UMD Theses and Dissertations
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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 Comparative genomic analysis of Vibrio cholerae O31: capsule, O-antigen, pathogenesis and genome(2006-11-21) Chen, Yuansha; Morris, J Glenn; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Vibrio cholerae is the causative agent of cholera. In order to understand the genetic basis underlying the emergence of novel epidemic strains of V. cholerae, the genetics of surface polysaccharide biogenesis, and the role of lateral gene transfer in the evolution of this species, we investigated. NRT36S and A5 are both NAG-ST producing, cholera toxin negative, serogroup O31 V. cholerae. NRT36S is encapsulated and causes diarrhea when administered to volunteers; A5 is acapsular and does not colonize or cause illness in humans. The structure of the capsular (CPS) polysaccharide in NRT36S was determined by NMR. The gene cluster of CPS biogenesis was identified by transposon mutagenesis combined with whole genome sequencing data. The CPS gene cluster shared the same genetic locus as that of the O-antigen of lipopolysaccharide (LPS) biogenesis gene cluster. The LPS biogenesis regions in A5 were similar to NRT36S except that a 6.5 kb fragment in A5 replaced a 10 kb fragment in NRT36S in the middle of the LPS gene cluster. The genome of NRT36S was sequenced to a draft containing 174 contigs plus the superintegron region. Besides confirming the existence of NAG-ST, we also identified the genes for a type three secretion system (TTSS), a putative exotoxin, and two different RTX genes. Four pili systems were also identified. Therefore, the genome of non-O1 Vibrio cholerae NRT36S demonstrates the presence of pathogenic mechanisms that are distinct from O1 V. cholerae. We conclude that lateral gene transfer plays a critical role in the emergence of new strains. The co-location of CPS and LPS could provide a mechanism for simultaneous emergence of new O and K antigens in a single strain. Our data also highlights the apparent mobility within the CPS/LPS region that would provide a basis for the large number of observed V. cholerae serogroups and the emergence of novel epidemic strains.Item What Makes a Pathogen? Genetic and Structural Heterogeneity of Neisserial Lipooligosaccharide(2006-01-24) O'Connor, Ellen Therese; Stein, Daniel C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The genus Neisseria consists of both pathogenic and commensal species that colonize mucosal niches. Specific structures of neisserial lipooligosaccharide (LOS) expressed by the gonococcus and meningococcus have been shown to play a role in pathogenesis. Commensal Neisseria also produce LOS, but can express additional structures not found in the pathogenic strains. N. sicca 4320 differs from other commensal Neisseria because it caused disease in a seemingly healthy individual. Therefore, this strain was used to research the extent to which a non-pathogen differs from a pathogen. A bioinformatic examination of the nucleotide sequence of the 4320 chromosome revealed strong homologies with N. meningitidis MC58. These findings suggest that 4320 is a member of the Neisseria and that commensal and pathogenic Neisseria have similar genomic content. Through MALDI-TOF, exoglycosidase digestion, and MSn analyses, 4320 was shown to express both LOS and LPS. The 4320 LOS resembles that of Haemophilus sp. in that it contained 3 heptoses. The LPS consists of a N-acetylglucosamine and rhamnose disaccharide repeat that is not attached to a lipid A, a novel molecule. PEA transfer onto pathogenic Neisseria LOS was examined, as strains expressing PEA at 3-HepII as opposed to 6-HepII are more serum resistant. Southern hybridization was used to identify the presence of phosphoethanolamine (PEA) transfer genes in N. sicca strains. 4320 showed differences in the presence of Lpt3 transfer genes compared to other strains within the species. Lpt3 was isolated and biochemically shown to mediate the addition of PEA to 3-HepII of LOS that was constructed to lack the 3-HepII decoration. lpt3 nucleotide sequence analysis was done on a diverse group of gonococcal isolates, and phylogenetic analysis showed a high degree of sequence divergence. These data support the idea that the presence of PEA at 3-HepII of LOS increases the pathogenic ability of the Neisseria, as lpt3 was only found in N. sicca strains isolated from fatal endocarditis cases.