Department: Integrated Biomedical Science Graduate Program ![[clear]](close-x.png "Remove this limiter")
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1.
Abbaoui, Besma.
BROCCOLI ISOTHIOCYANATES AS CHEMOPREVENTIVE AGENTS AND EPIGENETIC MODULATORS OF BLADDER CANCER.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2011, Ohio State University
► Bladder cancer is the fifth most common neoplasm and one of the…
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▼ Bladder cancer is the fifth most common neoplasm and one of the most expensive cancers to treat and monitor. Epidemiologic evidence suggests that cruciferous vegetables, particularly broccoli, may reduce bladder cancer risk. These vegetables contain high concentrations of glucosinolates (GLUs) that are enzymatically hydrolyzed to bioactive isothiocyanates (ITCs) by myrosinase. The most abundant ITC produced in broccoli, sulforaphane (SFN) is under investigation for its potential chemopreventive activity. Our objectives were to characterize GLUs and ITCs in broccoli and broccoli sprouts, determine their absorption and bioavailability in mice, define their bioactivity in bladder cancer in vitro and in vivo and define their potential mechanisms of action. Broccoli and broccoli sprouts GLUs and ITCs were characterized by HPLC-MS-MS. In vitro effects of non-hydrolyzed (GLUs) and hydrolyzed broccoli and broccoli sprouts (ITCs), and pure ITCs sulforaphane (SFN), erucin (ECN), iberin and allyl ITC, were studied on normal bladder urothelial cells and a panel of human bladder cancer cell lines, representing the spectrum of bladder cancer biology (RT4: non-invasive; J82 and UMUC3: invasive). Cell viability (MTS and SRB assays), apoptosis (caspase-3/7 activity and PARP cleavage) and cell cycle analysis (flow cytometry) were performed. For in vivo studies, female athymic nude mice were subcutaneously injected with UMUC3 cells and fed diets containing 4% freeze-dried broccoli sprouts, or 2% freeze-dried broccoli sprouts extract; or gavaged daily with 295 µmol/kg sulforaphane or 295 µmol/kg erucin (n=12/group). Tumor growth rate was monitored and final tumor weights, cell proliferation (Ki67) and apoptosis (PARP) were compared. Bioavailability of isothiocyanates was determined by extracting plasma, bladder and tumor metabolites and quantifying them by UPLC-MS/MS. The potential of SFN and ECN to act as histone deacetylase inhibitors (HDACI) was assessed by HDAC activity assay, HAT assay, biomarkers of HDACI: AcH3, p21 and TS (western blotting), and by LCMS. We found broccoli sprouts have higher levels of glucosinolates and isothiocyanates than broccoli. Broccoli sprout ITCs and SFN followed by ECN were the most potent inhibitors of bladder cancer cell viability and normal bladder urothelial cells were least sensitive to this inhibition. SFN and ECN resulted in dose-dependent induction of apoptosis and G2/M cell cycle accumulation. In vivo, a significant decrease in final tumor weight (36-60% reduction), decreased tumor cell proliferation (50%) and induction of apoptosis (2-fold) was observed in broccoli sprout and pure ITC gavage treatment groups when compared to control. Metabolites of SFN and ECN were present in plasma, tumor and bladder tissue of all treated groups (pico-micromolar range). Evidence of inter-conversion of SFN and ECN was observed. HDAC and HAT activity were significantly inhibited by both SFN and ECN treatment, AcH3 and p21 were increased and LCMS revealed decrease in phosphorylated histone H1, a plausible novel biomarker of bladder cancer progression. Our studies show that broccoli sprout isothiocyanates are abundant, bioavailable and have significant bioactivity in inhibiting bladder cancer in vitro and in vivo in association with epigenetic modulation. This work supports further preclinical and clinical studies to determine the potential chemopreventive and therapeutic effects of these compounds in bladder cancer.
Advisors/Committee Members: Clinton, Steven.
Subjects: Biomedical Research
Keywords: Broccoli, Bladder Cancer, Isothiocyanates, Proteomics, Metabolism, Transitional Cell Carcinoma
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2.
Aggarwal, Reeva.
Mechanisms of Human CD34+ Stem Cell-Mediated Regulation of Osteoporosis in a Preclinical Model.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2012, Ohio State University
► Osteoporosis is a systemic metabolic bone disorder characterized by low mineral density…
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▼ Osteoporosis is a systemic metabolic bone disorder characterized by low mineral density and micro architectural deterioration of skeleton. Osteoporosis or “porous bone” disease can progress asymptomatically until bones fracture. It is a major cause of morbidity and health care burden worldwide. Lack of physical activity, hormonal disturbances, age, gender, heredity and nutritional deficiency are some the factors implicated in causing the disease. Physiologically, bone is a dynamic organ that constantly undergoes remodeling. Bone consists of dense organic and inorganic components. At cellular level, balance/coupling between the activities of bone forming cells (osteoblasts) and bone resorbing cells (osteoclasts) maintains bone homeostasis. Increased bone loss due to increased osteoclast and decreased osteoblast activities is considered as an underlying cause of osteoporosis. Several therapies such as hormone replacement therapy in women, administration of anti-resorptives (bisphosphonates), implantation of osteoinductive device combined with change in lifestyle offers only temporary augmentation of bone mineral density with side effects. Consequently, the potential of stem cell based therapies is currently being considered. Adult stem cells expressing CD133/CD34 surface marker are multipotent cells that can be isolated from human umbilical cord blood and differentiated into many cell lineages by changing the molecular cues in their microenvironment. However, their limited numbers from a single unit restricts their experimental use. Our lab developed a nanofiber-based expansion technology to obtain adequate numbers of CD34+ cells isolated for experimental use and potential therapeutic applications. Herein, we show that nanofiber-expanded CD34+ cells could be differentiated into osteoblastic lineage, in vitro. Systemically delivered CD34+ cells home to the bone marrow and significantly improve bone deposition, BMD and bone micro-architecture in osteoporotic aged nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. The elevated levels of osteocalcin, IL-10, GM-CSF, and decreased levels of MCP-1 in serum parallel the improvements in bone micro-architecture. Furthermore, CD34+ cells improved osteoblast activity and concurrently impaired osteoclast differentiation, maturation, and functionality. Mechanistically, CD34+ cells offer osteoprotection via the upregulation of osteoblastic factors such as Runt-related transcription factor 2 (Runx2) and its downstream effectors such as collagen type I alpha1 and osteocalcin. Consistent with our in vivo observations of impaired osteoclastic activities after CD34+ stem cell therapy, CD34+ cells have the ability to regulate nuclear translocation of osteoclastogenic differentiation factor called nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), and thus negatively regulating its downstream transcriptional activity, and osteoclastogenesis. Taken together, these findings demonstrate a novel approach of utilizing nanofiber-expanded CD34+ cells as a therapeutic application for the treatment of osteoporosis in a preclinical model.
Advisors/Committee Members: Das, Hiranmoy.
Subjects: Biomedical Research
Keywords: "Umbilical Cord, CD34+ cells, Osteoblast, Osteoclast"
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3.
Alachkar, Houda.
Novel Biological Insights and Therapeutic Approaches in High-Risk Acute Myeloid Leukemia.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2012, Ohio State University
► Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal…
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▼ Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal proliferation of myeloid progenitors with a reduced ability to differentiate into more mature functional blood cells. Consequently, an accumulation of leukemic cells (blasts) in bone marrow and blood occurs and leads to hematopoietic failure. Even with recent progress in understanding the biologic and genetic changes that underlie the disease, most patients with AML fail to achieve long-term survival. This highlights the need for novel therapeutic strategies that would improve outcome. Cytogenetic aberrations have long been shown to be the most important risk factor for predicting outcome in AML and have therefore been used to guide treatment. However, each cytogenetic risk group presents with molecular heterogeneity, which may explain different outcome among patients with similar chromosome aberrations. Cytogenetically normal AML (CN-AML) is the largest cytogenetic group (45% of AML) and the best molecularly characterized among both younger and older AML patients. Frequent genetic mutations with prognostic significance have been identified in CN-AML. Patients with FLT3 internal tandem duplication (ITD) or IDH2-R172 have been shown to have worse outcome, while patients with CEBPA or NPM1 mutations are commonly reported to have more favorable prognosis. The cytogenetic and molecular aberrations associated with AML influence the expression of downstream target genes that encode proteins involved in complex biologic networks supporting leukemogenesis. Therefore, microarray genome-wide gene-expression profiling and microRNA-expression profiling assays provide molecular insight into the underlying biology of the different disease subsets, offer diagnostic and prognostic information, and potentially reveal novel therapeutic targets. The research we are presenting in the following chapters has two major aims. Aim 1: identify novel therapeutic targets and prognostic markers employing genome-wide analyses of gene expression signatures in high risk AML patients. Aim 2: investigate novel therapeutic approaches directed toward previously identified molecular targets in high risk disease. In chapter #2 we identified SPARC as a novel molecular and therapeutic target in AML. We demonstrated a functional, mechanistic and clinical implication of SPARC in AML, revealing that high SPARC expression promoted growth advantage in vitro, aggressive disease in vivo, and worse outcome in CN-AML patients. SPARC acts likely via activation of ILK/AKT/ï¢-catenin pathways. We also identified RUNX1 and IDH2-R172 mutations as an upstream upregulators of SPARC expression likely via NFκb/SP1 complex mediating mechanisms. In chapter #3 we investigated the preclinical and pharmacological activity of the natural initiation of translation inhibitor, silvestrol in FLT3-ITD positive AML denoting a large proportion of high risk AML patients. We demonstrated that silvestrol had a potent in vitro and in vivo anti-leukemia activity in FLT3-ITD and FLT3 overexpressing AML cells. Silvestrol inhibited FLT3 mRNA translation resulting in FLT3 protein down-regulation and in turn inhibition of aberrant tyrosine kinase activity. In conclusion, we identified an original molecular target with novel biological insights, and investigated innovative therapeutic approaches suitable for high risk subsets of AML. This research will significantly advance understanding of AML and open new avenues of treatment strategies, which will result in optimized patient care, and improved clinical outcome.
Advisors/Committee Members: Marcucci, Guido.
Subjects: Oncology; Pharmacology
Keywords: AML, SPARC, Silvestrol
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4.
Allen, Rebecca G.
Macrophage Microbicidal Activity is Enhanced by Stressor-Exposure.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2012, Ohio State University
► Exposure to social stressors is known to prime the innate immune system…
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▼ Exposure to social stressors is known to prime the innate immune system for enhanced reactivity to inflammatory stimuli, but the mechanisms by which stressor exposure can enhance immune activity are not well-defined. In mice, exposure to a social stressor called social disruption (SDR) increases circulating cytokines and primes splenic macrophages for an enhanced capacity to kill Escherichia coli, primarily through an increased production of the highly microbicidal compound peroxynitrite. Previous results demonstrate that the intestinal microbiota are in part responsible for the SDR-induced increase in circulating cytokines; reducing the microbiota through the use of a broad spectrum antibiotic cocktail prevented the SDR-induced increase in IL-6 and MCP-1. These studies tested the hypothesis that intestinal microbiota also contribute to the stressor-induced increase in the ability of splenic macrophages to kill E.coli. To test this hypothesis with SDR, groups of co-housed male mice were repeatedly defeated through direct interactions between the resident mice and an aggressive intruder. Following stress, E. coli were co-cultured with the splenic macrophages, and the number of bacteria within the macrophages was enumerated at 20 and 90 min (to determine the number of bacteria phagocytosed and then killed, respectively). We also measured changes in iNOS and pro-inflammatory cytokine expression as well as production of both superoxide anion and its reaction product with nitric oxide, peroxynitrite. When endogenous bacterial populations were eliminated through the use of germ free mice or reduced in mice treated with an antibiotic cocktail were stressed with SDR, we failed to observe the characteristic increases in pro-inflammatory cytokine and iNOS expression, superoxide anion, and peroxynitrite production. This lack of stressor-induced changes in splenic macrophage activity was associated with the failure of the stressor to enhance bacterial killing typically associated with SDR. However, when germ free mice were conventionalized to contain the normal gut microbiota and exposed to SDR, the enhanced pro-inflammatory cytokine and iNOS expression, superoxide anion, peroxynitrite, and ultimately the enhanced killing were restored. Additionally, the ability of the gut microbiota to prime splenic macrophages is associated with stressor-increased translocation of bacteria and their products from the intestinal lumen, as evidenced by an increase in circulating peptidoglycan. Importantly, the enhanced translocation of bacterial products is linked with stressor-induced mast cell degranulation and disruption of the intestinal epithelial barrier. When mast cells were inhibited from degranulating in vivo, stressor-exposure failed to enhance splenic macrophage microbicidal activity. Overall, these results indicate that the stressor-enhanced activity of CD11b+ splenic macrophages requires the degranulation of mast cells to disrupt the intestinal barrier and allow the translocation of bacteria and their products. These products stimulate production of pro-inflammatory cytokines which primes the macrophages for enhanced microbicidal activity through increased peroxynitrite production.
Advisors/Committee Members: Bailey, Michael.
Subjects: Biomedical Research; Immunology
Keywords: Stress; macrophage; bacterial killing; gut microbiota; peroxynitrite
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5.
Alvarez-Breckenridge, Christopher.
The Role of Natural Killer Cells in the Context of Oncolytic Herpes Simplex Virotherapy for Glioblastoma.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2011, Ohio State University
► It is controversial as to whether the host immune response hinders or…
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▼ It is controversial as to whether the host immune response hinders or improves the efficacy of oncolytic Herpes Simplex viral (oHSV) therapy of glioblastoma (GBM). Natural killer cells (NK) limit viral infections, and previous work suggests they may similarly attenuate virotherapy. Using both xenograft and syngeneic intracranial GBM tumor models, we used flow cytometry to evaluate the temporal pattern and phenotypic characteristics of NK cells present in the periphery and recruited to the site of oHSV infection. Within hours after infection and continuing through 72 hours following oHSV inoculation, NK cells were rapidly recruited to tumor bearing hemispheres. Moreover, these NK cells exhibited an activated phenotype, including enhanced CD69, CD62L, CD27, NKG2D, and Ly49D staining compared to vehicle treated mice. However, neither the number nor phenotype of peripheral NK cells was altered following oHSV infection. This robust NK response was confirmed to be detrimental to OV efficacy through the enhanced survival of NK depleted mice inoculated with oHSV compared to oHSV treated mice possessing NK cells. Interestingly, oHSV treated mice exhibited robust macrophage recruitment and activation at the site of infection. This was accompanied by the induction of macrophage/microglial associated inflammatory gene and protein expression, including CXCL9, CXCL10, CXCL11, iNOS, and TNF-α. However, when mice were depleted of their NK cells or IFN-γ knockout mice were used, their expression was abrogated. In vitro, human NK cells preferentially lysed oHSV-infected GBM in a cell contact, perforin, and DNAM-1 dependent manner. Fusion proteins were used to detect currently unknown ligands for the NK natural cytotoxicity receptors (NCR) and decipher the critical NK activating ligands that mediate this response. Following oHSV infection of a panel of GBM stem cells and cell lines, we detected robust up-regulation of ligands for NKp46 and NKp30. GFP expression was used to discriminate oHSV infected GBM cells and preferential NKp30/NKp46 ligand expression was found in the GFP+ population of cells. Additionally, blocking antibodies against either NKp30 or NKp46 abrogated NK mediated clearance of oHSV infected GBM, while antibodies against NKp44 did not inhibit killing. We have previously shown that immunomodulation with cyclophosphamide (CPA) and valproic acid (VPA) enhances oHSV efficacy. CPA administered prior to virus inoculation abrogated the oHSV induced NK and macrophage recruitment into the tumor at all time points tested compared to oHSV alone. Similarly, VPA treatment resulted in a decline in NK and macrophage recruitment at 6 and 24 hours post-oHSV; however, a robust increase at 72 hours-post-oHSV was seen, resembling the response seen with oHSV alone. VPA was also found to have a profound immunosuppressive effect on human NK cells in vitro. NK cytotoxicity was abrogated following exposure to VPA through down-modulation of cytotoxic gene expression and granzyme B protein levels. In addition, IFN-γ was suppressed in a Stat5/T-bet dependent manner. Collectively, these findings demonstrate that oHSV therapy for GBM is limited in part by a robust NK cell response mediated by specific NCRs, uncovering novel potential targets to enhance cancer virotherapy. Moreover, pharmacological co-therapies, such as CPA and VPA with oHSV, alter the host immune response to the virus albeit in differing ways. Future work will be needed to further define the nature of the innate immune response, how it coordinates downstream anti-tumor immunity, and how pharmacological agents can be optimized to modulate the host response to oHSV.
Advisors/Committee Members: Chiocca, E. Antonio.
Subjects: Oncology
Keywords: oncolytic virus; natural killer cell; glioblastoma
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6.
Anthony, Bryan Alan.
THE ROLE OF CD103 EXPRESSION IN PROMOTING INTESTINAL GRAFT VERSUS HOST DISEASE.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2011, Ohio State University
► The limiting factor to the broad use of bone marrow transplantation (BMT)…
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▼ The limiting factor to the broad use of bone marrow transplantation (BMT) to cure hematopoietic malignancy is graft verses host disease (GVHD). GVHD is caused by mature T cells present in the bone marrow graft that recognize and destroy host epithelial tissue. Mature T cells facilitate several beneficial effects including elimination of residual malignant cells, the so-called graft-vs-leukemia (GVL) effect. The goal of this work was to develop a strategy for attenuating GVHD pathology mediated by donor T cells without compromising beneficial GVL effects. During GVHD, integrins present on the surface of T cells dictate T cell localization and accumulation patterns. CD103 is an integrin that is upregulated on CD8 T cells during GVHD. Given that the only known ligand of CD103 is the epithelial-specific molecule E-cadherin, we postulated that CD103 expression is required for destruction of the gut epithelium during GVHD, but is not required for effective clearance of a blood malignancy. To test this overall hypothesis, we first characterized CD103 expression and its role in promoting GVHD pathology in the host gut in both an MHC-disparate and a clinically relevant MHC-matched GVHD model. We then assessed the capacity of donor T cells on the CD103-deficient background to mediate GVL effects. Our studies showed that CD103 expression was required for optimal accumulation of CD8 T cells in the gut epithelium. Co-depletion of iii CD4 and CD103 expressing cells completely prevented mortality following MHC-disparate BMT. Interestingly, CD103 depletion exacerbated GVHD pathology following MHC-matched GVHD induction. Adoptive transfer studies showed that T cells from CD103-deficient donors cleared malignant B cells as effectively as those from wild-type donors. Our data show a conflicting role for CD103 following BMT, however, our data suggest that CD103 expression on CD8 T cells promotes intestinal pathology, but is not required for host defense against hematopoietic malignancy. Together, these data suggest that CD103 blockade may have the potential to prevent gut GVHD pathology without compromising GVL effects.
Advisors/Committee Members: Hadley, Gregg.
Subjects: Immunology
Keywords: GVHD, CD8 T cell, CD103, GVL
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7.
Bevan, Adam Kimball.
AAV-based approaches to model and treat spinal muscular atrophy.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2012, Ohio State University
► The discovery of AAV9’s ability to transduce motor neurons along the entire…
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▼ The discovery of AAV9’s ability to transduce motor neurons along the entire length of the spinal cord has the potential to revolutionize the field of gene therapy. The most obvious disease to treat with this new technology is spinal muscular atrophy (SMA), a monogenetic disease where spinal motor neurons are the primary site of pathogenesis. Additionally, SMA is a good choice for gene therapy since all patients express at least some SMN, thereby decreasing the concern of anti-transgene immune responses. Currently, there is no therapy for children with SMA besides respiratory support and palliative care, so new therapies for this disease are desperately needed. After showing remarkable success in treating a severe mouse model of SMA, we have focused on answering the most important questions regarding how to best translate this potential therapy to the clinic. We have determined the age-related window of opportunity for targeting spinal motor neurons and assessed toxicity and safety of the proposed clinical doses in mice and nonhuman primates. In the course of our studies we discovered the surprising finding that SMA mice have a severe heart failure phenotype. Thus increased cardiac surveillance in SMA clinical trial patients is warranted. While the severe form of SMA is also the most common, we sought to assess an alternate muscle-enhancing therapy that successfully increased strength in a less-severe mouse model of SMA. Finally, we have created a model that accurately models less-severe SMA by using AAV9 to deliver a mouse-specific shRNA against Smn. This technique has allowed us to control the amount of Smn produced throughout the spinal cord more tightly than has been accomplished before, yielding a reliable model in which the full spectrum of SMA can be accurately modeled. Together, the data we have collected have helped to guide the design of the first-in-human clinical trial of AAV9, which will push the boundary of AAV-based medicine farther than any single trial has before.
Advisors/Committee Members: Kaspar, Brian.
Subjects: Molecular Biology; Neurobiology; Neurology; Neurosciences
Keywords: spinal muscular atrophy; SMA; AAV; gene therapy; follistatin
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8.
Beyer, Sasha Jasmine.
Modulation of Cell Surface Sodium/Iodide Symporter Expression and Activity in Breast Cancer.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2010, Ohio State University
► The sodium iodide symporter (NIS) mediates iodide uptake into thyroid follicular cells…
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▼ The sodium iodide symporter (NIS) mediates iodide uptake into thyroid follicular cells for the biosynthesis of thyroid hormones and also serves as the basis of radioiodine therapy of thyroid cancer. NIS is also expressed in the majority of breast tumors, suggesting that breast cancer patients may also benefit from radionuclide therapy. However, only a fraction of NIS-positive breast tumors have detectable radionuclide uptake that may or may not be sufficient for therapy. The objective of this study was to elucidate mechanisms of NIS regulation in breast cancer such that strategies for selectively increasing NIS-mediated radionuclide therapy can be devised. Previous studies have reported that predominant intracellular localization of NIS caused by cell surface trafficking impairments likely accounts for low radionuclide uptake in breast cancer, as NIS must be localized at the cell surface in order to confer functional iodide uptake. However, cross-reactivity of antibodies appears to account for some intracellular NIS immunostaining, suggesting that NIS cell surface trafficking impairments may be overestimated. Moreover, cell surface NIS protein levels among breast tumors are low, with less than 29% strongly expressing cell surface NIS protein. Whether cell surface trafficking impairments or low cell surface NIS expression account for undetectable radionuclide uptake must be determined such that appropriate strategies for increasing cell surface NIS are developed. Strategies that differentially regulate NIS expression/activity between thyroid and breast cancer may not only increase NIS expression/activity in breast cancer, but also minimize iodide uptake in thyroid. KT5823, a staurosporine-related protein kinase inhibitor, increases thyroid stimulating hormone-induced NIS expression/function in thyroid cells, but reduces iodide transport rate and NIS affinity for iodide in trans-retinoic acid and hydrocortisone (tRA/H)-treated MCF-7 breast cancer cells. Moreover, while KT5823 has little effect on NIS glycosylation in thyroid cells, longer KT5823 treatments result in accumulated hypoglycosylated NIS and further reduction in RAIU in MCF-7/tRA/H cells in addition to other breast cancer cells overexpressing exogenous NIS. KT5823 may help elucidate mechanisms of NIS regulation at multiple levels, including expression, glycosylation and activity. In order to identify potential biomarkers associated with NIS expression, published cDNA microarray data were analyzed to elucidate gene expression tightly correlated with NIS mRNA expression among human breast tumors. However, the sensitivity of oligonucleotide microarray technology was not sufficient to detect variability in NIS mRNA expression in breast cancers. We then immunostained a TMA composed of 28 human breast tumors which had corresponding oligonucleotide microarray data available for each tumor for NIS protein such that gene expression highly associated with cell surface NIS protein level could be identified. Despite a limited number of breast tumors, our analysis identified cysteinyl-tRNA synthetase as a biomarker that is highly associated with cell surface NIS protein level in ER-positive breast cancer subtype. Taken together, gene expression profiling for genes associated with cell surface NIS protein levels within each breast cancer subtype may lead to novel targets for selectively increasing NIS expression/function in breast cancers patients.
Advisors/Committee Members: Jhiang, Sissy.
Subjects: Molecular biology
Keywords: radionuclide therapy, breast cancer, sodium iodide symporter
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9.
Britt, Rodney Deon Jr.
The Role of Cyclooxygenase-2 in Newborn Hyperoxic Lung Injury.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2012, Ohio State University
► Development of respiratory distress syndrome (RDS) adversely affects patient populations in neonatal…
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▼ Development of respiratory distress syndrome (RDS) adversely affects patient populations in neonatal and pediatric intensive care units. Patients with RDS require ventilation and oxygen therapy to maintain adequate tissue oxygenation. Preterm infants who develop RDS are at risk of developing the chronic lung disease, bronchopulmonary dysplasia (BPD). Lung ventilation and exposure to supraphysiological concentrations of oxygen contribute to the risk of developing BPD. Preterm infants with BPD have reduced alveolar and vascular development. Survivors of BPD have diminished lung function and are at risk of developing additional lung diseases such as asthma. Dysregulation of the inflammatory response is a significant contributing factor to BPD. Previous studies showed that cyclooxygenase-2 (COX-2) expression and leukocyte infiltration are increased in the lung during newborn hyperoxic exposure in mice. To determine the role of COX-2 in newborn hyperoxic lung injury, newborn pups were injected with aspirin (non-selective COX -2 inhibitor) and celecoxib (selective COX-2 inhibitor) during exposure to hyperoxia. We tested the hypothesis that COX-2 inhibition would (1) reduce macrophage infiltration and chemokine expression, (2) improve lung alveolarization, and (3) improve lung function in newborn mice exposed to hyperoxia. Our data suggest that COX-2 has a pro-inflammatory role in macrophage infiltration but is not involved in lung alveolarization during hyperoxic lung injury. Understanding the role of COX-2 in the developing lung during hyperoxic exposure may lead to therapeutic strategies to improve clinical outcomes and prevent development of BPD. The role of nonciliated airway epithelial cells, or Clara cells, during inflammation remains poorly understood. Studies have suggested that Clara cells are critical for regeneration of the airway epithelium and produce mediators which regulate inflammation. Through immuohistochemical analysis, we have found that Clara cells express COX-2. Mouse transformed Clara cells (MTCC) were utilized as an in vitro model to assess Clara cell responses to pro-inflammatory stimuli. We tested the hypothesis that lipopolysaccharide (LPS) would increase COX-2 and chemokine expression in MTCC. Our data show that LPS stimulation increases COX-2 and chemokine mRNA and protein expression, while increasing prostanoid levels. LPS also stimulates phosphorylation of mitogen activating protein kinases: p38, JNK, and ERK. Our data suggest that Clara cells may produce prostaglandins and chemotactic factors during inflammation. We speculate that Clara cells produce mediators to modulate leukocyte infiltration and the progression of inflammation during the pathogenesis of acute lung injury. Further characterization of Clara cell function may help identify therapeutic strategies to enhance regeneration of the airway epithelium in patients with chronic inflammatory lung diseases.
Advisors/Committee Members: Rogers, Lynette.
Keywords: Bronchopulmonary Dysplasia, Cyclooxygenase-2, Clara cells, Aspirin
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10.
Buescher, Jessica L.
Structure-Function Analysis of GSK-3 Isoforms.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2010, Ohio State University
► Glycogen synthase kinase-3 (GSK-3) isoforms, GSK-3α and GSK-3β, are serine/threonine kinases involved…
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▼ Glycogen synthase kinase-3 (GSK-3) isoforms, GSK-3α and GSK-3β, are serine/threonine kinases involved in numerous cellular processes and diverse diseases, including Alzheimer’s disease, diabetes, and mood disorders. Accumulating evidence suggests that GSK-3 isoforms exhibit distinct activities and increasingly challenges the conventional belief that GSK-3 isoforms are functionally redundant. Despite abundant GSK-3-related research, the basis for differential functions of GSK-3 isoforms remains unresolved. Logically, the divergent regions of GSK-3, the N- and C-termini, might be predicted to mediate the differential activities of GSK-3 isoforms at the post-translational level. Herein we test the hypothesis that the divergent N- and C-terminal regions and conserved key residues of GSK-3 isoforms are functionally significant. To test our hypothesis we performed a structure-function analysis of GSK-3α and GSK-3β in mammalian cells. Deletion constructs of the non-catalytic N- and C-terminal domains in both GSK-3 isoforms were created as well as constructs containing point mutations of key regulatory residues. We examined the effect of these deletions and point mutations on GSK-3 activity, protein interactions, tyrosine autophosphorylation, and subcellular localization. We found that the N-termini of both GSK-3 isoforms are dispensable for activity, protein interaction with Axin GID, and tyrosine autophosphorylation, but are required for the GSK-3α-specific interaction with RACK1 WD4-7 and proper localization. In turn, progressive C-terminal deletions resulted in a loss of activity, impaired ability to interact with protein binding partners, a gradual loss of autophosphorylation, and mislocalization. Taken together, these observations imply that deletion of the C-termini of GSK-3 isoforms compromises proper protein folding and suggest that the C-terminal regions make significant contributions to the structural integrity of GSK-3 enzymes. Furthermore, our data predict that the development of therapeutic modulators targeting the C-terminus may result in isoform-specific GSK-3 inhibition through destabilization of GSK-3 structure.
Advisors/Committee Members: Phiel, Christopher.
Subjects: Biochemistry
Keywords: GSK-3, tau, Wnt, autophosphorylation, structure-function
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11.
Chen, Hui-Zi.
Mammalian Atypical E2Fs Link Endocycle Control to Cancer.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2011, Ohio State University
► The endocycle is a developmentally programmed variant cell cycle consisting of successive…
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▼ The endocycle is a developmentally programmed variant cell cycle consisting of successive S (DNA synthesis) and G (Gap) phases without an intervening M phase or cytokinesis. As a consequence of the regulated “decoupling” of DNA replication and mitosis, which are two central processes of the traditional cell division program, endocycling cells acquire highly polyploid genomes after having undergone multiple rounds of whole genome reduplication. Although essential for metazoan development, relatively little is known about the regulation of endocycle or its physiologic role in higher vertebrates such as the mammal. A substantial body of work in the model organism Drosophila melanogaster has demonstrated an important function for dE2Fs in the control of endocycle. Genetic studies showed that both endocycle initiation and progression is severely disrupted by altering the expression of the fly E2F activator (dE2F1) or repressor (dE2F2). In mammals, the E2F family is comprised of nine structurally related proteins, encoded by eight distinct genes, that can be classified into transcriptional activators (E2f1, E2f2, E2f3a and E2f3b) or repressors (E2f4, E2f5, E2f6, E2f7 and E2f8). The repressor subclass may then be further divided into canonical (E2f4, E2f5 and E2f6) or atypical E2fs (E2f7 and E2f8). Until this study, there has been sparse knowledge regarding the role of E2fs in the control of mammalian endocycle, or what the physiologic consequences might be of perturbed endocycles in tissues composed of polyploid cells such as the liver. Now using a combination of novel and established lineage-specific cre mice we identified that two opposing arms of the E2F program, one driven by canonical transcription activation (E2F1, E2F2 and E2F3) and the other by atypical repression (E2F7 and E2F8), converge on the regulation endocycles in vivo. Ablation of canonical activators in the two endocycling tissues of mammals, trophoblast giant cells (TGCs) in the placenta and hepatocytes in the liver, augmented genome ploidy, whereas ablation of atypical repressors diminished ploidy. Surprisingly, the severe reduction of ploidy caused by E2f7/E2f8 deficiency had no apparent adverse impact on placental and hepatic physiology. However, the sole inactivation of E2f8 in hepatocytes, which biases toward a diploid state, resulted in the development of spontaneous hepatocellular carcinoma (HCC). Loss of E2f8 also potentiated the development of carcinogen-induced HCC, suggesting that E2F8 functions to suppress tumor initiation and impede tumor progression. Taken together, the results presented within provide the first in vivo evidence for a direct role of E2Fs in regulating non-traditional cell cycles in mammals and genetically link endocycle control with cancer.
Advisors/Committee Members: Leone, Gustavo.
Subjects: Cellular Biology; Developmental Biology; Genetics; Molecular Biology
Keywords: mammlian endocycles, E2F transcription factors, cell cycle, liver cancer, mouse model
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12.
Cox, Gina Mavrikis.
Macrophage migration inhibitory factor: a study of the effects on the central nervous system microenvironment in experimental autoimmune encephalomyelitis.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2011, Ohio State University
► Multiple Sclerosis (MS) is a progressive immune-mediated demyelinating disease of the central…
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▼ Multiple Sclerosis (MS) is a progressive immune-mediated demyelinating disease of the central nervous system (CNS). Over time, permanent neurological disability accumulates as a result of the chronic activation of inflammatory and neurodegenerative pathways within the CNS. Macrophage migration inhibitory factor (MIF) is a multipotent cytokine that has recently been associated with clinical worsening and relapses in MS. The mechanism by which MIF promotes MS progression remains undefined. In an animal model of MS, experimental autoimmune encephalomyelitis (EAE), we identify a critical role for MIF in regulating CNS effector mechanisms necessary for the development of inflammatory pathology. Despite the ability to generate pathogenic myelin-specific immune responses peripherally, MIF-deficient mice have reduced EAE severity and exhibit less CNS inflammatory pathology. MIF-deficiency appears to mediate host protection from chronic EAE at the level of the CNS, since MIF-deficient mice exhibit significantly less disease following transfer of pathogenic myelin-specific cells. Upon examination of MS brain tissue, we found that MIF is highly expressed in regions of active inflammation, suggesting that MIF may have profound influence on the CNS inflammatory response. These studies highlight the ability of MIF to promote the activation and recruitment of CNS macrophages during EAE: both CNS effector mechanisms critical for EAE progression. MIF exerts potent proinflammatory effects on microglia and polarizes a proinflammatory gene expression profile associated with CNS autoimmune-mediated tissue damage. We propose a novel role for MIF in regulating the transcriptional activity of key mediators of myeloid cell function, including C/EBP-β, a transcription factor previously implicated in neuroinflammation. Lastly, we used intraspinal stereotaxic microinjection to examine the site-specific activity of MIF in the specialized CNS microenvironment. Intraspinal microinjection of MIF fully reconstitutes the ability of MIF-deficient mice to develop autoimmune-mediated inflammatory pathology, suggesting that MIF creates a CNS microenvironment permissive to the development of neuroinflammation. Collectively, these data demonstrate the ability of MIF to influence the CNS cellular and molecular inflammatory milieu in EAE and suggests that targeting MIF may represent a novel therapeutic approach for resolving CNS inflammation in MS.
Advisors/Committee Members: Whitacre, Caroline.
Subjects: Biomedical Research; Immunology
Keywords: macrophage migration inhibitory factor; multiple sclerosis; experimental autoimmune encephalomyelitis
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13.
Cush, Stephanie S.
Generation and Function of CD8 T Cell Memory to γ-Herpesviral Infection.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2010, Ohio State University
► γ-herpesviruses are a worldwide health concern, and result in a lifelong persistent…
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▼ γ-herpesviruses are a worldwide health concern, and result in a lifelong persistent infection that is associated with some degree of immune control preventing the virus from reactivating and causing disease. However, if this immune control is compromised, γ-herpesviruses can reactivate and cause a variety of tumors in the host. Thus, there is a great need to increase our understanding of the immune response to this family of oncogenic viruses. CD8 T cells have been shown to be critical for viral control. The generation, maintenance, and function of CD8 T cell memory during persistent viral infections is poorly understood. The overall objective of my dissertation is to gain an in depth understanding of CD8 T cell memory generation, maintenance, and function during low load persistent viral infections. We are using a well-established murine model of γ-herpes viral infection, murine gamma herpesvirus 68, or γHV68, to study the in vivo effects of a low load persistent infection on the generation and function of memory CD8 T cells. We determined that virus specific memory CD8 T cells are generated and maintained long term during γHV68 persistence through a variety of phenotypic and functional assays. We found that true bona fide memory CD8 T cells are fully functional during γHV68 latency, which is contrary to other chronic viral infections characterized by high antigenic load. We discovered that the γHV68 specific memory CD8 T cells are capable of surviving in a naïve mouse after adoptive transfer and subsequently proliferating homeostatically in response to cytokines. Additionally, the adoptively transferred memory CD8 T cells have the ability to protect against secondary infection with γHV68. We found heterogeneous virus-specific CD8 T cell memory populations during γHV68 latency, and demonstrated that the terminally differentiated CD8 T cell memory is also equally as functional as the central memory CD8 T cell memory. The current two dominating principles in the field of CD8 T cell memory are: 1) acute infection followed by viral clearance and stable memory formation and 2) persistent infection where there is no viral clearance which leads to T cell dysfunction. My data has contributed to the field of immunology by demonstrating that there is an additional option for how CD8 T cells function during persistent viral infections, where viral persistence does not always lead to memory CD8 T cell dysfunction. Our objective is to gain critical knowledge about CD8 T cell memory to elucidate the possible mechanisms of CD8 T cell function and dysfunction during γHV68 infection. The in vivo model provides relevant information for how memory is generated and maintained in the host, and hopes to offer insights to new therapies and prevention strategies, which is important for many diseases afflicting the human population.
Advisors/Committee Members: Flano, Emilio.
Subjects: Immunology
Keywords: T cell; T cell memory
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14.
Cyktor, Joshua Charles.
Immune Correlates of Resistance and Susceptibility to Tuberculosis.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2012, Ohio State University
► Tuberculosis (TB) has been a prevalent cause of morbidity and mortality in…
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▼ Tuberculosis (TB) has been a prevalent cause of morbidity and mortality in human beings for centuries, and is still a global health emergency today. Although not the headline of news in the United States, TB is rampant is sub-Saharan Africa and southeast Asia, where it is the number one killer of patients with acquired immunodeficiency syndrome (AIDS). Of the estimated 2 billion people infected with Mycobacterium tuberculosis (Mtb), the etiological agent responsible for TB, only a small percentage progress to active, contagious disease. This is because the immune system of most infected individuals is capable of effectively containing the infection, whereas in a percentage of individuals this containment will eventually fail, leading to reactivation of TB. Reactivation can arise due to numerous factors, including HIV infection, malnutrition, aging, or other immunosuppression. In some individuals, however, reactivation can occur without any overt immune deficiency. In this work, we sought to gain further understanding of the immunological mechanisms behind TB reactivation in susceptible individuals with otherwise intact immune systems. The differences in susceptibility of human populations are also observed in mice. Inbred strains of mice generally fall into two main categories: those that are relatively resistant to TB reactivation, and those that are relatively susceptible. In this work, we used C57BL/6 mice as a representative of the resistant strains of mice, and CBA/J mice to represent susceptible strains. Previous work by our laboratory has identified a main correlate of reactivation susceptibility in CBA/J mice to be overproduction of the immunosuppressive cytokine, interleukin-10 (IL-10). CBA/J mice genetically deficient in IL-10, were resistant to Mtb reactivation and developed human-like TB lesions, a finding that is unprecedented in the field. We also more clearly defined the susceptibility of CBA/J mice to be the result of significant CD8+ T cell dysfunction. Additional studies demonstrated ways to enhance the immune response to Mtb by removing the inhibitory receptor, killer cell lectin-like receptor G1 (KLRG1), which we hypothesize could significantly shorten the treatment for TB. Overall, this collective work expands our understanding of how susceptibility to Mtb is mediated, and how protective responses may be enhanced to ease the burden of this devastating disease on humanity.
Advisors/Committee Members: Turner, Joanne.
Subjects: Immunology
Keywords: Infectious Disease; Immunology
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15.
Donnelly, Dustin James.
Tailoring the heterogeneous macrophage response to spinal cord injury towards neuroprotection.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2011, Ohio State University
► After SCI, a heterogeneous macrophage response, comprised of resident microglia and blood…
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▼ After SCI, a heterogeneous macrophage response, comprised of resident microglia and blood monocytes, dominates the lesion environment and exerts divergent effects on indices of neurodegeneration and tissue repair. These CNS macrophages vigorously respond to injury, relying on environmental cues for chemoattraction and activation. These heterogenous macrophage response may be regulated by spatiotemporally distinct microenvironments to which each cell is subjected. At the molecular level, this regulation may involve signaling through CX3CR1. Fractalkine (CX3CL1) is a unique chemokine and immunoregulator that binds to the receptor CX3CR1, present on microglia and monocytes. In the healthy CNS, neuronal expression of CX3CL1 promotes a resting microglial phenotype. In the context of injury however, CX3CL1 shed by damaged neurons and endothelia promotes the recruitment and activation of microglia and monocytes. What role CX3CR1 signaling plays in the microglial and monocyte response to SCI has to this point been unexplored. Here we show that intraspinal CX3CL1 and CX3CR1 are dynamically regulated after SCI and that abolishing CX3CR1 signaling in SCI mice confers neuroprotection and promotes functional recovery. This is associated with a significant phenotypic alteration of responding macrophages. Deficient CX3CR1 signaling in CNS macrophages attenuates their ability to synthesize and release inflammatory cytokines and oxidative metabolites. CX3CR1-deficient mice had an increased accumulation of CCR2+Ly6Chi macrophages. Importantly, iNOS expression was virtually abolished on CX3CR1-deficient macrophages. In addition, MHC II expression was also decreased on CX3CR1-deficient macrophages, potentially decreasing harmful lymphocyte activation. These data suggest that CX3CR1 deficiency prevents or alters the recruitment and/or differentiation of harmful macrophages after SCI. Further experiments with chimeric mice showed that mice with wild-type microglia but CX3CR1-deficient monocytes had the highest degree of locomotor recovery after SCI. This work may potentially lead to development of cell-specific therapeutic strategies for tailoring the immune response towards neuroprotection after SCI. What role the spleen, and in particular splenic monocytes, play in the immunopathology associated with SCI has to this point also been unexplored. If different peripheral sources of monocytes contribute different monocyte subsets, then discerning the source of these monocytes may allow future therapies to target certain populations. We found that in normal wild-type mice after SCI, that the circulating monocyte pool remains relatively constant, even during robust intraspinal accumulation. Splenic numbers decrease immediately after injury, suggesting that the spleen may be responsible for a large degree of the initial buffering of the circulation. In addition, there is a colossal increase in the bone marrow population after SCI, suggesting injury-induced myelopoiesis. The monocyte/macrophage population in the spinal cord decreases through 14 days post-injury in splenectomized mice. The phenotype of intraspinal macrophages is also altered with splenectomy. However, we found no effect on locomotor recovery or tissue sparing resulting from splenectomy. Thus, a more extensive characterization of the role of the spleen in post-SCI inflammation is warranted.
Advisors/Committee Members: Guttridge, Denis.
Subjects: Biomedical Research; Immunology; Neurosciences
Keywords: spinal cord injury; microglia; macrophages; CX3CR1; fractalkine; spleen; neuroimmunology
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16.
Dunn, Melinda Carol Cox.
Inhibition of Respiratory Syncytial Virus In Vitro and In Vivo by the Experimental Immunosuppressive Agent Leflunomide.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2010, Ohio State University
► Respiratory syncytial virus (RSV) is the primary cause of bronchiolitis and pneumonia…
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▼ Respiratory syncytial virus (RSV) is the primary cause of bronchiolitis and pneumonia in infants and young children worldwide. RSV is an opportunistic pathogen that is often unsuspected as the cause of infections in solid organ and bone marrow transplant recipients, patients suffering cystic fibrosis or congenital heart disease, and the elderly. Ribavirin, the only currently approved antiviral therapy, is only marginally effective against RSV and has not been shown to reduce mortality due to infection. In addition, ribavirin must be administered by small-particle aerosol for 12 to 18 hours per day for 3 to 7 days, thus requiring hospitalization. The only option for prevention in at-risk infants and transplant patients is passive immunoprophylaxis with monoclonal antibody specific for the fusion protein of RSV. Leflunomide, an anti-inflammatory drug approved for treatment of rheumatoid arthritis, is an inhibitor of protein kinase activity and pyrimidine synthesis. It has been shown to be effective against acute and chronic transplant rejection in animal models and is currently showing promise as an immunosuppressant in clinical trials in human transplant recipients. Studies have also shown it to exert powerful antiviral activity against cytomegalovirus, herpes simplex virus, and polyomavirus BK. In the current study, we have tested the hypothesis that leflunomide possesses antiviral activity against RSV in vitro and in vivo. Phase contrast microscopy and immunohistochemical staining studies of RSV-infected cultured HEp2 cells and human small airway epithelial cells showed profound attenuation of virally-induced cytopathic change and syncytia formation in cultures incubated in the presence of A77 1726, the active metabolite of leflunomide. Data generated by quantitative plaque assay experiments demonstrated dramatic reduction in the production of infectious virus in the presences of pharmacologically relevant levels A77 1726, with an EC50 of ~15-20 μM. Data generated by identical experiments conducted in the presence of exogenous uridine, which restores intracellular pyrimidines to normal levels, suggested that the antiviral activity may be partially accountable to pyrimidine depletion at lower doses of A77, but not at higher, therapeutic doses. To determine whether leflunomide exerts antiviral activity in vivo, cotton rats were inoculated intranasally with each of 2 low-passage clinical isolates of RSV and treated daily with leflunomide or vehicle alone by gavage, either beginning on the day of inoculation or on day 3 post-inoculation (to more accurately simulate a clinical scenario). On day 5 post-inoculation animals were euthanized and lungs were harvested and homogenized for plaque assay. Viral loads in leflunomide-treated animals were >3 logs lower than those measured in vehicle-treated controls even when treatment was delayed, and were not restored by administration of exogenous uridine. To identify specific antiviral mechanisms of leflunomide, real time rt-PCR was used to quantitate viral genomic RNA and viral transcripts in RNA isolated from infected cultured cells incubated in the presence or absence of A77 1726 ± uridine. Data generated by these experiments demonstrated nearly complete inhibition of viral genomic RNA synthesis and gene transcription by A77 1726, which was only modestly restored by exogenous uridine. Western blot analysis of protein extracts from cells treated as above and probed with polyclonal RSV antibody likewise demonstrated attenuation of synthesis of several viral proteins in A77-treated infected cells, with viral protein synthesis partially restored by exogenous uridine. Collectively, these findings suggest promise for leflunomide, which can be administered orally, as a convenient addition to the growing arsenal of antiviral therapeutics.
Advisors/Committee Members: Waldman, W. James.
Subjects: Biomedical research
Keywords: Respiratory Syncytial Virus; Leflunomide; antiviral therapy
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17.
Fischer, Sara Nicole.
Electrospun Nanofiber Scaffolds as a Model of the Lung Microenvironment in Pulmonary Fibrosis.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2011, Ohio State University
► Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease of unknown…
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▼ Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease of unknown origin that is characterized by irreversible scar tissue formation within the lungs. Collagen deposition, myofibroblast expansion, and the development of fibroblastic foci are hallmark pathological events. The origin and mechanism of recruitment of myofibroblasts is unknown. We hypothesized that the fibrotic lung microenvironment causes the differentiation of bone marrow cells into myofibroblasts. In order to test this hypothesis, we developed a novel method of studying the effects of the fibrotic microenvironment. Poly (e-caprolactone) nanofiber scaffolds were electrospun and coated with lung extracts from bleomycin or PBS-treated mouse lungs. Also, nanofibers of varying moduli were employed to determine the effect of matrix stiffness on bone marrow cell differentiation in our model. Wild-type mouse bone marrow cells plated on the matrices coated with bleomycin- treated lung extract were observed by scanning electron microscopy to be secreting matrix materials and appearing more fibroblast-like after 8 and 14 days. These cells also had a significant increase in expression of myofibroblast genes, type-I collagen and alpha-smooth muscle actin, and a significant increase in expression of connective tissue growth factor and tenascin-C. Similar results were observed when the modulus of the matrix was increased, suggesting that stiffness of substrate plays a role in bone marrow cell differentiation. These data underscore the importance of bone marrow derived cells in mediating pulmonary fibrosis. We next determined the bone marrow-derived cell population that differentiated into myofibroblast-like cells when cultured in the ex vivo fibrotic lung microenvironment. Mesenchymal stem cells, and not hematopoietic stem cells, were able to differentiate into myofibroblast-like cells that were observed in previous studies. These data provide evidence that mesenchymal stem cells are capable of providing a source of myofibroblast precursor cells that traffic to the lung during pulmonary fibrosis. We have recently demonstrated that mice with an inactive form of ets-2 due to a mutation (ets-2 (A72/A72)) are protected from bleomycin-induced pulmonary fibrosis. To elucidate the cellular mechanisms of protection from pulmonary fibrosis due to the ets-2 mutation, ets-2 (A72/A72) bone marrow cells, fibroblasts, and mesenchymal stem cells were cultured in our ex vivo fibrotic lung microenvironment system. In each of these cell types, ets-2 (A72/A72) cells demonstrated a decreased response to the fibrotic microenvironment, as shown by morphology and real-time PCR. Fibroblasts generated from ets-2 (WT/WT) and ets-2 (A72/A72) mice were further tested with biomechanical assays to delineate how the ets-2 mutation alters responses to matrix proteins. Microarray analysis of ets-2 (WT/WT) and ets-2 (A72/A72) myofibroblasts exposed to the ex vivo fibrotic microenvironment revealed several new targets that may be related to the protective phenotype rendered by the ets-2 mutation. These studies provide evidence that ets-2 may be an attractive therapeutic target in IPF. The studies presented herein describe the development of an ex vivo fibrotic lung microenvironment nanofiber scaffold system and the validation of this system as a model for studying pulmonary fibrosis. With further study, the present work may result in the development of new therapies for IPF.
Advisors/Committee Members: Marsh, Clay.
Subjects: Biomedical Research
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18.
Gladman, Jordan Tanin.
Investigating the pre-mRNA splicing of the Survival Motor Neuron genes to model the Spinal Muscular Atrophy disease phenotype.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2010, Ohio State University
► Proximal spinal muscular atrophy (SMA) is a neurodegenerative disease caused by low…
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▼ Proximal spinal muscular atrophy (SMA) is a neurodegenerative disease caused by low levels of the Survival Motor Neuron (SMN) protein. In humans SMN1 and SMN2 encode the SMN protein. In SMA patients the SMN1 gene is lost and the remaining SMN2 gene only partially compensates. Mediated by a C>T nucleotide transition in SMN2, the inefficient recognition of exon 7 by the splicing machinery results in low levels of SMN. Exon 7 splicing is regulated by a number of splicing regulatory sequences. Here we show that mouse Smn and human SMN minigenes are regulated similarly by conserved elements within exon 7 and its downstream intron. Importantly, the C>T mutation is sufficient to induce exon 7 skipping in the mouse minigene as in the human SMN2 gene. Furthermore, by examining evolutionarily conserved sequences in the SMN genes from a number of different species we found two conserved elements that affect exon 7 splicing in our human minigene system. Additional analysis of one of these regions showed decreased inclusion of exon 7 in SMN transcripts when deletions or mutations were introduced. These results describe a novel intronic splicing enhancer sequence located in the final intron of the SMN genes. A better understanding of the way SMN pre-mRNA is spliced can lead to the development of new therapies aimed at correcting SMN2 splicing. Because the SMN2 gene is capable of expressing SMN protein, correction of SMN2 splicing is an attractive therapeutic option. While current mouse models of SMA characterized by Smn knockout alleles in combination with SMN2 transgenes adequately model the disease phenotype, their complex genetics and short lifespan have hindered the development and testing of therapies aimed at SMN2 splicing correction. When the mouse Smn gene was humanized to carry the SMN2 C>T mutation, keeping it under the control of the endogenous promoter and in the natural genomic context, the resulting mice exhibit exon 7 skipping and mild adult onset SMA characterized by muscle weakness, decreased activity, and an increased incidence of atrophic muscle fibers. This new Smn C>T mouse model represents an adult onset form of SMA in which therapies aimed at correction of SMN2 splicing can be easily tested.
Advisors/Committee Members: Chandler, Dawn.
Subjects: Molecular biology
Keywords: Spinal Muscular Atrophy; Survival Motor Neuron; pre-mRNA, RNA splicing
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19.
Gocha, April Renee Sandy.
Mechanisms of alternative telomere elongation in human cancer cells.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2012, Ohio State University
► Telomeres are DNA-protein structures that cap the ends of chromosomes and are…
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▼ Telomeres are DNA-protein structures that cap the ends of chromosomes and are important modulators of genomic stability. Telomeres shorten with each round of cell division and must be actively maintained in cells with high rates of proliferation, such as cancer cells. Cancer cells can elongate telomeres with the telomerase enzyme or in a telomerase-independent manner termed alternative lengthening of telomeres (ALT). ALT uses recombination to copy telomeric templates to lengthen shortened chromosome ends, although the exact functions and mechanisms of ALT are still loosely defined. While some proteins are essential for ALT, the role of other proteins implicated at the telomeres is still unknown. Due to structural and functional similarities to the BLM helicase, mutated in Bloom syndrome and required for ALT, this work asked whether the WRN helicase, mutated in Werner syndrome, is also required for ALT. Short interfering RNA knockdown of WRN in immortalized human cell lines demonstrates that WRN is necessary for ALT in some, but not all, ALT cell lines. In cells that require WRN for ALT, WRN knockdown results in telomere shortening and a loss of ALT characteristics. The requirement for WRN in ALT correlates with an interaction between WRN and the BRCA1 tumor suppressor. These results imply that ALT cells can use different mechanisms of alternative telomere maintenance and that these mechanisms make use of unique protein complexes. In a more clinical scope, this work also asked whether telomere maintenance mechanisms within human sarcoma tumors, which are known to have a high incidence of ALT, are variable. Both immunohistochemical and biochemical methods were used to evaluate human sarcomas for the presence of telomerase-expressing cells and cells with characteristics of the ALT pathway. This work has shown that ALT and telomerase are not mutually exclusive in some human sarcomas and that some tumors have a high degree of tumor heterogeneity in regards to telomere maintenance, with cells using either mechanism within the tumor. ALT is used by an estimated 15% of human tumors. Therefore, understanding ALT mechanisms will be important for the development of effective therapeutic strategies to inhibit ALT tumors. The clinical use of telomerase inhibitors in recent clinical trials warrants a close inspection of telomere maintenance in human tumors to get true measures of accurate therapeutic responses.
Advisors/Committee Members: Groden, Joanna.
Subjects: Biomedical Research
Keywords: telomere maintenance; telomere; ALT; telomerase; WRN; Werner syndrome; sarcoma
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20.
Green, Jill A.
Characterization of Neuronal Primary Cilia in Cellular Homeostasis and Disease.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2012, Ohio State University
► On nearly every mammalian cell, a tiny hair-like organelle known as a…
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▼ On nearly every mammalian cell, a tiny hair-like organelle known as a primary cilium protrudes from the cell surface. These cellular appendages act as specialized antennae to survey the extracellular milieu and transmit signals into the cell that are essential for cellular homeostasis. Although primary cilia were discovered over a hundred years ago and were originally considered evolutionary remnants, interest in these organelles has increased dramatically over the last ten years due to the recognized link between primary cilia and human disease. Improper formation or function of primary cilia can result in a myriad of human diseases and genetic disorders that are collectively called ciliopathies. Due to the ubiquity of cilia, ciliopathies can affect multiple organ systems and tissues and ciliopathy patients present with a wide range of clinical features including cystic kidney disease, retinal degeneration, anosmia, obesity, polydactyly, hypogenitalism, brain malformations, and intellectual disabilities. The pathophysiological consequences of primary cilia dysfunction highlight the important roles cilia play during development and in the normal function of most tissues. Although great progress has been made in understanding the functions of primary cilia on some cell types, primary cilia function on most cell types is still not known. This is particularly true for primary cilia on central neurons in the mammalian brain. Due to the enrichment of signaling machinery in neuronal cilia, such as G protein-coupled receptors (GPCRs) and type III adenylyl cyclase (ACIII) we hypothesize that neuronal cilia are specialized non-synaptic sensory and signaling organelles affecting neuronal function. To test this hypothesis we set out to determine the trafficking mechanisms responsible for mediating ciliary GPCR localization and determine whether proteins that localize to the ciliary membrane are functional. We have shown that a particular protein mutated in the human ciliopathy Bardet-Biedl syndrome (BBS), Bbs5, interacts with multiple ciliary GPCRs. This finding suggests BBS proteins regulate the trafficking of GPCRs into and out of the cilium by direct interaction. To determine whether ciliary GPCRs that are trafficked to the ciliary membrane are function, we analyzed components of the somatostatin (SST) signaling pathway. Although somatostatin receptor subtype 3 (Sstr3) was discovered to localize to neuronal cilia more than a decade ago, it has yet to be determined whether the receptor is functional within the cilium and can generate a signal. We have discovered that upon Sstr3 activation, the localization of SST signaling machinery is dynamic, suggesting neuronal cilia can sense and respond to neuromodulators by trafficking functional ciliary GPCRs and the appropriate signaling machinery to them. Furthermore, as the field of ciliary biology is ever changing, we have discovered that ciliary GPCRs can form heteromers within the mouse brain. As GPCR heteromerization can affect ligand binding properties and downstream signaling, these findings add a previously unrecognized layer of complexity to neuronal ciliary signaling. Taken together, this work presents a novel trafficking mechanism responsible for the tightly regulated localization of ciliary GPCRs and has highlighted how neuronal cilia function as specialized signaling organelles by orchestrating signal transduction cascades important for neuronal function.
Advisors/Committee Members: Mykytyn, Kirk.
Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology
Keywords: Primary cilia; neuronal cilia; ciliopathy; Bardet-Biedl syndrome; G protein-coupled receptors
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21.
Grierson, Patrick Michael.
The BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2012, Ohio State University
► The BLM helicase is a DNA repair protein mutated in the hereditary…
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▼ The BLM helicase is a DNA repair protein mutated in the hereditary condition Bloom’s syndrome (BS). BLM is best known for its roles in regulating homologous recombination-mediated DNA repair and telomere maintenance. A more limited body of work suggests that BLM regulates stability of the nucleolar ribosomal DNA. Previous work in our laboratory suggested that BLM might function in nucleolar ribosome biogenesis. In the present study, we investigated a role for BLM in nucleolar RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. We determined that the nucleolar localization of BLM is sensitive to inhibition of RNA polymerase I-mediated transcription by actinomycin D and 4-nitroquinoline-1-oxide, drugs that inhibit RNA polymerase I. We demonstrated that BLM facilitates 45S rRNA transcription using pulse-chase and nuclear run-on techniques and identified RNA polymerase I and DNA topoisomerase I as nucleolar protein partners. DNA topoisomerase I directly interacts with BLM, an interaction mediated predominantly by the C-terminus of BLM. The interaction of BLM with DNA topoisomerase I is functionally significant as it stimulates BLM unwinding activity in vitro using a RNA:DNA hybrid oligomer substrate that models rRNA transcription from rDNA. We established the biochemical requirement of a 3’ overhang of single-stranded DNA for BLM to unwind nucleolar-relevant RNA:DNA hybrids. Additionally, we discovered a physical interaction between BLM and the nucleolar protein nucleophosmin (NPM) both in nucleoli and nucleoplasm, suggesting a mechanism by which nucleolar trafficking of BLM may be mediated. Overall, this work demonstrates that BLM functions in a pathway of nucleolar ribosome biogenesis and suggests a mechanism by which it may do so. These findings may impact our ability to inhibit or promote cell growth in appropriate clinical settings.
Advisors/Committee Members: Groden, Joanna.
Subjects: Biochemistry; Biology; Genetics; Molecular Biology
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22.
Groer, Chad E.
Agonist-selective regulation of the mu opioid receptor by βarrestins.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2010, Ohio State University
► Morphine and other opiates mediate their effects through activation of the mu…
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▼ Morphine and other opiates mediate their effects through activation of the mu opioid receptor (MOR). Activation of the MOR results in recruitment of regulatory proteins, βarrestins, that can regulate how this receptor signals. In vivo studies suggest that disruption of βarrestin-mediated MOR regulation may enhance opiate-induced antinociception and reduce tolerance and certain unwanted side effects. Therefore, by understanding the cellular mechanisms by which this receptor is regulated, the development of analgesics which preserve the beneficial effects of opiates while eliminating unwanted side effects may be possible. In this dissertation we test the hypothesis that MOR agonists can bias MOR-βarrestin interactions, and that βarrestin recruitment profiles, in turn, may determine cellular responses evoked by these agonists. In the first data portion of this dissertation, we characterize several novel MOR agonists that are unable to promote βarrestin recruitment. Herkinorin is a moderately selective agonist at the MOR, based on the structure of a natural product, Salvinorin A. We find that herkinorin promotes very little MOR phosphorylation, does not recruit βarrestins, and does not induce receptor internalization in transfected cells. Herkinorin is unable to induce βarrestin recruitment or MOR internalization under conditions that facilitate receptor phosphorylation and subsequent βarrestin recruitment with other agonists. We also evaluated several derivatives of herkinorin with similar βarrestin recruitment and MOR internalization profiles. Therefore, herkinorin and its derivatives may be a promising step toward recapitulating morphine’s effects in βarr2-KO mice, which have been used to demonstrate that MOR activation without recruiting βarrestin2 may be therapeutically useful, by producing analgesia with reduced side effects. In the second data portion of this dissertation, we evaluate the interaction and functional consequences of MOR regulation by βarrestin1 and βarrestin2, in response to the classical agonists, DAMGO and morphine. Using both qualitative (microscopy) and quantitative (cell surface biotinylation and BRET) approaches, we have confirmed that DAMGO can induce robust interactions between the MOR and both βarrestins. Morphine, however, selectively promotes interactions with βarrestin2. Additionally, the agonist specific βarrestin interactions are required for internalization of the MOR. Finally, we show that βarrestin1 is required for agonist-induced MOR ubiquitination, such that only DAMGO, and not morphine, is able to promote MOR ubiquitination. Taken together, these data suggest that MOR regulation is highly dependent on the complement of proteins available to interact with the MOR, and that the nature of the ligand can determine how the MOR is regulated by the available proteins. Therefore, the development of biased ligands for the MOR should focus activation of the MOR, but circumventing βarrestin-mediated regulation. These concepts may be critical to consider in the development of opiate compounds designed to retain analgesic efficacy, while reducing the occurrence of unwanted side effects.
Advisors/Committee Members: Sadee, Wolfgang.
Subjects: Pharmacology
Keywords: opioid arrestin herkinorin morphine
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23.
Haizlip, Kaylan Michelle.
Manipulation and Alterations of the Force Frequency Response in Isolated Cardiac Muscle.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2012, Ohio State University
► There are three main physiological governing mechanisms for cardiac contractility; the force-frequency…
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▼ There are three main physiological governing mechanisms for cardiac contractility; the force-frequency response, the length-tension response, and the beta-adrenergic response. One of the major hallmarks of heart failure is a decrease in the force of contraction with increasing frequency. Here we utilize the ex vivo isolated cardiac muscle in an attempt to discuss the physiological role of the force-frequency response in health. We aim to determine the functional attributes of the normal heart by studying alterations in contractility, calcium transient amplitude, and phosphorylation status following changes in frequency, alterations in beta-adrenergic stimulation, and chronic stretch. All experiments were conducted on isolated muscle preparations extracted from the right ventricular free wall of the male New Zealand white rabbit. Our hope is to increase the understanding of the manipulations and alterations that occur in the context of the force-frequency response in health. We first determined the direct changes in the calcium and force response on a beat-to-beat basis during an instantaneous change in frequency. Because heart failure is rarely a spontaneous event, determining the minute changes in calcium transient amplitude as it relates to force production provides insight into the intricate balance that occurs at every beat. In this study we were able to highlight the dynamic relationship between force and calcium during the process of force stabilization at different frequencies. In additional studies, we determined the effects of beta-adrenergic stimulation in conjunction to the force-frequency response. This work aimed to combine the force-frequency response with the beta-adrenergic response in a controlled manner. These experiments highlighted a modulation of the force-frequency response during beta-adrenergic stimulation, suggesting an important role for beta-stimulation in immediate contractile alterations which have an inhibitory effect on the response to frequency. Finally, we determined the time dependent effects of stretch on alterations of the force-frequency response. We show that if a muscle is set to contract at a maintained stretch, there is a significant reversal of the force-frequency response that occurs in conjunction with alterations in the phosphorylation state of key myofilament proteins after 24 hours. Taken together, these results highlight potential modulators of the force-frequency relationship, which could be ideal targets for future work.
Advisors/Committee Members: Janssen, Paul.
Subjects: Physiology
Keywords: cardiac, force-frequency response, isolated muscle, contractility
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24.
Hanke, Mark L.
Sympathetic Nervous System Mediated Alterations in the Immunological and Behavioral Effects of Social Defeat.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2010, Ohio State University
► During physiological or psychological stress, catecholamines produced by the sympathetic nervous system…
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▼ During physiological or psychological stress, catecholamines produced by the sympathetic nervous system regulate the immune system. Previous studies report that the activation of β-adrenergic receptors (βARs) mediate the actions of catecholamines and increases pro-inflammatory cytokine production in a number of different cell types. Social disruption stress is a stressor that increases trafficking of immune cells from the bone marrow, induces glucocorticoid insensitivity of peripheral CD11b+ myeloid cells, alters the immune response to viral and bacterial challenges, and promotes long-lasting anxiety-like behavior in mice. The purpose of this study was to determine the degree to which the sympathetic nervous system affects the activation, priming and glucocorticoid insensitivity of splenocytes after repeated social defeat. Here we report that repeated social defeat increased catecholamine levels in the blood and spleen and that the increased anxiety-like behavior, splenomegaly, and plasma interleukin(IL)-6 induced by social disruption stress (SDR), were each reversed by pre-treatment with the β-adrenergic antagonist propranolol. Furthermore, propranolol pretreatment reversed the SDR-induced increase of TLR2 and TLR4 expression on the surface of splenic CD11b+ cells. In addition, supernatants from propranolol-treated socially defeated mice stimulated with lipopolysaccharide (LPS) contained less cytokines and propranolol pre-treatment abrogated the glucocorticoid insensitivity of CD11b+ cells ex vivo when compared to splenocytes from socially defeated vehicle mice. Furthermore we show that social defeat increased c-Fos staining in brain regions associated with fear and threat appraisal in a β-adrenergic dependent manner. Moreover, analysis by flow cytometry indicates that social defeat significantly increased the number of CD11b+/CD45high macrophages that traffic to the central nervous system. In addition, the surface expression of several inflammatory markers was increased on microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. mRNA analysis of the enriched microglia indicated that there was increased levels of inflammatory-related genes (IL-1β) and a reduction in glucocorticoid responsive genes (GILZ and FKBP51). These social defeat-dependent changes in enriched microglia were reversed by propranolol. To determine if the reactive phenotype of microglia predicted enhanced pro-inflammatory gene expression and glucocorticoid resistance, ex vivo cultures of enriched microglia were established. While glucocorticoids were anti-inflammatory in microglia cultures challenged with LPS, enriched microglia isolated from socially defeated mice produced significantly higher levels of MCP-1, IL-6, and TNF-α after LPS compared to control mice. Together, this study demonstrates that the anxiety-like behavior, immune activation and priming effects of social defeat result as a consequence of sympathetic nervous system activation in a β-adrenergic receptor dependent manner.
Advisors/Committee Members: Sheridan, John.
Subjects: Immunology
Keywords: Stress, Immunology, Behavior
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25.
Hardcastle, Jayson James.
Vstat120 modulates inhibits oncolytic viral therapy induced angiogenesis and innate pro-inflamatory response, augmenting oncolytic viral thereapy of glioblastom multiforme.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2011, Ohio State University
► Glioblastoma multiform is one of the deadliest cancers known and represents approximately…
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▼ Glioblastoma multiform is one of the deadliest cancers known and represents approximately 40% of all primary brain tumors diagnosed. A major histopathological hallmark of these tumors is increased vascularity and micro-vascular proliferation. Work recently completed by our lab showed increased micro-vascular density (MVD) following oncolytic virus (OV) therapy. To this end we created a novel armed OV termed RAMBO (Rapid Anti-angiogenesis Mediated By Oncolytic virus) that contains the transgene econding Vasculostatin (Vstat120). Vstat120 is the extracellular fragment of BAI1 that is proteolytically processed at a conserved G protein coupled receptor proteolytic cleavage site (GPS), resulting in the release of secreted Vstat120. Our work with RAMBO, in two GBM mouse xenografts studies, showed a significant enhancement of survival compared to the control virus, HSVQ. Additionally, subcutaneous GBM xenografts treated with RAMBO had a significant reduction in tumor MVD and vessel perfusion. In 2007 Park et. Identified BAI-1 as a phagocytic receptor expressed on cells of a monocytic lineage. Additionally, a synthetic peptide with 5 Thrombospondin Type-1 repeats and an integrin RGD binding domain was able to inhibit BAI-1 phagocytosis of apoptotic bodies. Our results with RAMBO showed that conditioned medium (CM), derived from infected glioma cells, significantly inhibits N13 and BV2 murine microglia phagocytosis of carboxylate coated beads and microglia migration. In vivo FACS analysis of microglia and monocytes isolated from mice bearing U87Delta-EGFR intracranial gliomas treated with RAMBO, HSVQ or PBS revealed a significant increase in monocyte infiltration into tumors treated by HSVQ; while no change in microglia or monocyte infiltration was found for mice treated with RAMBO compared to PBS treated tumors. FACS analysis for monocyte activation markers Ly6-C, MHC-II, CD86, and CD206 revealed a significant increase in receptor expression levels for monocytes issoalted from HSVQ treated mice compared to RAMBO and PBS treated mice. No significant difference was found for the receptor expression levels found for RAMBO treated mice compared to PBS treated mice. QPCR experiments using murine microglia specific primers for co-culture experiments with human glioma cells infected with RAMBO or HSVQ showed a significant down regulation of mRNA for all Type-1 and Type-2 IFNs and all inflammatory cytokines and chemokines tested for microglia co-cultured with RAMBO infected glioma cells compared to HSVQ. OV gene copy assays from microglia co-cultured with RAMBO or HSVQ treated glioma cells showed a significant increase in RAMBO gene copy vs. HSVQ gene copy 12 hrs post co-culture initiation. In-vivo QPCR for ICP4 mRNA and OV gene copy from intracranial U87Delta-EGFR and Gli26DeltaEGFR-H2B-RFP subcutaneous tumors, respectively, treated with either RAMBO or HSVQ showed a dramatic increase in ICP4 mRNA and OV gene copy for RAMBO treated tumors compared to HSVQ treated tumors. Our results indicate that Vstat120 is also able to modulate the monocytic/microglia innate pro-inflammatory response to OV therapy, resulting in increased OV replication in vivo and in vitro.
Advisors/Committee Members: Kaur, Blaveen.
Subjects: Biomedical Research; Immunology; Neurology; Oncology; Virology
Keywords: Oncolytic Virus, Glioma, Angiogenesis, Tumor Micro-environment, Vstat120, Microglia, Macrophages, Innate Immune Response, Inflammation, Anti-angiogenic, RAMBO
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26.
Herman, Sarah Elizabeth May.
MANIPULATION OF KINASE SIGNALING IN CHRONIC LYMPHOCYTIC LEUKEMIA: THE EFFECT ON DISEASE STATE.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2010, Ohio State University
► Chronic lymphocytic leukemia (CLL) has long been incorrectly labeled “the good leukemia”…
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▼ Chronic lymphocytic leukemia (CLL) has long been incorrectly labeled “the good leukemia” due to the age of onset and the low incidence of detectable symptoms. No leukemia; however, is “good,” and CLL is no exception. CLL is the most common type of adult leukemia in the United States and yet remains incurable by traditional therapies; this provides strong justification for developing additional types of therapeutics. Of particular interest are therapies that target signal transduction pathways essential to CLL cell survival mechanisms that are known to be aberrantly activated as these contribute not only to the inherent survival of transformed cells but also contribute to survival signaling received from the microenvironment. The focus of the work presented here is on altering kinase signaling in either the cancerous B-cell or the surrounding microenvironment to alter disease progression, duration, or state. Chapter one is a general introduction into B-cell biology and CLL: biology, diagnosis and treatment. The dependence of CLL cells on microenvironmental signaling is discussed with particular attention given to the bone marrow stroma and T-lymphocytes. In addition, key signaling pathways involved in B-cell survival and potential target kinases are discussed. Chapter two discusses the effects of inhibiting Phosphatidylinositol 3-Kinase (PI3-Kinase) for the treatment of CLL using a novel drug, CAL-101. We found that inhibition of PI3-Kinase signaling using a selective inhibitor resulted in not only direct cytotoxicity to CLL cells, but also altered the microenvironment in a way that was unfavorable to CLL cell survival. Chapter three discusses the effects of inhibition of B-cell receptor (BCR) signaling via inhibition of Tec family kinases (specifically BTK) using a novel drug, PCI-32765. Similarly to PI3-Kinase, we again found that inhibition of BCR signaling resulted in the induction of apoptosis in CLL cells and an alteration of microenvironmental signaling. Chapter four discusses the affects of activating PI3-Kinase for the treatment of CLL using the novel compound lenalidomide. The focus of this chapter; however, is on the contradictory roles of two therapies for the treatment of CLL and the potential downfalls of their combination. Chapter five discusses the conclusions and implications of our work; specifically the balance of kinase signaling involved in maintaining a disease state in CLL. In addition, chapter five discusses questions left unanswered and the future directions of this work. Together, this work provides valuable insight into the potential use of both kinase inhibitors and kinase activators for the treatment of CLL. This is important as unlike diseases such chronic myeloid leukemia (CML) which displays a targetable aberrant fusion protein kinase, CLL displays no such target, but does display altered kinase signaling as compared to normal B-cells. By utilizing the known differences between normal B-cells and transformed cells, such as CLL, we have been able to predict new target kinases. This work adds to our ability to develop new therapeutics as it suggests that inhibition or activation of any particular kinase may only be half the answer – with evidence suggesting a need for a set balance in kinase signaling.
Advisors/Committee Members: Byrd, John.
Subjects: Biology
Keywords: CLL; Phosphatidylinositol 3-kinase; Bruton's tyrosine kinase; lenalidomide; CAL-101; PCI-32765
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27.
Horvath, Dennis John Jr.
The Impact of Phagocyte-UPEC Interactions Upon Pathogenesis of Urinary Tract Infections.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2011, Ohio State University
► Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract…
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▼ Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). UTIs are considered to be the most common bacterial infection and account for a large degree of medical expenditures in the United States. UPEC possess numerous strategies to evade the innate immune system including: suppression of pro-inflammatory cytokines, invasion into bladder epithelial cells, development of intracellular bacterial communities, and filamentation. Filamentation, growth in the absence of cell division, has been shown to be essential for UPEC pathogenesis in a murine cystitis model. Furthermore, filamentous Gram-negative uropathogens have been identified in the urines of women with cystitis. Filamentous UPEC promote bacterial persistence within the urinary tract by contributing to the development of additional rounds of intracellular infection and the formation of a quiescent intracellular reservoir. However, the mechanism(s) by which filamentous UPEC are elicited and evade phagocytic killing remain unknown. Here we determined that shape-based inhibition of phagocytosis is one of the main mechanisms whereby UPEC is able to evade phagocytic uptake and destruction. Furthermore, we demonstrated that co-culture of UPEC with murine phagocytes (bone-marrow derived macrophages, peritoneal neutrophils, peritoneal macrophages, and RAW 264.7 cells) and the human cathelicidin, LL-37, are sufficient to elicit UPEC filamentation in vitro. Also, we determined that several two component systems (PhoPQ, PmrAB, SoxRS) are important to provide UPEC with a means to resist induction of filamentation. Finally, we determined that CX3CR1+low macrophages and CX3CR1+high dendritic cells contribute to the clearance of UPEC from the kidneys. In conclusion, we have demonstrated that filamentous morphology provides UPEC with an additional mechanism to evade innate immunity and that macrophages and dendritic cells contribute to the antibacterial defense of the urinary tract.
