Theses and Dissertations from UMD
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Item IDENTIFICATION OF A NOVEL ANTI-APOPTOTIC GENE OF MYCOBACTERIUM TUBERCULOSIS(2012) Hurley, Benjamin; Briken, Volker; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Over the next 20 years, more than 36 million people are expected to die of Mycobacterium tuberculosis (Mtb) related illness. We may prevent this only by learning as much as possible about the Mtb-mediated exploitation of the human immune system and successfully implementing that knowledge to combat the pathogen. One Mtb virulence mechanism involves inhibition of host apoptosis. An Mtb laden macrophage will attempt cell suicide, subsequently destroying any intracellular bacteria. In prior work, a large gain-of-function screen identified Mtb genomic regions involved in host apoptotic suppression; one such region is "K20." A loss-of-"gain-of-function" (LoGoF) screen involving in vitro transposon (Tn) mutagenesis of a K20 expressing vector identified individual gene(s) of K20 potentially responsible the virulence phenotype. This LoGoF screen found two unique K20 Tn-clones that consistently induced significant host apoptosis. These Tn's disrupted expression of K20 genes Rv2666 ( probable truncated transposase) and Rv2667 ( possible Clp ATPase). A Himar1 Tn mutant of Rv2666 was obtained through the TARGET mutant library project. Upon infection of human macrophages, TnRv2666 induced significantly more host apoptosis than wild type (WT) and complement, confirming Rv2666 as an anti-apoptotic gene of Mtb. There are no current publications suggesting a specific role for transposase-based virulence in Mtb; Rv2666 may affect an anti-apoptotic phenotype by modulating transcription of factors important for the suppression of host apoptosis. The second LoGoF identified gene, Rv2667/clpC2/clpX', was not available through TARGET; a recombinant ΔclpC2 mutant was generated. Comparing induced host apoptosis of ΔclpC2 infected macrophages to WT revealed Rv2667/clpC2 has no essential role as an anti-apoptotic gene of Mtb. ΔclpC2 was further characterized to explain the discrepancy between the initial LoGoF data and the ΔclpC2 Mtb results. Study of ΔclpC2 determined that it bears no significant differences with WT in terms of in vitro growth, host necrosis-induction, in vivo survival and induced host TNFα secretion levels. However, ΔclpC2 induces significantly more host IL-1β release than WT Mtb. The reason for this effect is unknown; ClpC2 may aid Mtb pathogenicity by limiting host inflammation, thus permitting infecting Mtb a "head start" against a host adaptive immune response.Item ENERGY METABOLISM IN DEVELOPING CHICKEN LYMPHOCYTES DURING THE EMBRYONIC TO POSTHATCH TRANSITION(2007-07-26) Rudrappa, Shashidhara Govindareddy; Porter, Tom E; Humphrey, Brooke D; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In chickens, the primary energy substrate is lipid during embryogenesis and carbohydrate after hatch. Accordingly, chicks adapt their metabolism to utilize glucose after hatch; however, little is known about metabolic adaptation in developing lymphocytes. Therefore, the objective of this dissertation was to examine metabolic adaptation in developing lymphocytes and the associated impact on their development. The first objective examined energy substrate utilization in bursacytes and thymocytes during the embryonic to posthatch transition. Glucose metabolism increased in both lymphocyte populations during the first two weeks posthatch due to increased glucose transporter-3 mRNA abundance, glucose uptake and hexokinase activity. Additionally, some of these metabolic markers were positively correlated with the serum glucose concentration. Glutamine metabolism increased in bursacytes only, and lipid metabolism was unaltered in both populations. Collectively, glucose is a preferred energy substrate for lymphocytes posthatch, and glucose utilization by developing lymphocytes may be related to the serum glucose concentration. The second objective determined the effect of glucose availability on thymocyte metabolism, energy status and survival. Embryonic thymic lobes were grown in culture in media containing varying glucose concentrations. Thymocyte glucose metabolism and mitochondria membrane potential were highest in 15 mM glucose and apoptosis was highest in 5mM glucose. Collectively, glucose availability regulates glucose metabolism in thymocytes, and these changes in glucose metabolism were related to thymocyte energy status and survival. The third objective determined the effect of glucose availability on T cell development. Thymocyte Interleukin-7Rα (IL-7Rα) mRNA abundance and CD4+ T cell numbers over the culture period were dependent upon glucose availability. Between 12 and 24 h, thymocyte IL-7Rα mRNA abundance increased in 5 mM increased 1.74-fold, while it decreased in 15 mM by 58.6%. CD4+ numbers decreased with time in 5 mM, whereas they increased with time in 15 mM. T cell receptor (TCR) β excision circles were higher in 15 mM compared to 5 mM at 12 h. Glucose availability alters TCR β rearrangement, IL-7Rα gene expression and CD4+ T cell development, which may influence naïve T cell generation. As thymocytes develop in a low glucose environment in ovo, this may be one factor that limits T cell development until hatch.