Doctor of Philosophy, Case Western Reserve University, 2006, Pharmacology

The 15-30% of human breast cancers that have upregulated HER2/ErbB2/Neu are highly aggressive and resistant to traditional treatments, resulting in poor prognosis. To identify novel therapeutic targets, we derived the transcriptomes associated with tumor progression in two independent mouse models of ErbB2/Neu-induced tumorigenesis. From MMTV-Neu mice, we identified 324 candidate genes unique to ErbB2/Neu-induced tumors relative to wild-type mammary glands. A subset of these genes also changed expression levels in preneoplastic mammary glands, indicating a pivotal role early in ErbB2/Neu-initiated tumorigenesis. Downregulation of several known transforming growth factor (TGF)-β target genes in the ErbB2/Neu molecular signature suggested attenuation of the TGF-β signaling cascade in these tumors. Analysis of TGF-β-Receptor-I/ALK5 by western blot and immunohistochemistry confirmed that Smad-dependent TGF-β signaling was inactive in these tumors. Although absent in most of the tumor, colocalization of phosphorylated Smad2 and Activin-Receptor-IB/ALK4 at the tumor periphery suggested functional Activin signaling at the leading edge of these tumors. Collectively, these data indicate intrinsic TGF-β pathway suppression in ErbB2/Neu tumors via loss of TGF-β-Receptor-I/ALK5. Recent studies have shown that pregnancy can accelerate ErbB2/Neu tumor development, inducing a susceptible cell population in MMTV-Neu mammary glands. The stochastic pattern of tumor development in multiparous MMTV-Neu mice suggests additional events are required for ErbB2/Neu oncogenesis. It remains unclear whether such events are genetic or reflective of the dynamic, pregnancy-associated hormonal control of the gland. Bitransgenic mice generated by breeding MMTV-Neu mice with a model of ovarian hyperstimulation developed multifocal mammary tumors in an accelerated, synchronous manner compared to virgin MMTV-Neu animals. This synchrony of tumor development suggests that trophic maintenance of the mamm (open full item for complete abstract)

Committee: Ruth Keri (Advisor) Subjects:

Bachelor of Science (BS), Ohio University, 2020, Biological Sciences

Adipose tissue (AT) is a unique energy storage tissue able to undergo significant hypertrophy and atrophy, dependent, in part, on nutrient status. When hypertrophy is in excess and sustained, the result is obesity, a common and widespread health problem. However, when AT stores are selectively depleted or abnormally deposited, the resultant condition known as lipodystrophy can lead to comparable health outcomes as obesity. That is, either extreme in AT mass results in similar metabolic outcomes (insulin resistance, increased immune cell infiltration, and increased production of inflammatory cytokines). With either chronic obesity or lipodystrophy, AT can undergo major remodeling, which can ultimately result in unresolved chronic inflammation and AT fibrosis. Growth hormone radically alters AT mass, and mice that overexpress growth hormone (bGH mice) are lipodystrophic with a notable increase in AT fibrosis. Key players in AT fibrosis could include fibroblast activation protein (FAP) and fibroblast growth factor 21 (FGF21), among others. FAP has been positively correlated with ECM remodeling and its knockdown has been associated with improved metabolism in diet induced obese mice, while FGF21 is associated with increased energy expenditure yet is cleaved and inactivated by FAP. Importantly, FGF21 and FAP levels were also recently assessed in patients with acromegaly, and these molecules were suggested as a marker of tissue fibrosis and positively correlate with GH action. [1] These results provide compelling evidence that assessing the FAP/FGF21 interaction in our bGH mice will help elucidate the mechanisms underlying the relationship between GH and fibrosis. Thus, the purpose of this study was to determine if these molecules are altered in bGH mice. Using serum and plasma collected from bGH and littermate controls and ELISA assays, our results indicate that FAP is not significantly altered in adult bGH mice, although sex s (open full item for complete abstract)

Committee: Darlene Berryman PhD, RD, LD (Advisor) Subjects: Biology; Biomedical Research

Master of Science (MS), Ohio University, 2008, Nutrition Science (Health and Human Services)

Microarray analyses were performed comparing mRNA expression levels in subcutaneous adipose tissue between mice with disruption in the growth hormone (GH) receptor gene (GHR-/- mice) and littermate controls. The data revealed that 87 genes were significantly upregulated and 72 genes were significantly downregulated in GHR-/- mice versus littermate controls. Among the 159 altered genes, a subgroup of five genes was chosen for confirmation by a second mRNA quantification method, real time reverse transcriptase polymerase chain reaction (RT-RT PCR). RT-RT PCR studies confirmed the altered gene expression for only three genes, angiotensinogen (Agt), adiponutrin (Adpn) and angiopoietin-like protein 4 (Angptl4). To better understand the role of GH in adipose specific expression of these three genes (Adpn, Agt, Angptl4), RT-RT PCR analysis was expanded to include an additional genotype of altered GH function, the bovine GH transgenic mice, an additional diet treatment high-fat diet, as well as two other adipose depots, the epididymal and retroperitoneal adipose depots. This thesis discusses in detail all gene alterations observed in the microarray analyses and the RT-RT PCR analyses for Adpn, Agt, Angptl4 in adipose tissue.

Committee: Darlene E. Berryman PhD (Advisor); Karen Coschigano PhD (Committee Member); Diana Manchester MS (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology; Nutrition

Master of Science, University of Toledo, 2018, Exercise Science

The transcription factor forkhead box O1 (FoxO1) is a downstream effector of insulin and insulin-like growth factor 1 (IGF-1) pathway and regulates various physiological processes including cell proliferation, differentiation, and metabolism. Though the role of FoxO1 in glucose metabolism is well chronicled, its function in lipid metabolism remains to be determined. It has been observed that one of the duties of FoxO1 is to act as a metabolic switch that shifts the metabolic response from utilizing glucose to fatty acids (FA) during times of diminished caloric intake. Downstream targets of FoxO1 aid in the transition from carbohydrate to FA metabolism, and research suggest cluster of differentiation 36 (CD36) to play a key role in the uptake of FA by skeletal muscle.16 The literature indicating a relationship between FoxO1 and the possibly increased expression of CD36 in the plasma membrane is limited and studies have primarily used in vitro models. Therefore, the purpose of this study was to use an in vivo model to elucidate the role of FoxO1 on the regulation of CD36 in skeletal muscle. Transgenic mice overexpressing the muscle-specific FoxO1 protein had their quadriceps muscles excised after an overnight fast. The muscle samples were homogenized, analyzed by western blotting, and quantified using densitometry. The transgenic mice overexpressing the FoxO1 protein had significantly (p<0.05) increased protein expression of FoxO1 compared to the wildtype (WT) mice. However, CD36 expression was not found to be significantly increased in FoxO1 transgenic samples compared with that found in the WT mouse samples. These findings indicate that in vivo increased FoxO1 expression in skeletal muscle is not associated with increased skeletal muscle expression of CD36.

Committee: Thomas McLoughlin Ph.D (Committee Chair); Francis X. Pizza Ph.D (Committee Member); Abraham D. Lee Ph.D (Committee Member) Subjects: Physiology

Doctor of Philosophy, The Ohio State University, 2014, Comparative and Veterinary Medicine

Chemotherapeutics remains a choice of treatment for several malignant diseases. However, selective cytotoxicity against cancer cells without compromising their normal counterparts pose a huge challenge for traditional drug design. Current therapies for chronic lymphocytic leukemia (CLL), the most prevalent adulthood leukemia in the western world are not curative rendering drug adverse effects and immunosuppression. Here we developed a novel non-immunosuppressive FTY720 derivative OSU-2S with potent cytotoxicity against leukemic B cells. OSU-2S induces activation of protein phosphatase 2A, phosphorylation and nuclear translocation of SHP1 S591 and deregulation of multiple cellular processes in CLL. Moreover, with relevant to CLL disease TCL1A expression that was identified to be down regulated in response to OSU-2S in the gene expression profile was independently confirmed to be significantly down regulated both at the mRNA and protein levels. Exposure of OSU-2S to unintended cells is expected to adversely affect physiological functions of these ubiquitous phosphatases. To selectively deliver OSU-2S to leukemic cells, we developed tumor antigen targeted delivery of immunonanoparticle carrying a OSU-2S (2A2-OSU-2S-ILP). 2A2-OSU-2S-ILP immunonanoparticles mediated selective cytotoxicity of CLL but not normal B cells through targeting receptor tyrosine kinase ROR1 expressed in leukemic but not normal B cells. Developing a novel spontaneous CLL mouse model expressing human ROR1 in all leukemic B cells, we demonstrate the therapeutic benefit of enhanced survival with 2A2-OSU-2S-ILP in-vivo. The newly developed non-immunosuppressive OSU-2S, its delivery using human CLL specific surface antigen ROR1 directed immunonanoparticles and the novel transgenic mouse model of CLL that expresses human ROR1 exclusively in leukemic B cell surface are highly innovative and can be applied to CLL and other ROR1+ malignancies including mantle cell lymphoma (MCL) and B-lineage acute lymph (open full item for complete abstract)

Committee: Natarajan Muthusamy DVM, PhD (Advisor); John Byrd MD (Committee Member); Ching-Shih Chen PhD (Committee Member); Cheryl London DVM, PhD (Committee Member) Subjects: Biomedical Research; Medicine; Nanotechnology; Therapy

Doctor of Philosophy (PhD), University of Toledo, 2013, College of Medicine

Human angiotensinogen gene locus is associated with hypertension. Human angiotensinogen gene (hAGT) has -6A/G polymorphism and -6A (rs5051) allele is associated with increased blood pressure. We have found that hAGT gene has three additional SNPs. Variants -1670A, -1562C, and -1561T almost always occur with -6A haplotype (Hap -6A); and variants -1670G, -1562G, and -1561G almost always occur with -6G haplotype (Hap -6G). We have shown that these polymorphisms affect the binding of glucocorticoid receptor to the promoter. Reporter construct containing 1.8 Kb of the hAGT gene promoter with Hap -6A has 4 fold increased glucocorticoid induced promoter activity as compared to Hap -6G. Therefore, we have generated transgenic (TG) mice containing either Hap -6A or Hap -6G of the hAGT gene to study the regulation of the hAGT gene in an in vivo situation. We have reported that transgenic mice with Hap -6A of hAGT gene has increased plasma angiotensinogen levels and significantly elevated blood pressure as compared to the Hap -6G. Since hAGT is not cleaved by mouse renin, we have generated double transgenic mice with human renin and hAGT gene of either Hap -6A or Hap -6G. We hypothesize that these three additional SNPs in the promoter of Hap -6A may be predisposing to hypertension in response to increased glucocorticoid levels. TG mice with Hap -6A and Hap -6G were treated with dexamethasone (DEX) (10µg/day in drinking water) for 72 hours. Our Q-RTPCR results show that mice with Hap -6A have significant increase in the mRNA expression of hAGT in the liver and the kidney on treatment with DEX whereas no significant changes were observed in Hap -6G. Q-RTPCR results were confirmed by western blots showing significant increase in the DEX induced expression of the hAGT protein in the liver and the kidney of the TG mice with Hap -6A. DEX treatment also increased the plasma hAGT protein and Ang-II levels in TG mice with Hap -6A whereas no significant changes were observed in TG (open full item for complete abstract)

Committee: Ashok Kumar PhD (Committee Chair); Bina Joe PhD (Committee Member); Andrew Beavis PhD (Committee Member); Nitin Puri MD PhD (Committee Member); Zahoor Shah PhD (Committee Member) Subjects: Biochemistry; Genetics; Molecular Biology; Pharmacology; Physiology

PhD, University of Cincinnati, 2001, Medicine : Cell and Molecular Biology

Voltage-dependent calcium channels (VDCC's) mediate the influx of Ca 2+ in response to membrane depolarization and regulate numerous intracellular pathways including: contraction, secretion, neurotransmitter release, and gene transcription. Calcium is the most ubiquitous signaling molecule, but elicits the most exquisitely specific responses. Therefore, proper regulation and control of Ca 2+ mobilization is essential to prevent aberrant signaling. Although a large body of knowledge has been accumulated on VDCC's, physiological characteristics in native tissue and the contribution of the channels to broad scope function remain vague. This study focuses on the cardiac-specific overexpression of the L-VDCC in the mouse which will further the understanding of the in vivo function and role of the L-VDCC in normal and diseased myocardium. In addition, compensatory mechanisms associated with the change in Ca 2+ homeostasis will be identified. Overexpression of the α1 subunit (Ca v 1.2) increased Ca 2+ influx into cardiomycytes, which resulted in an increase in cardiac contractile force. Interestingly, expression of the accessory subunits was unchanged compared to nontransgenic animals. Since no differences were observed in the activation or inactivation kinetics of the channel, the core stoichiometry remains intact between transgenic and nontransgenics. Two major findings in this transgenic model include an early loss of the β adrenergic receptor signaling pathway and a slow-progressing development of heart failure, both common characteristics of human pathology. Additionally, enhanced PKC α activation was an early event, which may serve as the common link between disease developmentand the defect in β adrenergic receptor signaling. Numerous signaling pathways have been identified to be involved in cardiac hypertrophy with no clear common member other than Ca 2+ . Clearly in this model, the increase ingress of Ca 2+ initiates the hypertrophic program with associated fibros (open full item for complete abstract)

Committee: Arnold Schwartz (Advisor) Subjects: Biology, Molecular

PhD, University of Cincinnati, 2000, Medicine : Molecular and Cellular Physiology

Annexin V is a promising candidate as an anticoagulant and anti-inflammatory agent due to its high affinity for anionic phospholipid surfaces. Its abundance and tissue localization in placental trophoblasts and vascular endothelial cells imply a physiological importance in these tissues. In the first section of this study, I demonstrate that annexin V is critical in maintaining murine placental integrity. I conclude that the protecting role of annexin V appears to be local because I did not detect the protein in culture media of either trophoblasts or endothelial cells. In the next portion of this study, I successfully targeted annexin V to the secretory pathway of mammalian cells and the protein was secreted into the medium. This unnatural targeting of annexin V to the lumen of the endoplasmic reticulum did not have significant effect on cellular function. Therefore, I concluded that targeting secretion of annexin V in transgenic animals should provide a valuable model for evaluating the therapeutic potentials of annexin V as anticoagulant and/or anti-inflammatory agent.

Committee: John Dedman (Advisor) Subjects: Biology, Molecular

PhD, University of Cincinnati, 2000, Medicine : Molecular, Cellular and Biochemical Pharmacology

Phospholamban is a phosphoprotein in the cardiac sarcoplasmic reticulum (SR), which regulates the apparent affinity of the SR Ca2+-ATPase (SERCA2) for Ca2+. Phospholamban, in its dephosphorylated form, inhibits SERCA2 activity, and phosphorylation removes this inhibition. Transgenic approaches were utilized in this dissertation to further elucidate the role of phospholamban's regulation of SERCA2, in vivo, under normal and pathophysiological conditions, both in the presence and absence of b-agonist stimulation. 1) Phospholamban ablation has been shown to be associated with an attenuated response to b-agonist stimulation. However, it is not known if the b-adrenergic response of phospholamban-deficient hearts would be affected under altered thyroid conditions. Hypo- and hyperthyroidism were induced in phospholamban-deficient and wild-type mice, and their responses to isoproterenol stimulation were examined in work-performing hearts. A close linear correlation was observed between the magnitude of the contractile parameter response and the PLB/SERCA2 ratio in hypo-, eu- and hyperthyroid wild-type hearts. However, no response to b-agonist stimulation was observed in phospholamban-deficient hearts with altered thyroid conditions, indicating that the changes in the thyroid states of these hearts do not influence the effects of isoproterenol on cardiac function. 2) The PLB/SERCA2 stoichiometric ratio under saturating conditions is not known. 1.8-fold, 2.6-fold, 3.7-fold and 4.7-fold overexpression of a non-phosphorylatable form of phospholamban, relative to SERCA2, resulted in maximal inhibition of SERCA2's apparent affinity for Ca2+ at phospholamban expression levels of 2.6-fold or higher, indicating that saturation of the PLB/SERCA2 ratio occurs at 2.6:1, in vivo. 3) The contribution of phospholamban versus the other key cardiac phosphoproteins during b-adrenergic stimulation is not known. To determine phospholamban's contribution indirectly, a non-phosphorylatable form (open full item for complete abstract)

Committee: Evangelia Kranias (Advisor) Subjects:

PhD, University of Cincinnati, 2009, Engineering : Biomedical Engineering

Tracking real-time changes in Col1 and Col2 gene expression could speed up the FTE process and could lead to new strategies to create zonal insertions in repair tendons. Hence double transgenic mice with fluorescent promoters for Col1 and Col2 gene expression were created and used to examine natural healing of patellar tendons, development of knee structures and the mechanical response of primitive MSCs as well as specialized chondrocytes and fibroblasts.The natural healing study sought to determine how full thickness, mid-substance punch defect injuries affect spatial and temporal patterns of Col1 and Col2 gene expression and biomechanics compared to age-matched, normal patellar tendons. Col1 expression in injured tendons was highest at two weeks after injury. No detectable Col2 gene expression was seen in the mid-substance healing wound site at any time point. Natural healing resulted in inferior repair properties at both 4 weeks and 6 weeks post injury compared to contralateral sham and age-matched tendons. Monitoring fluorescent changes in murine knees from embryonic through early post-natal development indicates that Col2 expression precedes Col1 expression. There were temporal and spatial changes in the expression of two related growth factors, TGF-β1 and BMP-2, and their downstream markers, pSmad1,5,8 and pSmad2,3 respectively. Exposing specialized and primitive cell-scaffold constructs to dynamic compressive strains produced temporal changes in gene, protein expression and biomechanics. Compression of chondrocyte-agarose scaffolds significantly increased ECFP fluorescence, Col2 mRNA expression, type II collagen content and aggregate modulus. ECFP fluorescence and Col2 mRNA expression were found to be positively correlated. Compression of MSC-agarose constructs significantly increased ECFP fluorescence, type II collagen content and aggregate modulus. Finally, 28 days of compressive stimulation of fibroblast-agarose constructs produced significant decrease (open full item for complete abstract)

Committee: David Butler PhD (Committee Chair); Jason Shearn PhD (Committee Member); Marepalli Rao PhD (Committee Member); Shawn Hunter PHD (Committee Member) Subjects: Biomedical Research

PhD, University of Cincinnati, 2007, Medicine : Neuroscience/Medical Science Scholars Interdisiplinary

