in its consideration of drug variation in time and space within a patient (pharmacokinetics) and variable pathogen response to drug (pharmacodynamics). In this dissertation, we describe novel computational and experimental approaches that integrate pharmacokinetics and pharmacodynamics to allow for more physically realistic models of the evolution of drug resistance. We apply these approaches to gain novel insights into drug dosing regimens and drug diffusion in tissue. In Chapters 1 and 2, we briefly review integrated pharmacokinetics and pharmacodynamics in the study of drug resistance and survey the current evidence of fitness costs to drug resistance in cancer. In Chapter 3, we developed a novel, fluorescence-based time-kill protocol for estimating drug dose-dependent death rates in bacteria. In Chapter 4, we described a software package, FEArS, that allows for efficient agent-based simulation of evolution under time-varying drug concentration. In Chapter 5, we leverage both of these methods to gain insight into why some antimicrobial treatments fail using computational modeling and simulated clinical pharmacokinetics. In Chapter 6, we use spatial agent-based modeling to examine how drug diffusion in tissue can promote tumor hetero (open full item for complete abstract)

Committee: Mark Chance (Committee Chair); Christopher McFarland (Committee Member); Jacob Scott (Advisor); Michael Hinzcewski (Committee Member); Drew Adams (Committee Member) Subjects: Bioinformatics; Biology; Biomedical Research; Biophysics

PhD, University of Cincinnati, 2023, Pharmacy: Pharmaceutical Sciences

HER2 is over-expressed in around 15% to 20% of breast cancers. HER3 plays a critical role in HER2 mediated tumorigenesis. Increased HER3 transcription and protein levels occur upon inhibition of HER2. We aimed to identify proteins that bound to HER3 upon inhibition of the HER family with the pan-HER inhibitor neratinib in HER2+ breast cancer cells. Immunoprecipitation of HER3 followed by mass spectrometry experiments found non-muscle myosin IIA (NMIIA) increased upon neratinib treatment relative to vehicle DMSO treatment. MYH9 is the gene that encodes for the heavy chain of NMIIA. Breast cancer patients with high MYH9 were significantly associated with a shorter disease specific survival compared to patients with low MYH9 expression from the METABRIC cohort of patients. In addition, high MYH9 expression was associated with HER2+ tumors from this cohort. RT-qPCR and immunoblots of whole cell lysates of BT474 and MDA-MB-453 HER2+ breast cancer cells demonstrated elevated HER3 and NMIIA mRNA and protein levels upon neratinib treatment for 24 hours. To examine the role of NMIIA in HER2+ breast cancer, we modulated NMIIA levels in BT474 and MDA-MB-453 cells using doxycycline inducible shRNA targeting MYH9. MYH9 knockdown reduces HER3 protein levels and concomitant reduction in downstream P-Akt. In addition, loss of MYH9 suppresses cell growth, proliferation, migration, and invasion. Our data reveals that NMIIA regulates HER3 and loss of NMIIA reduces HER2+ breast cancer growth.

Committee: Joan Garrett Ph.D. (Committee Chair); Nimita Dave PhD (Committee Member); Susan Waltz Ph.D. (Committee Member); Nalinikanth Kotagiri Ph.D. (Committee Member); Pankaj Desai Ph.D. (Committee Member) Subjects: Pharmaceuticals

