PHD, Kent State University, 2006, School of Biomedical Sciences

The significant variation within wildtype animals is an underutilized resource for studying skeletal development. In the mammalian metatarsal one end forms a growth plate and secondary center of ossification as in other long bones, however the opposite end undergoes direct ossification in a manner more similar to short bones. Thus, the metatarsal can serve as a model to explore the signals and factors that specify growth plate formation in the vertebrate skeleton. In pursuit of this goal I first compared the patterns of chondrocyte differentiation and proliferation during growth plate formation and direct ossification of an age series of mouse metatarsals. As in the human, both growth plate formation and direct ossification are easily discernable in the mouse metatarsal. In addition, growth plate formation is characterized by a region of peak of proliferation corresponding to reserve zone chondrocytes that distinguishes it from both established growth plates and direct ossification. Second, immunohistochemisty reveals that patterns of PTHrP and PTH/PTHrP-receptor are indistinguishable at each end of the metatarsal suggesting PTHrP signaling is not specific to growth plates. In contrast, the distribution of Patched, the Ihh receptor, is highly variable during growth plate formation and appears to co-localize to regions of increased proliferation, suggesting a potential role for Ihh signaling in specifying growth plate formation. Third, a comparative analysis of metapodial ossification between mice and alligators (Alligator mississippiensis) reveals similar proliferative patterns and protein expression during growth plate formation and within established growth plates, indicating that both species share common mechanisms of chondrocyte regulation. However, unlike mice, alligators establish growth plates at both ends of their metapodials. Phylogenetic analysis reveals that the direct ossification of one epiphysis and reliance on a single growth plate is restricted to e (open full item for complete abstract)

Committee: C. Lovejoy (Advisor) Subjects:

Doctor of Philosophy (PhD), Ohio University, 2024, Molecular and Cellular Biology (Arts and Sciences)

The increasing prevalence of type 1 and type 2 diabetes has made studying insulin secretion and production an urgent necessity. Parathyroid hormone-related protein (PTHrP) is a multifunctioning protein that is expressed in many cell types including pancreatic beta cells. It consists of an N-terminus, a mid-region which includes a nuclear localization sequence (NLS), and a C-terminus. PTHrP has been shown to increase insulin secretion and beta-cell proliferation. However, the mechanism underlying its actions is still unclear. Furthermore, other studies have shown unique roles of the NLS and C-terminus of PTHrP, but these regions of PTHrP have not been studied in beta cells. In this study, full-length murine PTHrP (-36-139) was overexpressed in isolated mouse islets and a beta-cell line Ins-GLuc. There was an increase in insulin content of islets due to PTHrP overexpression and an increase in intracellular calcium levels at higher extracellular glucose concentrations of 12-28mM. Transfecting Ins-GLuc cells with full-length PTHrP caused an increase in insulin secretion in 28mM glucose; however, this effect was reversed when cells were transfected with PTHrP lacking the NLS (-36-67…95-139) or PTHrP lacking the NLS and C-terminus (-36-67). Further studies with the PTHrP ∆/∆ mouse model which lacks the NLS and C-terminus of PTHrP revealed that the mice were both hypoglycemic and hypoinsulinemic, with smaller islets than control mice, increased intracellular calcium response to glucose and reduced pancreatic glucagon content. Subsequent RNA sequencing studies with a murine beta-cell line MIN6 revealed a downregulation of ER stress-related genes and an upregulation of genes involved in amino acid biosynthesis after transfection with full-length human PTHrP (-36-141). The data from the RNA sequencing studies suggested that PTHrP increased insulin production by enhancing protein processing in the ER and protecting the cells from ER stress. Collectively, this study showed (open full item for complete abstract)

Committee: Thomas Rosol (Advisor); Craig Nunemaker (Advisor) Subjects: Biology; Biomedical Research; Molecular Biology; Physiology