Activation of the hypothalamic-pituitary-adrenocortical (HPA) axis plays a role in regulating basic biological functions in the basal state. During homeostatic perturbations, it potentiates and induces physiological adaptations that are designed to aid an organism in overcoming challenges and regain homeostatic equilibrium. However, prolonged HPA axis activation can be deleterious to long-term survival, so the HPA axis is subject to autoregulation via a negative feedback loop. The endpoint of HPA axis stimulation is corticosterone (CORT) release, which travels through the general circulation and inhibits further HPA axis activation at the levels of the pituitary, hypothalamus, and extra-hypothalamic structures such as the hippocampus. Central to HPA axis response and regulation is the glucocorticoid receptor (GR), a ligand dependent transcription factor that is richly expressed in limbic forebrain regions such as the hippocampus, medial prefrontal cortex, and amygdala. These same regions are differentially affected in several psychiatric disorders, and many of these disorders feature HPA axis dysregulation that includes changes in GR signaling. Females are differentially affected by some psychiatric disorders, such as depression, and there are well-established sex differences in HPA axis responsivity, suggesting a possible link between sex and stress regulation. Dysregulation of the HPA axis can also occur during the aging process. Previous studies using lesions and antagonists have implicated GR in feedback inhibition, but to date, the role of forebrain GR has not been directly tested. Here we present a series of studies investigating the role of forebrain GR in basal HPA axis regulation, and after acute and chronic stress, taking into account sex and aging as factors. We employ a forebrain specific GR knockout mouse model (FBGRKO), in which GR is developmentally disrupted via the loxP-Cre-Recombinase system. We show that forebrain GR is necessary for basal regulat (open full item for complete abstract)

Committee: Dr. James Herman (Advisor) Subjects:

PhD, University of Cincinnati, 2007, Medicine : Molecular and Cellular Physiology

An important physiological role proposed for vascular smooth muscle Na +-K +ATPase (NKA) is the regulation of blood pressure via modulation of vascular smooth muscle contractility. To investigate the effects of a gain of function of NKA specifically in smooth muscle, we developed mice carrying a transgene for either the NKA α1- or α2-isoform (α1 sm+or α2 sm+mice) ligated to the smooth muscle specific α-actin promoter, SMP8. The α1 sm+mice displayed a 1.5-fold increase in the α1-isoform, while the α2 sm+mice had a 2-6-fold increase in the α2-isoform. Interestingly, the α-isoform not contained in the transgene was also increased to a similar degree for both transgenic lines, which was evident at the mRNA level as well. The increase in total NKA α-isoform protein was accompanied by a ~1.25- to 2.5-fold increase in NKA activity in gastric antrum from α1 sm+and α2 sm+mice respectively. The α2 sm+mice (high expression) were hypotensive relative to WT mice (109.9 ± 1.6 mmHg, n=13 vs. 121.3 ± 1.4 mmHg, n=11), whereas α1 sm+mice (low expression) were normotensive (122.7 ± 2.5 mmHg, n=11 vs. 117.4 ± 2.3 mmHg, n=12). Our results show that smooth muscle displays unique coordinate expression of NKA α-isoforms. Additionally, increasing smooth muscle NKA decreases blood pressure, which appears to be dependent on the degree of α-isoform expression. To elucidate mechanisms underlying the decrease in blood pressure, we examined pathways of intracellular Ca 2+regulation. Relaxation half-times from a KCl-induced contraction were significantly decreased in α2 sm+aorta but not α1 sm+aorta relative to paired WT aorta. Proteins involved in Ca 2+clearance (Na +-Ca 2+exchanger-NCX and plasma membrane Ca 2+ATPase-PMCA) and sequestration (sarcoplasmic reticulum Ca 2+ATPase-SERCA) were increased up to 5-fold in smooth muscle from α2 sm+mice, supporting enhanced Ca 2+clearance. Resting intracellular Ca 2+as well as sarcoplasmic reticulum store Ca 2+were significantly decreased in ASMCs from α2 s (open full item for complete abstract)

Committee: Dr. Richard Paul (Advisor) Subjects:

PhD, University of Cincinnati, 2005, Medicine : Molecular, Cellular and Biochemical Pharmacology

Heart failure is a complex syndrome characterized by left ventricular dysfunction, myocardial remodeling, and biochemical alterations. Several signaling pathways are involved in the induction of pathological remodeling and heart failure; many of these pathways are linked to cardiac sarcoplasmic reticulum (SR) Ca2+ cycling, as intracellular Ca2+ handling is the central coordinator of cardiac function. Since defects in SR Ca2+ cycling are a common pathophysiological characteristic of heart failure, targeting defects in Ca2+ handling is proposed as adjunctive therapy in end-stage heart failure. Further investigating the roles of known proteins and novel proteins in the Ca2+ cycling pathway is critical in identifying the best potential targets for therapeutic drugs. A recently discovered histidine-rich calcium binding protein (HRC) may play a role in Ca2+ homeostasis in the SR. However, its specific role in SR Ca2+ cycling remains to be elucidated. Our data indicate that: (a) HRC regulates SR Ca2+ uptake and is an integral regulatory protein in the cardiac muscle Ca2+ cycling cascade; (b) an increase in HRC protein levels results in cardiac dysfunction; and (c) HRC may be a therapeutic target for heart failure. We also further investigated the role of another important Ca2+ cycling regulatory protein, phospholamban (PLN), in the context of ischemia and reperfusion. Our data reveal that decreased particulate partitioning of protein kinase C epsilon (PKCε) contributes to the increased susceptibility to ischemic injury in PLN deficient hearts and that augmentation of PKCε particulate partitioning through myocardial specific expression of a selective PKCε translocation activator, ψεRACK, in hearts at an increased risk for ischemia is sufficient to confer cardioprotection. The data generated and implications of these data suggest a promising therapeutic target for heart failure may be the recently discovered regulator of SR Ca2+ uptake, HRC, while there should be caution in (open full item for complete abstract)

Committee: Dr. Evangelia Kranias (Advisor) Subjects: Biology, Molecular

Doctor of Philosophy, The Ohio State University, 2012, Molecular, Cellular and Developmental Biology

Psoriasin (S100A7) is expressed in several epithelial malignancies including breast cancer. Although S100A7 is associated with the worst prognosis in estrogen receptor α-negative (ERα-) invasive breast cancers, its role in ERα-positive (ERα+) breast cancers is relatively unknown. We investigated the significance of S100A7 in ERα+ breast cancer cells and observed that S100A7-overexpression in ERα+ breast cancer cells, MCF7 and T47D, exhibited decreased migration, proliferation, and wound healing. These results were confirmed in vivo in a nude mouse model system. Mice injected with S100A7-overexpressing MCF7 cells showed significant reduction in tumor size compared to mice injected with vector control cells. Mechanistic studies revealed that S100A7 mediates the tumor-suppressive effects by regulating β-catenin/TCF4 and p53 pathways. We observed down-regulation of β-catenin, p-GSK3β, TCF4, cyclin D1, and c-myc in S100A7-overexpressing ERα+ breast cancer cells. In addition, we observed increased expression of GSK3β. Treatment with GSK3β inhibitor CHIR 99021 increased the expression of β-catenin and its downstream target c-myc in S100A7-overexpressing cells. Tumors derived from mice injected with S100A7-overexpressing MCF7 cells also showed reduced activation of the β-catenin/TCF4 pathway. Our results also demonstrated that S100A7-overexpression in MCF7 cells increased the stability of p53 compared to vector control cells. Co-immunoprecipitation studies revealed that S100A7 binds to and co-localize with p53 in the nucleus of S100A7-overexpressing cells. In addition, we found that phosphorylation of p53 at Serine-15 residue was enhanced in S100A7-overexpressing MCF7 cells. Serine-15 is known to stabilize and activate p53 during cellular stresses. Further, real time PCR p53 array and immunoblotting experiments showed that S100A7-overexpression in MCF7 cells up-regulated ATR and its downstream molecules p-Chk1 and p-Chk2 which are known to phosphorylate serine-15 of p53. We (open full item for complete abstract)

Committee: Ramesh Ganju PhD (Advisor); Sujit Basu PhD (Committee Member); James Waldman PhD (Committee Member); Xue-Feng Bai PhD (Committee Member) Subjects: Oncology

Doctor of Philosophy, The Ohio State University, 2011, Neuroscience Graduate Studies Program

Drug dependence is a persistent problem throughout the world. Once addicted to a drug, many users have difficulty quitting use, even when they desire to stop using. This substance dependence is both psychological as well as neurophysiological. In 2008 the U.S. Department of Health classified 22 million Americans as having a significant degree of drug dependence. Additionally, the U.S. Office of National Drug Control Policy stated that the health care costs in 2004 for substance abuse was estimated to be comparable to cancer. There is therefore undoubtedly both an individual and societal need for therapeutic interventions in drug dependence. While a great deal is known about the molecular action of these addictive substances, very little is understood about the underlying neurocircuitry of addiction. The process of addiction is a learned behavior and is not solely driven by homeostatic adaptations to the drug itself. Psychoactive drugs overrun the natural reward circuitry of the brain, forcing maladaptive learning of drug associated rewards which then become overvalued in comparison to natural rewards. This dissertation approaches the study of drug addiction using two separate techniques: one regionally specific to the nucleus accumbens, and one cell type specific to dopaminergic and GABAergic neurotransmitter systems of the central nervous system. All drugs of abuse increase striatal dopamine release despite their widely varied mechanisms of action. The ventral striatum primarily consists of the Nucleus Accumbens (NAc), which has long been thought of as the origin of addiction-like behaviors. As such, the NAc has been proposed to be one of the primary reward centers of the brain. Utilizing viral delivery of short-hairpin RNA (shRNA) into the NAc, knockdown of specific subtypes of receptors can be achieved. In this manner, behavioral testing can investigate changes in rodent addiction-like behavior following region specific knock down of dopamine receptor isoforms. (open full item for complete abstract)

Committee: Howard Gu PhD (Advisor); Wolfgang Sadee PhD (Committee Member); Rene Anand PhD (Committee Member); John Oberdick PhD (Committee Member); Susan Cole PhD (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology; Neurobiology; Neurosciences; Pharmacology

Doctor of Philosophy, The Ohio State University, 2010, Biochemistry Program, Ohio State

Hypertension represents a major risk factor for cardiovascular diseases. Increased mechanical stress/hypertension in the vessel wall triggers the hypertrophic signaling pathway, resulting in structural remodeling of vasculature. We have developed a novel transgenic mouse model by overexpressing the cDNA of human profilin1 in the blood vessels of transgenic mice. We showed that increased expression of profilin1 protein in the medial layer of the aorta induces stress fiber formation, triggering the hypertrophic signaling resulting in vascular hypertrophy and, ultimately, hypertension in older mice. Our results showed significant increases in the expression of alpha1integrin (280%), beta1 integrin (325%) and the activation of Rho/ROCKII pathway (260% and 350%, respectively) in profilin1 mesenteric arteries. The activation of Rho/ROCK led to the inhibition of eNOS expression (39%) and phosphorylation (35%), while an increase in myosin light chain phosphorylation (p-MLC20) (372%). There were also increases in hypertrophic signaling pathways i.e. phospho-ERK1/2 (p-ERK1/2) (312.15%) and phospho-JNK (p-JNK) (232.5%). Functional analyses of profilin1 mesenteric arteries showed significant increases in the vascular responses towards phenylephrine, while significant decreases in response towards ROCK inhibitor Y27632, acetylcholine (Ach), sodium nitrite NaNO2 and cytochalasin D. In addition, we assessed the effects of losartan, amlodipine or atenolol on vascular hypertrophy-associated hypertension, by treating the profilin1 transgenic mice for 4 weeks. Our myograph results showed improvement in the contraction response towards phenylephrine and in the relaxation response towards Ach and NaNO2 in losartan- and amlodipine-treated profilin1 mice. Western blot analyses using mesenteric arteries of lasortan and amlodipine-treated profilin1 mice showed significant decreases in their signaling respectively as follows: the expression of alpha1 integrin (104% and 93%) and beta1 int (open full item for complete abstract)

Committee: Altaf Wani (Advisor); Hamdy Hassanain (Committee Member); Arthur Strauch (Committee Member); Robert Snapka (Committee Member) Subjects: Biochemistry; Pharmacology; Pharmacy Sciences

Doctor of Philosophy, The Ohio State University, 2010, Molecular, Cellular and Developmental Biology

B-cell chronic lymphocytic leukemia (B-CLL), the most common human leukemia in the world, is a malignancy of mature B-lymphocytes. Deregulation of the TCL1 oncogene is a causal event in the pathogenesis of the aggressive form of this disease as was verified by using animal models. To study the mechanism of TCL1 regulation in CLL, we carried out microRNA expression profiling of three types of CLL: indolent CLL, aggressive CLL, and aggressive CLL showing 11q deletion. We identified distinct microRNA signatures corresponding to each group of CLL. We further determined that TCL1 expression is regulated by miR-29 and miR-181, two microRNAs differentially expressed in CLL. Expression levels of miR-29 and miR-181 generally inversely correlated with TCL1 expression in the CLL samples we examined. Our results suggest that TCL1 expression in CLL is, at least in part, regulated by miR-29 and miR-181 and that these microRNAs may be candidates for therapeutic agents in CLLs overexpressing TCL1. Human B-CLL occurs in two forms: aggressive (showing high ZAP-70 expression and unmutated IgH VH) and indolent (showing low ZAP-70 expression and mutated IgH VH). We found that miR-29a is upregulated in indolent human B-CLL compared to aggressive B-CLL and normal CD19+ B-cells. To study the role of miR-29 in B-CLL, we generated Eµ-miR-29 transgenic mice overexpressing miR-29 in mouse B-cells. Flow cytometric analysis revealed a markedly expanded CD5+ population in the spleen of these mice starting at 2 months of age. 85% (34/40) of miR-29 transgenic mice exhibited an expanded population of CD5+ B-cells, a characteristic of the B-CLL phenotype. An average of 50% of the B-cell population in these transgenics was CD5 positive. At the age of 2 years these mice showed significantly enlarged spleens and an increase in CD5+ B-cell population of up to 100% of B-cells. Of 20 Eµ-miR-29 transgenic mice followed up to the age of 24-26 months, 4 (20%) developed frank leukemia and prematurely died from (open full item for complete abstract)

Committee: Carlo Croce MD (Advisor); Kay Huebner PhD (Committee Member); Denis Guttridge PhD (Committee Member); Amanda Toland PhD (Committee Member) Subjects: Biomedical Research; Genetics; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2005, Molecular, Cellular, and Developmental Biology

In this dissertation, we examine the in vivo functions of two cell junction proteins, CASK and Claudin-5. CASK is a member of the membrane associated guanylate kinase family of proteins, while Claudin-5 is a protein that has been most thoroughly characterized as a component of tight junctions. This dissertation describes investigations of the CASK protein in skeletal muscle at the neuromuscular junction (NMJ) and Claudin-5 in cardiomyocytes We show that the CASK protein is present in skeletal muscle and is predominantly localized to the primary gutter of the NMJ, with a small amount of presynaptic localization. Immunoprecipitations reveal that CASK interacts in vivo with Dlg. We also examined the CASK protein in the C2C12 myogenic cell line and found that CASK localization is developmentally regulated and determined that CASK is recruited to the NMJ by a mechanism with is independent of that which recruits acetylcholine receptors. Finally, we generated two lines of transgenic mice, which overexpress a full-length or truncated version of the CASK protein. Overexpression of either the full-length or truncated CASK protein does not result in any morphological or functional abnormalities of the skeletal muscle or NMJ. However, overexpression of either CASK protein results in a loss of CASK protein presynaptically at the NMJ. Finally, overexpression of either CASK protein did not alter the expression or localization of Dlg. This dissertation also details an examination of the Claudin-5 protein in cardiac muscle from wild-type mice and two mouse models of cardiomyopathy, the mdx and dko mouse. We show that the Claudin-5 protein is present in normal cardiac muscle and is localized to the lateral membranes of cardiomyocytes. We also demonstrate that Claudin-5 protein expression and localization is unaltered in hearts from mdx mice, but is downregulated in dko hearts, as compared to normal hearts. Expression levels of cell junction proteins present at the intercalated disc w (open full item for complete abstract)

Committee: Jill Rafael-Fortney (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2004, Molecular, Cellular, and Developmental Biology

Ets transcription factors have been implicated in the development, regulation, and survival of numerous hematopoietic derived cells, including B and T-lymphocytes. E26 avian leukemia oncogene-2 (Ets-2) is an Ets family member transcription factor. Strong expression of Ets-2 is observed in developing thymocytes from the DN1 stage and in B-cells from the pro-B stage. A conserved sequence within the Ets-2 pointed domain amino acids 69 to 73 PLLTP is able to interact with the Map kinase ERK. Threonine seventy-two is phosphorylated by ERK1, and permits transactivation by Ets-2. Mutation of this residue from a threonine to an alanine, Ets-2 T72A, abrogates Ras mediated transactivation of Ets-2. Based on this evidence, we hypothesized that Ets-2 was important in T- and B-cell development, activation, and function. To test this hypothesis we analyzed Ets-2 activation in T-cell lines, and created two transgenic mice lines that express Ets-2 T72A in either developing T-cells or B-cells. Transactivation of Ets-2 can be induced by both Ras activation and by phorbol 12-myristate 13-acetate and ionomycin stimulation in the Jurkat T-cell line. In-vitro analysis of Ets-2 activation by ionomycin results in a rapid, but transient Ets-2 band shift as evidenced by western blot analysis of protein extracts from thymocytes. Transgenic mice that over-express Ets-2 T72A in the thymus displayed a dramatic reduction in thymus size associated with hypocellularity. This reduction was associated with a partial developmental block at the double negative 2 and double negative 3 stage of development, a twenty-fold increase in c-Kit+ expression, and a five-fold increase the CD5low population. Further, thymocytes from the transgenic mice have increased apoptosis both in-vitro and in-vivo compared to non-transgenic littermates. Transgenic mice that over-express Ets-2 T72A in B-lymphocytes revealed a loss of the B220Hi population of B-cells in the bone marrow. This population consists of mature B-cell (open full item for complete abstract)

Committee: Natarajan Muthusamy (Advisor) Subjects: Biology, Cell

Doctor of Philosophy, The Ohio State University, 2003, Microbiology

Human T-cell lymphotropic virus type I (HTLV-1) is a C type retrovirus that is the causative agent of an aggressive T-cell malignancy, adult T-cell leukemia/lymphoma (ATLL). The virus is also implicated in a number of inflammatory disorders, the most prominent among them being HTLV-1 associated myelopathy or tropical spastic paraparesis (HAM/TSP). HTLV-1, like many viruses that cause chronic infection, has adapted to persist in the face of an active immune response in infected individuals. The viral transactivator Tax is the primary target of the cellular immune response and humoral responses are mainly directed against the envelope protein. Vaccination against HTLV-1 is a feasible option as there is very little genetic and antigenic variability. Vaccination regimes against chronic viruses must be aimed at augmenting the immune response to a level that is sufficient to clear the virus. This requires that the vaccine delivers a potent stimulus to the immune system that closely resembles natural infection to activate both the humoral arm and the cellular arm. It is also clear that multicomponent vaccines may be more beneficial in terms of increasing the breadth of the immune response as well as being applicable in an outbred population. As a step in this direction, this dissertation work first describes the identification and evaluation of human T-cell and B-cell epitope based vaccines that are capable of inducing robust immune responses. A novel strategy was designed for delivery of multiple Tax protein derived CTL epitopes into the same antigen presenting cell for the simultaneous priming of anigen specific T-cells. This design allowed antigen processing by cellular proteasomes to efficiently liberate the individual minimal epitopes endogenously for presentation via MHC Class I. This type of construct was effective in vivo in HLA-A*0201 transgenic mice in inducing cellular immune responses against all individual epitopes. A statistically significant reduction in vir (open full item for complete abstract)

Committee: Pravin Kaumaya (Advisor) Subjects: