published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

This research initiated an investigation of the structural relationships between melanocyte stimulating hormone (α-MSH) and its melanin dispersion on lizard (Anolis carolinensis) and frog (Rana pipiens) skins bioassays as representing models for mammalian and amphibian melanocytes, respectively. From previous extensive structure-activity relationships of α -MSH together with the theoretical modeling we were able to design a group of linear and cyclic peptides related to "4-10" fragment analogues of α -MSH. The solid phase synthesis of α -MSH and its related analogues using the p-methyl-benzhydrylamine resin was accomplished. The C-terminal carboxamide and N-terminal acetylamide were maintained in all peptides synthesized. The cyclic peptides were prepared in solution phase using the linear peptides generated by solid phase. All the cyclization were done by using the hydrochloride salts of the peptide and DMF as solvent with diphenylphosphoryl azide (DPPA) as a coupling reagent in the presence of K₂HPO₄ as a base. The yields of the cyclic peptides were in the range of 30-40 percent. In all the synthesized peptides the replacement of D-Phe⁷ with L-Phe⁷ causes reduction in the potency of the peptide on lizard or frog skins bioassays. Also, the reduction or increase in ring size in the cyclic peptide from a 23 membered ring diminishes the biological effect of the peptide under testing.

Continuous-time Markov chains (CTMC's) form a convenient mathematical framework for analyzing random systems across many different disciplines. A specific research problem that is often of interest is to try to predict maximum probability sequences of state transitions given initial or boundary conditions. This work shows how to solve this problem exactly through an efficient dynamic programming algorithm. We demonstrate our approach through two different applications - ranking mutational pathways of HIV virus based on their probabilities, and determining the most probable failure sequences in complex fault-tolerant engineering systems. Even though CTMC's have been used extensively to realistically model many types of complex processes, it is often a standard practice to eventually simplify the model in order to perform the state evolution analysis. As we show here, simplifying approaches can lead to inaccurate and often misleading solutions. Therefore we expect our algorithm to find a wide range of applications across different domains.

Resistance to antimalarial drugs continues to be a major obstacle in controlling and eradicating malaria. The identification of genetic markers of resistance is vital for disease management but they can be difficult to predict before resistance arises in the field. This thesis describes an alternative approach to gene identification, combining an in vivo experimental evolution model, Linkage Group Selection (LGS) and Solexa genome re-sequencing. Here this model was used to resolve the genetic basis of chloroquine and artemisinin resistance in the rodent malaria parasite Plasmodium chabaudi. AS-30CQ is a parasite with high resistance to chloroquine and resistance to artemisinin. It was crossed with the genetically different drug-sensitive strain AJ. The resulting progeny were selected with drugs and backcrossed to the sensitive parent. Both crosses were treated with increasing concentrations of chloroquine and artemisinin. The frequency of markers from the sensitive parasite were analysed in order to characterize the signatures of drug selection. Three loci involved progressively in chloroquine resistance were identified on chromosomes 11, 3 and 2. One main locus on chromosome 2 was identified with artemisinin selection. The Solexa platform was used to re-sequence the genomes of both AS-30CQ and its sensitive progenitor, AS-sens. The differences between the two genomes were integrated with the LGS data to identify: 1) a strong candidate for the main CQresistance determinant - a putative amino acid transporter on chromosome 11 (aat1) 2) two candidates for high level chloroquine resistance on chromosome 3. and 3) a mutation in ubp1 gene on chromosome 2 that is likely to contribute to the highest level of chloroquine resistance and be main determinant of the artemisinin resistance phenotype. In addition the last section of this thesis describes two otherwise isogenic clones showing low- and high levels of chloroquine resistance were grown competitively to evaluate the effect of these mutations on parasite fitness. The highly resistant strain demonstrated a loss of fitness in relation to its more sensitive progenitor and was outcompeted in untreated and low-treated infections.

Submitted in fulfillment for the Degree of Doctor of Philosophy (Biotechnology), Durban University of Technology, Durban, South Africa, 2016. / Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB) has infected approximately one-third of the world population, with 9.6 million TB cases in 2014. The emergence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains of MTB has further complicated the problem of TB control. It is now imperative that novel antimycobacterial compounds are discovered in order to treat infections and reduce the duration of current TB therapy courses. For centuries, medicinal plants have been used globally worldwide for the treatment and prevention of various ailments. This occurs particularly in developing countries where infectious diseases are endemic and modern health facilities and services are inadequate. In recent years, the use and search for plant drug derivatives have been fast-tracked. Ethnopharmacologists, botanists, microbiologists, and natural product chemists are trying to discover phytochemicals which could be developed for the treatment of infectious diseases, especially TB. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimycobacterial activity. In the search for new lead compounds, nine medicinal plant species, Buddleja saligna, Capparis tomentosa, Carpobrotus dimidiatus, Dichrostachys cinerea, Ekerbergia capensis, Ficus Sur, Gunnera perpensa, Leonotis leonurus and Tetradenia riparia were collected in Kwa-Zulu Natal (KZN) following report of their therapeutic use in traditional medicine to treat symptoms and infections related to TB. They were tested in vitro for their activity against Mycobacterium smegmatis, Mycobacterium tuberculosis H37Rv (ATCC 25177) and three well-characterized clinical isolates of MDR-TB and XDR-TB using the agar incorporation method. The minimum inhibitory concentration of the active plant extracts was determined using the broth microdilution method. Our findings show that five of the nine plants screened have antimycobacterial activity with concentrations ranging from 125 µg/ml to 1000 µg/ml. The aqueous extracts of G. perpensa and T. riparia; and the methanolic extracts of B. saligna, C. tomentosa, and C. dimidiatus possessed significant activity against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177) and the three well-characterized clinical isolates of MDR-TB and XDR-TB. The cytotoxic effect of the active plant extracts was evaluated against the mouse BALB/C monocyte-macrophage (J774.2) and peripheral blood mononuclear cells (PBMCs). The toxic effects of the active plant extracts were evaluated using the brine shrimp lethality assay. Except for a high concentration of G. perpensa none of the other plants which possessed antimycobacterial activity showed any toxic or cytotoxic activity. The active plant extracts were thereafter assessed to determine if they had any effect on the survival or death of mycobacterial species, M. smegmatis, bound within the macrophage (J774.2) cell line at a concentration of 100 µg/ml. B. saligna had inactivated most of the phagocytosed bacilli after 24 hours of treatment therefore, it has a bactericidal effect on the mycobacteria located within the mouse macrophage. A phytochemical investigation of the leaves of B. saligna led to the isolation of two isomeric pentacyclic triterpene compounds namely Oleanolic Acid (OA) and Ursolic Acid (UA) using thin layer chromatography followed by silica gel column chromatography. The structures of these compounds were fully characterized by detailed NMR investigations, which included 1H and 13C NMR. Ursolic acid was isolated from this plant for the first time. Two-dimensional (2D) and three-dimensional (3D) quantitative structure-activity relationship (QSAR) studies were carried out to provide insight on the interaction of the compounds with the enzyme. Molecular docking studies predicted the free binding energy of the triterpenes inside the steroid binding pocket of Mycobacterium tuberculosis fadA5 thiolase compared to a reported inhibitor. Thus, their ability to inhibit the growth of Mycobacterium tuberculosis was predicted and was confirmed to possess significant antimycobacterial activity when tested against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177), clinical isolates of MDR-TB and XDR-TB using the Microplate Alamar Blue Plate (MABA) assay. The present study has scientifically validated the traditional use of medicinal plant B. saligna. / D

Antibiotic resistance in bacteria is a growing threat to global health. Many of the genes responsible for resistance are carried on mobile genetic elements which can be transferred laterally between strains and species. The most important of these are conjugative and mobilisable elements including plasmids and integrating and conjugating elements, ICEs. Haemophi/us influenzae is an important human pathogen, which was first identified as carrying antibiotic resistance genes in the 1970s. Much of this resistance is encoded by ICEHin1056, which is present in H. influenzae strains worldwide. The aims of this study were to describe features of the biology of ICEHin1056, with particular reference to the genetic site and control mechanisms responsible for instigating conjugative transfer. The origin of transfer has been localised to a sequence on ICEHin1056 and an environmental stressor initiating conjugative transfer, oxidative stress, has been identified. In addition, detailed phylogenetic analysis has demonstrated ICEHin1056 to be part of a much larger family of mobile genetic elements, widely distributed in proteobacteria and carrying accessory genes responsible for survival in adverse environments, virulence and antibiotic resistance. The ICEs in the family have conserved homology of gene content and synteny of gene arrangement over deep evolutionary time, challenging the accepted paradigm of modular mosaicism of mobile genetic elements. A key event in increasing dissemination of the ICE, acquisition of a phage type integrase gene has also been identified. The findings presented provide significant insight into the behaviour of ICEs and may in future allow predictions about the spread of virulence factors and antibiotic resistance genes, with important implications for human and animal health.

MSc (Med), Clinical Microbiology and Infectious Diseases, Faculty of Health Sciences, University of the Witwaterstrand / Emergence and spread of resistance to ciprofloxacin among Neisseria gonorrhoeae strains has reduced the options of effective treatment for gonococcal infections and has become a concern worldwide. Up until 2008, ciprofloxacin was recommended first-line therapy for treatment of presumptive N. gonorrhoeae infections in South Africa. At the time this MSc project was conceived, ciprofloxacin was still used as first-line therapy for presumptive gonococcal infections. A real-time polymerase chain reaction (PCR) assay was used to detect ciprofloxacin-resistant N. gonorrhoeae in DNA extracted from non-invasive urine samples collected as part of the national microbiological surveillance (NMS) programme during 2006-2007. The molecular epidemiology of ciprofloxacinresistant Neisseria gonorrhoeae was investigated by sequencing the quinolone resistance determining regions (QRDR) of the gyrA and parC genes of N. gonorrhoeae and performing N. gonorrhoeae multi-antigen sequence typing (NGMAST). As part of the NMS program for sexually transmitted infections (STIs) urine and urethral swabs were collected from men presenting with urethral discharge at primary health care clinics in Johannesburg (Gauteng), Cape Town (Western Cape) and Kimberley (Northern Cape). Urine samples and cultured N. gonorrhoeae isolates from 2006-2007 were stored at -700C and available for this study. Gonococci, previously isolated from urethral swabs, were subcultured directly onto New York City media. Isolate identity was re-confirmed by typical colony morphology and biochemical tests. Urine samples from Johannesburg were tested in order to develop the real-time PCR protocol. Subsequently, paired urethral swab DNA and N. gonorrhoeae cultures were tested from NMS patients recruited in Kimberley and Cape Town. Where possible, the PCR assay results were compared with paired antibiotic susceptibility data for ciprofloxacin. Quinolone resistance determining regions (QRDR) for gyrA and parC were screened for known point mutations associated with resistance to ciprofloxacin. Detection of mutations by the real-time PCR assay generally agreed with the phenotype of either decreased susceptibility or resistance to ciprofloxacin. All ciprofloxacin resistant gonococcal isolates had the same gyrA and parC mutations, which initially suggested that quinolone resistant N. gonorrhoeae (QRNG) in Kimberley, Cape Town and Johannesburg, may be attributed to the spread of a single clone. The use of a more discriminatory typing scheme, Neisseria gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST) genotyping, revealed that ciprofloxacin resistant gonococcal isolates in Johannesburg and Cape Town were heterogeneous, with sequence type (ST) 217 being most prevalent in both cities (5/16, Johannesburg; 7/11, Cape Town). In contrast, all eight QRNG isolates from Kimberley were typed as ST 533. The use of molecular methods allowed ciprofloxacin antimicrobial susceptibility determination by PCR in non-invasive specimens. This is useful in situations where bacterial cultures are unavailable or die before antimicrobial susceptibility testing can be performed. Molecular assays to detect ciprofloxacin resistance may guide physicians as to the most ideal antimicrobial combinations for individual patient treatment. As a result of emerging widespread resistance gonococci to ciprofloxacin, in 2008, the Department of Health recommended that ciprofloxacin be removed as a first line therapy in the South African national sexually transmitted infections treatment guidelines for treatment of urethritis, cervicitis and their complications. Although ciprofloxacin is no longer used as a first-line therapy to treat gonorrhoea within our country, it may still be used in cases of severe penicillin allergy or as part of multi-drug therapy for gonococcal infections in the future. The ability to detect ciprofloxacin resistance by real-time PCR will be a useful technique in such situations.

by Tsang Wing Pui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 195-215). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.