Advisors/Committee Members: Justice, Sheryl.
Subjects: Biomedical Research; Immunology; Microbiology
Keywords: urinary tract infections; Uropathogenic E. coli; innate immunity; phagocytosis; flow cytometry; macrophages; dendritic cells
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28.
Hughes, Tiffany L.
The stage-specific effects of IL-1β on human natural killer cell development.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2011, Ohio State University
► Our laboratory previously demonstrated that natural killer (NK) cell developmental intermediates can…
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▼ Our laboratory previously demonstrated that natural killer (NK) cell developmental intermediates can be found in secondary lymphoid tissue (SLT) (Freud, Becknell et al. 2005). Our subsequent study detailed the surface phenotype of SLT NK progenitor and precursor populations, provided a paradigm for NK ontogeny, and demonstrated that NK development progresses in a unidirectional manner through four discrete developmental stages (Freud, Yokohama et al. 2006). Stage 3 cells, which are exclusively committed to the NK lineage, were characterized as a population of immature NK (iNK) cells because they lack certain characteristics of mature NK cells, including some NK cell surface receptors, cytolytic activity, and the capacity for interferon-gamma (IFN-γ) production. It has already been documented that interleukin (IL)-15 is crucial for mature NK cell function (Carson, Giri et al. 1994), survival (Cooper, Bush et al. 2002), and for normal NK cell development (Yu, Fehniger et al. 1998); however, only a small fraction [~2-20%] of stage 3 iNK cells become stage 4 mature NK cells in response to IL-15 in vitro (Freud, Yokohama et al. 2006). Thus, the identity of the immunomodulatory factors regulating stage 3 iNK cell expansion and differentiation may not yet be fully elucidated. For the past four years, this topic has been the focus of my research. Through the course of many studies, we (myself along with my collaborators) have discovered that the IL-1 receptor (IL-1R1) is selectively expressed by a subpopulation of stage 3 iNK cells. We also discovered that the vast majority of stage 3 iNK cells in SLT constitutively and selectively express aryl hydrocarbon receptor (AHR) and IL-22, a TH17 cytokine which may have an important role in maintaining mucosal immunity. IL-1R1, AHR, and IL-22 expression were absent during earlier and later stages of NK development, and expression of AHR and IL-22 were largely restricted to the IL-1R1hi subpopulation of stage 3 iNK cells. These IL-1R1hi stage 3 iNK cells localized proximal to IL-1β-producing conventional dendritic cells (cDC) within SLT, proliferated in direct response to cDC-derived IL-15 and IL-1β, and required continuous exposure to IL-1β to retain AHR and IL-22 expression. In the absence of IL-1β, a substantially greater fraction of IL-1R1hi stage 3 iNK cells differentiated to stage 4 mature NK cells, and acquired the ability to kill and secrete IFN-γ. Thus, cDC-derived IL-1β preserves and expands IL-1R1hiIL-22+AHR+ stage 3 iNK cells, potentially influencing human mucosal innate immunity during infection. These data support a role for IL-1β as an immunomodulatory factor capable of regulating the development of human NK cells in vivo, and also suggest that these “immature” NK cells may have a unique functional role within SLT. Herein, I present these findings and provide an extended discussion relating our new data to current concepts in the field of NK cell developmental biology.
Advisors/Committee Members: Caligiuri, Michael.
Subjects: Immunology
Keywords: natural killer; development; IL-15; IL-1
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29.
Jensen, Jordan Royce.
Development of Tau-Selective Imaging Agents for Improved Diagnosis of Alzheimer’s Disease and Other Tauopathies.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2011, Ohio State University
► Currently, definitive AD diagnosis is made post-mortem by the appearance of extracellular…
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▼ Currently, definitive AD diagnosis is made post-mortem by the appearance of extracellular amyloid-beta peptide plaques and intracellular tau tangles. These lesions are proteinaceous aggregates in the form of in-register cross-beta-sheet fibrils. For pre-mortem diagnosis, sensitivity and specificity are 80% and 70%, respectively. CSF biomarkers and radiopharmaceutical imaging could improve AD diagnostic accuracy. The lumbar puncture procedure needed for CSF sampling is more painful and risky than whole brain PET imaging. Although imaging of both amyloid-beta and tau lesions could provide diagnostic information, tau-selective imaging agents should be superior to amyloid-beta-selective or pan-binding agents because the appearance of tangles in certain brain regions precedes plaque formation by decades, tangle presence correlates better than plaques with neuronal loss, and tangles develop in a more stereotypical spatial pattern over time than do plaques. Lewy bodies, seen in PD and LBD, are composed mainly of alpha-synuclein protein, and could interfere with tau lesion imaging. Therefore, compounds are needed which bind tau, but not amyloid-beta or alpha-synuclein filaments. ThS and ThT are well-characterized probes of cross-beta-sheet structures and are commonly used in displacement assays to identify fibril-binding compounds. ThS and ThT displacement from recombinant tau filaments and PHFs was similar for several compound classes. Multimodal compound binding to 0N4R tau fibrils was observed with ThT displacement by BTA-2. Certain BTAs and oxindoles displaced ThT from 2N4R tau, 0N4R tau, and alpha-synuclein fibrils more potently than from 1N3R tau, amyloid-beta1-40, and amyloid-beta1-42 filaments, despite frequent incomplete ThT displacement by BTAs at high compound concentration. However, a few potent (AC50 < 10 nM) BTA compounds displaced ThT from 2N4R and 0N4R tau isoforms ~10-fold better than from alpha-synuclein. BTA compound potency was proportional to percentage of observed ThT displacement for all fibril types. Binding potency of BTAs and oxindoles was increased by maximizing the number of intervening aromatic electrons between the electron donating and electron acceptor groups (polymethine system), with mono- and dimethyl amines being the strongest donors. For oxindoles, the presence of a chlorine or bromine atom at position 5 of the oxindole ring and/or having a double ring system for the aryl group increased potency. Increased compound width, such as “E-form” oxindoles and BTA 23, seemed to increase compound selectivity for 2N4R and 0N4R tau fibrils over amyloid-beta fibrils. I attribute this selectivity to double glycine channels found in tau fibrils that are not present in amyloid-beta filaments. Future BTAs and oxindoles should be wide, have large aryl groups (such as double ring systems), and contain mono- or di-methyl amino donor groups in positions that maximize the number of polarizable electrons. QSAR methods may uncover other important compound design factors. Additional future work for BTA and oxindole compound development that must precede imaging agent trials in humans includes displacement of radiolabeled probes in a filter-based assay, staining post-mortem AD patient brain tissue sections with radiolabeled BTA and oxindole compounds, identifying appropriate 18F labeling methods, and testing of 18F-labeled imaging agents in AD animal models for appropriate PK/PD properties and PET efficacy.
Advisors/Committee Members: Kuret, Jeffrey.
Subjects: Biomedical Research
Keywords: tau; amyloid beta; alpha-synuclein; alzheimer's disease; polarizability; dispersion; selectivity; PET; radioligand; diagnosis; thioflavin T; polymethine; dementia; oxindole; benzothiazole
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30.
Kwilas, Anna R.
Respiratory Syncytial Virus Based Vectors for the Treatment of Cystic Fibrosis.
Degree: PhD, Integrated Biomedical Science Graduate Program, 2010, Ohio State University
► Cystic fibrosis (CF) is the most common lethal recessive genetic disease in…
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▼ Cystic fibrosis (CF) is the most common lethal recessive genetic disease in the Caucasian population. It is caused mutations in the CF transmembrane conductance regulator (CFTR) gene which lead to the production of a partially or completely non-functional protein. Lack of CFTR in epithelial cell membranes results in abnormal ion transport and dehydration of mucosal surfaces. In the respiratory tract this leads severe lung disease, the leading cause of morbidity and mortality in the CF population. Since CFTR was first cloned in 1989, a major goal of CF research has been to develop effective gene therapy for CF lung disease. Respiratory syncytial virus (RSV) naturally infects the ciliated cells that line the human respiratory tract, cells that express CFTR and play a major role in determining the hydration of the airway mucosal surface. In addition, although an immune response is mounted against RSV, this does not prevent subsequent infections suggesting that an RSV-based vector might be effectively re-administered a requirement since the apical ciliated cells of the respiratory tract are terminally differentiated and have a defined life span. To test whether the large, 4.5 kb, CFTR gene could be expressed by a recombinant RSV and whether infectious virus could be used to deliver CFTR to ciliated airway epithelium derived from CF patients, we have inserted the CFTR gene into four sites in the recombinant, green fluorescent protein-expressing (rg)RSV genome to generate virus expressing four different amounts of the CFTR protein. Two of these rgRSV-CFTR viruses were capable of expressing CFTR with little effect on viral replication. rgRSV-delivered CFTR was functional in primary CF airway epithelial cultures. Infection with these viruses resulted in chloride channel function similar to that seen in non-CF cultures and correction of additional characteristic defects seen in CF airway epithelia. Unfortunately, most ciliated airway cells infected with RSV die within 5 days. To create an improved RSV-based vector we generated an RSV “replicon” by removing the three glycoprotein genes from a full-length RSV cDNA. The replicon is able to replicate in cells without killing them or producing infectious virus, allowing the isolation and expansion of replicon-containing cells. Providing the viral glycoproteins in trans enables the production of “one-step virus” (OSV) which is capable of delivering a replicon to fresh cells where it produces its encoded proteins, but again cannot spread. We inserted the CFTR gene into the RSV replicon genome in four positions. All of these replicons were capable of producing the CFTR protein. When CFTR-expressing replicon OSV were used to infect primary human airway epithelial cultures, replicon containing cells remained within the cultures for 12-20 days, indicating that replicon containing cells have a survival advantage over those infected with complete virus. Our results indicate that RSV is capable of expressing CFTR, supplying enough functional CFTR to primary CF airway cells to correct their physiologic defects and with modification can remain in primary airway cells for a prolonged period of time. Collectively, our data support further investigation of RSV as a potential vector for CF gene therapy.
Advisors/Committee Members: Peeples, Mark.
Subjects: Virology
Keywords: cystic fibrosis; respiratory syncytial virus; gene therapy
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