3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Structure-function studies of a putative ribonuclease HI from Mycobacterium tuberculosis.(1999) Thomsen, Michelle LesleyBacterial Ribonuclease HI, which ensures that initiation of DNA replication occurs at the unique oriC; locus, is encoded by rnhA. The rnhA gene from Mycobacterium smegmatis encodes a protein that is closely related to other bacterial RNases HI (Dawes et al., 1995). Activity gel analysis rletected RNase HI activity associated with proteins in whole-cell extracts of Mycobacterium tuberculosis in the 14-25 kDa size range. A putative rnhA homologue was identified in M. tuberculosis and sequence analysis revealed that the rnhA open reading frame contains an apparent fusion of two genes (Cole et al., 1998). The 5' -region of the ORF corresponds to an rnhA homologue, whereas the 3'-region contains a gene, annotated herein as pgm, which encodes a protein belonging to the phosphoglycerate mutase (POM) family of proteins. The full-length ORF, as well as the individual mhA and pgm segments, were cloned into the pMAL-c2 expression vector and recombinant proteins were overexpresssed in E. coli as maliose binding fusion proteins. Recombinant proteins were purified and rabbit polyclonal antisera raised against each one were used to probe whole cell extracts of M. tuberculosis. Cross-reaction with polypeptides of unknown identity was observed. Limited proteolysis of the recombinant proteins suggest an instability of folding in E. coli. Functional investigation of the M. tuberculosis RNase HI included complementation of an E. coli RNase HI-defective mutant, and an M. smegmatis strain carrying a defective rnhA allele integrated at its rnhA locus, with M. tuberculosis rhns-pgm supplied in trans. No complementation in either hosts was observed. Upon completion of the genome sequence of H37Rv (Cole et al., 1998), it became apparent that the rnh/i-pgm ORF is the fourth gene in an operon which includes a gene known to be involved in cobalamin biosynthesis. Significant homlogy of the PGM to CobC phosphatase of Salmonella typhimurium implicates a role for rnh/: pgm in the cobalamin biosynthetic pathway of M. tuberculosis.Item Design and development of multifunctional Raman active noble metals nanoprobes for the detection of malaria and tuberculosis biomarkers(2016) Mlambo, MbusoSurface enhanced Raman spectroscopy (SERS) has emerged as a surface sensitive vibrational technique that leads to the enhancement of the Raman scattering molecules on or close to the surface of a plasmonic nanostructure. The enhancement is found to be in orders of 104 to 1015, which allows the technique to be sensitive enough to detect a single molecule. In this study, we report on the synthesis of different sizes of gold and silver nanoparticles (AuNPs and AgNPs) and gold nanorods (AuNRs). These are functionalized or co-stabilized with different stoichiometric ratios of HS-(CH2)11-PEG-COOH and alkanethiols (Raman reporters), i.e.; HS-(CH2)11-NHCO-coumarin(C), HS-(CH2)11-triphenylimidazole (TPI), HS- (CH2)11-indole (HSI), HS-(CH2)11-hydroquinone (HQ) to form mixed monolayer protected clusters (MMPCs). The alkanethiols were chosen as Raman reporters to facilitate the selfassembled formation of monolayers on the metal surface, thus resulting in stable MMPCs. The optical properties and stability of MMPCs were obtained using ultraviolet-visible (UVvis) spectrophometry and a zeta sizer. Size and shape of the as-synthesized nanoparticles were obtained using transmission electron microscopy (TEM). The tendency of thiolcapped nanoparticles to form self-assembled ordered superlattices was observed. Their Raman activities were evaluated using Raman spectroscopy, with the enhancement factor (EF) being calculated from the intensities of symmetric stretch vibrations of C-H observed in the region of about 2900 to 3000 cm-1 in all SERS spectra. In all four different alkanethiols (Raman reporters), smaller size metal nanoparticles (14 nm for AuNPs and 16 nm AgNPs) showed higher EF compared to 30 and 40 nm metal nanoparticles. The EF was observed to increase proportionally with stoichiometric ratios of alkanethiols from 1% iv | P a g e to 50%. The prepared MMPCs with small sizes were used as a SERS probe for the detection of malaria and tuberculosis biomarkers.Item Characterization of resuscitation promoting factors in Mycobacterium smegmatis(2012-09-12) Mapela, Lusanda ThatoMycobacterium tuberculosis, the causative agent of tuberculosis (TB) has infected one third of the world’s population and continues to claim more lives annually than any other infectious disease agent. A significant proportion of individuals carry latent TB infection (LBTI) which is characterized by the absence of clinical symptoms and it has been postulated that the tubercle bacilli are in a dormant-like state during this type of infection. Resuscitation promoting factors (Rpfs) are cell wall hydrolases which cleave glycosidic bonds within the peptidoglycan (PG), a mechanism thought to result in reactivation of bacteria from the state of dormancy. M. tuberculosis encodes five rpf-like homologues which are collectively dispensable for growth but are required for reactivation from dormancy in vitro, and for virulence in the mouse model of infection. LTBI thus poses a huge threat to the global burden of active disease. The purpose of this study was to further investigate the biological roles of Rpfs by assessing the effects of rpf gene deletion in M. smegmatis, a model organism used for TB research. M. smegmatis encodes four rpf-like genes designated rpfA, rpfB, rpfC and rpfE, and deletion mutants that lack one or more of these genes were constructed by allelic exchange mutagenesis. M. smegmatis mutant strains that lack either rpfA or rpfB display no significant differences in growth both on solid and in liquid medium when compared to wild type. However, loss of rpfA resulted in bacterial clumping during stationary-phase growth in broth culture and changes in cell morphology. Moreover; the rpfA rpfB double mutant and its derivative strain lacking rpfC, rpfA rpfB rpfC displayed a ca. 2-4 log increase in susceptibility to erythromycin and vancomycin. Furthermore, unusual colonial morphologies with reduced serpentine cording and smooth peripheries were observed for these multiple mutants. Cell surface defects and cell distortions were evidenced as wrinkle-textured, bent cells and polar tip bulges for the abovementioned mutants. The multiple deletion strains also displayed a defect in biofilm formation revealing an inability for the mutants to form complex cell-cell interactions. Collectively, the data are suggestive of a loss of bacterial cell wall integrity due to rpf gene deletion and it is proposed that rpfA, in combination with other genes, is largely responsible for cell wall integrity maintenance. Our data indicate that these factors play an important role in cell growth and division and therefore represent an untapped source of novel targets for anti-tubercular drug discovery.