Doctor of Philosophy, University of Akron, 2023, Biomedical Engineering

Cancer is the second most common cause of death in the United States, with an estimated 1,958,310 new cases and 609,820 deaths in 2023. Cancer is a complex disease with different sub-populations of genetically diverse cells forming tumors. Despite advancements in identifying molecular drivers of cancer to develop new therapies, drug resistance remains a major barrier against effective cancer treatments. A key driver of drug resistance is a population of tumor cells known as cancer stem cells (CSCs), which possess the ability to change their state under drug pressure and evade drug toxicity. Identifying approaches to target CSCs is expected to improve treatment outcomes. Preclinical models provide a major tool in cancer research. Three-dimensional (3D) tumor models have gained significant momentum in the past decade due to their ability to recapitulate the biological characteristics of tumors and facilitate mechanistic studies and drug testing applications. Here, we developed and used 3D spheroids and organoids of both cancer cell lines and primary patient-derived tumor cells to study drug resistance of colorectal cancer cells with mutations in KRAS and BRAF kinases. We used tumor spheroid cultures in long-term cyclic treatments with targeted therapies against MAPK/MEK pathway to model adaptive drug resistance of cancer cells. We found that inhibiting MAPK/MEK pathway led to the stemness of cancer cells and activated several oncogenic signaling pathways. A combination treatment using a MEK inhibitor and a CSC inhibitor blocked stemness and oncogenic signaling. We also developed an organotypic model consisting of organoids of primary tumor cells and specific components of the tumor microenvironment such as patient-derived cancer-associated fibroblasts (CAFs) and the extracellular matrix. We used this model to elucidate the role of stemness, Wnt/ß-catenin pathway, and signaling with CAFs in the organoid formation of tumor cells and their responses and resistance to ME (open full item for complete abstract)

Committee: Hossein Tavana (Advisor); Yang H Yun (Committee Member); Ronaldo J.J Ramirez (Committee Member); Nic Leipzig (Committee Member); Ge (Christie) Zhang (Committee Member) Subjects: Biomedical Engineering; Oncology

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

Doctor of Philosophy, Case Western Reserve University, 2023, Systems Biology and Bioinformatics

The current maximum tolerated dose treatment paradigm for cancer and bacteria fails to account for the capacity of these disease agents to evolve. When treatment fails to achieve rapid extinction, drug resistant clones rapidly proliferate into an uncontrollable tumor. To make significant progress for cancer patients, we need to better understand the evolutionary processes that drive cancer, and design treatments that explicitly account for them. In this dissertation, I will describe 3 projects that support the design of evolutionary therapies that explicitly account for the capacity of cancer (and bacteria) to evolve resistance in response to drug therapy. In Chapter 2, we developed two novel methods to support precision targeting of tumors; a novel leave-one-out style method for node ranking and a novel algorithm for ranking miRNA combinations that maximizes tumor disruption while minimizing toxicity. In Chapter 3, we described crosstalkr, an open-source software package to facilitate interactomic analyses. In Chapter 4, we described a novel approach for designing evolutionary therapies that leverages reinforcement learning to learn drug cycling policies given only limited information about an evolving system.

Committee: Mehmet Koyuturk (Committee Chair); Jacob Scott (Committee Member); Michael Hinczewski (Committee Member); George Dubyak (Committee Member) Subjects: Bioinformatics; Computer Science

Doctor of Philosophy (PhD), University of Toledo, 2022, Biomedical Sciences (Cancer Biology)

Prostate cancer (PCa) has a unique metabolic profile where it relies on different forms of glucose metabolism at different stages of disease progression. In its early stages, prostate cancer uses the more efficient TCA cycle, but switches to glycolysis when it becomes metastatic (Warburg effect), leading to accumulation of lactate, which then gets exported out of the cells to maintain redox balance. Such metabolic reprogramming can lead to gain-of-function mutations and affect drug sensitivity. Enzalutamide is a second-generation antiandrogen used for the treatment of metastatic castration resistant PCa. However, resistance to Enzalutamide develops in most patients within 9-15 months. Since Warburg effect is a hallmark of metastatic prostate cancer, we examined the expression of monocarboxylate transporters (MCTs) that transport lactate in parental and Enzalutamide-resistant C4-2B and 22RV1 cells and found that they are overexpressed in the Enzalutamide resistant subtypes. Based on this observation, we hypothesized that targeting lactate transport might be a potential strategy to overcome Enzalutamide-resistance in prostate cancer cells. Using cell survival and cell proliferation assays, we found that MCT antagonists resensitized Enzalutamide resistant C4-2B and 22Rv1 cells to treatment with Enzalutamide. Using the Seahorse based glycolytic rate assay, we found that these combination treatments significantly reduced the extracellular acidification rate by reducing the level of glycolysis in the Enzalutamide-resistant prostate cancer cells. We also found that treatment with MCT antagonists either singly, or in combination with Enzalutamide suppressed prostate cancer cell xenograft growth in SCID mice bearing subcutaneously injected parental or Enzalutamide-resistant C4-2B cells. Based on these observations, we concluded that targeting MCT inhibition could be an attractive strategy to overcome Enzalutamide resistance in prostate cancer.

