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In response to the urgent need for an effective vaccine against tuberculosis (TB), mass spectrometry-based immunopeptidomics was employed to identify novel mycobacterial antigens. Human macrophages (differentiated THP-1 cells) were utilised as an in-vitro model of TB, in which mycobacterial peptides presented on major histocompatibility complex (MHC) molecules were identified. As a result, 64 and 81 mycobacterial proteins derived from the Bacille Calmette-Guérin (BCG) vaccine and Mycobacterium tuberculosis (MTB) were discovered, respectively. The identified proteins were prioritised based on their T-cell epitope repertoire through the use of immunoinformatics tools in silico, resulting in a list of potential candidate antigens. This approach efficiently facilitated the identification and prioritisation of novel mycobacterial antigens, in which T-cell responses and the diversity of human leukocyte antigen (HLA) alleles were considered. The top candidate antigens (hisD, metE and mmpL12) were selected for screening in mice through the application of mycobacterial growth inhibitory assays (MGIAs) to evaluate their protective properties. MGIA was used as an ex-vivo surrogate assay for an in-vivo MTB challenge experiment. The mouse groups that were vaccinated with metE (either in the form of plasmid DNA or as a protein with adjuvant) conferred protection compared with unvaccinated mice. The metE-DNA vaccine induced polyfunctional CD4+ T cells expressing interferon-gamma (IFNγ), tumour necrosis factor-alpha (TNFα), and interleukin-2 (IL-2), whereas the metE-AddaS03 vaccine elicited a range of CD4+ T cells that expressed TNFα, IL-2, IL-4, and IL-17. Both the metE-DNA and metE-AddaS03 vaccines significantly induced the production of immunoglobulin G (IgG) antibodies compared with the naïve group; when the vaccines were compared, the antibody concentration that was induced by the metE-AddaS03 vaccine was 16-fold higher than that induced by the metE-DNA vaccine. The findings of this work show that the metE protein is a novel protective candidate that merits further development for inclusion in a subunit vaccine against TB. Interestingly, IFNγ was found to enhance the expression and the total number of identified peptides that were associated with HLA-DR significantly compared with HLA-DQ and HLA-DP. The effects of mycobacterial infection and IFNγ treatment on the surface expression of HLA-DR alleles (HLA-DRB1*0101, -DRB1*1501 and - DRB5*0101) expressed by THP-1 cells were investigated quantitatively. Mass spectrometry-based quantitative proteomics was used to analyse the expression levels of HLA-DR proteins obtained from the same samples used for immunopeptidomics. Without IFNγ treatment, DRB1*0101 was dominantly expressed compared with DRB1*1501 and DRB5*0101. In contrast, when the sample was treated with IFNγ, DRB1*1501 was up-regulated more than DRB1*0101. Compared with uninfected samples, the levels of HLA-DRA1 and -DRB1*0101 showed a decreasing trend upon infection, whereas levels of DRB1*1501 and DRB5*0101 increased or remained unchanged. When the mycobacterial peptides were predicted according to their binding affinity, 55% of peptides were assigned as ligands for DRB1*0101, compared with about 24.3% and 20.7% assigned as ligands for DRB1*1501 and DRB5*0101, respectively. To expand this further to the whole sequence, 76% of the tested antigens (47 antigens) were predicted to contain more peptides for DRB1*0101 than for DRB1*1501. The data collected in this work suggest that mycobacteria may evade Tcell recognition as they target HLA-DR alleles that are more likely to present their antigens (i.e. they inhibit the HLA-DRB1*0101 allele and avoid being presented by the DRB1*1501 allele).

Tuberculosis (TB) remains one of the leading infectious disease causes of death at a global level, only surpassed in 2020-21 by COVID-19. Airborne infection prevention and control (IPC) measures in health facilities play an important role to reduce the spread of TB and make health care facilities safer for patients and health workers. My overall aim with the programme of research was to explore health worker and patient experiences of using TB IPC measures in healthcare facilities from an interdisciplinary perspective. I conducted a systematic review and qualitative evidence synthesis to better understand the existing academic literature on TB IPC implementation and develop implementation considerations that could support TB IPC guideline implementation. I found that health workers often focussed their TB IPC efforts on patients who are known with TB and are already on treatment, who only pose a small risk of infection. Existing research also highlighted stigma as an important impediment to TB IPC implementation, with sparse evidence about how this could be mitigated. I responded to these evidence gaps by including this as focus areas for my fieldwork. I looked at how a district hospital refined where it focussed it TB IPC efforts and aimed to explore ways in which the stigma associated with TB IPC measures could be better understood and potentially mitigated. Using the rural Eastern Cape in South Africa as setting for in-person data collection, I draw on in-depth individual interviews with 18 health workers and 15 patients conducted between 2019 and 2020 as well as fieldnotes and document reviews. During 2021 I conducted 11 remote follow-up interviews during the COVID-19 pandemic. My analysis makes an original contribution to understanding the complexity involved in using TB IPC measures in health facilities through exploring a positive deviant organisational case study. My main findings are that TB IPC use can be understood as a process of organisational sensemaking, where what was 'necessary' and 'effective' TB IPC are contested and negotiated between health workers. In my study context, I found that TB IPC measures both reflected and contributed to the stigma that patients with TB experience. I recommend viewing TB IPC implementation in health facilities through the perspective of the broader cultural setting and adapting IPC campaigns based on contextual factors such as local explanatory models of illnesses. Using a pragmatist philosophical approach, I focus on how these findings could be used to support TB IPC implementation and relate to the clinical context. The position I develop in this thesis is to view TB IPC implementation as an example of a social practice: with reciprocal interactions between broader societal social structures, norms in healthcare organisations, health worker and patient knowledge, beliefs and values, and the social meanings and material properties of TB IPC technologies. Viewing TB IPC implementation as a social practice offers new ways of understanding why TB IPC has been difficult to implement and presents novel possibilities for an interdisciplinary approach to support TB IPC implementation.

Biallelic mutations in the glucocerebrosidase (GBA1) gene cause Gaucher disease, characterized by lysosomal accumulation of glucosylceramide and glucosylsphingosine in macrophages. This and other lysosomal diseases occur with high frequency in Ashkenazi Jews. It has been proposed that the underlying mutations confer a selective advantage, in particular conferring protection against tuberculosis. Here, using a zebrafish Gaucher disease model, I find that the mutation GBA1 N370S, predominant among Ashkenazi Jews, increases resistance to tuberculosis through the microbicidal activity of glucosylsphingosine in macrophage lysosomes. Consistent with lysosomal accumulation occurring only in homozygotes, heterozygotes remain susceptible to tuberculosis. Thus, our findings reveal a mechanistic basis for protection against tuberculosis by GBA1 N370S and provide biological plausibility for its selection if the relatively mild deleterious effects in homozygotes were offset by significant protection against tuberculosis, a rampant killer of the young in Europe from the Middle Ages through the 19th century.