Bachelor of Science (BS), Ohio University, 2024, Biological Sciences

Parathyroid hormone-related protein (PTHrP) is a hormone originally discovered in cancer patients as a cause of cancer-associated hypercalcemia. PTHrP was then unexpectedly found to be in many normal tissues of the body. PTHrP is similar in function and structure to the well-known hormone, parathyroid hormone (PTH). Previous studies showed that removing the nuclear localization sequence (NLS) and the C-terminus domains of PTHrP in mice (PTHrP 1- 66) caused severe abnormalities including lack of skeletal formation and decreased hematopoiesis (Toribio et al., 2010). These mice usually die within 5 days. Due to the dwarfism of these mice, this thesis explores the relationship of how reduced PTHrP affects the growth hormone (GH) and insulin-like growth factor 1 (IGF-1) axis in the liver of mice. Using quantitative polymerase chain reaction (qPCR) and RNA-sequencing, we found that IGF-1 was decreased in the liver and serum. We also found that GH in serum and its mRNA was normal to increased. The RNA-seq data also showed that the growth hormone receptor (GHR) was slightly downregulated, but genes downstream from the GHR were variably modified. Unexpectedly, the RNA-seq data showed that the most downregulated pathway was the cell cycle pathway.

Committee: Thomas J. Rosol (Advisor) Subjects: Biology

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

Parathyroid hormone-related protein (PTHrP) was initially discovered as the causative agent of hypercalcemia of malignancy. It has since been determined to be an essential regulator of many physiologic processes, including skeletal development. The primary effects of PTHrP are mediated through its amino-terminus, where it is equipotent with N-terminal parathyroid hormone (PTH) at the common PTH/PTHrP receptor. However, unlike PTH, PTHrP has multiple additional functional domains capable of eliciting biological effects distinct from the N-terminus and independent of PTH1R. Our laboratory and others have identified that two regions of PTHrP, the nuclear localization sequence (NLS) and C-terminus, have bone-forming effects distinct from, or complementary to, the N-terminal region of PTHrP. This dissertation focused on 1) the role of the NLS and C-terminus of PTHrP on the multilineage differentiation of mesenchymal stem cells (MSCs) and 2) the effects of N-terminal PTHrP 1-34 on skeletogenesis and engraftment using the vossicle ectopic vertebral transplantation model. The following work demonstrates two major cumulative findings. First, the NLS and C-terminus of PTHrP regulate multiple genes, microRNAs (miRs), and signaling pathways central to the normal differentiation of MSCs into osteoblasts, chondrocytes, adipocytes, and myocytes and function of these differentiated progeny. The NLS and C-terminus complement the pro-osteochondrogenic actions of the N-terminus. In addition, they complement the inhibitory effects of the N-terminus on adipogenesis and myogenesis. This was the first study 1) demonstrating that the NLS and C-terminus of PTHrP are involved in regulating adipogenesis and myogenesis and 2) identifying miRs regulated by any region of PTHrP. Second, PTHrP 1-34 enhances the engraftment of and skeletogenesis in the vossicle model of ectopic vertebral transplantation. In addition, bioluminescence and fluorescence imaging can be used as non-invasive methods t (open full item for complete abstract)

Committee: Thomas Rosol DVM, PhD (Advisor); Ramiro Toribio DVM, MS, PhD (Advisor); Matthew Allen Vet MB, PhD (Committee Member); Lawrence Kirschner MD, PhD (Committee Member) Subjects: Biology; Endocrinology; Molecular Biology; Radiology

