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

Bachelor of Science (BS), Ohio University, 2023, Biological Sciences

Fibrosis, a pathological process characterized by excess extracellular matrix (ECM) deposition, can occur in many internal organs and tissues in response to various stimuli. As fibrosis progresses, scarring occurs, which ultimately leads to tissue dysfunction and organ failure. Patients with acromegaly, a rare disease usually caused by a benign, GH-producing pituitary tumor, have been reported to have prominent ECM deposition and scarring in certain tissues, which is indicative of fibrosis. In bGH transgenic mice, which express high levels of bovine growth hormone, several tissues [white adipose tissue (WAT), heart, intestine, and kidney] demonstrate a fibrotic phenotype. However, there is no previous research that investigates various bGH tissues – particularly from mice derived from a single cohort – for fibrosis. Additionally, WAT fibrosis is associated with obesity and lipodystrophy, and seems to be particularly associated with excess GH. This study aims to investigate the role of different cell types and genes involved in the development and progression of WAT fibrosis and determine if fibrosis is increased in BAT, liver, quad, kidney, lung, and spleen of aged bGH mice. Results of this thesis included a striking observation of increased fibrosis in all bGH tissues examined. For WAT, decreases in fibrosis-associated RNA expression in 3-month-old bGH mice via qPCR analysis was only observed in the perigonadal depot and not the subcutaneous depot that has more prominent collagen deposition. Interestingly, we observed an intriguing increase in fibrosis-associated RNA expression in a population of adipose stem and progenitor cells in 6-month-old mice within subcutaneous bGH WAT. These results indicate a potential common GH-induced mechanism of fibrosis across bGH tissues and pave the way for future research into WAT fibrosis.

Committee: Darlene Berryman (Advisor) Subjects: Biology; Biomedical Research

Doctor of Philosophy (PhD), Ohio University, 2018, Molecular and Cellular Biology (Arts and Sciences)

Growth Hormone (GH) is well known to have prominent anabolic effects on bone, muscle and fat, but its effects on the gastrointestinal tract are less well studied. The fact that recombinant GH therapy has been used to treat intestinal diseases such as short bowel syndrome and inflammatory bowel disease indicates that GH can exert effects in the intestines. However, little is known about the basic biological effects of GH on this organ. The focus of this study was to examine the effects of GH in the absence of intestinal disease using genetically altered mice. Mice with systemic alterations in GH activity displayed morphological and metabolic changes that correlated with levels of GH action. In a mouse model with the GH receptor (GHR) gene disrupted in intestinal epithelial cells, there were sex specific changes in intestinal length, glucose metabolism, barrier function, and fat absorption, but no apparent changes in mucosal morphology. In other experimental models, genetically engineered mice with chronically increased GH showed increases in collagen protein content with no detectable change in collagen gene expression. Mice with chronically decreased GH showed a decrease in collagen protein content with no significant change in collagen gene expression. An increase in collagen gene expression was observed in wild-type mice after one month of daily injections of GH. These findings establish some effects of GH action on the intestines and encourage further examination of intestinal GH induced signaling on overall animal physiology.

Committee: John Kopchick PhD (Advisor); Darlene Berryman PhD (Committee Member); Mark Berryman PhD (Committee Member); Fabian Benencia PhD (Committee Member) Subjects: Biology; Cellular Biology; Endocrinology; Genetics; Molecular Biology

Master of Science (MS), Ohio University, 2017, Food and Nutrition Sciences (Health Sciences and Professions)

White adipose tissue (WAT) is a complex and dynamic endocrine organ that is most commonly recognized for its energy storage capacity. Brown adipose tissue (BAT) functions to dissipate stored energy in the form of heat through a process known as nonshivering thermogenesis. This process is aided by the mitochondrial matrix protein, uncoupling protein 1 (UCP1), which creates a proton leak across the inner mitochondrial membrane causing chemical energy to be released as heat. More recently, clusters of brown-like or “beige” adipocytes have also been identified in WAT. These adipocytes have the capacity to interconvert between the two phenotypes, in part, through increasing their expression of UCP1. This “beiging” is dependent on environmental and chemical conditions present within the cell. Growth hormone (GH), a protein secreted from the anterior pituitary, has been positively correlated with increased BAT mass. However, this evidence is controversial, and the effects of GH on BAT and WAT beiging are not well defined. Bovine growth hormone transgenic (bGH) mice have increased GH action, are giant and lean, yet develop insulin resistance and have shortened lifespans compared to their wild-type (WT) littermates. The purpose of the current study was to compare the expression of beiging-associated factors at both the RNA and protein levels in bGH mice and WT littermate controls. To accomplish this, several different methods were used to measure expression of beiging-associated gene expression in the WAT and BAT depots of these mice. A previously collected RNA-Seq dataset revealed significant genotype and depot differences, with the greatest number of expression changes detected between genotype in the subcutaneous depot and between depot in the bGH mice. Additionally, qPCR, Western blot analysis, and immunohistochemistry with confocal imaging revealed that UCP1 RNA and protein expression were undetectable in the WAT depots of these mice. No significant differences in UCP1 (open full item for complete abstract)

Committee: Darlene Berryman PhD, RD (Advisor); Cheryl Howe PhD (Committee Member); Edward List PhD (Committee Member) Subjects: Biology; Cellular Biology; Nutrition; Physiology

Doctor of Philosophy (PhD), Ohio University, 2016, Molecular and Cellular Biology (Arts and Sciences)

Human melanomas are one of the most therapy resistant types of cancer, expressing a repertoire of mechanisms for drug resistance. Intracellular signaling networks used by the melanoma cells for active proliferation, migration, invasion, therapy resistance and metastases happen to strongly overlap with those regulated by the human growth hormone (hGH). Indeed consistently high levels of GH receptor (GHR) expression have been observed in almost all melanoma cell lines in the NCI60 human cancer panel. There are no comprehensive studies investigating the effects of GH action or GHR antagonism on the GH-responsive intracellular signaling pathways and downstream effects in human melanoma. Here we report for the first time, a detailed analysis of the effect of siRNA mediated GHR knock-down (KD) and effect of hGH on the intracellular signaling and downstream phenotypes in human melanoma cells. We report the existence of an autocrine loop of hGH-GHR in the aforesaid human melanoma cell lines, changes observed in relevant intracellular signaling pathways, RNA transcript level changes observed in multiple key modulators of the GH/GHR axis. Phosphorylation states of the JAK2, SRC, STAT1, 3, and 5, p44/42-MAPK, AKT and mTOR increased in a dose-dependent manner with hGH stimulation and were significantly attenuated by GHR-KD. Differential yet significant changes were observed in the relative mRNA transcript levels of prolactin (PRL), insulin and related growth factors (IGF1, IGF2) and their receptors, following hGH treatment or GHR-KD. Thus, the GH/GHR interaction can influence the levels of the JAK2, SRC, STAT1, 3, and 5, p44/42-MAPK, AKT and mTOR in human melanoma cells and may provide leads as to potential targets for therapeutic intervention.

Committee: Shiyong Wu (Advisor); John Kopchick (Committee Member) Subjects: Biochemistry; Biology; Endocrinology; Molecular Biology; Oncology

Bachelor of Science (BS), Ohio University, 2016, Biological Sciences

Objective: Although growth hormone (GH) and fibroblast growth factor 21 (FGF21) have a reported relationship, FGF21 and its receptor fibroblast growth factor 1 (FGFR1) and cofactor ß-Klotho (KLB) have not been analyzed in states of altered GH action. This study measured circulating FGF21 and tissue expression of Fgf21, Fgfr1, and Klb in mice with modified GH action to further elucidate the regulation of FGF21 action by GH. Design: 7 month old male mice bovine GH transgenic (bGH) mice (n = 9) and wild type (WT) controls (n = 10), and GH receptor knockout (GHR-/-) mice (n = 8) and WT controls (n = 8) were used for all measurements. Body composition was performed before dissection and tissue weights were measured at the time of dissection. Serum FGF21 levels were evaluated by ELISA. Expression of Fgf21, Fgfr1, and Klb mRNA in white adipose tissue (AT), brown AT, and liver were evaluated by real-time quantitative PCR. Results: As expected, bGH mice had increased body weight (p = 3.70E-8), but decreased percent fat mass (p = 4.87E-4). Likewise, GHR-/- mice had decreased body weight (p = 1.78E-10), but increased percent fat mass (p = 1.52E-9), due to increased size of the subcutaneous AT depot when normalized to body weight (p = 1.60E-10). Serum FGF21 levels were significantly elevated in bGH mice (p = 0.041) and unchanged in GHR-/- mice (p = 0.88). Expression of Fgf21, Fgfr1, and Klb mRNA in white AT and liver were downregulated or unchanged in both bGH and GHR-/- mice. The only exception was Fgf21 expression in brown AT of GHR-/-, which trended toward increased expression (p = 0.075). Conclusion: We provide evidence that circulating FGF21 is increased in bGH animals, but remains unchanged in GHR-/- mice. Downregulation or no change in Fgf21, Fgfr1, and Klb expression are seen in white AT, brown AT, and liver of bGH and GHR-/- mice when compared to their respective controls, except for an increase in brown AT Fgf21 expression in GHR-/- mice, which could sugge (open full item for complete abstract)

Committee: Darlene Berryman PhD (Advisor); Soichi Tanda PhD (Advisor) Subjects: Biology; Biomedical Research; Molecular Biology

Doctor of Philosophy (PhD), Ohio University, 2014, Biological Sciences (Arts and Sciences)

Growth hormone (GH) and insulin-like growth factor I (IGF-I) are thought to play key roles in the development and maintenance of the heart. Evidence of the profound effect GH has on the heart can be found in patients with diseases that disrupt GH action. In pathological states of acromegaly (caused by oversecretion of GH), GH deficiency, and Laron Syndrome (a disease caused by mutations in the GH receptor resulting in systemic lack of GH induced action), patients exhibit unique cardiovascular phenotypes of altered structure and function. Despite these observations, the exact role of GH in the cardiovascular system is not well characterized. To better understand the effects that GH action has on the cardiovascular system, we studied the well-established bovine GH (bGH) transgenic mouse and developed a novel tamoxifen-inducible, cardiac-specific GH receptor (GHR) gene disrupted mouse (iC-GHRKO). We find that the bGH mice exhibit an age-dependent elevation of systolic blood pressure that is correlated with changes in both the brain natriuretic peptide and renin angiotensin systems. Our initial characterization of the iC-GHRKO mice surprisingly reveals that cardiac GH signaling is not necessary for maintenance of baseline cardiac function, but does appear to be required for normal glucose homeostasis in older mice. These studies take us a step closer to understanding the role of GH in the cardiovascular system and imply complex hormonal regulation of both cardiovascular function and whole body metabolism.

Committee: John Kopchick (Advisor); Darlene Berryman (Committee Member); Shigeru Okada (Committee Member); Calvin James (Committee Member) Subjects: Endocrinology; Molecular Biology

Bachelor of Science (BS), Ohio University, 2010, Chemistry

Obesity is characterized by a constant state of low-grade inflammation accompanied by elevated levels of macrophages in the adipose tissue itself. This phenomenon appears to be dependent on the anatomical location of the adipose depot. Previously our laboratory has demonstrated that growth hormone (GH) has the ability to decrease fat mass in a depot specific manner; however, its role in adipose tissue inflammation or macrophage infiltration has not been evaluated. Using a diet-induced obese mouse model, the purpose of this study was to determine the effects of GH or IGF-1 administration on gene expression of macrophage and inflammation markers in different depots of adipose tissue. Eighty-four C57BL/6J mice were placed on a high fat diet for 16 weeks to induce obesity. Mice were then separated into 4 groups (n=7), based on experimental treatment and length of treatment. The four experimental treatments included a control, a GH treated, an IGF-1 treated, and a diet reversal group. The control group and the diet reversal group received twice daily injections of phosphate buffered saline (PBS), and the experimental groups received twice daily injections of either GH (5.0 μg/g body weight) or IGF-1 (2.5 μg/g body weight). Groups were treated for 2, 7, or 21 days, and at the end of each time period, the mice were sacrificed and dissected. The fat pads collected and analyzed include subcutaneous, retroperitoneal, mesenteric, and epididymal depots. There was no difference in depot mass among the experimental groups following 2 days of treatment. After 7 days of treatment, significant differences were present among the diet reversal group and either the control or IGF-1 treated groups within the retroperitoneal and brown adipose depots. Finally, 21 days of treatment resulted in significantly lower fat masses in the GH and diet reversal treatments as compared to the control and IGF-1 treated group. Expression levels of the macrophage F4/80 were determined by real-time PCR. R (open full item for complete abstract)

Committee: Darlene Berryman PhD (Advisor); Edward O List PhD (Advisor) Subjects: Biomedical Research

Master of Science (MS), Ohio University, 2010, Biological Sciences (Arts and Sciences)

Growth hormone (GH) is a peptide hormone that is secreted from the anterior pituitary under the regulation of different proteins, hormones, amino acids and nutrients.GH and its signaling pathways regulate cellular growth, development and metabolism.Involvement of GH in damaging the kidney, especially in diabetes, is a matter of concern as diabetic nephropathy has been regarded as an increasing health threat. Establishment of mesangial cell lines could provide an effective in vitro model to investigate a connection between individual/multiple downstream signaling pathway/s of GH signaling and damage of kidney mesangium and thus was the topic of this thesis. The primary step of establishing this in vitro model was to characterize the presence or absence of functional GH signaling in mesangial cell lines established from the nontransgenic (NT) and growth hormone receptor gene-disrupted or knockout (GKO) mice. Glomeruli from the kidneys of NT and GKO mice were isolated and used to establish two mesangial cell lines. Specific morphology, selective media and immunohistochemistry confirmed the identity of the cells as mesangial cells. Next, expressed GHR transcripts were characterized in the mesangial cell lines by performing RT/PCR and gel electrophoresis. Expression of an intact GHR transcript in NT mesangial cells and a stable but mutated GHR transcript in GKO cells was demonstrated. Finally, the presence or absence of GH signaling in NT and GKO mesangial cell lines was tested by assaying the induction of RNA transcripts or phosphorylated proteins of downstream signaling pathways in response to exogenous GH stimulation using real-time RT/PCR, western blotting analysis or ELISA. The only response observed was GH stimulation of iNOS and STAT5b mRNA expression in both the NT and GKO mesangial cell cultures, suggesting that mesangial cells from the NT mice might be useful for studying the GH-stimulated expression of specific GH responsive genes but the cells from the GKO mi (open full item for complete abstract)

Committee: Karen Coschigano PhD (Advisor); Kenneth Goodrum PhD (Committee Member); Ramiro Malgor PhD (Committee Member); Darlene Berryman PhD (Committee Member) Subjects: Biology

Master of Science (MS), Ohio University, 2007, Nutrition Science (Health and Human Services)

A better understanding of the storage and release of neutral lipids from adipocytes is vital to understanding obesity and diabetes. Work in recent years has revealed a central role for perilipin, a lipid droplet surface protein, for optimal triacylglycerol storage and fatty acid release. Perilipin belongs to the PAT family of proteins, including perilipin, ADRP, and TIP47. Perilipin and ADRP are most closely related and are responsible for controlling lipase access to TAG. Growth hormone (GH) has repeatedly been shown to induce lipolysis and inhibit lipogenesis although the mechanism is not well understood. The potential relationship between GH and the PAT family has yet to be examined. To address this, we utilized two mouse lines with altered GH signaling: 1) GH receptor gene disrupted (GHR -/-) mice; and 2) mice expressing bovine GH (bGH). Perilipin, ADRP, and TIP47 mRNA expression was compared in three adipose depots (subcutaneous, epididymal and retroperitoneal) in GHR -/-, bGH, and wild type (WT) mice on a high fat (HF) or low fat (LF) diet using quantitative real time PCR. Results showed a significant decrease in perilipin mRNA in the subcutaneous and retroperitoneal depots of bGH mice on the LF diet compared to GHR -/- and WT mice. However, there was no significant difference in perilipin mRNA expression between the GHR -/- and WT mice in any of the fat depots. ADRP mRNA expression levels are more variable than perilipin mRNA levels with respect to genotype and diet treatment. This research may provide insight into the relationship between GH and the lipid packaging functions of the PAT family and may aid in discovering a prospective target to aid in the treatment of obesity.

Committee: Darlene Berryman (Advisor) Subjects: Health Sciences, Nutrition