Tuberculosis (TB) remains a major cause for morbidity and mortality in children, and the very young and immunocompromised are especially at high risk. A major hurdle is a timely diagnosis, as microbiological confirmation - regarded as the reference standard - remains uncommon and the available tools are neither child-friendly, user-friendly, nor sufficiently accurate. This thesis presents results from a large TB diagnostic cohort of children younger than 15 years recruited in five countries (South Africa, Tanzania, Mozambique, Malawi, India), aiming to evaluate the diagnostic accuracy of three novel tests for childhood TB. Participants suspected of having TB underwent thorough clinical and microbiological testing, and reference standard investigations (culture and GeneXpert® MTB/RIF Ultra, "Ultra®") were conducted alongside the novel tests. The latter included a novel immunoassay using whole-blood, two urinary tests detecting mycobacterial antigen, and a stool processing kit allowing for detection of mycobacterial DNA. Clinical case definitions for TB applied followed published consensus statements. Of 974 children recruited over 30 months, 732 were eligible for assignment to clinical case definitions, being confirmed TB (32.0%, 234/732), unconfirmed TB (36.2%, 265/732), and unlikely TB (31.8%, 233/732). Of the whole cohort, 46.4% (340/732) were younger than five years, with median ages of 6.1 years (IQR 1.8-11.3) in children with confirmed TB, 4.6 years (IQR 1.7-8.2) in children with unconfirmed TB, and 5.5 years (IQR 2.5-8.0) in children with unlikely TB. HIV-coinfection was reported in 16.2% (117/732) of the overall cohort, with most cases being classified as unconfirmed TB (26.0%, 68/117). Tuberculin skin test positivity, chest radiography findings, and number of symptoms suggestive of TB were strongly associated with the diagnosis of TB when conducting logistic regression. Microbiological confirmation was achieved via culture and Ultra® in 46.6% (109/234), via culture alone in 17.1% (40/234), and via Ultra® alone in 36.3% (85/234). Of the latter, 73.0% (62/85) were only confirmed by the lowest semi-quantitative readout Trace call. Diagnostic accuracy of the T-cell activation marker for TB (TAM-TB) was assessed, using phenotypic characterisation of MTB-specific CD4+-cells. In the overall cohort, the sensitivity was determined at 51.3% (95% CI 41.8-60.7) and specificity at 88.2% (95% CI 81.3-93.2), comparing children with confirmed TB to those with unlikely TB. When using a microbiological reference standard requiring TB-culture positivity, the performance indicators were pronouncedly higher, with increases in sensitivity of more than 25% for certain subgroups. The urinary tests evaluated here were AlereLAM and FujiLAM, both being lateral flow assays targeting the mycobacterial cell-wall protein lipoarabinomannan (LAM). Comparing children with confirmed TB to those with unlikely TB, sensitivity was determined at 31.6% (95% CI 25.2-38.6) for FujiLAM and 13.2% (95% CI 8.9-18.6) for AlereLAM, with determined specificities of 89.1% (95% CI 83.8-93.1) and 94.1% (95% CI 90.0-96.9). Applying regression analyses, test accuracy for both tests was strongly associated with clinical study centre and age of the participant, more pronouncedly so for FujiLAM. Stool was processed using a kit and subsequently analysed using Ultra®. Determined sensitivity across the cohort was 34.2% (95% CI 27.5-41.4) with a specificity of 85.4% (95% CI 79.6-90.1), when comparing children with confirmed TB to those with unlikely TB. The sensitivity was significantly increased in female children (47.3%, 95% CI 36.7-58.0), compared to male (22.5%, 95% CI 14.9-31.9). Test accuracy was associated with proxies of TB bacillary load and extensive pathological findings on chest radiography. In conclusion, all tests evaluated here showed modest accuracy to diagnose children with TB. Although neither of the novel tests evaluated did achieve a level of accuracy usually aimed for a diagnostic, it was sufficient to be of value in a setting where easy-to-obtain specimens can be used, circumventing the need for elaborate sampling infrastructure. If implemented in settings with limited infrastructure, their impact might still be considerable. The findings of this thesis suggest that an approach with multiple tests might be more suitable to diagnose children with TB and highlights the need for further evaluations of diagnostic multi-step algorithms based on both clinical data and novel tests.

Tuberculosis (TB) is a major cause of death worldwide. We have a limited understanding of how the human immune system responds to Mycobacterium tuberculosis (M.tb), the main infectious agent that causes TB. In this project, non-human primates (NHPs) were exposed by aerosol to 1x107 colony forming units (CFU) of Bacille Calmette-Guérin (aeBCG). BCG is a live attenuated strain of Mycobacterium bovis, which can be used as a model of M.tb infection, and was safe to deliver to NHPs in a controlled manner. The immune response to aeBCG was characterised across different tissue compartments: blood, broncho-alveolar lavage (BAL), lymph nodes (LNs) and lung. There was little overall change in the immune parameters measured in either rhesus or cynomolgus macaques (RM or CM) at Day 2 (D2), D7 or D14 after aeBCG. Expression of cluster of differentiation-161 (CD161) on lymphocyte subsets was an early marker of this response at Week 2 (Wk2). Purified Protein Derivative (PPD)-specific interferon-gamma (IFNg)-producing spot forming unit (SFU) frequency increased from Wk4 to Wk16 after aeBCG. The peak immune response, measured by flow cytometry was at Wk12. PPD-specific immunoglobulin-G (IgG) titre increased at Wk9 in serum. The kinetic of the immune response to aeBCG measured was similar, but delayed, compared to the previously published immune response to intradermal (ID) BCG6, 8. The aeBCG delivery method (mask or mouthpiece) had no effect on the observed changes in immune parameters. Data from animals in both delivery groups were combined (n=16). A parallel experimental clinical medicine study showed the peak immune response to 1x107 CFU aeBCG in humans was at D7 (McShane group, Oxford - ClinicalTrials.gov ID: NCT03912207). The differences in immune response kinetic detected between NHPs and humans shown in this project have not been previously described and merit further study.

