Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item INVESTIGATION OF DISRUPTED INSULIN SIGNALING IN A SWINE MODEL(2024) Markley, Grace Irene; Stahl, Chad H; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Insulin is an anabolic hormone involved in glucose uptake and synthesis of fats, proteins, and glycogen. Domesticated livestock species such as swine require efficient insulin signaling to meet production demands across the world. Insulin signaling is tightly regulated and acts on metabolic tissues such as the liver, skeletal muscle, and adipose tissue. The most well characterized disruption of insulin signaling is insulin resistance that is often caused by obesity induced inflammation. However, insulin signaling can be disrupted via atypical mechanisms such as immune response to a pathogen and adaptor proteins. We aimed to evaluate the impact of pathogen exposure and intrinsic adaptor proteins on insulin signaling in pigs. The first study focuses on the impact of growth factor receptor bound protein 10 (GRB10) as an inhibitor of insulin signaling. In commercial swine, the presence of GRB10 has been linked to growth, reproduction, feed efficiency and lean muscle growth. While insulin induces glucose uptake in typical tissues, such as the liver and skeletal muscle, insulin also acts on other cell types including mesenchymal stem cells (MSC). MSC are adult multipotent stem cells that can self-renew and differentiate into multiple cell types including adipocytes. The process of adipogenesis requires insulin signaling to synthesize new triglycerides and store them in lipid droplets. While GRB10 has been established as a regulator of insulin signaling, the role of GRB10 in swine MSC has yet to be firmly established. We generated GRB10 knockdown (GRB10-KD) MSC to evaluate the impact of GRB10 on insulin signaling and glucose uptake. We observed reduced glucose utilization under basal conditions and reduced insulin signaling when incubated with insulin over 48 hours. We also noticed a two-fold reduction in proliferation rate among GRB10-KD MSC. When differentiated into adipocytes, we observed an increase in transcript abundance with genes associated with insulin signaling and adipogenesis. GRB10 has the potential to regulate insulin signaling in swine MSC and contribute to overall growth and development. The second chapter focuses on the impact of lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, which can induce a severe, systemic immune response. In pigs, chronic LPS exposure has induced insulin resistance. However, the effects of acute exposure to LPS on insulin signaling and resistance have not been elucidated. We found that acute exposure to LPS in crossbred post-weaning pigs induced changes in insulin signaling and glucose metabolism. There was an LPS induced decrease in insulin two hours after injection which was paired with hyperglycemia. At 24 hours post LPS there was a marked insulin resistance indicated by hyperinsulinemia and hyperglycemia. We also noted that there were liver specific decreases in genes associated with glucose metabolism, insulin signaling and fatty acid metabolism. As well as reduction in protein abundance such as protein kinase B (AKT) and phosphoinositide-3 kinase (PI3K) in the liver after LPS administration. During an acute exposure to endotoxin, insulin signaling, and glucose metabolism is reduced in the liver. These results highlight that insulin signaling is a complex and dynamic process that can be controlled through a variety of mechanisms and swine can serve to model these disruptions.Item MODULATING KEY GENES INVOLVED IN PANCREAS FORMATION AND INSULIN SIGNALING USING CRISPR/CAS9 IN THE PIG(2019) Sheets, Timothy P; Telugu, Bhanu P; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Among the metabolic diseases, diabetes remains a “pressing problem” as recognized by World Health Organization, not only due to the impact on individuals’ lives, but also because of the rapid increase in newly diagnosed patients. To better understand the mechanisms of diabetes, this dissertation investigates the role of NGN3 in pancreas development using CRISPR/Cas9 gene targeting in the pig model. NGN3 was selected for study because of its critical role in endocrine pancreas formation. Our research demonstrates that the targeted ablation of NGN3 blocks development of the endocrine pancreas, a finding supported through gene expression analysis. Furthermore, follow-up studies show that clonal piglets derived from NGN3-ablated animals lack the major endocrine islet cell types and subsequent expression of key endocrine hormones. This porcine model provides valuable insights into the study of type 1 diabetes in early post-natal life and future applications of human-to-pig chimeric organ development for transplant surgery. Expanding upon this porcine model for diabetes, we sought to apply this approach to the study of type 2 diabetes using a novel pig model, thus bridging the gap between mouse and human. For this endeavor, we identified GRB10 as a potential critical mediator in insulin signaling, development, and growth potential following an extensive literature review. The potential for dual applications in both agriculture and medicine was also identified as an objective. Analysis of qPCR data from in vitro overexpression studies supports that GRB10 modulates insulin signaling through the canonical insulin pathway. Additional data from two in vivo gene editing trials targeting the GRB10 locus in both Ossabaw and domestic pig breeds show a supportive qualitative trend towards growth regulation in the Ossabaw pig breed. Further evidence is required to determine whether GRB10 plays the same role in the domestic pig, as a limited cohort size of mutants precluded an extensive analysis of phenotypes. Together, our assessment of NGN3 and GRB10 offer significant potential for modeling of both type 1 and type 2 diabetes as well as modeling of growth traits in the pig through application of advanced genome engineering technology.