Doctor of Philosophy, The Ohio State University, 2009, Veterinary Biosciences

Human T-lymphotropic virus type 1 (HTLV-1) is a delta retrovirus that causes several diseases including adult T-cell lymphoma/leukemia (ATLL). The prognosis of ATLL patients is poor with a median survival of six months. This poor prognosis is due to the limited responsiveness to standard chemotherapy and severe paraneoplastic syndromes, including humoral hypercalcemia of malignancy (HHM), which results from an increase in osteoclastic bone resorption induced by humoral factors secreted from the tumor cells in ATLL patients. Therefore, it is urgent to develop new therapeutic strategies for ATLL with HHM and to understand the pathogenesis of osteoclastic bone resorption induced by HTLV-1 infection. The development of new drugs for ATLL with HHM has been hampered by the lack of relevant animal models which are able to mimic the human disease manifestations, such as HHM and lymphoma. A xenograft mouse model of ATLL with HHM using RV-ATL cells, a cell line derived from an ATLL patient, has been developed and the HHM and lymphoma development has been characterized in mice in our laboratory. In this study, we transduced RV-ATL cells with a lentiviral vector containing the luciferase gene (luc) in order to perform non-invasive in vivo imaging, which allows longitudinal monitoring of tumor growth without sacrifice of animals. Mice developed severe tumor burden and HHM within 4 weeks. Therefore, this bioluminescent animal model is suitable for preclinical and pathogenesis studies of HHM in ATLL. To investigate new treatments for ATLL with HHM, we selected two drugs that have shown their clinical benefits in other cancers but have not yet been tested for their combinatorial effects in ATLL. PS-341 is a selective proteasome inhibitor that is able to inhibit cell proliferation and induce apoptosis mainly through the inhibition of NF-κB activity. Zoledronate is a third generation bisphosphonate that is able to inhibit osteoclast function. We found that both PS-341 and zoledronat (open full item for complete abstract)

Committee: Thomas Rosol (Committee Chair); Patrick Green (Other); Lawrence Mathes (Other); Michael Lairmore (Other) Subjects: Molecular Biology; Oncology

Doctor of Philosophy, The Ohio State University, 2009, Veterinary Biosciences

Humoral hypercalcemia of malignancy (HHM) is a debilitating syndrome seen in patients with neoplasia of squamous epithelial cell origin. The highest prevalence of HHM is associated with squamous-cell carcinoma of the lung (SCCs) and ranges from 27-66%. HHM results from increased synthesis and secretion of parathyroid hormone-related hormone (PTHrP). The mechanisms that activate PTHrP gene expression in tumors associated with HHM have yet to be identified. The contribution of the epidermal growth factor receptor (EGFR) to HHM in the human lung SCC cell lines, RWGT2 and HARA was investigated. To test the relationship between EGFR activity and PTHrP gene expression, PTHrP mRNA levels were measured by Q-RT-PCR following treatment of lung SCC lines with the EGFR tyrosine kinase inhibitor (TKI) PD153035, anti-amphiregulin antibodies as well as with EGF-ligands. Overall, PTHrP expression was significantly increased with EGF-ligand treatment. The in vivo relationship between EGFR and PTHrP gene expression was investigated using xenograft HARA and RWGT2 HHM models. Hypercalcemic mice were treated with the TKI, gefitinib. RWGT2 plasma calcium levels were significantly reduced at all time points when compared to pretreatment and control values. In conclusion, autocrine activation of PTHrP gene expression is mediated through the EGFR in the RWGT2 line, however, our results indicated that the major mechanism of HHM induction in the HARA model was not through EGFR but rather the high concentrations of PTHrP secreted by the HARA line were significantly influenced by exogenous factors. The role of a known regulator of calcium homeostasis in humans, the calcium-sensing receptor (CaR) was investigated. Our experiments evaluate evidence for the expression of the CaR in human lung SCC. We examined if PTHrP secretion and HHM occurs in response to CaR stimulation in the RWGT2, HARA and BEN Australia SCCs. We find that CaR is expressed in lung SCCs and stimulation with extracellular calci (open full item for complete abstract)

Committee: Thomas J. Rosol PhD (Advisor); John Foley PhD (Committee Member); Michael Lairmore PhD (Committee Member); Michael Oglesbee PhD (Committee Member) Subjects: Oncology

Doctor of Philosophy, The Ohio State University, 2007, Veterinary Biosciences

Although adult T-cell leukemia/lymphoma (ATLL) is caused due to infection with human T-lymphotropic virus type-1 (HTLV-1), cellular events besides HTLV-1 infection are required for transformation of T-cells. HTLV-1 Tax plays an important role in the transformation of lymphocytes; however, the exact mechanisms remain unclear. Parathyroid hormone-related protein plays an important role in the pathogenesis of humoral hypercalcemia of malignancy observed in the majority of ATLL patients. PTHrP is also up-regulated in HTLV-1-carriers and normocalcemic ATLL patients. Using long-term co-culture assays, herein we reported that PTHrP and its receptor (PTH1R) were highly expressed during HTLV-1-mediated immortalization of lymphocytes. Co-transfection assays with HTLV-1 and PTHrP reporter plasmids showed that HTLV-1 mildly up-regulated PTHrP expression indicating that other cellular factors or events are required for increased expression of PTHrP in ATLL cells. The PTHrP gene is regulated by three promoter regions (P1, P2, and P3). We characterized an NF-eB binding site in the P2 promoter of human PTHrP and detected a specific complex in Tax-expressing human T-cells composed of p50/c-Rel, and two distinct complexes in ATLL cells consisting of p50/p50 homodimers and a second unidentified protein(s). Furthermore, we showed inhibition of NF-eB activity with Bay 11-7082 decreased PTHrP P2 promoter-initiated transcripts. Finally we developed a comparative xenograft model of canine T-cell lymphoma with hypercalcemia. Quantitative RT-PCR of T-cell lymphoma samples from hypercalcemic canine patients showed that PTHrP likely plays a central role in the pathogenesis of HHM and that hypercalcemia is the result of a combinatorial effect of different hypercalcemic factors. We monitored in vivo tumor progression and metastases in the mouse model by transducing the lymphoma cells with a lentiviral vector that encodes a luciferase-yellow fluorescent protein reporter and showed that in vivo tr (open full item for complete abstract)

Committee: Thomas Rosol (Advisor) Subjects: Biology, Veterinary Science

Doctor of Philosophy, The Ohio State University, 2003, Veterinary Biosciences

Parathyroid Hormone-related Protein (PTHrP) is the causative factor of humoral hypercalcemia of malignancy (HHM) observed in patients with adult T-cell leukemia/lymphoma (ATLL). Human T-Lymphotropic Virus Type-1 (HTLV-1) is the etiology of ATLL. We developed a SCID/beige mouse model of HHM to study the pathogenesis of HHM in ATLL. SCID/beige mice inoculated intraperitoneally with human RV-ATL cells developed lymphoma in the mesentery and multiple organs. RV-ATL cells were immunohistochemically positive for PTHrP and expressed high levels of PTHrP mRNA in a HTLV-1 Tax-independent manner. Mice with lymphoma developed severe hypercalcemia, and their plasma PTHrP concentrations correlated positively with calcium concentrations. Bone densitometry and histomorphometry in lymphoma-bearing mice revealed significant bone loss due to marked increase osteoclastic bone resorption. There was a strong inverse relationship between PTHrP and tax/rex mRNA expression using quantitative RT-PCR in RV-ATL and other HTLV-1 cell lines. The PTHrP gene is regulated by three promoter regions (P1, P2, and P3). Quantitative RT-PCR for alternative PTHrP promoter usage in RV-ATL and HTLV-1 cell lines revealed that P3-initiated transcripts were the most abundant in cells with low tax and high PTHrP mRNA expression. We observed constitutive and specific binding of ETS-1 to an ETS binding site (P3 EBS) in the P3 region using nuclear extracts from RV-ATL cells. Transfection experiments in Jurkat T cells resulted in increased transcription of P3-luciferase constructs in response to PMA and ionomycin mediated through P3 EBS. Stimulation with PMA and ionomycin also induced a protein/DNA complex formation on P3 EBS identical to that observed in RV-ATL cells. Co-transfection with Ets-1 and constitutively active Mek-1 expression vectors in Jurkat T cells resulted in a robust induction of luciferase activity under the regulation of P3. The activation was mediated by P3 EBS. Nuclear extracts from Jurkat T c (open full item for complete abstract)

Committee: Thomas Rosol (Advisor) Subjects: