MS, University of Cincinnati, 2018, Arts and Sciences: Physics

This thesis presents the numerical simulation and 3D representation of the “track model” [1, 2], a statistical ionization model developed to describe the ionization distribution at sub-molecular level in biological materials by energetic charged particles. The track model is based on the theory of stopping power [3] and is used to calculate the rates of clustered ionization in DNA. In the numerical simulations, ionization patterns from charged particles are shown for three different levels of linear energy transfer (LET) - Low, medium (10 times Low-LET) and high (100 times Low-LET), corresponding to 1 Gray (Gy) radiation dose for different charged particles. For the graphical visualizations, human DNA genome is represented as cubic cells and the clustered ionization is shown as cluster of holes (created by the ionization) on linear tracks. All holes occurring on the same track within a 3 nm radius is considered to form a cluster. The visualization shows how the clustered ionization depends on LET and the probability of occurrence of clusters with higher complexity (defined as the number of holes in a cluster) increases from low to higher LET radiation.

Committee: Kay Kinoshita Ph.D. (Committee Chair); Leigh Smith Ph.D. (Committee Member); L. C. R. Wijewardhana Ph.D. (Committee Member) Subjects: Physics

Master of Sciences, Case Western Reserve University, 2018, Physics

There are numerous applications of liquid crystals spanning many fields of technology. There are also many applications for detecting patterns of ionizing radiation such as X-rays. This project involves using well known properties of liquid crystals to design a detector to display two dimensional patterns of ionizing radiation. Polymerized liquid crystal films were fabricated with one surface being a transparent electrical conductor, and the other open to air. These films were designed so that an optical change in the layers occurs when charged particles are deposited on the surface. This allows for the creation of displays that show the spatial arrangement of deposited charges. It is well known that a gas with appropriate electrical fields can convert patterns of ionizing radiation into patterns of charge. We have explored different liquid crystal and polyimide combinations to create stable hybrid aligned and homeotropically aligned open cells. We have developed an electron source which generated the electrons needed to test the liquid crystal layers. Finally, we have done several tests to better understand the mechanism for inducing a transition in the layers, and to make the liquid crystal films more useful for charged particle detection.

Committee: Charles Rosenblatt (Advisor); Rolfe Petschek (Advisor); Benjamin Monreal (Advisor) Subjects: Physics

Doctor of Philosophy (PhD), Ohio University, 2016, Chemistry and Biochemistry (Arts and Sciences)

Cell adhesion is an important early step of cancer metastasis, yet the roles of sialylation in regulating integrin-mediated breast cancer cell adhesion in comparison to migration and invasion are not well-understood. The role of sialylation on alpha5 beta1 and alpha2 beta1 integrins in the regulation of adhesion between breast cancer cells and extracellular matrix (ECM) was studied. Our data showed that alpha2, alpha5 and beta1 integrins had considerable alpha 2,6 sialylation on MDA-MB-231 cells, whereas signals from MCF-7 cells were undetectable. Cleavage of alpha 2,6 sialylation increased adhesion of MDA-MB-231 cells to ECM, while adhesion of MCF-7 cells was unaffected, consistent with the latter's lack of endogenous alpha 2,6 sialylated surface integrins. Neither surface expression of alpha5 beta1 and alpha2 beta1 integrins, nor activated beta 1 integrin, changed in MDA-MB-231 cells after sialidase treatment. However, sialidase treatment did not have significant impact on migration or invasion of MDA-MB-231 cells. Integrins not only play an important role in adhesion of cancer cells, but also have a direct connection with ionizing radiation-induced atherosclerosis, which is an adverse effect observed after radiotherapy. However, minimal attention has been given to monocytes/macrophages, which are exposed to the radiation at the same time. Under flow conditions using a parallel plate flow chamber to mimic physiological shear stress, we demonstrate here that the avidity between very late antigen-4 (VLA-4) of RAW264.7 cells and its ligand vascular cell adhesion molecule-1 (VCAM-1), was increased after low dose (0.5 Gy), but was reduced after higher dose (5 Gy) treatment of ionizing radiation. Treating the cells with free radical scavenger N-acetyl-L-cysteine reduced the avidity between RAW264.7 cells and VCAM-1 to a similar level. These results suggest that ionizing radiation regulates adhesive interactions between VLA-4 and VCAM-1, and that reactive oxygen spe (open full item for complete abstract)

Committee: Shiyong Wu (Advisor) Subjects: Biochemistry

Bachelor of Science (BS), Ohio University, 2016, Biological Sciences

Metastasis is involved in 90% of cancer deaths. Ionizing radiation (IR), which is a common cancer treatment, has been shown to alter migration and invasive potential in multiple cancer cell lines in previous research. The tumor suppressor protein, p53, is mutated in about 50% cancers. This project investigated the role of p53 mutation in a cancer's migratory response and epithelial-mesenchymal transition (EMT) after treatment with a single dose of IR (4 Gy or 8 Gy) or with a fractionated treatment (4 treatments of 2 Gy). A single dose of 8 Gy increased migration in H1299 cells expressing p53-R280K and also slightly increased expression of EMT marker proteins. No other significant differences occurred after treatment with 4 Gy or 8 Gy. Fractionated treatment increased migration in H1299 p53-null cells, but did not alter migration or EMT in H1299 cells expressing p53-WT, p53-R175H, or p53-R280K.

Committee: Shiyong Wu Ph.D. (Advisor) Subjects: Biology; Radiation

Doctor of Philosophy, The Ohio State University, 2015, Comparative and Veterinary Medicine

Human norovirus (NoV) is responsible for more than 95% of nonbacterial gastroenteritis and over 60% of foodborne illness each year in the US. However, it has been a challenge to conduct research on human NoV because it cannot be grown in cell culture and lacks a small animal model. Despite major efforts, there is no antiviral therapeutic or vaccine for human NoV. In addition, the survival of human NoV is poorly understood. Fresh produce is major high risk food for human NoV-related outbreaks of gastroenteritis because it can easily become contaminated at any point during pre-harvest and post-harvest stages of production. However the ecology, persistence, and interaction of human NoV in produce systems is poorly understood. Determining the mechanisms underlying human NoV fresh produce contamination can facilitate the development of effective preventative and control measures to limit human NoV outbreaks. The objectives of this research are to determine whether human NoV and its surrogates become internalized via the root of growing produce and disseminated to the edible portion of the plants grown in soil; to determine whether biotic and abiotic stress can affect the rate of internalization and dissemination of human NoV in fresh produce; and to determine whether non-thermal food processing technologies can effectively inactivate internalized viruses while maintaining the fresh quality of produce. Romaine lettuce grown in soil was inoculated with 2 × 108 plaque forming unit (PFU) of human NoV surrogates [Murine norovirus-1 (MNV-1); Tulane virus (TV)] via the roots of plants. Plants were grown for 14 days and leaves, shoots, and roots of each plant were harvested and homogenized and viral titer was determined by plaque assay. It was found that both MNV-1 and TV can efficiently be internalized via plant roots and disseminated to shoots and leaves of lettuce. At day 14 post-inoculation, the titer of MNV-1 and TV in leaves of lettuce plants reached a level of (open full item for complete abstract)

Committee: Jianrong Li PhD (Advisor); Melvin Pascall PhD (Committee Member); Mark Peeples PhD (Committee Member); Wondwossen Gebreyes (Committee Member) Subjects: Food Science; Virology

Doctor of Philosophy, The Ohio State University, 2014, Molecular, Cellular and Developmental Biology

In this dissertation study, we have investigated the protein functions in DNA double-strand break (DSB) repair of three important factors, BRCA1, 53BP1 and SUMO isoforms, at levels of biochemical activity, protein dynamics and chromosomal DNA repair. Our work reveals novel mechanisms of these proteins functioning in response to DSB damage, hence providing insights of where and how they are actively involved in each subpathway of DSB repair. In the first part of our work, we studied BRCA1, a tumor suppressor important for the maintenance of genomic stability including centrosome control and DSB repair, and found that a putative enzymatic mutant of BRCA1— BRCA1(I26A), which had been thought to disrupt its E3 ligase activity, was still functional in the cellular processes of regulating centrosome number and homologous recombination-dependent DSB repair, thereby raising a question of whether I26A mutant is indeed inert. Reevaluation of the ubiquitination activity of this BRCA1(I26A) mutant revealed that it is an active E3 ubiquitin ligase when associated with the appropriate E2 factor. We then think that conclusions about the dispensability of the BRCA1-dependent enzymatic activity in various cellular processes should be reconsidered. Next we studied the unique function of 53BP1, a known NHEJ factor for DSB repair. We found that 53BP1 specifically promotes the error-free conservative-NHEJ (C-NHEJ) mechanism, dependent on its upstream recruiters RNF8 and RNF168. 53BP1 has no effect on the highly mutagenic and deletional alternative-NHEJ (Alt-NHEJ) pathway or on homology-directed repair (HDR), but it suppresses single-strand annealing (SSA). We discovered that the localization of 53BP1 at sites of DSBs is accompanied by its bulk removal from the nucleus except at sites of DNA damage. And the degradation of bulk 53BP1 upon DNA damage is due to each action of RNF8 and RNF168. Further, we showed that failure to degrade bulk 53BP1 results in the failure for its downstream (open full item for complete abstract)

Committee: Jeffrey Parvin (Advisor); Altaf Wani (Committee Member); Qianben Wang (Committee Member); Robin wharton (Committee Member) Subjects: Molecular Biology

MS, University of Cincinnati, 2000, Medicine : Environmental Health Sciences

Both the Geographic Information Systems (GIS) and the Statistical Analysis System (SAS) developed by geographers and statisticians, respectively, were employed to detect the impact of the previous Feed Materials Production Center (FMPC) at Fernald, OH on urinary cancer incidence in community residents participating in the Fernald Medical Monitoring Program (FMMP). To explore the spatial pattern of urinary cancer incidence, GIS was employed to conduct address matching and create quadrats. Moran's Index, a measurement of spatial autocorrelation, was calculated in SAS. Since the spatial autocorrelation was not significant, a backward elimination strategy followed by a stratification analysis of weighted logistic models with DISTANCE of each participant to FMPC as the main factor of interest was employed. Among the people who drank well water, the nearer they lived to FMPC, the more likely it was for them to get urinary cancer (Risk Ratio = 0.703, 95% CI = (0.56, 0.88) ). AGE, GENDER and SMOKING were also included in modeling as covariates. The data indicate that GENDER may be a potential effect modifier. In order to show how to incorporate the spatial autocorrelation into a regular logistic regression model, autologistic regression models were built using PROC GENMOD in SAS, which confirmed that urinary cancer incidence presented no obvious spatial pattern in the Fernald vicinity. Key Words: urinary cancer incidence, Fernald Medical Monitoring Program, spatial autocorrelation, Moran's Index, GIS, weighted logistic model, autologistic regression model.

Committee: Paul Succop (Advisor) Subjects:

MS, University of Cincinnati, 2000, Medicine : Environmental Health Sciences

The role of environmental radiation in the etiology of birth weight and sex ratio was evaluated for 10,105 live births, among a population living near a former uranium processing facility near Fernald, Ohio. Complete pregnancy histories and residential histories were collected by a medical monitoring program. Using a retrospective cohort study, the pregnancies were analyzed controlling for gravidity, gestational age of the fetus at birth, sex, cigarette smoking, alcohol consumption, maternal age, maternal height, work during pregnancy, race and diabetes. There were no significant associations found between ionizing radiation, birth weight or sex ratio. Cigarette smoking, gestational age of the fetus, maternal height and sex were significant risk factors in all regression models used to analyze birth weight, and maternal age was significant in some regression models used to analyze sex ratio. Although these analyses found no association between the exposure metric, birth weight and sex ratio, more precise exposure modeling is needed before a definitive answer to the scientific question can be found.

Committee: Susan Pinney (Advisor) Subjects:

PhD, University of Cincinnati, 2011, Medicine: Epidemiology (Environmental Health)

Context: Nearly four million U.S. workers are potentially exposed to ionizing radiation in the course of their employment each year. Although, evidence exists that unequivocally establishes ionizing radiation as a human carcinogen, little is known about the effects of protracted low-dose exposures. Objective: Conduct a case-control analysis examining the relation between protracted low-dose ionizing radiation exposure and leukemia in a cohort of U.S. nuclear workers. Methods: Each case was matched to four controls on attained age. Ionizing radiation exposures were assessed using measurements and employment histories. Conditional logistic regression analyses were conducted using general relative risk models to estimate the excess relative risk (ERR) of all leukemia, leukemia excluding CLL, AML, CML, and CLL while controlling for potential confounding by race, sex, benzene exposure, social economic status, and either hire date or birth date. Results were tested under differing exposure lag assumptions and time windows of exposure. Results: There were 369 leukemia cases identified in a population of 105,245 U.S. nuclear workers. Positive, but imprecise risk estimates were observed for all outcomes excluding CLL, although the observed dose response for non-CLL leukemia, AML, and CML showed attenuated risk in the low dose (<10 mGy) and high dose (>100 mGy) regions. The linear ERR per 10 mGy absorbed dose to bone marrow was 0.009 (95% CI:-0.014, 0.051). A three-piece linear spline model best fit the non-CLL data, where slope estimates (ERR·10 mGy-1) were statistically significant in the first two segments: -0.68 (95% CI: -0.92, -0.33) for doses =8.0 mGy; 0.20 (95% CI: 0.082, 0.35), dose=8<-46 mGy; and -0.016 (95% CI: <-0.022, 0.018), dose=46+ mGy. Leukemia risks were characterized by a “wave-like” function of time, where peak risks were observed from exposures occurring from five to ten years prior to the age at death of the index AML case (ERR·10 mGy-1 = 0.76; 95% CI: 0 (open full item for complete abstract)

Committee: Susan Pinney PhD (Committee Chair); Mary Schubauer-Berigan PhD (Committee Member); Charles Ralph Buncher ScD (Committee Member); Richard Hornung DrPH (Committee Member); Henry Spitz PhD (Committee Member) Subjects: Epidemiology

PhD, University of Cincinnati, 2007, Medicine : Molecular Genetics, Biochemistry, and Microbiology

Bloom's syndrome (BS) is an autosomal recessive inherited disorder characterized by small size, immunodeficiency, and a striking predisposition to cancer. Cells from persons with BS demonstrate genomic instability, characterized by chromosome breaks, gaps, quadraradials, and a marked elevation in SCE. The gene mutated in BS has been identified and encodes a DNA helicase known as BLM. The mechanism by which BLM deficiency leads to cancer predisposition in persons with BS remains to be elucidated. My work focuses on understanding the role of the BLM helicase in the cellular response to DNA damage induced by diverse genotoxins. Using lymphoblast cell lines isolated from persons with BS, and from age- and sex-matched control individuals, I tested growth capacity in response to several genotoxic agents. I demonstrated BS cell sensitivity to mitomycin C (MMC), hydroxyurea (HU), and a low sensitivity to ionizing radiation (IR). To test sensitivity in vivo, Blm heterozygous mice were treated with large doses of IR and MMC. These experiments demonstrated that heterozygous mice have a slight sensitivity to IR, but surprisingly not to MMC. To confirm that BLM is involved in the response to DNA damage induced by these agents, I demonstrated that a GFP-BLM fusion protein could co-localize with phosphorylated H2AX at sites of DNA damage, and that this co-localization was much greater following DNA damage induced by MMC and HU than damage induced by IR. Lastly, I used flow cytometry to demonstrate that the kinetics of H2AX phosphorylation and de-phosphorylation are largely normal in BS cells, with only a slight delay in cell cycle progression following MMC exposure. I also investigated the formation of foci of H2AX phosphorylation in the breast cancer cell line MCF7 following treatment with the histone deacetylase inhibitors (HDI) trichostatin A (TSA) and sodium butyrate (NaB). I demonstrated that these foci were correlated with double strand DNA breaks as treatment of MCF7 cells (open full item for complete abstract)

Committee: Dr. Joanna Groden (Advisor) Subjects: Health Sciences, Oncology

Bachelor of Science (BS), Ohio University, 2011, Chemistry

There were over 207,000 new cases of invasive breast cancer and nearly 40,000 deaths from the disease in the U.S. in 2010. It is, therefore, critical to develop a better understanding of the disease process. Invasive cancer is marked by the loss of integrity of the basement membrane of a tissue, a specialized form of extracellular matrix (ECM) that normally separates a tissue from its surroundings; however, a complex, dynamic interaction involving many overlapping signaling pathways exists between breast tissue and its neighboring ECM. Invasion is initiated when the tumor cells have increased adhesion to the ECM, which is mainly modulated by interactions with the integrin family of adhesion proteins on the surface of the tumor cells. Each integrin heterodimer has a different adhesive affinity for each of the ECM proteins, namely fibronectin, vitronectin, collagen I, and collagen IV. This project examined the relative expression of integrins following exposure to ionizing radiation, which is commonly used for cancer therapy. Ionizing radiation forms free radicals and reactive oxygen species, which then cause damage within the cell. Although the exact mechanism is unknown, the hypothesis of this study was that exposure to high dose ionizing radiation would change the expression of the integrins and change integrin-mediated adhesion to the ECM proteins. Results showed that in the MDA-MB-231 cell line, ionizing radiation induces significant changes in both integrin expression and integrin-mediated adhesion to fibronectin, laminin, and collagens I and IV. After gathering further evidence from this and other cell lines, this information could potentially be used to optimize treatment for patients with invasive cancers.

Committee: Shiyong Wu PhD (Advisor) Subjects: Biochemistry; Biomedical Research; Cellular Biology; Chemistry

Doctor of Philosophy, Case Western Reserve University, 2006, Environmental Health Science

The E2F family of transcription factors is comprised of nine active members to date, which activate or repress genes. Well-studied members, such as E2F1 and E2F4, are known to play key roles in G1/S transition, S-phase progression, and mitosis. E2F1 is generally regarded as a transcriptional activator and E2F4 is a transcriptional repressor; the balance of their expression levels may promote apoptosis or cell cycle arrest, respectively. Functioning together with or independently of p53 in cell cycle regulation, E2F1, has been implicated in G1 cell cycle arrest following DNA damage; they share similar target genes. Retinoblastoma (Rb) and related pocket proteins p130 and p107 play important roles in cell cycle control. However, because of multiple interacting partners, their specific roles have not been clear. Following genotoxic stress, p130 interacts with E2F4. An ionizing radiation-induced G2-phase arrest was characterized by decreased expression of MPM-2, a mitosis marker, and Cyclins A2 (early G2) and B1 (late G2 and M). Concomitant with this G2-arrest, E2F4 cellular localization was redirected to the nucleus, similar to that of p130. Knock-down of E2F4 by siRNA elicited persistent cellular DNA damage and sensitization following irradiation. Flow cytometry analyses revealed an increased population of cells with an apparent S-phase content following this treatment, but these cells were not actively dividing. Downstream E2F4 targets potentially involved in the exit from G2 arrest were identified by expression-profiling. Chromatin immunoprecipitation localized E2F4 at promoter regions of the Bub3 and Pttg1 genes following irradiation. This work indicates that E2F4 expression, nuclear localization, and target gene repression following irradiation play crucial roles in mediating the exit from G2 arrest in cells that sustain irreparable DNA damage. As the E2F4/p130 complex has been implicated in G0 control, its translocation leading to the repression of genes with G2/ (open full item for complete abstract)

Committee: Alex Almasan (Advisor) Subjects:

Doctor of Philosophy, Case Western Reserve University, 2004, Pathology

Radiation therapy is a common treatment for many types of tumors. Therefore, it is vital to understand the cellular responses to radiotherapy in malignant cells, as well as the surrounding normal tissues for optimizing the efficacy of this therapy. Our laboratory identified clusterin (CLU) as a protein/transcript that could be induced by doses of ionizing radiation (IR) as low as 0.02 Gy, suggesting a role for CLU in the cellular response to IR. CLU is a secreted glycoprotein that has been implicated in many normal biological processes as well as many pathological processes (Chapter 1). While the exact function of CLU still remains unknown, it has been suggested that secretory CLU (sCLU), the fully processed and glycosylated form of CLU, plays a role in cytoprotection after cellular stress. This thesis characterizes the regulation of sCLU after IR exposure. We observed that sCLU protein levels were low and only marginally induced after IR in cell lines containing functional wild-type p53. In contrast, cell lines expressing mutant p53 or null p53 had high basal levels of sCLU. This led us to investigate the role of p53 as a transcriptional repressor of sCLU (Chapter 2). Using genetically matched cell lines, we demonstrated that sCLU is transcriptionally repressed by p53. 12 Further work was done to characterize the signaling pathways required for sCLU induction after IR. We demonstrated that sCLU is involved in cell survival after IR using sCLU specific siRNA (Chapter 3). Furthermore, we demonstrated that the Src-Mek-Erk signaling cascade is reactivated 24-72 h after IR, which corresponds to the late induction of sCLU, and that the signal for sCLU induction after IR is generated by activation of the insulin-like growth factor-1 receptor (IGF-1R). Finally, we demonstrate that this signaling cascade culminates in the activation of Egr-1, which is required for the induction of sCLU after IR.

Committee: David Boothman (Advisor) Subjects:

Doctor of Philosophy, Case Western Reserve University, 1994, Environmental Health Science

Eukaryotic chromatin is folded into topologically constrained loops by the attachment of DNA to the nuclear matrix. The sequences which anchor DNA, matrix attachment regions (MARs), can be identified by their specific and preferential binding to the matrix. Previous evidence from our laboratory (Chiu et al. 1986) suggested that the close association of MARs with the nuclear matrix confers a high susceptibility to radiation-induced DNA-protein crosslink (DPC) formation. We demonstrate both by in vivo and in vitro assays the presence of a DNA region (750-bp) 3′ to the β-major globin gene that is associated with the nuclear matrix of a murine erythroleukemia cell line. The in vivo assays indicated the presence of an attachment region in a 2.7- kb DNA fragment 3′ to the poly (A) site of the β-major globin gene domain. Visual analysis of the sequence of the 2.7-kb fragment revealed sequence-motifs characteristic of a MAR on a 750-bp stretch of DNA within the 2.7-kb fragment. The 750-bp DNA fragment was amplified by PCR, end-labeled and subjected to in vitro binding assays. In the in vitro binding assays, the binding of a MAR fragment was competed at high concentrations of sonicated E. coli DNA, and more effectivel y by poly(dAT), but not by pBR322 fragment. The in vitro assays confirmed the presence of a MAR in the 750-bp fragment. To examine the induction of DPC by radiation, we further developed the in vitro binding assay. The crosslinking of nuclear matrix protein to four end-labeled DNA sequences was studied in vitro: κ-Ig MAR (2.8-kb) and the hsp MAR and two pBR322 fragments as non-MAR-containing DNA controls of similar size. When isolated nuclear matrices and end-labeled DNA fragments were irradiated (0-100 Gy) and poly(dAT) was added after irradiation, in addition to elimination of non-specific binding, a dose-dependent increase in the binding was revealed for both the κ-Ig MAR and hsp MAR. Such an increase did not occur for either of the pBR322 fragments. When nu (open full item for complete abstract)

Committee: Nancy Oleinick (Advisor) Subjects: Biology, Molecular