Committee: Nagalakshmi Nadiminty (Advisor); Viviana Ferreira (Committee Chair); Shi-He Liu (Committee Member); Saori Furuta (Committee Member); Lirim Shemshedini (Committee Member) Subjects: Biomedical Research

Master of Arts (MA), Bowling Green State University, 1966, Biological Sciences

Committee: Shirley Harmon (Advisor) Subjects: Biology

Master of Arts (MA), Bowling Green State University, 1965, Biological Sciences

Committee: Shirley A. Harmon (Advisor) Subjects: Biology

Bachelor of Science, Wittenberg University, 2022, Biochemistry/Molecular Biology

Antibiotic resistance in bacteria is becoming a significant concern, originating from the increased amount of antibiotics prescribed by doctors and patients failing to complete their course of treatment. Resistant bacteria are quickly adapting to new forms of antibiotics and are becoming more deadly. Based on the Centers for Disease Control and Prevention's 2019 estimate, in the United States approximately 2.8 million antibiotic resistant infections occur each year and about 90,000 deaths can be attributed to bacterial infections1. Therefore, it is essential that high school and undergraduate students interested in going into the medical field are introduced to the idea of antibiotic resistance. Several different methods of growing resistance to kanamycin and ampicillin in E. coli were created and tested to provide a laboratory experience to students at an introductory chemistry level. Multiple procedures were created using either solid or liquid medium, however, the most practical and successful procedure involved growing resistance in liquid medium in a single flask, which was left to grow over the course of a week. Additionally, the ability for already resistant strains to lose their inherent resistance was also tested through continually propagating cultures in liquid medium without antibiotic. It was found that, through this method, kanamycin resistance in E. coli could be lost in about three weeks or less.

Committee: Daniel Marous (Advisor); Michelle McWhorter (Committee Member); Margaret Goodman (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Microbiology; Molecular Biology

Bachelor of Arts, Wittenberg University, 2022, Chemistry

According to the CDC, in the United States, about 3 million antibiotic resistant infections occur each year with 35,000 deaths from bacterial infections. The large quantity of antibiotics that are prescribed to patients increases the ability of bacteria to develop this resistance, especially when patients do not complete their full course of medication. With the development of bacterial resistance becoming a global phenomenon, it is crucial that the public become more educated on this issue. Toward this end, a method has been developed that demonstrates the growth of antibiotic resistance and could be incorporated into a high school or undergraduate laboratory curriculum. This lab experience would be ideal for students who are considering a medical or scientific research career, allowing them to witness resistance firsthand. It would also serve to educate other individuals, making them aware of the general issues associated with bacterial resistance. Over the course of a week, resistance is gradually evolved in E. coli. The students would then determine the minimum inhibitory concentration of the resistant strain and compare their value to the wildtype level.

Committee: Daniel Marous (Advisor); Justin Houseknecht (Committee Member); Matthew Collier (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Microbiology; Molecular Biology

Master of Science (MS), Bowling Green State University, 2021, Biological Sciences

The overarching menace of drug resistance of Cystic Fibrosis (CF) pathogens, especially by members of Burkholderia cepacia complex and P. aeruginosa strains is a matter of grave concern. However, there is a glimmer of hope in the discovery of novel therapeutics; exploitation of naturally existing competition for survival through antagonism among the microbes could provide an effective therapeutic alternative. Stress response-mediated antagonistic activity could be triggered by variety of selection pressures. In this study, stress was induced in CF pathogens in vitro by exposure to either ultraviolet (UV) irradiation or the DNA cross-linker mitomycin C. Induction by UV irradiation was far more effective in triggering the induction of genes encoding antagonistic elements. Additional experiments optimized the recovery of functional lytic elements. Production requirements, as part of custom-optimization of the method, and identification of nature of the substances have been assessed systematically by different qualitative and semi-quantitative metrics such as timed early production, clear zone of inhibition (ZOI) formation tracked by serial-fold dilution or MIC. Moreover, the lytic substances have been partially identified as non-bacteriophages but more likely as heat labile proteins having a single-hit mechanism of lytic properties similar to that of bacteriocins and tailocins. Target host range susceptibility and panel-wide screening for recovery of more putative toxins with cross-sensitivity tests was also performed. In total, two strains from Burkholderia cepacia complex panel and eight from P. aeruginosa panel have been identified as producers of such putative toxins.

Committee: Raymond Larsen (Advisor); Christopher Ward (Committee Member); Hans Wildschutte (Committee Member) Subjects: Biology; Health; Microbiology; Molecular Biology

Doctor of Philosophy in Biomedical Sciences (Ph.D.), University of Toledo, 2021, Biomedical Sciences (Cancer Biology)

Cancer remains a significant public health burden worldwide, and advances in treatment are aimed at delivering a deathblow to cancer cells, while sparing normal cells. Hence, the ongoing search for novel targets has led to the discovery of a cytoplasmic cis ATR (Ataxia Telangiectasia and Rad3 Related) protein whose function is normal in cells at homeostasis, but when predominant in the cytoplasm, it provides an oncogenic drive through evasion of apoptosis. ATR is a member of the PIKK (Phosphatidylinositol 3-kinase-related kinase) family of protein kinases and nuclear ATR plays a crucial role in DNA damage responses (DDR) by phosphorylating hundreds of downstream proteins. However, ATR is also present in the cytoplasm as either a cis or trans isomeric form, depending on Pin1 which converts cis ATR to trans ATR. Following DNA damage, Pin1's isomerization of cytoplasmic ATR is inhibited leading to an increase in cytoplasmic cis ATR levels. This DNA damage can be from an acute insult or from effects of accumulated damage from the aging process. Following DNA damage, cytoplasmic cis ATR, via its BH3 domain, binds to t-Bid at the outer mitochondrial membrane and sequesters t-Bid to prevent t-Bid binding of pro-apoptotic Bax/Bak protein thus, suppressing apoptosis. Therefore, the levels of cis ATR in the cytoplasm could determine cell fate: death or immortality. With a predominance of cis ATR in cell cytoplasm, there is an inability of the cells to be killed by apoptosis, leading to DNA damage accumulation, genomic instability, and thus oncogenesis. This phosphorylation-dependent peptidyl-prolyl isomerization of ATR by Pin1 serves as a regulatory tool that can be taken advantage of since PP2A can dephosphorylate the key phosphorylated residue in ATR that Pin1 recognizes. With PP2A regulation of ATR, it is possible to shift the balance between the cis and trans forms of cytoplasmic ATR. This is particularly important because it provides a potential strategy in enhancing can (open full item for complete abstract)

Committee: Yue Zou Ph.D. (Advisor); Kathryn Eisenmann Ph.D. (Committee Chair); Caitlin Emily Baum Ph.D. (Committee Member); Saori Furuta Ph.D. (Committee Member); Phillip R. Musich Ph.D. (Committee Member) Subjects: Biochemistry; Biomedical Research; Cellular Biology; Molecular Biology; 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