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Whitson, Jeremy ALens Adaptation to Glutathione Deficiency: Implications for Cataract
Doctor of Philosophy, Case Western Reserve University, 2017, Pathology
The antioxidant glutathione (GSH) protects lens proteins from post-translational modifications that result in their aggregation and cataract formation. With age, the human lens becomes increasingly depleted of GSH, which contributes to the development of age-related cataract. In order to gain a comprehensive understanding of the role of GSH in the pathogenesis of age-related cataract, I set out to study the consequences of and adaptations to GSH-deficiency in the lens using the Lens Glutathione Synthesis Knockout (LEGSKO) mouse model of cataract. The questions addressed in this thesis are: 1) How does the LEGSKO lens maintain >1 mM GSH despite a complete lack of GSH synthesis? and 2) What gene expression and signaling changes are associated with lens GSH deficiency? The first of these questions was addressed by measuring lens uptake of isotopically-labeled GSH using an LC-MS/MS system. I determined that mouse lenses obtain exogenous GSH in two ways: from the aqueous humor via an active transport mechanism and from the vitreous humor via passive diffusion. It was found that mouse eyes have a high concentration of GSH in their vitreous humor and a low concentration of GSH in their aqueous humor and, because of this, nearly all the GSH in the LEGSKO lens is derived from equilibration with the vitreous pool. It was also found that the eyes of humans and other large animals lack this high vitreous GSH concentration. The second question of this thesis was addressed by comparing the transcriptomic profiles of wild-type control lenses, chronically GSH-deficient LEGSKO lenses, and acutely/severely GSH-deficient buthionine sulfoximine-treated (BSO;GSH synthesis inhibitor) LEGSKO lenses using RNA-Seq technology. These data show that the most robust responses to GSH-deficiency in the lens are upregulation of detoxifying genes, including metallothioneins, aldehyde dehydrogenases, and carboxylesterase, activation of epithelial-mesenchymal transition (EMT) signaling, and alterations to lipid homeostasis and transport systems. These findings suggest that GSH plays a role in EMT-mediated posterior secondary cataract and implicate new potential targets for cataract therapeutics. This body of work greatly expands knowledge of the benefits and regulation of GSH in the lens, consequences of its loss, and other genes which promote lens clarity.

Committee:

Vincent Monnier (Advisor); Xingjun Fan (Advisor); Alan Tartakoff (Committee Chair); John Mieyal (Committee Member); Maria Hatzoglou (Committee Member); Xiongwei Zhu (Committee Member); Clive Hamlin (Committee Member)

Subjects:

Biology; Biomedical Research; Pathology

Keywords:

cataract, glutathione, EMT, lens, transcriptome, homeostasis

Broutian, Tatevik RafikThe Study of HPV Integration as a Means for Discovery of Candidate Driver Genes in HNSCC
Doctor of Philosophy, The Ohio State University, 2017, Biomedical Sciences
Head and neck squamous cell carcinoma (HNSCC) is sixth in cancer incidence worldwide. Human papillomavirus (HPV) accounts for ~5% of the world’s cancer burden, including a distinct subset of oropharyngeal HNSCCs rising in incidence in numerous developed countries. The transforming ability of HPV is primarily attributed to the viral oncoproteins E6 and E7 which inactivate the tumor suppressor proteins p53 and pRb, respectively. While E6 and E7 expression are sufficient for immortalization, poorly defined, secondary genetic events are necessary for cellular transformation. Whole genome sequencing of HPV-positive HNSCC cell lines and primary tumors demonstrated a direct association between HPV integrants and focal host genomic instability, including amplification, rearrangement, deletion, and translocation. HPV integrants frequently disrupted expression of host genes with established roles in cancer pathogenesis. We hypothesized that host genomic alterations caused by HPV integration are critical secondary genetic events necessary for the pathogenesis of HPV-caused cancers and that identification of HPV integration sites can serve as a tool for discovery of candidate driver genes targetable for HNSCC therapeutics. To test this hypothesis we extensively characterized two integration events at TP63 and PIM1genes in UM-SCC-47 and UPCI:SCC090, respectively. Both genes were candidates due to their extensive links to cancer. In UM-SCC-47, HPV integration resulted in disruption of wild-type p63 transcripts and detection of unique fusion transcripts that served as a template for translation of a novel truncated p63 protein (p63T). Although morpholino mediated knockdown of p63T did not affect UM-SCC-47’s growth and viability, p63T displayed novel localization in the cytoplasm indicative of possible new protein interactions not previously characterized. Furthermore, its stability was shown to be modulated by p53 and the proteasome. In UPCI:SCC090, HPV integration resulted in a 16-fold amplification of the PIM1 oncogene. This gene amplification was accompanied by high levels of PIM1 transcripts and protein as detected by RNAseq and Western blot, respectively. Genetic and pharmacologic knockdown of PIM1 using CRISPR technology, shRNA, and pan-PIM inhibitors greatly compromised the growth of UPCI:SCC090 cells demonstrating that the HPV mediated PIM1 amplification significantly contributes to this cell line’s growth and viability. High PIM expression was also observed in other HNSCC cell lines and primary tumors. PIM expression is regulated through the JAK/STAT, PI3K-AKT, and NFKß pathways, which are frequently activated in HNSCCs. Pan-PIM inhibitor studies extended to other cell lines demonstrated sensitivity in the low µM range and synergistic cell death in combination with epidermal growth factor receptor (EGFR) inhibitors. PIM kinases are rational and promising new targets for development of HNSCC therapeutics. Combined together, our work demonstrates that HPV is a direct insertional mutagen that significantly alters gene structure, expression, and function. Comprehensive understanding of the functional consequences of HPV integration will expand our understanding of HPV malignancies and provide insight on the design of novel therapeutics for all HNSCCs.

Committee:

Maura Gillison (Advisor); Quintin Pan (Committee Member); Dawn Chandler (Committee Member); Nyla Heerema (Committee Member)

Subjects:

Biomedical Research

Keywords:

HPV; HNSCC; PIM Kinase; Integration; Prevalence; TP63

Schrock, Morgan SWwox deficiency in human cancers: Role in treatment resistance
Doctor of Philosophy, The Ohio State University, 2017, Biomedical Sciences
WWOX gene deletions occur in a variety of human cancer types, and reduced Wwox protein expression can be detected early during cancer development. In this study, loss of expression of the fragile site-encoded Wwox protein was found to contribute to radiation and cisplatin resistance of cells, responses that could be associated with cancer recurrence and poor outcome. We found that Wwox loss is followed by mild chromosome instability in genomes of mouse embryo fibroblast cells from Wwox-knockout mice. Human and mouse cells deficient for Wwox also exhibit significantly enhanced survival of ionizing radiation and bleomycin treatment, agents that induce double-strand breaks (DSBs). In a xenograft tumor model of irradiated cells, Wwox-deficient cancer cells exhibited significantly shorter tumor latencies, suggesting that Wwox-deficiency facilitates radiation resistance in vivo. In examining mechanisms underlying Wwox-dependent survival differences, we found that Wwox-deficient cells exhibit enhanced homology directed repair (HDR) and single strand annealing (SSA) repair pathways, but reduced nonhomologous end-joining (NHEJ) and Alternative-NHEJ (Alt-NHJE) repair, suggesting that Wwox contributes to DNA DSB repair pathway choice. We also demonstrated interaction of Wwox with Brca1, a driver of HDR, and show via immunofluorescent detection of repair proteins at ionizing radiation-induced DNA damage foci that Wwox expression suppresses DSB repair at the end-resection step of HDR. We propose a genome caretaker function for Wwox, in which Brca1–Wwox interaction supports NHEJ as the dominant DSB repair pathway in Wwox-sufficient cells. This Wwox effect has important consequences in human disease: in a cohort of brain, ovarian and non-small cell lung cancer patients treated with radiation or cisplatin, Wwox deficiency significantly correlated with shorter overall survival times, indicating that dysregulation of DSB repair by Wwox-deficiency worsens patient outcome via treatment resistance.

Committee:

Kay Huebner (Advisor); Paul Goodfellow (Committee Member); Jeffrey Parvin (Committee Member); K. John McLaughlin (Committee Chair)

Subjects:

Biomedical Research; Molecular Biology

Keywords:

WWOX, FRA16D, chromosome fragile site, tumor suppressor, double strand break repair

Seshadri, Dhruv RamakrishnaImmuno-nanotherapeutics to Inhibit Macrophage Polarization for Non-Small-Cell Lung Cancers
Master of Sciences, Case Western Reserve University, 2017, Biomedical Engineering
Lung cancer is the leading cause of cancer-related mortalities in the USA with a five-year survival rate of ~15%. For patients with Non-Small-Cell Lung Cancer (NSCLC), chemotherapy, oncogene targeted therapy, or immunotherapy are the primary modes of treatment. Response rates to immunotherapies for NSCLCs are < 20%, due to the tumor micro-environment (TME) that favors immune-evasion and pro-tumorigenic pathways such as macrophage polarization from a pro-inflammatory (M1) to a pro-tumorigenic/angiogenic (M2) phenotype. Additionally, the TME is compromised by the chronic enzymatic breakdown of the elastic matrix which catalyzes polarization. Exogenous delivery of Doxycycline (DOX) has shown to inhibit the M1-M2 phenotypic switch. We explored the utility of antibody-conjugated DOX-poly(ethylene glycol)-poly(lactic glycolic-acid) (PEG-PLGA) nanoparticles (NPs) to inhibit macrophage polarization and demonstrate that steady-state release of DOX from these NPs is possible in a low dose range to inhibit polarization and repolarize macrophages back to the M1 phenotype.

Committee:

Anand Ramamurthi (Advisor); Eben Alsberg (Committee Member); Colin Drummond (Committee Member)

Subjects:

Biomedical Engineering; Biomedical Research; Polymer Chemistry; Polymers

Keywords:

Tumor Microenvironment, Nanomedicine, Nanoparticles, Polymers, Lung Cancer, Macrophages, Immunotherapy, Extracellular Matrix, Elastin

ALBOSLEMY, TALIBKruppel-like factor 2: A regulator of macrophage-mediated innate immune response against Staphylococcus aureus biofilm.
PHD, Kent State University, 2018, College of Arts and Sciences / School of Biomedical Sciences
Complications arising from cutaneous and soft tissue infections with Staphylococcus aureus (S. aureus) are a major clinical problem due to the high incidence of these infections and the widespread emergence of antibiotic-resistant bacterial strains. In particular, S. aureus infection involving the formation of biofilm is clinically challenging due to antibiotic resistance and immune evasive characteristics of biofilm-associated infection. Accumulating evidences support that macrophages associated with S. aureus biofilms have decreased secretion of cytokines and chemokines, which might have contributed to the impaired bactericidal activity. A series of recent studies have demonstrated that S. aureus biofilms skew macrophage polarization towards an anti-inflammatory phenotype that persists bacterial survival. However, the detailed mechanism underlying the evasion of macrophage-mediated innate immune response against S. aureus biofilm is poorly understood. The central hypothesis of this study is that S. aureus biofilm environment skews macrophage polarization toward the anti-inflammatory phenotype by interfering with a pattern recognition receptor (PRR)-dependent positive regulatory mechanism or aberrantly amplifying a negative regulatory mechanism of NF-kB activation. In this study, using an in vitro model of murine macrophages exposed to the secreted factors from S. aureus biofilm, we have determined the impact of the S. aureus biofilms on macrophage-mediated innate immune response and elucidated its associated signaling mechanism. The major finding of this study is that S. aureus biofilm environment associated with alpha toxin skews macrophage polarization toward the anti-inflammatory phenotype by aberrantly increasing the expression of krupper-like factor 2 (KLF2) in macrophages, which in turn negatively regulate the activation of NF-kB pathways for host innate immune responses. Our study provides an important insight into the mechanism by which S. aureus biofilm impair macrophage-mediated innate immune defense functions and is the first to identify KLF2 in macrophage as a molecular target by S. aureus biofilm to evade innate immune attack by macrophages. Understanding the nature of host-microbial interactions, in particular in the context of the mechanisms used by S. aureus biofilm to evade host innate immune defense, is the key to develop an effective immunotherapy for combating infectious diseases. Our study provides a new opportunity for immunotherapy for targeting macrophage polarization toward the anti-biofilm phenotype by targeting KLF2 and its associated signalling in macrophages against S. aureus biofilm infection.

Committee:

Min-HO Kim (Advisor)

Subjects:

Biomedical Research

Keywords:

Staphylococcus aureus; Biofilms; Macrophage; Inflammation; innate immune response; KLF-2

Alswillah, Turkeyah STHE USP14 DEUBIQUITINASE REGULATES NHEJ DNA REPAIR IN PROSTATE CANCER
Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2018, College of Sciences and Health Professions
Ionizing radiation (IR), a prototypical DNA damaging agent, induces DNA double-strand breaks (DSBs). IR-induced DNA-DSBs are predominantly repaired by the non-homologous end joining (NHEJ) pathway. IR-induced DNA damage has been shown to induce autophagy, an intracellular degradation process that delivers cytoplasmic components to the lysosome. Here, we examined the interplay between autophagy and DNA damage response (DDR), with a particular emphasis on an E3 ubiquitin ligase RNF168, which is negatively regulated by a deubiquitinase (DUB) enzyme, USP14. Akt, a serine/ threonine protein kinase, is known to mediate the phosphorylation of USP14 on Ser432. Loss of the tumor suppressor PTEN, phosphatase and tensin homolog, results in hyperactivated Akt signaling, commonly reported in human malignancies, including prostate cancer (PCa). Autophagy inhibition was coupled to diminished RNF168-mediated histone H2A ubiquitination. The E3 ubiquitin ligase RNF168 is essential for recruitment of the DNA-DSBs signaling proteins, including 53BP1, a critical DDR signaling effector at the DNA-DSBs sites. IR induced sustained ¿-H2AX, and reduced 53BP1 in autophagy-deficient PCa cells. Autophagy-deficient cells showed also reduced activity for DNA-PKcs, a kinase that is required for the NHEJ repair pathway. A defect in recruitment of DNA repair proteins could be caused by hyperactivity of the deubiquitinase USP14. In response to IR, we show that USP14 was upregulated and generated foci in autophagy-deficient cells. Importantly, inhibiting autophagy following radiation-induced recruitment of USP14 onto chromatin. There was an inverse correlation between USP14 and RNF168 expression in autophagy-deficient PCa and non-small cell lung cancer (NSCLC) cells, indicating that USP14 may contribute to the aberrant inhibition of the NHEJ-DDR signaling pathway by targeting RNF168. USP14 inhibition by IU1, an enzymatic activity inhibitor or Akt inhibition rescued the activity of NHEJ-DDR proteins in autophagy-deficient cells. These findings uncovered a novel negative NHEJ regulation mechanism by USP14, which provided unique insights into the link between autophagy and NHEJ-DDR.

