Master of Science (MS), Wright State University, 2023, Pharmacology and Toxicology

Increased vulnerability to the DNA-damaging effects of ultraviolet (UV) radiation is a hallmark of the uncommon genetic disorder Xeroderma Pigmentosum (XP). The Nucleotide Excision Repair (NER) route uses the DNA Damage Recognition and Repair component XPA to identify and get rid of damaged DNA segments. Although the solar UVB (290-320 nm) radiation is necessary for humans to produce vitamin D, it may also induce erythema and inflammatory reactions, and in some pathological situations, such as XPA deficiency, results in increased UV responses (photosensitivity). Our group has previously shown, using cell lines and mice, that increased synthesis of the lipid mediator Platelet-activating factor (PAF) is what causes UVB-induced amplified effects linked to XPA deficiency. Importantly, our team has recently found that PAF-receptor (PAFR) signaling induces the activation of the enzyme acid sphingomyelinase (aSMase), resulting in the release of tiny subcellular membrane-bound particles known as microvesicle particles (MVPs) from the plasma membrane of keratinocytes. The objective of the ongoing research is to ascertain whether XPA deficiency stimulates increased MVP synthesis in response to UVB and more clinically significant solar-stimulated light (SSL; includes all UV associated with sunlight) via the PAFR signaling pathway and whether inhibiting PAFR or aSMase can significantly decrease photosensitivity. Studies using a keratinocyte cell line deficient in XPA showed more MVP release when exposed to UVB and SSL radiation than XPA-positive cells. UVB and SSL treatment of XPA KO mice resulted in increased MVP release, exaggerated erythema, and cytokine production in comparison to wild-type mice. It was discovered through genetic methods utilizing mice lacking in PAFRs and aSMase that these heightened UV responses depended on a new route involving PAFR-mediated aSMase activation. Finally, experiments on mice showed that administration with the aSMase inhibitor imipramine (open full item for complete abstract)

Committee: Jeffrey B. Travers M.D., Ph.D. (Advisor); Ravi P. Sahu Ph.D. (Committee Member); Yanfang Chen M.D., Ph.D. (Committee Member) Subjects: Pharmacology; Toxicology

Master of Science (MS), Wright State University, 2023, Pharmacology and Toxicology

The skin is exposed to a variety of external stressors, from irritants to toxic agents to ultraviolet radiation (UVR). While mice and human volunteers can be used for research studies, there are limitations in the range of environmental stressors that can be studied using these models. Therefore, our research team has been investigating the use of human skin explants as a model to study the immediate effects of environmental stressors ex vivo. Although this method has been successful, obtaining fresh human skin for these studies is difficult due to limited availability. To overcome this issue, we are evaluating the possibility of using porcine skin explants in our model systems. The primary advantage of using porcine skin is that it is readily available in large quantities, and its structure bears many similarities to that of human skin. In our research, we have found that various stressors, including UVB radiation, can trigger the production of Platelet-Activating Factor (PAF) in the skin. This, in turn, activates the enzyme acid sphingomyelinase (aSMase), leading to the shedding of subcellular microvesicle particles (MVP). In our research studies, we have used various models, such as cell lines, mice, human skin explants, and limited studies in human volunteers. Currently, we are examining the possibility of using porcine skin explants as a model to study MVP release in response to different types of stressors that are relevant to the skin. The application of the carbamyl PAF agonist PAF (CPAF) or a phorbol ester topically to the skin leads to an increase in MVP release. Similarly, exposing the skin to different UVB fluences also results in a higher release of MVP. Our experiments on porcine skin explants show similar results to human skin explants in terms of MVP release, although porcine skin appears to be less reactive to these agents. Although porcine skin shares many structural similarities with human skin, unlike human skin and cell lines, the combined impac (open full item for complete abstract)

Committee: Jeffrey B. Travers M.D., Ph.D. (Advisor); David Cool Ph.D. (Committee Member); Yangfang Chen M.D., Ph.D. (Committee Member) Subjects: Pharmacology; Toxicology

Master of Science (MS), Wright State University, 2021, Pharmacology and Toxicology

The functions of Ataxia telangiectasia and Rad-3 related protein (ATR) is very much important in a cell, as it is a DNA damage response protein, which plays an important role in cell division, DNA repair and apoptosis. This protein helps in proliferation in the actively DNA dividing normal cells and in cancer cells. The functions of ATR in a proliferating cell are well studied and known to involve regulation of replication fork and cell cycle progression after DNA damage. Whereas, in a non-replicating cell, the functions of ATR are not so well known. In the human body, most of the cells are in a non-replicating state, which do not actively replicate DNA, and include cells in a quiescent, senescent, and terminally differentiated state. What could be the function of ATR in these cells is something that nobody has ever looked at and is important because differentiated cells are routinely exposed to DNA damaging agents. ATR inhibitors are used as combination treatments in DNA damage-based anti-cancer therapies to inhibit pro-survival functions of ATR in cancer cells. Some of the studies show that, inhibition of ATR in non-diving cells would show an opposite effect than in the diving cells in response to DNA Damage caused by UVB. Hence, we have conducted experiments to test if inhibition of ATR would show a pro-survival role in differentiated keratinocytes. DNA damage has been induced using UV-B radiation and ATR is activated in both differentiated N-TERT keratinocytes in vitro and in human skin.

Committee: Michael G. Kemp Ph.D. (Advisor); Jeffrey B. Travers Ph.D., M.D. (Committee Member); Yong-Jie Xu Ph.D., M.D. (Committee Member) Subjects: Aging; Atmosphere; Atmospheric Sciences; Biomedical Research; Climate Change; Clinical Psychology; Environmental Science; Environmental Studies; Health Care; Health Education; Health Sciences; Medicine; Molecular Biology; Oncology; Pharmaceuticals; Pharmacology; Pharmacy Sciences; Toxicology

Master of Science (MS), Wright State University, 2021, Pharmacology and Toxicology

Cytokines play a pivotal role in regulating inflammation, which is a condition that makes the tissue vulnerable to different pathological and physiological conditions. Thus, how cytokines are regulated is an important area of study. Skin that receives ultraviolet B radiation (UVB), a major pro-oxidative stressor, results in the release of multiple cytokines and chemokines like tumor necrosis factor (TNF)-alpha and interleukin (IL)-8. Previous studies from our group and others have demonstrated synergistic release of TNF-alpha when UVB is combined with IL-1 or the lipid mediator Platelet-activating factor (PAF). Of interest, subcellular microvesicle particles (MVP) have been proposed to play an important role in intercellular communication. Moreover, UVB and PAF agonists cause MVP release in keratinocytes. Therefore, we believe that understanding the role of MVP in these inflammatory responses could be insightful for photosensitivity mechanisms and to suppress inflammation. The current study focuses on the combination of low concentrations of PAF agonist and UVB in-vitro and ex-vivo to observe potential synergism in the release of cytokines and MVP. We also studied the effects of acid sphingomyelinase (aSMase) inhibitor imipramine, for its ability to modulate both MVP and cytokine release. The application of aSMase inhibitor inhibited the synergistic response of MVP and cytokines allows us to conclude the potential involvement of MVP in the exaggerated response of cytokines from combining UVB and PAF. These studies have potential relevance in understanding abnormal skin reactions such as photosensitivity.

Committee: Jeffrey B. Travers M.D., Ph.D. (Advisor); Mike Kemp Ph.D. (Committee Member); Ji Chen Bihl M.D., Ph.D. (Committee Member) Subjects: Pharmacology

Master of Science (MS), Wright State University, 2021, Pharmacology and Toxicology

Xeroderma Pigmentosum is a genetic disorder in which ability to repair DNA damage such as from UV radiation is decreased. Nucleotide excision repair is known for repairing DNA damage caused by UV radiation and XPA plays a major role in recognizing and eliminating abnormal section of DNA. Therefore, XPA deficiency decreases repair efficiency of DNA. Of note, XPA deficiency is linked with photosensitivity. Microvesicle particles are membrane-bound particles which are released into the extracellular environment in response to multiple stimuli including the lipid Platelet activating factor (PAF). Previous studies have shown that XPA deficiency can induce increase production of reactive oxygen species and generates large amounts of PAF agonists produced non-enzymatically. Hence, the present studies are designed to study if XPA deficiency induces higher UVB-MVP release via PAF-R signaling pathway. Studies involving a XPA- deficient keratinocyte cell-line were able to show that UVB irradiation can cause increase MVP release. Similarly, XPA knockout (KO) mice generated increased MVP with UVB irradiation both in skin as well as plasma in comparison to wild-type mice. Increased production of cytokines (TNF-alpha and IL-6) were also seen in XPA KO mice. However, absence of XPA did not affect MVP release when treated with PAF-R agonist or phorbol ester TPA. Topical application of the acid sphingomyelinase (aSMase) inhibitor imipramine was able to inhibit UVB induced MVP release and pro-inflammatory cytokines. Likewise, genetically knocking down aSMase affected MVP release by UVB irradiation in comparison to wild-type and XPA KO mice. As MVP been involved in UVB signaling, inhibiting MVP release by pharmacological means might be a novel therapeutic approach in photosensitive conditions.

Committee: Jeffrey B. Travers M.D., Ph.D. (Advisor); Michael G. Kemp Ph.D. (Committee Member); Ji Chen Bihl M.D., Ph.D. (Committee Member) Subjects: Pharmacology; Toxicology

Master of Science (MS), Wright State University, 2020, Pharmacology and Toxicology

Skin cancer is the most prevalent human malignancy and is primarily caused by ultraviolet (UV) wavelengths of sunlight. However, the fact that most skin cancers occur in people over the age of 60 indicates that advantaged age is a second skin cancer risk factor. Why geriatric skin is prone to developing skin cancers is not clear, but several studies have shown that dermal fibroblasts in geriatric skin express lower levels of the hormone insulin-like growth factor-1 (IGF-1) than young adult skin and that deficient IGF-1 signaling negatively impacts how epidermal keratinocytes respond to UVB radiation. A major regulator of the cellular response to UVB-induced DNA damage response is the tumor suppressor protein p53, and a previous study indicated that p53 is not properly activated in UVB-irradiated human keratinocytes deficient in IGF-1 signaling. Using cultured human keratinocytes in vitro, we show here that several downstream transcriptional targets of p53, including the cell cycle-dependent kinase inhibitor p21, the translesion synthesis polymerase pol eta, and the DNA repair factors XPC and DDB, are not properly induced following UVB exposure in cells deprived of IGF-1. Using discarded human skin from routine panniculectomies, we show that the iv topical application of an IGF-1 receptor antagonist similarly abrogates the ability of UVB exposure to properly induce pol eta and p21. Because these various p53 target gene products are involved in delaying entry into S phase, accurately replicating UVB photoproducts, and removing UVB photoproducts from genomic DNA, our findings indicate that the inability of IGF-1-deficient geriatric skin properly activate p53-response genes may predispose geriatric skin to mutagenesis and carcinogenesis.

Committee: Michael G. Kemp Ph.D. (Advisor); Jeffrey B. Travers M.D., Ph.D. (Committee Member); Young-jie Xu M.D., Ph.D. (Committee Member) Subjects: Pharmacology; Toxicology

Doctor of Philosophy, The Ohio State University, 2007, Integrated Biomedical Science

Skin cancer is the most common type of cancer in the United States and it is primarily caused by the exposure to ultraviolet-B radiation. Non-melanoma skin cancers, such as basal cell carcinoma and squamous cell carcinoma comprise the majority of skin cancer cases. Ultraviolet-B radiation induces a host of changes in the skin, the primary target of UVB, including inflammation, DNA damage, and eventually skin carcinogenesis. UVB radiation can also induce immunosuppression. This immunosuppression makes it less likely that the immune system will be able to respond efficiently to immunogenic tumors such as squamous cell carcinomas. Therefore, we would predict that factors that can affect cell-mediated immunity would also have a high likelihood of affecting SCC tumor development as well. Psycho-physiological stress is powerful modulator of immune system function as well. While short-term (acute) stress enhances cell-mediated immunity long-term (chronic) stress suppresses cell-mediated immunity. We have used these stressors in combination with UVB radiation to understand the effects of psychological stress on UVB induced inflammation, DNA damage, and ultimately SCC skin carcinogenesis. Using techniques such as immunohistochemistry, real-time PCR, flow cytometry, and ELISA, we found that acute stress reduced DNA damage, enhanced gene expression of Th1 cytokines and chemokines in the skin, and enhanced leukocyte traffic, primarily T cell, into the skin and sites of tumor formation. As a result of this, acute stress reduced SCC tumor development. In contrast to these results, we found that chronic stress increased DNA damage, decreased the gene expression of Th1 cytokines and chemokines in the skin, and decreased T cell infiltration into the skin and sites of tumor formation. As a result of this, chronic stress increased SCC tumor development. Finally, we examined anxiety levels in our mice and because we considered high anxiety as a type of innate chronic stress, we predict (open full item for complete abstract)

Committee: Tatiana Oberyszyn (Advisor) Subjects: