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No description available.

No description available.

No description available.

Fork-head associated (FHA) domains function in the assembly of signalling complexes through specific interactions with phospho-threonine motifs. FHA domains are found in eukaryotes, and intriguingly in a subset of bacterial species including Mycobacteria tuberculosis. In addition, the M. tuberculosis genome encodes 11 eukaryotic-like serine/threonine protein kinases (STPKs). In M. tuberculosis, the co-expression of FHA domain-containing proteins and STPKs strongly suggest that these bacterial FHA domain-containing proteins engage in phospho-dependent protein-protein interaction, and FHA dependent processes in bacteria are controlled by STPK-dependent phosphorylation. This study describes a body of biophysical and biochemical experiments which characterises the interactions between two STPKs, PknA and PknB, and three FHA domain-containing proteins, Rv0019, Rv0020 and Rv1827. Isothermal titration calorimetry and surface plasmon resonance combined with site-directed mutagenesis are used to investigate interactions of kinase domains with FHA domains. These experiments reveal that the FHA domains interact with specific phospho-threonine residues located within the kinase domain activation loop. Additionally, in vitro kinase assays demonstrated that the interactions also involve phosphorylation of the FHA domains and/or adjacent segments of the protein. These data suggest that STPK-mediated signalling in M. tuberculosis involves a complex series of interactions and suggests a 'molecular docking' model for the phosphorylation of FHA domain-containing proteins. Finally, high-throughput assays have been used to identify several inhibitors of PknB which will serve as the basis for development of novel antimicrobial therapies.

The recent resurgence of drug resistant strains of Mycobacterium tuberculosis, the causal agent of tuberculosis (TB) has strengthened the need for new anti-TB drugs. However, the accompanying experiments are lengthy due to the slow growth rate and pathogenic nature of the tubercle bacillus. Consequently a faster growing, nonpathogenic Mycobacterium would be ideal as a first screen in drug discovery. This project investigated metabolic features of M smegmatis to establish its utility as a first screen in the discovery of metabolic drug targets.

The aim of this thesis was to investigate the mechanisms by which Mycobacterium tuberculosis develops resistance. Resistance depends entirely on genetic alterations within the genome as a consequence of spontaneous mutations. It is hypothesized that an isoniazid resistant strain, deficient in its response to oxidative damage through a mutation in katG, demonstrates a hypermutator phenotype which leads to multiple drug resistance. However there was no evidence of an increased mutation rate. It is likely that compensatory mutations are occurring allowing the bacteria to adapt to its surroundings. The hypothesis that oxidative damage is the major force driving mutations in M. tuberculosis was tested by analyzing reported mutations in rpoB and pncA. The lack of evidence of this type of damage indicates that M. tuberculosis is sufficiently competent in repair. This leads to the conclusion that oxidative damage is not the primary mechanism for mutation in the M. tuberculosis genome and instead it is the relative fitness of the mutant strain coupled with the resistance phenotype that fixes mutations and permits survival and detection. Sub-optimal therapy can lead to the selection of mutant strains. It has been shown previously that exposure to sub-inhibitory concentrations of quinolone can increase the mutation rate of mycobacteria. Gene expression profiling was used to determine whether this increase in mutation rate was due to an induction of the SOS repair genes among other DNA repair systems and found that some of these genes were differentially expressed during treatment. Sub-populations of resistant bacteria may exist in clinical specimens prior to drug exposure and this population may be likely to pre-dominate leading to heterogeneous resistance. This phenomenon called heteroresistance was demonstrated in laboratory strains of Mycobacterium fortuitum and Mycobacterium smegmatis and in M. fortuitum clinical isolates.

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Text in English / Aim: The aims of this study were to investigate the treatment outcomes of patients with MDRTB and its determinants at referral hospitals in Ethiopia. The study also aims to develop a conceptual model for enhancing treatment of patients with MDR-TB in Ethiopia. Design and methods: A concurrent mixed methods design with quantitative dominance was used to investigate treatment outcomes of patients with MDR-TB and its determinants. Results: A total of 136 (n=136) patients with MDR-TB participated in the study, 74 (54%) were male and 62 (46%) were female. Forty-one (31%) of the patients had some co-morbidity with MDR-TB at baseline, and 64% had body mass index less than 18.5kg/m2. Eight (6%) of the patients were diagnosed among household contacts. At 24 months, 76/110 (69%) of the patients had successfully completed treatment, but 30/110 (27%) were died of MDR-TB. Multivariable logistic regression revealed that the odds of unfavourable treatment outcomes were significantly higher among patients with low body mass index (BMI <18.5kg/m2) (AOR=2.734, 95% CI: 1.01-7.395; P<0.048); and those with some co-morbidity with MDR-TB at the baseline (AOR=4.260, 95%CI: 1.607-11.29; p<0.004). The majority of the patients were satisfied with the clinical care they received at hospitals. But as no doctor was exclusively dedicated for the MDR-TB centre, patients could not receive timely medical attention and this was especially the case with those with emergency medical conditions. The caring practice of caregivers at the hospitals was supportive and empathic but it was desperate and alienating at treatment follow up centres. Patients were dissatisfied with the quality and adequacy of the socio-economic support they got from the programme. Despite the high MDR-TB and HIV/AIDS co-infection rate, services for both diseases was not available under one roof. Conclusions: Low body mass index and the presence of any co-morbidity with MDR-TB at the baseline are independent predictors of death among patients with MDR-TB. Poor communication between patients and their caregivers and inadequate socio-economic support were found to determine patients’ perceived quality of care and patients’ satisfaction with care given for MDR-TB. / Health Studies / D. Litt et Phil. (Health Studies)