Committee:

Alexandru Almasan (Committee Chair); Aimin Zhou (Committee Co-Chair); Girish Shukla (Committee Member); John F Turner (Committee Member)

Subjects:

Biochemistry; Biology; Biomedical Research; Cellular Biology

Idippily, NethrieEVALUATION OF A SMALL MOLECULE AGONIST OF EPHA2 RECEPTOR TYROSINE KINASE AND COPALIC ACID ANALOGS AS PROSTATE CANCER THERAPEUTICS
Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2018, College of Sciences and Health Professions
Project I: Chemotherapeutic drugs have many side effects that are undesirable and are highly toxic. Therefore, there is a growing need for the development of drugs with enhanced efficacy, specificity, and potency to provide cancer patients with a better prognosis. It was discovered that a member of the Receptor Tyrosine Kinase family, EphA2, may prove to be a viable target in developing anti-cancer agents. In the presence of its ligand, EphA2 receptor is responsible for apoptotic and anti-migratory activity. However, in the absence of ligand, EphA2 is able to stimulate cell migration and therefore tumorigenic activity. These conflicting roles of EphA2 and the upregulation of this receptor that is seen in many cancers have provided a novel strategy in designing therapeutic agents. Therefore, small molecules can be used to stimulate ligand-dependent pathways of EphA2 that induce anti-migratory and anti-proliferative effects in cancer cells in order to inhibit metastasis and tumor progression. A small molecule, doxazosin, was identified as an EphA2 agonist in a recent study. It demonstrated positive results in that its actions were similar to those of the natural ligand. Subsequently, a library of compounds was generated using doxazosin as the lead compound in order to improve its activity. These compounds were tested for their activity in stimulating EphA2 receptor. Two of them showed improved activity compared to doxazosin while mimicking a mechanism similar to the native ligand. The structure activity relationship of these derivatives, the in vitro mechanisms and pharmacokinetic profiles were also analyzed, which provides a basis for further optimization and subsequent in vivo studies of these compounds in the future. Project II: Copalic acid which is one of the diterpenoid acids in copaiba oil inhibited the chaperone function of ¿-crystallin and heat shock protein 27 kDa (HSP27). It also showed potent activity in decreasing the level of an HSP27 client protein, androgen receptor (AR), which makes it useful in prostate cancer treatment. In order to develop potent drug candidates to decrease the AR level in prostate cancer cells, copalic acid analogs were synthesized. Using AR level as a readout 15 copalic acid analogs were screened, which showed that two of those compounds were much more potent than copalic acid. They inhibited AR positive prostate cancer cell growth in a dose-dependent manner. Furthermore, they also inhibited the chaperone activity of a-crystallin.

Committee:

Bin Su, Ph.D. (Advisor); Aimin Zhou, Ph.D. (Committee Member); Yan Xu, Ph.D. (Committee Member); Yana Sandlers, Ph.D (Committee Member); Andrew Resnick, Ph.D. (Committee Member)

Subjects:

Analytical Chemistry; Biochemistry; Biology; Biomedical Research; Organic Chemistry; Pharmacology

Uddin, Muhammad ErfanCharacterization and Quantitation of Collagen-I Oxidation in TGF-β Stimulated Fibroblast Culture
Master of Science in Biological Sciences, Youngstown State University, 2017, Department of Biological Sciences
Incisional hernia is one the most common postoperative complications of abdominal surgery. Wound healing studies detailing the events of scar formation have shown that scar collagen composition and structure changes with the maturation of the scar. Cytokines, such as TGF-β, and reactive oxygen species (ROS), such as H2O2, present in the environment during the wound healing process can influence the events of scar formation and the properties of scar tissue. Few studies, however, have examined how these factors can influence the structure and stability of collagen fibers. This study uses a MRC-5 fibroblast culture preparation, and 2D gel profile of a CNBr digested cells and media preparations to identify ROS-oxidation changes in collagen structure, characterize the collagen structure of TGF-β-treated cultures, and evaluate the ROS sensitivity of this collagen in TGF-β-treated cultures. Results showed that MRC-5 cultures treated with TGF-β (0.5%) displayed a 2D gel collagen profile distinct from their untreated counterparts. The collagen content in both MRC-5 cells and media was greater and displayed the presence of the collagen fragment, α1CB6. Results showed that oxidation of TGF-β-treated cultures also produced a 2D gel collagen profile distinct from the untreated controls. Oxidation yielded more high molecular weight collagen peptide fragments and generated the appearance of an additional collagen CNBr fragment, α1CB8. The results of these studies suggest that oxidation and TGF-β treatment can alter the collagen fiber structure and organization in the MRC-5 culture.

Committee:

Johanna Krontiris-Litowitz, PhD (Advisor); Diana Fagan, PhD (Committee Member); Gary Walker, PhD (Committee Member); Mark Womble, PhD (Committee Member)

Subjects:

Biochemistry; Biomedical Research; Molecular Biology; Surgery

Keywords:

Collagen-I; TGF-B; Wound Healing; Reactive Oxygen Species

Carey, Heather ATranscriptional Control of Osteoclast Differentiation by Myeloid Master Regulator PU.1 and Its Co-partners MITF and EOMES
Doctor of Philosophy, The Ohio State University, 2017, Integrated Biomedical Science Graduate Program
Osteoclasts are bone resorbing cells that differentiate from myeloid precursors. The crosstalk between bone-forming cells (osteoblasts) and osteoclasts tightly regulates the dynamic and continuous process of bone remodeling. Deregulation of this delicate balance is implicated in debilitating diseases such as osteoporosis, rheumatoid arthritis, and bone metastasis in cancer. A variety of transcription factors including PU.1, MITF, NFATc1, and c-FOS are essential for osteoclast differentiation. However, the interplay between these factors is not fully defined. We have previously shown that the transcription factors PU.1 and MITF act as a complex to regulate multiple genes required for osteoclast function. In this study, we examined global transcriptional regulation of target genes by PU.1 and MITF in myeloid precursors and osteoclasts using chromatin immunoprecipitation coupled with next generation sequencing (ChIP-Seq) to map the genome-wide binding of these factors. In parallel, microarray analysis was performed to monitor target gene expression changes over the course of differentiation. We also queried the sequences jointly bound by PU.1 and MITF to search for conserved binding sequences potentially indicating novel co-partners in osteoclasts. We used micro-computed tomography and histological analysis to examine the effects of myeloid lineage-specific and osteoclast-specific deletion of PU.1, and its copartners in vivo and in vitro. ChIP-Seq and microarray profiling revealed that PU.1 and MITF jointly regulate the transcription of over 1000 genes in developing osteoclasts. Most of the PU.1/MITF co-bound regions were found in distal enhancer-like elements at the same sites in both myeloid precursors and osteoclasts. Overlap of our ChIP-Seq and microarray data sets utilizing Gene Set Enrichment Analysis (GSEA) revealed that transcription factor genes were significantly enriched in genes with PU.1/ MITF co-bound regions. These PU.1/ MITF target transcription factors include Nfatc1, cFos, Fosl2, and Prdm1, which are already known to be essential for osteoclast differentiation. Additionally, 38% of genomic regions jointly bound by PU.1 and MITF in developing osteoclasts contained the binding motif of the T-box transcription factor EOMES. Conventional ChIP assays validated binding of EOMES to PU.1/MITF bound regions. Micro-computed topography analysis of murine bone has demonstrated that the loss of PU.1 or EOMES in osteoclasts and their myeloid precursors leads to an osteopetrotic (dense bone) phenotype in neonatal mice due to significantly deficient osteoclast differentiation and function. Further, neonatal mice harboring Mitf mutant alleles, which either ablate MITF’s ability to bind to DNA or respond to RANKL signaling, also display a severe osteopetrotic phenotype due to defective osteoclastogenesis. Our results demonstrate that PU.1, MITF, and their novel co-partner, EOMES, regulate a network of transcription factors in myeloid precursors that triggers osteoclast differentiation in response to cues from the bone microenvironment. Ablation or mutation of PU.1, MITF, or EOMES results in disruption of this transcription factor network and thereby hampers the differentiation program. Characterization of the complex mechanisms that control osteoclast differentiation is necessary for the development of better prognostic markers and targeted therapies for diseases involving deregulated osteoclast function.

Committee:

Michael Ostrowski, PhD (Advisor)

Subjects:

Biomedical Research

Ratliff-Rang, Christine AnnetteThe Hypercapnic Ventilatory Response and Behavior in Ca2+-Activated K+ (BK) Channel Knock Out Mice And T-Cell Death-Associated Gene 8 (TDAG8) Receptor Knock Out Mice
Master of Science (MS), Wright State University, 2017, Physiology and Neuroscience
Some acid sensing areas in the brain control the expression of breathing and anxiety/fear including the locus coeruleus (LC) (Redmond & Huang, 1979) and the nucleus tractus solitarius (NTS). It has been found that knocking out T-cell death-associated gene 8 (TDAG8), a chemosensor, attenuates CO2 induced fear phenotypes in mice. However their hypercapnic ventilatory response (HCVR) has not yet been looked at. Also, BK channels are large-conductance, calcium-activated potassium channels that are activated by increases in concentration of intracellular calcium ions. It has been found that BK KO rats have an increase in their HCVR (Patrone et al., 2014) however their CO2 induced anxiety/fear has not been looked at yet. In this thesis, we found that the BK channel is involved in the HCVR in mice and that the CO2 induced anxiety/fear pathway and the HCVR pathway are separate pathways in the BK and the TDAG8 mice.

Committee:

Christopher Wyatt, Ph.D. (Committee Co-Chair); Adrian Corbett, Ph.D. (Committee Co-Chair); Larry Ream, Ph.D. (Committee Member)

Subjects:

Behavioral Sciences; Biology; Biomedical Research; Neurosciences

Keywords:

Neuroscience; BK channel; TDAG8

Daubenspeck, April ArnoldProteomic Analysis of Ischemic Stroke Blood Biomarkers
Doctor of Philosophy (PhD), Wright State University, 2017, Biomedical Sciences PhD
Stroke is a global burden that claims 6 million lives and permanently disables another 5 million each year. The risk of morbidity and mortality following stroke decreases when early diagnosis and treatment are achieved. However there is no blood test to diagnose ischemic stroke because biomarkers to date have not shown adequate sensitivity and specificity to be adopted into hospital protocols. The overall objective of this dissertation was to identify novel blood protein biomarkers of ischemic stroke. We achieved this by using a number of proteomic and statistical techniques to investigate the differential abundance of proteins in the blood of stroke and non-stroke groups. We identified four high-abundance serum proteins differentially expressed between the ischemic brain of stroke patients undergoing mechanical thrombectomy and the same patient’s circulating arterial blood. Three proteins were significantly higher in the circulating blood, and a fourth protein had a large fold change in abundance in the ischemic blood of a few patients. We then identified an additional fourteen serum proteins with significant differences between the venous blood of stroke patients and that of healthy individuals. To expand these findings in a more clinically relevant patient group, protein concentrations for these eighteen individual proteins were evaluated in stroke patients and patients with stroke-mimicking symptoms. Separate analysis of each protein revealed a low diagnostic accuracy. A new analysis was therefore initiated to compare the concentrations of 1310 serum proteins in stroke patients and stroke-mimicking patients. We identified 27 additional proteins that were significantly different between stroke and stroke-mimicking patient groups, and used machine learning to determine combinations of these proteins that could constitute a stroke biomarker panel. Random Forest Analysis identified three protein panels which differentiated stroke from non-stroke groups. Thus, consistent with previous research, we did not find an individual blood protein that was likely to be clinically useful as a diagnostic biomarker. However, three protein biomarker panels successfully differentiated stroke patients from stroke-mimicking patients which may lead to the development of a validated point-of-care diagnostic test.

Committee:

James Olson, Ph.D. (Advisor); Brian Ludwig, M.D. (Committee Member); David Cool, Ph.D. (Committee Member); Yanfeng Chen, Ph.D. (Committee Member); Adrian Corbett, Ph.D. (Committee Member)

Subjects:

Biomedical Research

Keywords:

biomedical research

Hinzey, Adam HSocial Influences on Breast Cancer Pathophysiology and Allodynia Following Nerve Injury: Mechanisms and Mediators
Doctor of Philosophy, The Ohio State University, 2017, Biomedical Sciences
The social environment within which we live can have a profound influence on physiology, both in normal functioning and disease processes. The effects of both social support and isolation have been demonstrated in a wide variety of disease processes, including breast cancer and pain development following nerve injury. In both models, I sought to identify key biologic mediators of the effects of isolation on pathophysiology. In a mouse model of breast cancer, I identified a role for oxytocin as a necessary mediator of the protective effects of social housing on tumor suppressor phosphatase and tensin homolog (PTEN) gene expression within tumors, along with a shift in natural killer (NK) cell developmental stage toward a more immature phenotype in isolated mice. In a model of neuropathic pain development post- nerve injury I identified interleukin-1B (IL-1B) and interleukin-6 (IL-6) as key mediators of the effect of isolation on mechanical paw withdrawal threshold, a surrogate measure of pain. In addition, a population of macrophages within the brain is differentially activated in isolated mice following injury, providing insight into the immune effectors of changes in the social environment. The data elaborate upon the extensive identified roles of the innate immune system in modulating the effects of social milieu on the disordered physiologic processes associated with disease or injury.

Committee:

Ann Courtney DeVries, PhD (Advisor); Gustavo Leone, PhD (Committee Member); Balveen Kaur, PhD (Committee Member); Maryam Lustberg, PhD (Committee Chair)

Subjects:

Biomedical Research; Neurosciences; Psychobiology

Keywords:

social isolation; breast cancer; psychoneuroimmunology

Kohnken, RebeccaMicroRNAs in Cutaneous T-cell Lymphoma Pathogenesis
Doctor of Philosophy, The Ohio State University, 2017, Comparative and Veterinary Medicine
Cutaneous T-cell lymphoma (CTCL) is a rare form of non-Hodgkin’s lymphoma that is characterized by mature CD4+ T-cells that home to and proliferate within the skin. In the majority of patients, the disease eventually progresses to involve the blood and viscera. This is a heterogenous and complex cancer, and there are few effective therapies available for patients with advanced-stage disease. A hallmark of CTCL progression is epigenetic dysregulation, particularly involving abnormal expression of microRNAs (miRs). Novel therapeutic compounds with some promise in CTCL are epigenetic modifying drugs, such as histone deacetylase inhibitors (HDACi). However, despite initial responses in some patients, many will eventually relapse or develop clinical resistance. This failure highlights the urgent need for better understanding of the underlying pathogenesis of CTCL progression as well as identification of novel therapeutic targets. Therefore, we hypothesized that miRs play a central role in the pathogenesis of CTCL, by acting as both oncogenic and tumor suppressive factors, and that therapeutic targeting of miRs may represent an effective treatment strategy for CTCL patients. The first part of this thesis investigates an oncogenic miR, miR-181, in CTCL patient-derived cell lines as well as in primary CTCL patient cells. Previous work from our lab had shown decreased expression of sterile alpha motif (SAM) and histidine/aspartic acid (HD) domain-containing protein 1 (SAMHD1) in CTCL patients. SAMHD1 is a deoxynucleoside triphosphate hydrolase with established roles in nucleic acid metabolism, restriction of human immunodeficiency virus -1 replication, regulation of innate immune signaling, and modulation of DNA damage signaling. Recently, this gene has been implicated as a tumor suppressor gene in several types of cancer. The regulation of SAMHD1 and its potential role in the pathogenesis of CTCL was not previously known. We discovered that SAMHD1 protein levels were consistently and significantly decreased in CTCL patient-derived cell lines and primary patient cells. We were able to demonstrate regulation of SAMHD1 protein level by miR-181, a miR that was consistently upregulated in these samples. This work expanded on a novel means of regulating SAMHD1 expression in CD4+ T-cells and further implicated miR-181 dysregulation in the pathogenesis of CTCL. In the second portion of this thesis, we investigated an epigenetic miR, miR-29b, in CTCL patient-derived cell lines, primary patient cells, and a novel mouse model of CTCL. Previous work in our lab had shown suppression of miR-29b in interleukin-15 (IL-15)-driven large granular lymphocyte leukemia. We identify a novel target of miR-29b, a bromodomain and extra-terminal motif (BET) protein, bromodomain-containing protein 4 (BRD4). We elaborated on a regulatory pathway by which IL-15 signaling results in decreased miR-29b expression. This repression allows increased BRD4 chromatin binding across the genome, which results in increased oncogene expression in CTCL patient cells. Using the IL-15 transgenic mouse as a preclinical model of CTCL, we show that directly targeting BRD4 binding or indirect inhibition of BRD4 through rescue of miR-29b expression, results in decreased clinical disease severity. This work therefore identifies a novel regulatory loop in CTCL and provides insight into potential therapeutic targets to guide development of more effective treatment strategies for CTCL patients. Lastly, another oncogenic miR, miR-214, was investigated as a therapeutic target in CTCL. Previous work to describe the miR-nome of CTCL patients had identified miR-214 as upregulated in CTCL patients, a dysregulation that correlated with poor clinical outcome. We describe the epigenetic mechanism that upregulates miR-214 expression in CD4+ T-cells. Furthermore, we utilized the IL-15 transgenic mouse model of CTCL to evaluate the clinical efficacy of targeting miR-214 in vivo with an antagomiR-214. This work represents one of the first successful uses of antagomiR therapy in CTCL and provides further support to the notion of using miR-based therapies in this disease. This thesis addresses the complex epigenetic dysregulation critical to the development and progression of a rare form of lymphoma. These studies demonstrate the wide-ranging and varied function of miRs in CTCL, and provide mechanistic insight into regulation of miR expression as well as their downstream effects. Further, we provide preclinical data to support the development of miR-based therapies for CTCL patients, which may provide more effective and safer therapeutic options in the future.

Committee:

Anjali Mishra (Advisor); Michael Caligiuri (Committee Member); Amy Johnson (Committee Member); Robert Baiocchi (Committee Member); Krista La Perle (Committee Member)

Subjects:

Biomedical Research

Keywords:

Cutaneous T cell lymphoma

Waghulde, Harshal BMapping and CRISPR/Cas9 Gene Editing for Identifying Novel Genomic Factors Influencing Blood Pressure
Doctor of Philosophy (PhD), University of Toledo, 2016, Biomedical Sciences (Cardiovascular and Metabolic Diseases)
Hypertension is a complex polygenic trait and a significant risk factor for cardiovascular and metabolic diseases. Rodent models serve as tools to identify causal genes for complex traits. This dissertation is comprised of two projects. Project 1 utilizes substitution mapping as an approach to locate blood pressure quantitative trait loci (BP QTLs) on rat chromosome 5 (RNO5) and project 2 utilizes Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated proteins 9 (Cas9) genetic engineering as an approach to explore the physiological function of G-protein coupled estrogen receptor (Gper1) in a rat model of hypertension. Previously, using linkage analysis and substitution mapping, two closely-linked interactive blood pressure quantitative trait loci (QTLs), BP QTL1 and BP QTL2, have been defined within 117894038bp-131853815bp region (RGSC 3.4 version) on rat chromosome 5 (RNO5). This was done by using a series of congenic strains consisting of genomic segments of the Dahl salt-sensitive (S) rat substituted with that of the normotensive Lewis (LEW) rat. Through the construction and characterization of a panel of S.LEW bicongenic strains and corresponding S.LEW monocongenic strains, definitive evidence of epistasis (genetic interaction) between BP QTL 1 (7.77Mb) and BP QTL 2 (4.18Mb) has been documented. In order to further map these interacting QTLs, we constructed a new panel of 7 bicongenic strains and monitored their blood pressure by radiotelemetry. The data obtained from these new strains further resolved BP QTL1 from 7.77Mb to 2.93Mb. It was also evident that the QTL2 is not a single QTL, but consists of at least 3 QTLs (2.26Mb, 1.31Mb and 175kb) with contrasting effects on blood pressure. In the second project, we utilized CRISPR/Cas9 genetic engineering approach to study the physiological role of G-protein coupled estrogen receptor (Gper1) in the Dahl-salt sensitive (S) rat. A link between gut microbiota and blood pressure (BP) regulation was previously demonstrated in our laboratory. Gut microbiotal transplantation from Dahl-salt resistant (R) rats into genetically hypertensive Dahl-salt sensitive (S) rats caused an elevation in BP, which was associated with an increase in plasma acetate. Acetate is a short chain fatty acid, which is a known ligand for two of the G-protein coupled receptors, Gpr41 and Olfr78. Deletion of either Gpr41 or Olfr78 is reported to affect BP. Because S and R rats do not have allelic variations of Gpr41 and Olfr78, the observed increased plasma acetate being associated with elevated blood pressure cannot be attributed to these two receptors alone. This led us to hypothesize that yet unknown receptors of acetate exist on the rat genome to regulate BP. To test this hypothesis, we focused on a more recently discovered G-protein coupled estrogen receptor (Gper1) which belongs to the same class of orphan receptors as Gpr41. To completely disrupt Gper1 in S rats, we employed clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) approach with two gRNAs each targeting one end of the rat Gper1 gene. The resultant Gper1-/- rats had significantly lower BP and increased vasorelaxation to acetylcholine compared to wild type S rats. Further, to examine whether the presence or absence of Gper1 influence vascular response to short chain fatty acids (acetate, propionate and butyrate), wire myograph studies were conducted using small mesenteric arteries (SMAs). While a rapid contraction effect of acetate and butyrate in phenylephrine pre-contracted arteries were similar, the sustained relaxation following rapid contraction was significantly decreased in vessels from Gper1-/- rats. Because gut microbiota is the source of short chain fatty acids, we conducted microbiotal transplantation studies, data from which demonstrated that the observed BP lowering effect of Gper1-/- was abolished. Collectively, the results point to Gper1 as a novel short chain fatty acid receptor.

Committee:

Bina Joe, PhD (Committee Chair); Guillermo Vazquez, PhD (Committee Member); Kathryn Eisenmann, PhD (Committee Member); Jennifer Hill, PhD (Committee Member); Jiang Tian, PhD (Committee Member)

Subjects:

Biomedical Research; Genetics; Health Sciences; Physiology

Keywords:

Blood pressure; quantitative trait loci; rat chromosome 5; epistasis; congenic; G-protein coupled estrogen receptor; CRISPR; gut microbiota; acetate; butyrate

Wu, XiaojunIdentification of two novel in vivo-upregulated Francisella tularensis proteins involved in metal acquisition and virulence
Doctor of Philosophy (PhD), University of Toledo, 2016, Biomedical Sciences (Infection, Immunity, and Transplantation)
Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs) are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal binding and uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. We have identified two new proteins, FTT0267 (named fmvA, for Francisella metal and virulence) and FTT0602c (fmvB), which are homologs of those iron acquisition genes and demonstrated that both are upregulated during mouse infections. Based on sequence homology and in vivo upregulation, we hypothesized that FmvA and FmvB are OMPs involved in metal acquisition and virulence. Despite sequence similarity to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither fmvA nor fmvB mutants exhibited growth defects in iron limitation. However, among other metals examined in this study, magnesium limitation significantly induced fmvB expression, fmvB mutant was found to express significantly higher levels of lipopolysaccharide (LPS) in magnesium-limiting medium, and increased numbers of surface protrusions were observed on fmvB mutant in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of fmvB mutant revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in fmvB mutant, compared with wild-type F. tularensis. To provide further evidence for the potential role of FmvB in magnesium uptake, we demonstrated that FmvB was outer membrane-localized. Finally, both fmvA and fmvB mutants were found to be significantly attenuated in mice and cytokine analyses revealed that fmvB mutant-infected mice produced lower levels of pro-inflammatory cytokines, including GM-CSF, IL-3, and IL-10, compared with mice infected with wild-type F. tularensis. Taken together, these studies have characterized two previously-unstudied F. tularensis proteins, have shown that both play roles in F. tularensis virulence, and provide new insights into the importance of magnesium for intracellular pathogens.

Committee:

Jason Huntley (Committee Chair); Robert Blumenthal (Committee Member); William Maltese (Committee Member); Kevin Pan (Committee Member); R.Mark Wooten (Committee Member)

Subjects:

Biology; Biomedical Research; Health Sciences; Immunology; Microbiology

Keywords:

Francisella tularensis; outer membrane proteins; virulence; metal acquisition; hypothetical proteins

Paliobeis, Andrew SEffects of Pramlintide on Mitochondrial Dynamics and Health in the Alzheimer's Disease APP/PS1 Mouse Model
BS, Kent State University, 2017, College of Arts and Sciences / Department of Chemistry
Alzheimer’s disease (AD) is typically characterized by its hallmark pathological features including amyloid plaques and neurofibrillary tangles. However, several lines of evidence suggest that a more fundamental insult, including oxidative stress and mitochondrial dysfunction, is responsible for the disease. Oxidative stress has been shown to be an early predictor and key pathological feature of neurodegenerative diseases including AD. Previous work in our laboratory has demonstrated that administration of pramlintide, the rodent-derived analog of the hormone amylin, can reduce levels of oxidative stress in a transgenic mouse model of AD (APP/PS1) as well as Neuroscreen-1 cultures (neuronal cell model). Although it is apparent that pramlintide has antioxidant activity, its mechanism of action remains unclear. Because mitochondria are the main producers of reactive oxygen species (ROS) that account for oxidative stress in a cell, they have been a target of our investigation, specifically whether pramlintide treatment regulates mitochondrial protein changes associated with AD and present in the APP/PS1 mice. Our data show that pramlintide treatment improves mitochondrial health by regulating mitochondrial dynamics-associated proteins. By regulating dynamics-associated proteins, pramlintide can potentially restore a proper fission/fusion equilibrium of these organelles and provide membrane stability. We also demonstrate that pramlintide protects neuroblastoma cells against mitochondrial membrane potential loss in an oxidative stress event. Taken together, our data suggest that some of the effects of pramlintide on oxidative stress regulation may stem from improving mitochondrial health and function.

Committee:

Gemma Casadesus, PhD (Advisor); Ernest Freeman, PhD (Committee Member); Alexander Seed, PhD (Committee Member); Rodger Gregory, PhD (Committee Member)

Subjects:

Biochemistry; Biology; Biomedical Research

Keywords:

Alzheimers disease; Mitochondrial dynamics; Oxidative stress; APP-PS1 mouse model

McBeth, Lucien ReiterGlucocorticoid Receptor beta Increases the Migration of Human Urothelial Carcinoma Cells
Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2016, Biomedical Sciences (Cardiovascular and Metabolic Diseases)
Urothelial carcinoma is one of the most prevalent cancers encountered in the country, and recent investigations demonstrate the important role of glucocorticoid signaling in the disease. We have shown that an alternate isoform of the glucocorticoid receptor (GR), GRß, causes migration of human urothelial carcinoma cells. We begin with a literature review of the role of GRß and another nuclear receptor, the androgen receptor, in urothelial carcinoma. Next, we investigate the role of GRß in the migration of human urothelial carcinoma cells in two transitional human uroepithelial carcinoma cells, UMUC-3 and T24. We found that the T24 cells have higher GRß expression compared to the UMUC-3, and that both cell lines had a similar GRa level. Interestingly, the higher GRß expression was correlated with enhanced migration rates, which was reduced with GRß inhibition. In-silco analysis of the 3’ untranslated region (3’UTR) of human GRß revealed a potential micro-RNA (miRNA) binding site for miR33a, miR144, and miR181. Therefore, we cloned the 3’UTR of human GRß and mutated the miRNA binding sites, which showed that miR144 positively regulates GRß expression. In addition, miR144 and GRß expression were increased during migration of uroepithelial carcinoma cells. Therefore, we constructed a peptide nucleic acid (PNA) conjugated to a cell penetrating-peptide (CPP) that we termed Sweet-P to inhibit the miR144 binding site in the 3’UTR of human GRß. Furthermore, Sweet-P decreased GRß expression and, as a result, inhibited migration of uroepithelial carcinoma cells, demonstrating its potential as a therapeutic. We then complete the thesis with a discussion of the potential for Sweet-P in cancer therapy and other GRß-related diseases, which may serve as the first anti-GRß drug.

Committee:

Terry Hinds, Jr (Committee Chair); Edwin Sanchez (Committee Member); Beata Lecka-Czernik (Committee Member); Nitin Puri (Committee Member)

Subjects:

Biomedical Research

Keywords:

Glucocorticoid receptor; GR; GR alpha; GR beta; glucocorticoids; androgens; androgen receptor; AR; cancer; bladder cancer; males; growth; inflammation; microRNA; miRNA; migration; asthma; Sweet-P

Nyaboke, RoselineThe Role of N2A and N2B Titin Isoforms in Muscle Cell Development
Master of Science in Biological Sciences, Youngstown State University, 2016, Department of Biological Sciences
The process of myogenesis is essential in life since it is initiated immediately after conception and continues throughout life time. It enables the formation of muscle tissues during embryonic development, and there are three types of muscle tissues that are formed during this process. The three types of muscles are skeletal, cardiac and smooth muscles. Skeletal muscle is the most abundant and is important in the movement of the body, and is formed by the muscle precursor’s cells called the myoblasts. Muscle cells consist of sarcomeres that consists of giant protein called titin that are embedded in the sarcomere. Titin is encoded by a single gene TTN that has 363 exons that undergoes alternative splicing to produce the two isoforms; N2A which is located in skeletal muscle and N2B isoform is located in the cardiac muscle. The process of muscle cell development are regulated by growth factors that results to a maturation of a myofiber. In vivo culturing of muscle cells, the cells are fed with medium that is supplemented with growth factors and is controlled during different stages of myogenesis. We conducted this experiment in different time points using the C2C12 cell line from a mouse myoblast, with our main focus on how these two genes, N2A and N2B are expressed throughout the process of myogenesis. The cells were fed with high growth serum during the first phase of myogenesis and during differentiation, the medium was supplemented with low growth serum. RNA was collected in 6 different time points. Analysis of gene study was made using RT-qPCR, and N2A and N2B genes were normalized against actin gene to bring out which gene is expressed higher than the other. The genes were expressed highly in different time points and this did not agree with our hypothesis and we therefore concluded that low serum medium does not trigger myogenesis.

Committee:

Gary Walker, PhD (Advisor); Jonathan Caguiat, PhD (Committee Member); David Asch, PhD (Committee Member)

Subjects:

Biology; Biomedical Research; Genetics; Molecular Biology

Keywords:

N2A; N2B; titin; isoforms; muscle; myogenesis

Agans, Richard ThomasModeling Effects of Diet on Human Gut Microbiota
Doctor of Philosophy (PhD), Wright State University, 2016, Biomedical Sciences PhD
The human gut microbiota is integrally involved in the metabolism of nutrients contained within the human diet. Studies into human nutrition have primarily been carried out using human and animal models. These studies are extremely important in our understanding of human nutrition, however, suffer from inherent limitations including unique microbial compositions between individuals, compliance in human studies, inability to carry out mechanistic studies, and inability to interrogate proximal regions of the gut without applying invasive techniques. In vitro gut simulator systems circumvent many of these limitations in animal and human models by allowing control of gut environmental conditions, decreasing variability observed between subjects, and enabling mechanistic investigations and interrogations of inaccessible regions of the gut. In this work a custom biofermentation system, the human gut simulator, was designed, validated, utilizing previously reported gut conditions, capable of temperature, pH, and atmosphere regulation, nutrient transit, and it allows real-time sampling of vessel contents or addition of exogenous agents. The human gut simulator was further employed to the study of gut microbiota response to dietary long chain fatty acids as a sole nutrient source, following stabilization on a rich `western’ medium. Microbiota showed rapid responses to the transition from western to fat medium; where a lack of carbohydrates and proteins resulted in decreased community density. Specific members of the microbiota were capable of utilizing long chain fatty acids, including Bilophila, Alistipes, and Escherichia/Shigella. Interestingly, members of the microbiota incapable of metabolizing long chain fatty acids included beneficial microbes Roseburia, Bifidobacterium, and Akkermansia. Ordination and principal response curves analyses highlighted a significant effect of medium change on shifts in microbial composition over time. In conjunction with in vitro studies, human volunteers were enrolled to assess responses of microbiota to diets high in proteins, carbohydrates, or fats. Microarray analysis revealed specific individual host responses to test diets with smaller community wide effects. Increasing the amount of protein in the diet had a positive impact on relative abundance of Akkermansia, Alistipes, Enterococcus, and Lactococcus, while higher carbohydrates and fats resulted in higher abundances of Bifidobacterium, and Alistipes and Escherichia/Shigella, respectively. Together these results indicate that the Human Gut Simulator allows for robust studies of the human gut microbiota, and offers a foundation for conducting nutritional interventions in human subjects.

Committee:

Oleg Paliy, Ph.D. (Advisor); Volker Bahn, Ph.D. (Committee Member); Nancy Bigley, Ph.D. (Committee Member); Mill Miller, Ph.D. (Committee Member); Lawrence Prochaska, Ph.D. (Committee Member)

Subjects:

Biomedical Research; Ecology; Microbiology; Nutrition

Keywords:

Microbiota, in vitro gut simulator, bioreactor, nutrition, fatty acid

Mao, LiangyanAssessment of Bone Regeneration in a Rat Femur Defect Model Following Recombinant Human Bone Morphogenetic Protein 2 Delivery from Keratin Hydrogels with Tunable Rates of Degradation: Micro-CT Analysis and Histology
Master of Science, Miami University, 2016, Chemical, Paper & Biomedical Engineering
Bone injury is remains a worldwide clinical challenge from which 6 millions of adults in United States suffer every year. A well-known growth factor that can promote healing in bone defects is bone morphogenetic protein 2 (BMP-2). This molecule stimulates the activity of osteoblasts and other cells to promote bone formation. The release of recombinant human bone morphogenetic protein 2 (rhBMP-2) to the local tissue should be controlled by delivery from an osteoconductive biomaterial. Collagen is used as the biomaterial in current clinically-approved products, but this material degrades rapidly and leads to various side-effects such as ectopic growth. We propose to continue our investigation of an alterative carrier system: keratin biomaterials. We have previously established that levels of disulfide cross-linking can be controlled by formulating mixtures of oxidatively extracted keratin (keratose) and reductively extracted keratin (kerateine). Previous studies by our group indicate that the degradation rate of the keratin materials as well as release of rhBMP-2 from the materials in vivo depends on the level of disulfide cross-linking. In this study, we evaluate the effect of modulating the rate of rhBMP-2 delivery from keratin biomaterials on bone regeneration in a critically-sized rat femur defect model. In this research, micro computed tomography (µ-CT) analysis of the defect site for volume of regeneration and measures of bone mineral density were used in conjunction with and histology as outcome measures to assess bone healing. The results of this research should specifically provide a better understanding of the effects of modulating keratin biomaterial degradation rates on bone regeneration.

Committee:

Justin Saul (Advisor)

Subjects:

Biomedical Research

Hottenstein, Kristi NA Qualitative Case Study on Human Subject Research Public Policy Implementation at One Council on Undergraduate Research Institution.
Doctor of Philosophy, University of Toledo, 2016, Higher Education
Regulations for research involving human subjects in higher education have long been a critical issue. Federal public policy for research involving human subjects impacts institutions of higher education by requiring all federally funded research to be passed by an IRB. Undergraduate research is no exception. Given the literature on the benefits of undergraduate research to students, faculty, and institutions, how human subject research public policy is being implemented at the undergraduate level was a significant gap in the literature. This qualitative single case study examined the human subject research policies and practices of a selective, Mid-western, Council on Undergraduate Research institution. The purpose of the study was to determine how this institution implemented human subject research public policy to benefit its students. This institution used a hybrid approach of public policy implementation that met federal requirements while capitalizing on the role local actors can play in the implementation process. This model resulted in a student friendly implementation emphasizing various learning outcomes and student mentoring. Although there is considerable research and public discussion on the negative aspects of IRBs, if approached in a manner that embraces student learning, the IRB experience can be an extremely beneficial aspect of the institution’s learning environment.

Committee:

David Meabon (Committee Chair)

Subjects:

Biomedical Research; Education; Education Policy; Educational Leadership; Educational Theory; Higher Education Administration; Operations Research; Organization Theory; Social Research

Keywords:

IRB; institutional review board; CUR; council on undergraduate research; undergraduate research; UR; public policy; implementation; human subject research; implementation theory; hybrid theories; student mentoring; benefits of undergraduate research

Clever, David CT Cell-Intrinsic PHD Proteins Regulate Pulmonary Immunity
Doctor of Philosophy, The Ohio State University, 2016, Biomedical Sciences
Local immunity is an important feature of metastatic sites. Circulating tumor cells must evade secondary site immune responses for successful metastasis. The lung is a common metastatic site for numerous cancer types including malignant melanoma. While the diffuse pulmonary vascular architecture contributes to metastatic seeding, we hypothesized that organ-specific immunoregulatory mechanisms establish the lung as an immunologically permissive niche for tumor colonization. T lymphocytes play a critical role in coordinating organ-specific immune responses. Pulmonary T cell responses are restrained despite continuous exposure to innocuous foreign antigens. Excessive T cell effector activity within the pulmonary environment can result in adverse immune-mediated pathology. Thus, T cells must possess an intrinsic mechanism to sense their entry into the lungs and subsequently suppress responses against harmless self and foreign antigens. Consequently, however, such mechanisms might also repress T cell responses against infiltrating metastatic tumor cells. In the lung parenchyma T cells are exposed to localized concentrations of molecular oxygen (O2) as much as 2 to 3-fold higher than other lymphoid and non-lymphoid tissues. The prolyl-hydroxylase (PHD) family of proteins forms the cellular oxygen sensing machinery. We hypothesized that oxygen sensing by T cell-intrinsic PHD proteins coordinates an immunosuppressive program in the lung. Utilizing a mouse model with a T cell-specific deletion of all three PHD proteins (PHD-tKO), we found that T cell-intrinsic oxygen sensing is required to prevent mild autoimmune inflammation of the lungs. PHD proteins enable environmental oxygen to limit pulmonary type helper (Th)-1 responses, promote induction of CD4+-regulatory T (Treg) cells, and restrain CD8+ T cell effector differentiation and function in the steady state and following exposure to innocuous environmental antigens. Consequently, T cell-intrinsic PHD proteins establish the lung as an immunologically favorable metastatic niche and powerfully license colonization by circulating tumor cells. Tumor infiltration is accompanied by PHD protein-dependent induction of pulmonary Treg cells and suppression of IFN-γ-dependent tumor clearance. Strikingly, T cell-intrinsic deletion or pharmacological inhibition of PHD proteins limits tumor colonization of the lung. Thus, the PHD proteins represent a novel therapeutic target to enhance anti-tumor T cell-mediated immunity. Adoptive cell transfer immunotherapy (ACT) is an emerging therapeutic strategy that harnesses the power of tumor specific T cells to mediate extensive tumor regression. Pharmacologic inhibition of PHD proteins using the small molecule DMOG promotes the effector capacity of tumor-specific CD4+ and CD8+ T cells. Importantly, following transfer into tumor bearing hosts, DMOG treated tumor-specific T cells mediated superior tumor regression at multiple sites of disease compared to control treated cells. Collectively, our results provide the first demonstration of an oxygen-dependent immunoregulatory program in the lung. We identify a non-redundant role for the oxygen-sensing PHD proteins in T cell biology. We also identify an immunological basis for preferential hematogenous metastasis of cancer cells to the lung and importantly elucidate a novel targetable pathway to enhance the efficacy of immune-based therapy by modulating the activity of the PHD proteins.

Committee:

Schlesinger Larry, MD (Advisor); Restifo Nicholas , MD (Committee Member); Caligiuri Michael , MD (Committee Chair); Byrd John, MD (Committee Member); Benson Don, MD (Committee Member)

Subjects:

Biomedical Research; Immunology

Keywords:

Tumor Immunology, Cancer Immunology, Immunotherapy, T cell differentiation, Pulmonary immunity, Mucosal immunology, Autoimmunity

Paul, Anup KAssessment of the Severity of Aortic Stenosis using Aortic Valve Coefficient
PhD, University of Cincinnati, 2016, Engineering and Applied Science: Mechanical Engineering
Introduction. Accurate assessment of the severity of stenosis is critical in patients with aortic stenosis (AS). The ambiguities and imprecisions of the current diagnostic parameters can result in sub-optimal clinical decisions. In this research, we investigated the feasibility of using the functional diagnostic parameter AVC (Aortic Valve coefficient: ratio of the total transvalvular pressure drop to the proximal dynamic pressure) in the non-invasive assessment of AS and also for improving the concordance between non-invasive and invasive assessment of AS severity. Methods. AVC was calculated using Doppler (non-invasive) and cardiac catheterization (invasive) measured parameters obtained from retrospective chart reviews. Linear regression analysis was performed to assess any significant correlations between AVC and the measured parameters and also between the Doppler and catheterization derived parameters. To accurately evaluate the hemodynamics for diseased aortic valves using patient-specific computational formulations it is necessary to determine the pre-stressed condition of the in-vivo geometry. The previously developed optimization based inverse algorithm was improved to evaluate the pre-stress due to the change in arterial property of the tapered femoral artery. The compliance of the artery for a range of systemic pressures was also computed. Subsequently, a hybrid inverse algorithm was developed to determine the load-free and pre-stressed condition of patient-specific arterial geometries obtained from clinical MRI. The algorithm included the in-vivo axial stretch, lumen pressure and the patient-specific tissue properties determined from clinical data. <I>Results. A statistically significant and strong combined linear correlation (r = 0.93, p &lt 0.001) of AVC with the transvalvular pressure drop and the left ventricular outflow tract velocity was observed. The mean values of AVC were shown to better delineate moderate and severe stenosis (54% difference). An improved significant correlation was observed between Doppler and catheter derived AVC (r = 0.92, p &lt 0.05) when compared to the correlation between Doppler and catheter measurements of mean pressure drop (r = 0.72, p 0.05) and aortic valve area (r = 0.64, p &lt 0.05). The results obtained from the optimization based inverse algorithm showed that the change in arterial wall property caused significant variation in the dimensions of the load-free artery and insignificant variation in the dimensions and the circumferential stress of the pre-stressed artery. Further, the computed compliance of the artery was significantly influenced by the change in the average arterial pressure. The results obtained from the hybrid inverse algorithm showed that the radial shrinkage and thickening of the load-free patient-specific arterial wall was non-uniform. The load-free inner and outer diameters of the patient-specific artery were 33-38% and 22-25% smaller, respectively, than the corresponding in-vivo</I> diameters. The variation of the pre-stressed diameters from the in-vivo geometry was less than 5.1%. <I>Conclusions. This research has confirmed the feasibility of using both pressure drop and flow in a single combined non-dimensional non-invasive diagnostic index, AVC, for the assessment of AS severity. The long-term goal of this research is to evaluate the specificity and sensitivity of AVC for the diagnosis of AS using patient-specific computational formulations and prospective studies under clinical setting.

Committee:

Rupak Banerjee, Ph.D P.E. (Committee Chair); Mohamed Effat, M.D. (Committee Member); Frank Gerner, Ph.D. (Committee Member); Kumar Vemaganti, Ph.D. (Committee Member)

Subjects:

Biomedical Research

Keywords:

Aortic stenosis;aortic valve disease;inverse algorthm;pre-stressing;Doppler assessment of aortic stenosis;finite element modeling

Pozsgai, Eric RAdeno-Associated Virus Mediated ß-Sarcoglycan Gene Replacement Therapy for the Treatment of Limb Girdle Muscular Dystrophy Type 2E
Doctor of Philosophy, The Ohio State University, 2016, Biomedical Sciences
One major class of muscular dystrophy is limb-girdle muscular dystrophy (LGMD), encompassing many different subtypes. LGMD2E is one of the most severe forms of LGMD, resulting from recessive mutations in the ß-sarcoglycan (SGCB) gene, causing loss of functional protein. SGCB is a structural protein component of the dystrophin-associated protein complex (DAPC), which as a whole provides structural and mechanical stability to the sarcolemma. Due to the loss of protein, a devastating disorder ensues with widespread progressive muscle wasting, leading to loss of function. Disease progression is directly correlated with age, where the most severe cases have symptom onset in early childhood and many patients are rendered non-ambulant by their teens. In addition to skeletal muscle weakness, significant respiratory failure and fatal cardiomyopathy are common features in more severe LGMD2E patients. To date, no effective therapy exists to treat this debilitating disease. Thus, with an urgent need for a viable treatment option for LGMD2E patients, this work attempted to develop the first viral-mediated approach to restore wild-type (WT) SGCB. Providing a relevant model to test therapeutic efficacy, the Sgcb-null (sgcb-/-) mouse recapitulates the clinical phenotype and shares many of the pathologic features of LGMD2E patient biopsies including myofiber necrosis, central nucleation, inflammation, and fibrosis. We have engineered a viral mediated gene replacement therapy using adeno-associated virus (AAV) carrying a codon optimized human SGCB gene (hSGCB) driven by one of two different muscle specific promoters. We first established proof-of-principle efficacy of scAAVrh.74.tMCK.hSGCB by intramuscular (IM) injection and isolated-limb perfusion (ILP) in young and aged mice, which resulted in long-term widespread transgene expression accompanied by histological and function benefits. Notably, we saw a considerable reduction in fibrotic tissue, a significant component of the disease and a potential major obstacle of gene transfer. After switching promoters to the MHCK7 promoter which provides robust expression in cardiac tissue, we then demonstrated efficacy of systemic delivery of scAAVrh.74.MHCK7.hSGCB in treating skeletal and cardiac muscle deficits in sgcb-/- mice to provide a potential rationale for meaningful results in a clinical trial. This led to nearly 100% transgene expression in numerous muscles throughout the limbs, torso, and the heart, which was again accompanied by improvements in muscle histopathology and function, as well as increased overall activity. Importantly, a formal GLP toxicology study of AAV.hSGCB gene transfer in WT mice showed no adverse effects. In this well-defined model of LGMD2E, we have established a clinically relevant path for AAV mediated SGCB gene replacement therapy that has great promise for LGMD2E patients. After demonstrating the importance of functional deficits in dystrophic muscle to serve as outcome measures for functional recovery from SGCB gene therapy, we applied these concepts to the investigations of several other forms of LGMD, Types 2B and 2L, involving dysferlin (DYSF) and anoctomin5 (ANO5), respectively. In these studies, we optimized and validated muscle fiber isolation with a laser induced membrane repair assay to aid in thoroughly characterizing these diseases and test therapeutic interventions like viral-mediated gene transfer. Using this technique, we are able to directly measure a functional parameter of both DYSF and ANO5 and quantify the membrane repair ability in different physiological settings. Taken together, we report here an experimental method that can be a powerful tool for pre-clinical studies of muscular dystrophy. We also emphasize the importance of the pre-clinical efficacy study with SGCB gene replacement therapy and the impact it has in the translational pathway in providing a clinically meaningful therapy for LGMD2E patients.

Committee:

Louise Rodino-Klapac (Advisor); Jerry Mendell (Committee Member); Scott Harper (Committee Member); Paul Janssen (Committee Member)

Subjects:

Biomedical Research

Keywords:

Gene Therapy, Muscular Dystrophy, Beta-Sarcoglycan, AAV

Johnson, Travis SteeleEstimation of Neural Cell types in the Allen Human Brain Atlas using Murine-derived Expression Profiles
Master of Science, The Ohio State University, 2016, Public Health
Spatial patterns of gene expression in brain tissue have been observed in both human and more thoroughly in mouse. Because of the increased complexity and data availability, these spatial gene expression patterns in human have not been biologically explained in the same way that they have in murine models. In mice they can be explained by the spatial distribution of cell types. Using this insight, we address the human biology behind these spatial transcription changes by informing the human model with a-priori mouse transcriptional profiles. We apply clustered single cell RNA sequencing (scRNA-Seq) derived expression profiles across transcriptomics platforms to show: i) ordinary least squares regression can accurately predict the cell types within a tissue sample (validated through biomarkers and training/test data), ii) the distribution of cell types in the human brain vary between different locations in the brain, and iii) the distribution of human and mouse neural densities correlate between human and mouse. We developed a methodology to deconvolute cell types within heterogeneous tissue samples that can be applied to disparate data and achieve meaningful results. We then map these results to the large Allen Brain microarray database to evaluate changes in tissue between brain location.

Committee:

Kun Huang, PhD (Advisor); Po-Yin Yen, PhD (Advisor); Raghu Machiraju, PhD (Committee Member); Albert Lai, PhD (Committee Member)

Subjects:

Biomedical Research

Keywords:

Deconvolution, Cross-species, RNA-Seq, Microarray, Expression profiles, Brain structure, Neuron, Mouse brain, Human brain, Neural cell types, Linear modeling

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