Master of Science, University of Akron, 2024, Biology

LeptinA-F0 knockout Danio rerio were generated using Crispr/Cas9 to test the effects of leptin expression on bone development and growth into adulthood. Within one clutch, 3 mutants were generated for a 25% successful-mutation rate. Using calcein staining and fluorescence microscopy, calcium deposition in vertebrae #3-5 was measured at days 12 and 17 post-fertilization (dpf). Results showed faster calcification rates in leptinA mutants compared to wildtype. Mutants exhibited decreased weight and shorter longitudinal length at onset of adulthood. MicroCT analysis revealed smaller vertebral column volumes per body length in mutants, with individual pre-caudal vertebrae showing no significant difference in volume between mutants and wildtype. Mutant bone density was lower than wildtype. Otoliths showed no significant differences in volume nor density. These findings suggest leptin influences bone remodeling early in vertebral body formation, with effects varying among bones.

Committee: Richard Londraville (Advisor); Qin Liu (Committee Member); Rachel Olson (Committee Member) Subjects: Biology

Doctor of Philosophy (PhD), University of Toledo, 2022, Experimental Therapeutics

Hemorrhagic stroke is the second most frequent cause of stroke after ischemic stroke. It accounts for 10-15% of all strokes and has the highest mortality rates. About 50% of patients die within the first month of the attack, and survivors suffer from long-term disabilities and post-hemorrhagic stroke cognitive impairment (PSCI). There is no specific therapy for intracerebral hemorrhage (ICH), and the management remains only supportive and surgical treatment. To develop new therapeutic alternatives, it is necessary to understand molecular mechanisms involving secondary brain damage, particularly microglial activation, neuroinflammation, and PSCI. Microglia are the primary immune cells in the brain and play a key role in responding to injury. Cofilin-1, encoded by the CFL1 gene, is a non-muscle isoform that regulates actin dynamics and plays a critical role in actin filament turnover. Previous studies suggested that cofilin-1 is overexpressed during stressful conditions and forms cofilin-actin rods/aggregates that initiate apoptosis and are involved in neurodegenerative diseases. Recent studies from our lab showed that knockdown of cofilin-1 in mice using siRNA improved neurobehavioral parameters and decreased hematoma volume. This dissertation investigates the role of cofilin-1 signaling in microglial activation, neuroinflammation, and PSCI following ICH using human autopsy brain sections, animal models of collagenase-induced ICH in mice, and a novel microglial cofilin-1 specific gene deleted mouse model (cofilin-1 mutant mice). We observed widespread cofilin-1 expression in microglia in the human autopsy brain sections with hemorrhagic stroke. The novel findings support the scientific premise that cofilin-1 plays a critical role in secondary injury after ICH. We subjected different cohorts of mice to intrastriatal collagenase injection-induced ICH and sacrificed them at various time points to study cofilin-1 signaling for up to 28 days. The results showed that cofil (open full item for complete abstract)

Committee: Dr. Zahoor A. Shah (Committee Chair); Dr. F. Scott Hall (Committee Member); Dr. William S. Messer (Committee Member); Dr. Hermann V. Grafenstein (Committee Member); Dr. Caren Steinmiller (Other) Subjects: Aging; Behavioral Sciences; Molecular Biology; Neurobiology; Neurosciences; Therapy

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

Multiple sclerosis (MS) is a chronic inflammatory disease that affects the brain and spinal cord leading to demyelination. Symptoms in patients can present in a broad range. Mild symptoms may manifest as peripheral tingling or numbness, while severe symptoms may present as severe motor disabilities or sudden blindness. A number of disease modifying therapies are available to help improve quality of life, but there is presently no cure available. Per the current numbers, there are nearly 1 million adults in the US who suffer from MS. Proinflammatory T helper (Th) cells that infiltrate the central nervous system (CNS) are thought to be the initial mediators of disease. Findings from the mouse model of MS, experimental autoimmune encephalomyelitis (EAE) lead to the understanding that MS is an autoimmune disease where T cells are reactive to myelin antigens. Controlling the biology of these T cell responses could bring therapeutic benefits to patients. Previous work from our lab has helped establish the role of the type II arginine methyltransferase, PRMT5 (protein arginine methyltransferase 5) in Th cell expansion and EAE autoimmunity using selective inhibitors. However, the role and mechanism by which PRMT5 modulates T cell responses and EAE progression haven't been determined. During my time in the lab, we developed two conditional knockout mouse models to evaluate PRMT5's function in T cells. In my thesis work I used our mouse models, exploratory RNA sequencing (RNA-Seq) and mass spectrometry techniques to uncover the mechanisms of action that drive T cell differentiation, expansion and the PRMT5 mediated symmetric dimethylation (SDM) targets of T cells. We identified a link between PRMT5 and cholesterol metabolism in Th17 cell development. We found that cholesterol pathway intermediates act as agonists for ROR-γt, the signature transcription factor encoding Th17 cells. PRMT5 expression was necessary for proper activity of the enzymes driving cholester (open full item for complete abstract)

Committee: Mireia Guerau-de-Arellano (Advisor); Stephen Kolb (Committee Member); Federica Accornero (Committee Member); Robert Baiocchi (Committee Member) Subjects: Biomedical Research; Immunology

Master of Science (MS), Ohio University, 2021, Food and Nutrition Sciences (Health Sciences and Professions)

Growth hormone receptor knockout mice (GHRKO) have a complete ablation of growth hormone's (GH) action. These mice are small and obese but paradoxically have increased insulin sensitivity and increased lifespan when compared to wild type (WT) controls. This phenotype is believed to be due to the lack of GH's insulin desensitizing or diabetogenic effects as well as a resistance to cancer and tumor formation in these mice. Another method of extending lifespan in mice is via the use of the compound rapamycin. Rapamycin is noted for its ability to target the mechanistic/mammalian target of rapamycin (mTOR) pathway, a central regulator of cell metabolism, growth, and survival. Surprisingly, treatment with rapamycin also correlates with decreased glucose tolerance and worsened insulin sensitivity. Rapamycin treatment is one of the few observed phenomena where insulin sensitivity is decreased, but lifespan is increased. GHRKO mice are similarly glucose intolerant but have increased insulin sensitivity and decreased insulin secretion. My project is part of a larger study that will determine if rapamycin feeding reverses or amplifies the positive health effects seen in GHRKO mice as well as alter longevity. The specific goal of my thesis was to utilize a cohort of GHRKO mice and examine the short-term effect of rapamycin treatment on the GH/IGF-1 axis (IGF-1) and on glucose homeostasis. This was assessed by taking various blood glucose measurements (e.g. fasting glucose, glucose tolerance, insulin tolerance) or by evaluating serum levels of various proteins associated with glucose homeostasis (e.g. insulin, C-peptide, adiponectin, high molecular weight (HMW) adiponectin, resistin, TNF-α, MCP-1). We found no changes in glucose or insulin tolerance in response to rapamycin feeding. In addition, IGF-1 levels were not significantly different due to rapamycin feeding although both groups of GHRKO mice had undetectable levels. Activity of mTORC1 complex was assessed via western bl (open full item for complete abstract)

Committee: Darlene Berryman PhD (Committee Member); Edward List PhD (Committee Member); Robert Brannan PhD (Committee Chair) Subjects: Biology; Molecular Biology; Nutrition

Master of Sciences, Case Western Reserve University, 2021, Biology

ACF7 (actin crosslinking family protein-7) is a large actin-bundling and microtubule/actin crosslinking protein that is primarily responsible for cytoskeletal organization and integrity. Hair cells contain specialized subcellular structures to enable hearing, including the actin-rich cuticular plate and the circumferential band. ACF7 localizes to the cuticular plate and circumferential band of both mice and zebrafish. Due to known roles of ACF7 in the cytoskeleton of cell types throughout metazoans, it is plausible that ACF7 plays a vital role in the subcellular architecture of hair cell. To determine the function of ACF7 in hair cells, we used a Pax2-Cre/loxP system that removes three exons of the Macf1 actin binding domain in all descending cells of Pax2-expressing otic progenitors. Surprisingly, our data in four-week old conditional knockout mice suggests that hair cells have normal survivability, morphology, polarity, and hearing capabilities, demonstrating that the loss of ACF7 does not impact hearing in adult mice.

Committee: Brian McDermott Jr. (Advisor); Martín Basch (Committee Member); Ruben Stepanyan (Committee Member); Yolanda Fortenberry (Committee Chair) Subjects: Audiology; Biology; Biomedical Research; Genetics; Molecular Biology; Physiology

Master of Science (M.S.), University of Dayton, 2020, Biology

Calcium (Ca2+) ions comprise critical second messengers for a wide variety of cellular processes, including gene expression, cell proliferation and death, and metabolism; neurons are no exception to this. Intraneuronal Ca2+ handling regulates processes such as long-term potentiation (LTP), synaptic transmission, and generation of firing patterns. Subsequently, disruptions of neuronal Ca2+ has been implicated in several neuropsychiatric and neurodegenerative disorders. A key component of the neuronal Ca2+ handling toolkit is the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump, specifically the SERCA2 isoform. We have recently identified a novel SERCA regulator to be expressed in the central nervous system. In the context of the current thesis, we assessed how loss of this gene may affect the neurochemical status and the cortical oscillatory activity upon genetic ablation of this gene in mice. Specifically, in the first part of the study an ex vivo neurochemical screening in distinct brain regions was conducted using high performance liquid chromatography (HPLC) with coulometric detection focusing on monoaminergic (i.e., noradrenaline, serotonin, dopamine) and aminoacidergic neurotransmission (i.e., glutamate, aspartate, γ-aminobutyric acid); in the second part of the study an electroencephalogram (EEG)-based power spectral analysis was conducted in order to assess how loss of this molecular player affects cortical oscillations in the different vigilance states in mice lacking this gene and in their wild type controls. Overall, current findings show that ablation of this gene results in sex-dependent and brain region-specific endophenotypic alterations and further provide valuable insights to understanding the role of this novel player in brain physiology and pathophysiology.

Committee: Pothitos Pitychoutis Ph.D. (Advisor); Karolyn Hansen Ph.D. (Committee Member); Amit Singh Ph.D. (Committee Member) Subjects: Biology; Neurobiology; Neurosciences

PhD, University of Cincinnati, 2019, Medicine: Neuroscience/Medical Science Scholars Interdisciplinary

Parkinson's disease (PD) is a complex neurodegenerative disorder with a plethora of symptoms categorized as motor and non-motor. Historically, research on PD has focused mainly on degradation of the nigrostriatal pathway. However, degeneration of additional brainstem regions, including the locus coeruleus (LC) and dorsal raphe nucleus (DRN), and dysfunction of their associated noradrenergic and serotonergic neurotransmitter systems, respectively, also contribute to disease pathology. Nevertheless, the etiology of the vast majority of PD cases remains unknown. The overall goal of our work was to examine the relatively novel DJ-1 knockout (KO) rat model of PD in an effort to expand our knowledge of the manifestation and progression of PD. Complete loss of the protein DJ-1 leads to an autosomal recessive form of PD. The DJ-1 KO rat was created with the objective of generating an animal model with a more robust PD phenotype than seen previously in DJ-1 KO mice. Although a few studies on these mutant rats have noted some promising PD-like features, they have not always been in agreement. Here, we conducted a more thorough analysis of the DJ-1-deficient rat model, hypothesizing that we would uncover aberrant motor and non-motor behaviors, altered neurotransmitter levels and reduced neuronal survival in PD-relevant brain regions. In Aim 1, we performed a battery of motor and non-motor tasks at various ages to expand the behavioral characterization of the DJ-1 KO rat. Consistent with a PD phenotype, the DJ-1 KO rats demonstrated a reduction in rears, stride length, and grooming time, compared to wild-type (WT) control rats. However, the DJ-1-deficient rats took more steps than WT controls in several motor tasks, inconsistent with PD-like behavior. The non-motor findings were also mixed, in that DJ-1 KO rats exhibited deficits in short-term memory, but also better olfactory detection and increased sucrose intake during the sucrose preference task. In Aim 2, we e (open full item for complete abstract)

Committee: Mark Baccei Ph.D. (Committee Chair); Christina Gross Ph.D. (Committee Member); Kim Seroogy Ph.D. (Committee Member); Michael Williams Ph.D. (Committee Member); David Yurek PhD (Committee Member) Subjects: Neurology

Doctor of Philosophy, The Ohio State University, 2019, Molecular, Cellular and Developmental Biology

Duchenne muscular dystrophy is a severe childhood-onset striated muscle disease that has no cure. The current treatment for skeletal muscle weakness has substantial side-effects. We have previously shown that treatment with drugs that inactivate the mineralocorticoid receptor (MR) improves skeletal muscle function in muscular dystrophy mice. To determine if these MR antagonists work through direct mechanisms in the skeletal muscle, we conditionally knocked out the myofiber MR in muscular dystrophy mice. Genetic ablation of MR improved skeletal muscle force and reduced fibrosis in muscular dystrophy mice similar to that observed with MR antagonist drugs. Additionally, MR antagonists stabilize fragile dystrophic skeletal muscle membranes in a MR independent manner, suggesting that these drugs have many benefits for skeletal muscle in muscular dystrophy. We then evaluated previously identified candidate MR responsive genes for their role in muscular dystrophy. None of the evaluated genes appeared to be direct myofiber specific MR targets. To investigate the role of the myofiber MR in normal muscle biology, we acutely injured the skeletal muscle of myofiber MR conditional knockout mice on a wild-type background. We found for the first time that acutely injured skeletal muscle has MR hormonal regulation and genetic ablation of the MR temporarily stabilized damaged myofibers at four days after acute muscle injury. Pharmacological inhibition of the MR with MR antagonist treatment delayed normal muscle repair in acute injury, suggesting additional roles for MR in other cell types in skeletal muscle contribute to regeneration after acute injury. These results have implications for MR modulation after acute and chronic skeletal muscle injuries.

Committee: Jill Rafael-Fortney (Advisor); Lawrence Kirschner (Committee Member); Sharon Amacher (Committee Member); Paul Martin (Committee Member) Subjects: Cellular Biology; Molecular Biology; Physiology

Master of Science in Biological Sciences, Youngstown State University, 2018, Department of Biological Sciences and Chemistry

The lactose operon is responsible for the import and metabolism of lactose. β- galactosidase is a lactose operon enzyme that hydrolyzes lactose into glucose and galactose. Enterobacter sp. YSU, which was isolated from a heavy metal contaminated site in Oak Ridge, Tennessee, contains a lactose operon. Many studies used β- galactosidase as a reporter gene to study the expression levels of other genes. This was accomplished by replacing the gene of interest with the gene for β-galactosidase, lacZ. Thus, the gene of interest controls the expression of lacZ. Then, LacZ activity is detected using a color indicator such as 5-bromo-4-chloro-3-indolyl- β-D-galactopyranoside (X- gal) or o-nitrophenyl-β-D-galactopyranoside (ONPG) which turn blue or yellow, respectively, when hydrolyzed by ß-galactosidase. The overall goal is to use lacZ as a reporter gene to study metal resistance genes in Enterobacter sp. YSU. Before lacZ can be used as a reporter, it must be removed from the genome of Enterobacterr sp. YSU to eliminate background activity. A large section of the lac operon was cloned into a suicide plasmid and sequenced. Then, PCR was used to amplify the whole plasmid lacking the lacZ gene. The DNA was ligated to produce a new recombinant plasmid lacking lacZ or it was ligated with a kanR/sacB DNA fragment to produce a new recombinant plasmid with lacZ replaced by the kanR/sacB genes. KanR is a selectable marker for kanamycin resistance and sacB is a counter-selectable marker. Cells containing the sacB gene die when spread on agar plates containing 5% sucrose. Deletion of the lacZ gene in Enterobacter sp. YSU will be a two-step process. First, the recombinant with the kanR/sacB replacement will be electroporated into Enterobacter sp. YSU. Homologous recombination into the chromosome of Enterobacter sp. YSU will allow it to grow on kanamycin plates. Second, the lacZ deletion plasmid will be electroporated into the new strain. Homologous recombination will remove the sacB gen (open full item for complete abstract)

Committee: Jonathan Caguiat Ph.D. (Advisor); David Asch Ph.D. (Committee Member); Xiangjia Min Ph.D. (Committee Member) Subjects: Biology; Genetics; Microbiology; Molecular Biology

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

The inner ear hair cells are mechanosensitive and significant for hearing. In the actin-based cuticular plate (CP) of hair cells, microtubule and actin cross-linking factor 1 (Macf1) has been hypothesized to form the linker bridging the microtubule and the actin. To further understand the role of Macf1 in hearing, knockout mice need to be obtained. As null mice of Macf1 is lethal at an early developmental stage, my work mainly focused on generating the conditional knockout of Macf1 via Cre-loxP system in mouse inner ear. In addition, the actin-binding protein dematin is also reported to exist in the inner ear hair cells and be involved in regulating the actin cytoskeleton dynamics. However, the expression pattern of dematin remains unknown in the hair cells. I characterized the expression pattern of dematin in mouse inner ear hair cells, and this will help better understand the function of dematin in hair cells in the future.

Committee: Brian McDermott Ph.D. (Advisor); Ruben Stepanyan Ph.D. (Committee Member); Deborah Harris M.S. (Committee Member); Jessica Fox Ph.D. (Committee Chair) Subjects: Audiology

Doctor of Philosophy (PhD), University of Toledo, 2017, Biomedical Sciences (Molecular Medicine)

Atherosclerosis is the main cause of coronary heart disease, the leading cause of mortality in developed countries. Development of atherosclerotic lesions is in part, associated with increased apoptosis of endothelial cells and disturbance of endothelial integrity. By virtue of its important role and strategic anatomic location in the arterial tree, the endothelium plays a major role in all phases of lesion development. In endothelial cells, a number of atherorelevant systemic and local factors induce endoplasmic reticulum (ER) stress by causing disturbance in endothelial cell ER homeostasis which activates the unfolded protein response (UPR). Although this concerted and adaptive process is initially aimed at protecting the cell, sustained and unresolved ER stress can lead to endothelial cell apoptosis. In a number of cell types, persistent and/or exacerbated Ca2+ influx has also been shown to promote ER stress. In previous studies from our laboratory it was shown that in human coronary artery endothelial cells (HCAECs) constitutive Ca2+ influx plays a critical role in inflammatory signaling, and that the Transient Receptor Potential Canonical 3 (TRPC3) channel mediates such constitutive Ca2+ entry, which is exacerbated under atherorelevant conditions. It was also observed in these studies that TRPC3 constitutive function couples to Ca2+/calmodulin-dependent protein kinase II (CAMKII) to drive inflammatory signaling. In other cell types, TRPC3 has been found to contribute to UPR signaling and apoptosis, and CAMKII is shown to act as a mediator in the signaling pathway that results in ER stress-mediated apoptotic cell death. However, whether a similar role of TRPC3 exists in HCAECs remained unknown. In this work, we first examined whether inhibition of constitutive Ca2+ influx in general and TRPC3 in particular, plays a role in ER stress-induced apoptosis in HCAECs. Our results show that global blockade of constitutive Ca2+ influx or buffering of intracellular Ca2+ (open full item for complete abstract)

Committee: Guillermo Vazquez Ph.D. (Committee Chair); Bina Joe Ph.D. (Committee Member); David Giovannucci Ph.D. (Committee Member); Viviana Ferreira Ph.D. (Committee Member); Sivarajan Kumarasamy Ph.D. (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, Miami University, 2016, Biology

The ocular lens is an excellent model to study cell signaling, cell survival and cell differentiation. The lens is comprised of only two cell types: proliferative lens epithelial cells and terminally differentiated lens fiber cells. The lens fiber differentiation process involves specific changes in gene expression between the two cell types. However, a comprehensive understanding of gene expression changes during lens fiber differentiation remains incomplete. Furthermore, despite the wealth of knowledge of transcription factors involved in lens cell proliferation, survival and lens fiber differentiation, little information exists about the role of DNA methylation and miRNAs in these processes. This study presents the first application of RNA-seq to provide a comprehensive view of both the relative abundance and differential expression of mRNAs and long intergenic non-coding RNAs from lens epithelial cells and lens fiber cells. We also investigated the role of DNA methylation in lens development. We found that while Dnmt1 inactivation at the lens placode stage led to lens DNA hypomethylation and severe lens epithelial apoptosis, lens fiber cell differentiation remained largely unaffected. The simultaneous deletion of phosphatase and tensin homolog (Pten) elevated the level of phosphorylated AKT and rescued many of the morphological defects and cell death in DNMT1- deficient lenses. With a different Cre driver (MLR10) we demonstrated that a small number of lens epithelial cells escaped Dnmt1-deletion and over-proliferated to compensate for the loss of Dnmt1-deleted cells, suggesting that lens epithelium possess a substantial capacity for self- renewal. Inactivation of both Dnmt3a and Dnmt3b by either the Le-Cre or MLR10-Cre transgene did not result in any obvious lens phenotype prior to 10 months of age, indicating that de novo DNA methylation, at least as mediated by both DNMT3A and DNMT3B, is dispensable for normal lens development. Our comparative miRNA-Seq data a (open full item for complete abstract)

Committee: Michael Robinson (Advisor) Subjects: Biology; Biomedical Research; Genetics; Zoology

Master of Science (MS), Ohio University, 2016, Food and Nutrition Sciences (Health Sciences and Professions)

White adipose tissue (WAT) is a complex endocrine organ composed of mature adipocytes and a variety of cells that compose the stromal vascular fraction (SVF). The SVF includes preadipocytes, fibroblasts, endothelial cells, and immune cells such as macrophages, T cells, natural killer cells, dendritic cells, and B cells. The cellular composition of the SVF and specific function of WAT depots depends on anatomical location. Growing evidence suggests that the immune cell populations present in WAT are intimately linked with the inflammatory, endocrine, and metabolic function and dysfunction of the individual WAT depots. Growth hormone (GH) impacts adiposity and immune function in a depot-dependent manner and is well known for its ability to decrease WAT mass by promoting lipolysis and inhibiting lipogenesis. To date, no one has reported the influence of a reduction of GH action on the immune cell profile in different WAT depot. Therefore, the purpose of the current study was to examine the WAT immune cell populations in dwarf growth hormone receptor knockout (GHR-/-) mice, which have no GH induced signaling. To this end, immune cell populations of three distinct WAT depots were characterized using flow cytometry. Although marked genotype differences in WAT immune cell populations were found in epididymal and mesenteric WAT, the immune cell profile of the subcutaneous depot was not different between genotypes. Interestingly, only subcutaneous fat was preferentially increased in GHR-/- mice, suggesting that immune cells do not contribute to the expansion of this depot. Our results also indicate that a lack of GH alters WAT immune cell populations ina depot-specific manner.

Committee: Darlene Berryman PhD (Advisor); Fabian Benencia PhD (Committee Member); Robert Brannan PhD (Committee Member) Subjects: Biology; Nutrition

Doctor of Philosophy, The Ohio State University, 2015, Molecular, Cellular and Developmental Biology

Both epidemiological and empirical data have demonstrated the association between long-term exposure to fine particulate matters (PM2.5) and the burden of atherosclerosis. Strawberry, a rich source of micronutrients and several bioactive phytochemicals, has been implicated in the prevention of cardiovascular diseases and metabolic syndromes. This study was designed to explore the effects of freeze-dried strawberry supplementation on PM2.5-potentiated atherosclerosis in apoE-/- mice, which is a spontaneous atherosclerotic model. Mice, fed with high-fat chow or high-fat chow supplemented with 10% strawberry powder, were exposed to either filtered air or concentrated ambient particles using “Ohio Air Pollution Exposure Systems for Interrogation of Systemic Effects” for 6 months. After PM2.5 exposure, plaque area and lipid area of aorta were evaluated using hematoxylin & eosin staining and oil-red O staining, respectively. In addition, total cholesterol, high-density lipoprotein and fasting glucose were measured using diagnostic kits. Furthermore, the levels of inflammatory cytokines in plasma were determined via ELISA kits, and the mRNA expressions of inflammatory cytokines within lung were quantitated using real-time PCR. Our data showed that long-term PM2.5 exposure potentiated atherosclerosis and inflammation in these mice; strawberries reduced inflammation and improved lipid profile, but did not attenuate atherosclerosis after PM2.5 exposure. These findings indicate that other mechanistic pathways, e.g. systemic oxidative stress, autonomic nervous system imbalance, and the direct toxic effects of particles, may diminish the effects of strawberries on PM2.5-potentiated atherosclerosis.

Committee: Qinghua Sun (Advisor); Loren Wold (Committee Member); Kamal Mehta (Committee Member); Zhenguo Liu (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy, The Ohio State University, 2015, Integrated Biomedical Science Graduate Program

Genome instability, which can be defined as an increase in changes at both the nucleotide and chromosomal level (e.g. point mutations, chromosomal translocations), results from errors in normal biological processes that function to repair, replicate, and segregate the genome during cell division. Genomic instability is a hallmark of human neoplasia, present in varying degrees in all stages of cancer, from precancerous to advanced cancer. Genome instability is initiated due to loss of expression of the FHIT gene, located at 3p14.2; the murine Fhit gene is located at 14A2. Fhit loss occurs early in human cancer development and is frequently observed in preneoplastic lesions. The Fhit protein is a tumor suppressor and genome caretaker that modulates genome stability and level of DNA damage that accumulates beginning in precancerous lesions. To investigate the contribution of loss of Fhit expression to the generation of mutations and to define mechanisms that underlie these genome alterations, we performed an analysis of whole exome sequences from cell lines and tissues, from wildtype and Fhit-/- mice. -/- cells and tissues demonstrated increased numbers of C>T and T>C mutations and Fhit-deficient kidney cell cultures that survived dimethylbenz(a)anthracene treatment exhibited increased numbers of T>A mutations. Following determination of trinucleotide contexts of mutations, a Fhit-loss signature was proposed to consist of C>T and T>C mutations that may be due to respective increased spontaneous deamination (C>T mutations) and deoxyribonucleotide triphosphates pool imbalance (T>C mutations), a signature similar to the 'age at diagnosis' signature identified in human cancers. Increased T>C mutations in -/- exomes may be due to the imbalance in deoxyribonucleotide triphosphates, particularly thymidine triphosphate, resulting from decreased expression of Thymidine Kinase 1 in Fhit-deficient cells and tissues. Fhit-deficient kidney cell cultures that survived in vitr (open full item for complete abstract)

Committee: Kay Huebner PhD (Advisor); Vincenzo Coppola MD (Committee Member); Sissy Jhiang PhD (Committee Member); Yuri Pekarsky PhD (Committee Member) Subjects: Genetics

PHD, Kent State University, 2015, College of Arts and Sciences / School of Biomedical Sciences

Osteoporosis impacts 55% of the population aged 50 and older, with post-menopausal, thin women of Caucasian descent at the highest risk for developing this disease. As the median age of the population rises, so does the incident of fracture caused by osteoporosis. Currently, only one bone anabolic factor is available on the market, PTH1-34, with a therapeutic window of only 2 years; new novel bone anabolic factors are needed. One protein with the potential for osteoporosis therapy is osteoactivin (OA). Previous studies have demonstrated the role of OA in osteoblast and osteoclast differentiation and function. In this study, we utilized global OA knockout mice to explore the physiological role of OA in bone homeostasis. We also characterized the role of osteoactivin in osteoblasts and osteoclasts by studying OA-/- cells ex vivo. In addition, we tested how the skeleton responded to stress induced by estrogen-deficient osteoporosis model in OA-mutant (D2J), OA knockout, and their respective wildtype controls. Additionally, we investigated the functional role of osteoactivin-derived peptide OA-D. We hypothesized that osteoactivin was critical for postnatal bone growth and development, and that OA-D would be capable of stimulating bone formation and rescuing osteoporotic phenotypes. We demonstrated that the OA-null animals had increased bone mass. Ex vivo analysis of osteoblast differentiation and function revealed defects in mineralization and differentiation. Next, we observed that osteoclast size due to increased fusion of large osteoclasts was significantly increased in the OA-/- animal. However, osteoclast resorption was significantly decreased. Next, OA-D was tested in C57/Blk6 mice for its ability to stimulate bone mass. We showed that OA-D injection in mice resulted in a trending increase in bone mass. Alternative administration of OA-D showed similar results in bone mass. Ovariectomy of OA mutant and OA-/- animals failed to induce significant bone loss (open full item for complete abstract)

Committee: Fayez Safadi PhD (Committee Chair); Werner Geldenhuys PhD (Committee Member); Chris Malcuit PhD (Committee Member); Moses Oweyumi PhD (Committee Member); Walt Horton PhD (Committee Member); Srinivasan Vijayaraghavan PhD (Committee Member) Subjects: Biology; Cellular Biology; Pharmacology

Doctor of Philosophy, The Ohio State University, 2014, Biochemistry Program, Ohio State

Clostridium acetobutylicum JB200, a mutant strain of C. acetobutylicum ATCC 55025 obtained through strain evolution in a fibrous bed bioreactor, had high butanol tolerance, and produced up to ~21 g/L butanol from glucose in batch fermentation, an improvement of ~67% over the parental strain (~12.6 g/L). To explore the genetic differences between the three C. acetobutylicum strains JB200, ATCC 55025, and the type strain ATCC 824, the whole genomes of ATCC 55025 and JB200 were sequenced using high-throughput Illumina sequencing technology. Comparative genomic analysis performed for the three strains identified many variations that may contribute to the non-spore-forming and enhanced butanol-producing characteristics in both ATCC 55025 and JB200. In addition, the comparative analysis revealed only seven point mutations in the genome of JB200, which might contribute to the greatly improved butanol production and tolerance of JB200 compared to ATCC 55025. Among the seven mutations, a single base deletion in the coding region of the cac3319 gene causes a large portion (~75% of the original length) of truncation at the C-terminus of its encoding histidine kinase. The inactivation of cac3319 in ATCC 55025 indicated the important role of cac3319 in regulating butanol tolerance and production of C. acetobutylicum. This study provides a molecular-level understanding of non-sporulation, higher butanol tolerance and production in C. acetobutylicum mutants, revealing possible regulatory factors involved in butanol tolerance and production.

Committee: Shang-Tian Yang (Advisor); Jeffrey Chalmers (Committee Member); Andre Palmer (Committee Member); Hua Wang (Committee Member) Subjects: Biochemistry; Bioinformatics; Chemical Engineering

Master of Science (MS), Ohio University, 2014, Biological Sciences (Arts and Sciences)

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease and renal failure in humans. The molecular pathways that lead to DN are not well known. This research investigates possible roles of several signal transducers and activators of transcription (STAT) proteins in this disease using a STAT5A/B knockout (SKO) mouse model. Based on previous observations of increased inflammation-related gene expression in the kidneys of diabetic SKO mice, the hypothesis of the current project was that the combination of the loss of STAT5 repression and increase of STAT3 activity escalates inflammation-related gene expression in the kidneys of diabetic SKO mice. In support of this hypothesis, an increase of IRF-1 RNA expression, reflective of the loss of STAT5 repression, was observed in the kidneys of diabetic SKO mice. Levels of phosphorylated STAT3 were also increased in the kidneys of diabetic SKO mice. These results suggest that STAT5 acts as a repressor of inflammation-related genes in DN and, in its absence, expression of these genes is no longer repressed, either due to direct loss of the STAT5 repression or due to increased STAT3 activity which could potentially increase their expression.

Committee: Karen Coschigano PhD (Advisor); Calvin James PhD (Committee Member); Ramiro Malgor MD (Committee Member) Subjects: Animals; Biology; Biomedical Research; Immunology; Molecular Biology

Doctor of Philosophy, Miami University, 2014, Zoology

We investigated the interaction between prolactin (PRL) and the HPA axis. A physiologically relevant, hyperprolactinemic model, i.e. postpartum female rats, or PRL knockout mice were used. Animals housed with or separated from their pups for 1 or 24 hours or 8 days were acutely stressed. Circulating PRL and corticosterone (CORT) and PRL-R mRNA in the choroid plexus were determined. PRL returned to pre-pregnancy values by one hour after pup removal. The HPA axis response was restored after 24 hours of separation, although basal CORT levels remained elevated for 8 days after terminating lactation. PRL-R expression decreased 24 h after pup separation, but returned to pre-pregnancy levels by 8 days. Stress-induced HPA axis activation occurred only when PRL-R expression levels were similar to or lower than levels in virgins indicating PRL-R up-regulation contributes to an attenuated HPA response. The sustained, elevated basal CORT levels suggest the metabolic demands and/or stress of lactation persist in the absence of suckling. Additionally, differential effects of gender, diet and PRL on HPA axis activation were studied using wild-type (wt) and PRL knockout (KO) mice. Male and female mice fed normal chow (C) or high-fat diet (HFD) for 12 weeks were acutely stressed. CORT levels were quantified under basal conditions, during stress and at the time of sacrifice. Serial sampling acted as a non-specific stressor, regardless of gender, genotype or diet; PRL dampened this response in males only. HFD increased body weight and fat accumulation in males, but not in females, regardless of genotype. In chow fed animals, stress increased CORT in both males and females, regardless of genotype, indicating that PRL is not involved in activating the HPA axis. HFD increased PRL-R mRNA in wt and KO females, but only in wt males indicating factors other than PRL regulate PRL-R expression in females. HFD increased leptin levels only in wt males and females, suggesting PRL influences lepti (open full item for complete abstract)

Committee: Phyllis Callahan PhD (Committee Chair); James Janik PhD (Committee Co-Chair) Subjects: Biology; Endocrinology; Neurosciences

Bachelor of Science (BS), Ohio University, 2011, Biological Sciences

Global Growth Hormone Receptor (GHR) knockout mouse models show increased insulin sensitivity and extended longevity. It is theorized that the lack of growth hormone signaling in a single tissue is responsible for these effects. The most likely tissues include liver, adipose, and muscle. To examine that theory, three tissue specific GHR knockout mouse models were created using tissue specific promoters. This thesis focuses on methods to verify that those models lack GHR in, and only in, the intended tissue. The best confirmation of knockout would be to test for the functional GHR protein, however, western blot analysis demonstrated problems with specificity of the antibodies even with immunoprecipitation to increase protein concentration. PCR, however, shows promise and warrants further investigation.

Committee: Darlene Berryman (Advisor); Edward List (Advisor) Subjects: Biology; Microbiology; Molecular Biology