DRUM Collection: Cell Biology & Molecular Genetics Theses and Dissertations

IDENTIFICATION OF A NOVEL ANTI-APOPTOTIC GENE OF MYCOBACTERIUM TUBERCULOSIS

2012-01-01T00:00:00Z

Title: IDENTIFICATION OF A NOVEL ANTI-APOPTOTIC GENE OF MYCOBACTERIUM TUBERCULOSIS Authors: Hurley, Benjamin Abstract: 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.

CHARACTERIZATION OF THE N-ACETYLGLUTAMATE SYNTHASE KNOCKOUT MOUSE, A NOVEL MODEL OF HYPERAMMONEMIA

2012-01-01T00:00:00Z

Title: CHARACTERIZATION OF THE N-ACETYLGLUTAMATE SYNTHASE KNOCKOUT MOUSE, A NOVEL MODEL OF HYPERAMMONEMIA Authors: Senkevitch, Emilee Abstract: All knockout mouse models of urea cycle disorders die in the neonatal period or shortly thereafter. Since N-acetylglutamate synthase (NAGS) deficiency in humans can be effectively treated with N-carbamyl-L-glutamate (NCG), we sought to develop a mouse model of this disorder that could be rescued by biochemical intervention, reared to adulthood, reproduce, and become a novel animal model for hyperammonemia. NCG and L-citrulline (Cit) were used to rescue the NAGS knockout homozygous (Nags-/-) pups and the rescued animals were characterized. This regimen has allowed for normal development, apparent health, and reproduction. Interruption of this rescue intervention resulted in the development of severe hyperammonemia and death within 48 hours. We have developed a home cage behavioral system that allows to monitor and analyze the chronology of animal behaviors during healthy and hyperammonemic states. Data collected from this study reveals that mice decrease their normal activity around 12 hours and become severely lethargic by 20 hours following NCG withdrawal. Understanding the chronology of hyperammonemia will aid in future studies to discover neuro-protective drugs for treating hyperammonemia. This mouse model will also allow studies of the pharmacokinetics and pharmacodynamics of NCG, much of which is still unknown. Understanding how NCG is transported into cells and then cleared is clinically relevant and can potentially lead to more efficient administration of this drug. We conclude that a novel NAGS deprived mouse model has been developed which can be rescued by NCG and Cit and reared to reproduction and beyond. This biochemically salvageable mouse model recapitulates the clinical phenotype of proximal urea cycle disorders and can be used as a reliable model of induced hyperammonemia by manipulating the administration of the rescue compounds.

B Cell Memory, CD23, and Lipid Metabolism: A Preliminary Study

2012-01-01T00:00:00Z

Title: B Cell Memory, CD23, and Lipid Metabolism: A Preliminary Study Authors: Wiggins, Melvin Daniel Abstract: We each receive vaccinations throughout our lives, which protect us from many pathogens and gives long-term protection through generating a subset of memory lymphocytes. This study explores whether CD23 (Fcε receptor) and high fat diet have roles in regulating memory B cells. CD23 in B cells was examined using CD23 transgenic mice. My data show that, after antigenic stimulation, CD23 co-aggregates with the BCR. The percentages of isotype switched B cells as well as other peripheral B cell subsets in the spleen are not altered in unimmunized CD23 transgenic mice, implicating that CD23 does not have any significant role in the generation of memory B cells. High fat diet with and without high cholesterol led to increased numbers of mature follicular B cells and decreases in transitional B cells in a NPC1L1 independent manner.The marginal zone B cells numbers are increased in the mice fed high fat/high cholesterol diets. This suggests a possible role of high fat/high cholesterol diet in regulating the peripheral development of B cells.

Developing Tools for Investigating Chemotaxis Signal Clusters in Bacillus subtilis

2012-01-01T00:00:00Z

Title: Developing Tools for Investigating Chemotaxis Signal Clusters in Bacillus subtilis Authors: Rogers, James Allen Abstract: Many bacteria make use of a set of dedicated chemoreceptor proteins to control a His-Asp signaling system; this control converts environmental sensory information into instructions that regulate flagellar rotation, enabling chemotaxis. This thesis summarizes my investigations of some of the chemotaxis signaling proteins in Bacillus subtilis, particularly coupling proteins CheW and CheV. Proteins CheA, CheW, CheV, CheY, and FliM were each expressed in B. subtilis as translational fusions with either YFP or CFP. These fusion proteins were then shown to fluoresce in living bacterial cells. Motility experiments were conducted to compare the function of these fusion proteins to their wild type counterparts. This thesis proposes a series of experiments that would use these fluorescent fusion proteins to further explore the idea that these chemotaxis proteins change position when B. subtilis encounters chemostimuli.