This study has been developed from the perspectives of both an artist and an educator so as to create an immersive, memorable and instructive experience for audiences through the creation of interactive replica dress pieces. The research explores how its practical outcome - a multisensory, narrative, dress-based artwork: A Conversation with Mary's Dress - could be used in a museum education context, alongside or even replacing the study of original items of dress in order to research and experience fashion as social history, while protecting fragile originals. This artwork allows viewers to investigate the dynamic interaction between dress and disease as revealed by deep investigation of a surviving Eighteenth Century gown worn by Mary Graham during her final illness and just before her death from tuberculosis in 1792. The replica dress that constitutes the artwork allows viewers to handle, touch and explore interactively a dress that essentially reproduces the original. This process engages, educates and inspires the viewer in ways that are simply not possible when only the original is available for examination. In addition to and like the dress, a fabric based pocketbook and heart and lungs contain embedded sound-spots that allow viewers to experience and follow the full story of the making of the replica artwork, Mary Graham's treatments and her subsequent death from tuberculosis. The entire work, then, rewards curious hands and minds through sharing its rich history by way of a multisensory experience. This project has developed from and builds upon the researcher's past work in this field as a costume designer making items for a wide range of productions, including costumes for museums and heritage sites based on the study of collected originals from across the world. This is to complement current and past practice within the field of dress in the context of history, art and the museum. The enhanced sensory experience for audiences created by this interactive replication of original fashion, was tested and evaluated, as demonstrated here in the audience survey responses. The approach has facilitated highly positive experiences and, at the same time, passed on to audience's new information in innovative ways. That has also created a tool, which has the potential to shape future dress handling educational programs.

Tuberculosis (TB) is still considered part of the top 10 mortal diseases caused by a single infectious agent. Thus, understanding the immune mechanisms during TB infection is essential to find new therapeutic and prophylactic approaches. Different immune cells have been widely studied to understand TB pathology. However, mast cells (MCs), which are important innate immune cells, have been little explored. Their strategic location and ability to secrete a wide repertoire of pro and anti-inflammatory molecules make MCs relevant against infections. To better define general strategies used by MCs against bacterial threats, using an in vitro model of human MCs (hMCs), we first characterized MC functions by means of degranulation, cytokine secretion, MC extracellular traps (MCETs) formation and production of reactive oxygen species (ROS) upon Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus pneumoniae stimulation. Our data showed that while L. monocytogenes induce degranulation, MCET formation and cytokine release in the absence of ROS in MCs, S. pneumoniae promote ROS production without inducing MCET and degranulation and while E. coli promote MC degranulation in the absence of MCET and ROS, S. aureus induce a selective release of prostaglandin D2 without degranulation. Thus, MCs display an individualized pattern of response depending on the bacterial type. Next, we characterized MC location, numbers and predominant phenotype in human lung post-mortem samples of patients affected by TB. Using fluorescent staining to identify MCs in lung infected tissue, we found MCs located at inflammatory sites, close to periphery of granulomas and predominantly abundant at fibrotic sites. We also found a co-localization of MCs with IL-17 and TGF-β which are relevant for granuloma formation and fibrogenesis respectively. Thus, we proposed MCs as contributors to the early inflammatory stage and the late fibrotic phase of TB pathology. Finally, to better define the means of interaction between MCs and mycobacteria, we investigated the outcome of direct crosstalk between MCs and BCG, which is an attenuated strain of Mycobacterium bovis that fails to protect the adult infection. We found that MCs only respond to BCG when exposed to IL-33 pre-treatment by the release of IL-8 and MCP-1. Furthermore IL-33 enhanced both CD48-MC expression and number of MC-BCG interactions. Thus, IL-33 may serve to pre-activate MCs and potentiate inflammatory responses against BCG. Altogether the current study proposed MCs as important mediators during TB and as potential targets for vaccine boosters.

A bacterial species' genetic content can be remarkably fluid. The collection of genes found within a given species is called the pan-genome and is generally much larger than the gene repertoire of a single cell. A consequence of this pan-genome is that bacterial genomes are highly adaptable and thus variable. The dominant paradigm for analysing genetic variation relies on a central idea: all genomes in a species can be described as minor differences from a single reference genome, which serves as a coordinate system. As an introduction to this thesis, we outline why this approach is inadequate for bacteria and describe a new approach using genome graphs. In the first chapter, we present algorithms for de novo variant discovery within such genome graphs and evaluate their performance with empirical data. The remaining chapters address a question relating to a critical bacterial pathogen: can Nanopore sequencing of Mycobacterium tuberculosis provide high-quality public health information? We collect data from Madagascar, South Africa, and England to help answer this question. First, we assess outbreaks identified using single-reference and genome graph methods. Second, we evaluate antimicrobial resistance predictions and introduce a framework for using genome graphs to improve current methods. Lastly, we train an M. tuberculosis-specific Nanopore basecalling model with considerable accuracy improvement. Together, this thesis provides general methods for uncovering bacterial variation and applies them to an important global public health question.

Multidrug-resistant tuberculosis (MDR-TB) has been an emerging public health problem. MDR-TB is a fatal disease whilst treatment of MDR-TB is prolonged but less effective and toxic. Inter-individual variations in clinical presentation and treatment response have been reported in tuberculosis (TB). Identifying host determinants for MDR-TB is important in the search for adjunctive therapy to improve treatment outcome for MDR-TB. This thesis aims to evaluate the impacts of three host factors of leukotriene A4 hydrolase (LTA4H) genotype, diabetes mellitus (DM) and vitamin D on clinical presentation, treatment response and treatment outcome of pulmonary TB patients, particularly MDR-TB. The first results chapter, chapter 3, illustrates the picture of increasing MDR-TB in Ho Chi Minh City, Vietnam, from 2011 to 2015 and its risk factors for poor treatment outcomes. In chapter 4, LTA4H genotype showed no significant association with clinical presentation, bacterial burden, extent of lung damage, treatment response or outcome in 312 MDR-TB and 600 drug susceptible (DS) TB patients. Although a potential marker for host-targeted therapy for TB meningitis, this genotype does not appear to be such for pulmonary TB. In chapter 5, the detrimental effect of DM on clinical presentation, bacterial load, disease severity, increased inflammation and treatment response was clearly observed in pulmonary TB. Although not a significant risk factor for mortality and poor treatment outcome, DM prolonged sputum culture conversion of MDR-TB and should be considered as an important host factor to adjust for. Chapter 6 reveals the association between vitamin D and extent of lung damage, severity and inflammatory response through blood biomarkers among 309 MDR-TB and 301 DS-TB patients. Vitamin D deficiency caused an adverse effect on MDR-TB, but not on DS-TB during treatment. The work of this thesis has provided more evidence on host factors in efforts to improve treatment outcomes for pulmonary MDR-TB.

Drug-induced liver injury is one of the leading causes of acute liver failure and is a serious barrier to drug development. Furthermore, it is a frequent adverse reaction to the antimicrobials used to treat tuberculosis, resulting in reduced treatment effectiveness, incomplete treatment, relapse and the development of antimicrobial resistance. The aim of the studies in this thesis were threefold, to investigate biomarkers of liver injury in patients receiving anti-tuberculosis medication, to investigate miRNA changes in zebrafish larvae due to drug-induced liver injury, and to develop a novel point of care diagnostic test for miR-122, a miRNA biomarker of liver injury. The novel biomarkers, microRNA-122 (miR-122) and cytokeratin-18 (K18) have been found to be diagnostic and prognostic in patients who overdose on paracetamol. A key question is, do these biomarkers retain their efficacy in the presence of infection? The circulating concentrations of miR-122 and K18 were defined in the following populations: healthy volunteers, active tuberculosis patients, latent tuberculosis patients, patients with non-tuberculous mycobacterial infection and HIV-tuberculosis coinfected patients. Circulating concentrations of miR-122 and K18 were not significantly different across these groups, indicating infection does not affect concentrations of miR-122 and K18. Furthermore, concentrations of miR-122 and K18 did not rise upon starting treatment. There was a significant correlation between the gold standard marker alanine aminotransferase (ALT) and both miR-122 and K18 demonstrating these biomarkers rise with elevations in ALT. In two individuals who developed drug-induced liver injury, miR-122 and K18 rose with ALT, suggesting these biomarkers have diagnostic potential for anti-tuberculosis drug-induced liver injury. To reduce the need for rodent studies a zebrafish larvae model of liver injury was developed. Reducing the number of rodent studies carried out reduces experimental costs and the use of zebrafish larvae enables rapid high-throughput experiments. Furthermore, using zebrafish larvae is in line with the "3Rs" (reduce, refine, replace) approach of animal use in science, with the replacement of rodents, a higher-order mammal, with the lower-order zebrafish larvae. Zebrafish larvae exposed to isoniazid and pyrazinamide developed liver toxicity as determined by mortality, fluorescent imaging and histology. In order to investigate pathways altered in anti-tuberculosis drug-induced liver injury and identify potential novel biomarkers small RNA sequencing was undertaken. Pathways were altered in anti-tuberculosis drug induced liver injury, including those associated with the metabolism of xenobiotics. Building on previous work by my group, zebrafish larvae were exposed to triptolide, a Chinese herbal medicine which induces liver injury. A method was developed to collect specific cell populations, hepatocytes and immune cells, from transgenic zebrafish larvae using fluorescence activated cell sorting (FACS). The miRNA changes in isolated cells populations were determined using qRT-PCR, and in whole fish using small RNA sequencing. Novel miRNA biomarkers of liver injury were identified from the small RNA sequencing data and translated in plasma samples from patients with anti-tuberculosis drug-induced liver injury. Two of these miRNAs rose with ALT in liver injury. A point of care test is needed for drug-induced liver injury, suitable for use in resource poor settings. A novel RNA toehold switch sensor was combined with a fluorescent and colorimetric outputs to provide a quantitative response to miR-122 concentrations. Toehold switches with a range of structures were developed and tested. Switches demonstrated varying levels of sensitivity and specificity for miR-122.

Drug-resistant tuberculosis (TB), one of the leading causes of death worldwide, arises mainly from spontaneous mutations in the genome of Mycobacterium tuberculosis (M. tb). There is an urgent need to understand the mechanism by which the mutations confer resistance in order to design new drugs to reduce its emergence in the future. The present study involves analyses of drug-resistant mutations in M. tb using both computational and experimental approaches. In Chapter 2 in-silico analyses of mutations linked to isoniazid (INH) and rifampicin (RIF) resistance were performed in seven proteins. These proteins included the catalase-peroxidase (KatG), Enoyl-Acyl Carrier Protein reductase (InhA), β-ketoacyl ACP synthase (KasA), alkyl hydroxide reductase (AhpC) and NADH dehydrogenase (Ndh) for INH resistance and RpoB and RpoC for RIF resistance. The impacts of mutations on the structure and function of these proteins were predicted using software developed in the Blundell group, including impacts on stability using SDM, a statistical approach, and mCSM, a machine learning approach, while the effects of mutations on protein-protein interactions and protein-ligand affinity were predicted using mCSM-PPI and mCSM-lig respectively. The models of mutants were built using Modeller, and the atomic interactions of wild-type and mutant residues were analysed by Intermezzo and Arpeggio. Around 80% of the mutations that were analysed were predicted to be destabilizing for the respective protein. Mutations were found predominantly to target the active sites and interfaces of the proteins, and 100 % of the mutations present at the interface of the studied proteins were predicted to be destabilizing for protein-protein interactions by mCSM-PPI. Moreover, all the mutations present in the active sites of the proteins were predicted to decrease the affinity of the ligand for the protein. KatG (a heme-dependent catalase peroxidase) and two KatG mutants W107R and T275P were selected for experimental analyses. KatG activates the prodrug INH and mutations in KatG are likely to be the first step in conferring resistance against the drug. The structures of wild-type KatG protein (with and without the addition of INH) were obtained using X-ray crystallography and cryo-EM and potential INH binding sites in KatG were identified using cryo-EM and illustrated in Chapter 4. The structure of KatGINH revealed the dynamic binding ability of INH and the power of cryo-EM as a technique to visualise small drug molecules Two KatG mutants, W107R and T275P were created in Chapter 5 and the structures were obtained using cryo-EM. In combination with cryo-EM, the wild-type and mutant proteins were also characterised using UV-visible spectroscopy, circular dichroism and biochemical methods. The analyses revealed that the variants cause disorder in the heme environment preventing heme uptake and retention by the KatG protein likely to be the cause of INH resistance. This study helped in providing new insights into INH resistance caused by resistance mutations in KatG.

The overall aim of this study was to employ a biomolecular technique - ancient DNA (aDNA) - to study two ancient diseases that were endemic in Europe (and therefore Britain) during the medieval period: tuberculosis and leprosy. In humans, the diseases are caused by M. tuberculosis and M. leprae, respectively - both of which are members of the M. tuberculosis complex (MTBC). Skeletal manifestations of both diseases may develop in bone remains, which can be recognized using osteological analysis. In some cases, however, the skeletal changes are ambiguous. Ancient DNA methods are used for case confirmation and to answer historical questions such as the spread, origin and evolution of disease. The first objective of this thesis was to determine whether the MTBC aDNA detection frequency is high enough to plan a larger study to test hypotheses such as possible strain differences in urban and rural areas, as it has been suggested that urbanization assists the spread of tuberculosis, enhancing its virulence. To meet this objective, 60 skeletal remains from 16 different locations in Yorkshire, England were studied. All samples were screened for MTBC aDNA presence and 8 samples were selected for next-generation sequencing (NGS). In the PCR assay screening, only 1 sample produced a positive MTBC amplification. However, when subjected to NGS, this sample together with the other 7 samples did not produce enough sequence reads to allow genome comparisons. An attempt to compare metagenomic content between urban and rural sites was also performed. There was no specific difference in metagenomic content between urban and rural samples. Based on the PCR analysis, the sample St Andrew Fishergate 6, dated to the early 14th century AD, showed evidence of possible tuberculosis infection. NGS analysis further revealed a possible M. tuberculosis and M. leprae mixed infection, albeit with insufficient read coverage to determine genome sequence polymorphism. The second objective was to use NGS to determine the genotype of the M. leprae strains present in skeletons from two mediaeval sites, at Chichester and Raund Furnells, both in England. This study served as a continuation for the previous confirmation by PCR of leprosy in these skeletons. The samples were further subjected to whole M. leprae genome target enrichment before subsequent high-throughput sequencing. For all 3 historical M. leprae isolates, at least 70% genome sequence coverage was obtained, with a mean read depth of 4-10x. The near-complete genome sequences that were obtained allowed subtype identification for each of the ancient M. leprae isolates. Two mediaeval samples from Chichester belonged to the 3I subtype, which is typical of ancient Northern European and contemporary North American isolates. Meanwhile, an M. leprae isolate from Raunds was identified as belonging to the 3K subtype - the first example of this subtype identified in Britain. Transmission of the M. leprae 3K subtype to Britain is suggested to have been associated with the travels of crusaders and pilgrims to the Holy Land during the mediaeval period. The overall conclusion of the work is that although M. leprae aDNA is well preserved in skeletal remains showing osteological signs of leprosy, the same is not true for MTBC preservation in skeletons showing indications of tuberculosis. To test hypotheses such as the effect of urbanisation on tuberculosis, a high frequency of MTBC detection must be achieved, but this is complicated by the very nature of ancient DNA itself - highly fragmented, low endogenous DNA copy, presence of environmental contaminants - and by the possibility of low bacterial load in skeletons at the time of death. In projects where the testing of a high number of samples is required, more stringent selection criteria must be imposed to minimize the impact of destructive analysis.

To end Tuberculosis, point-of-care diagnostic tests accessible by all isolated communities worldwide are required. Using ELISA platform, this study evaluates the use of synthetic lipidsas antigens in diagnostic testsfor the detection of TB (pulmonary, extra-pulmonary, latent TB infection) in patient sera and plasma samples, with and without HIV. Taking the most promising synthetic lipid antigen forwards, a point-of-care flow through assay is developed for the detection of pulmonary TB, with a smartphone application. Using ELISA and the point-of-care flow through assay, the role of synthetic lipid antigens for the detection of Buruli Ulcer is evaluated, with promising results (sensitivity 63%, specificity 80%for ELISA).ELISA with a single synthetic cordfactor (alpha, keto or methoxytrehalose dimycolate) antigen can detect pulmonary TB from non-TB samples from an endemic population with and without HIV. In terms of detection of disease state, synthetic keto TDM antigen can distinguish pulmonary TB from latent TB infection using the IgG antibody response in both ELISA (sensitivity 90%, specificity 80%) and the flow through assay (sensitivity 63%, specificity 90%), with sera or plasma samples. The simple 10 minute flow through assay for less than $10 per test with almost equivalent performance to the laboratory-restricted 3 hour ELISA has reached a new milestone in the development of point-of-care diagnostic tests using synthetic lipid antigens, and the smartphone application opens up the possibility for monitoring of TB patients in isolated communities. Although the performance of ELISA and the Flow Through Assay is not as strong as the leading TB diagnostic Xpert; itis envisaged that with further optimisation the novelflow through assay using synthetic lipid antigens will meetthe World Health Organisation requirements for a point-of-care sensorand makeTB diagnostic testing more accessible to isolated communities most in need.

Excavations at the Catholic Third Street Cemetery in Dubuque, Iowa (2007-2011), uncovered 939 unmarked graves dating to the period between 1833 and 1880. Skeletal analysis identified 43 individuals whose age estimate ranges overlapped with osteological (12 to 20 years) and social (13 to 19 years) adolescence, as defined for this cultural context. The current research design focused on mortality patterns and mortuary preparations of these individuals and highlights differences between teenagers and the rest of the cemetery population. This interdisciplinary project utilised data from osteological analyses and archival research to explore health and mortality among adolescent non-survivors. Palaeopathological observations reflecting early life health insults (linear enamel hypoplasias, cribra orbitalia, porotic hyperostosis) and later illness (labyrinthine endocranial lesions, periosteal new bone formation, tubercular lesions) were studied to explore the potential vulnerability of adolescents with frailty acquired through earlier health stresses. Perimortem trauma and local death records were examined to determine the proportion of teenage mortality due to external causes such as accidents, homicide, and suicide. The lack of perimortem trauma observed in the Third Street adolescent sample was explained to some extent by the number of teenagers who perished from, but were unmarked by, a single accidental cause – drowning. The investigation of mortuary treatment examined combinations of burial attributes – including coffin hardware, burial clothing, religious objects, and nonreligious grave goods – and demonstrated how age-related patterns reflect an increase in socially acceptable sentimentality and changing views of the afterlife, with preferential treatment afforded to some adolescents. Comparative pathological marker and burial attribute data were gathered from publications on nine additional nineteenth-century burial populations, and death records from a tenth were consulted. Despite issues with inconsistent data collection procedures for parts of the comparative sample, results tentatively support the observations from Third Street. The proportion of adolescents with both early-life stress markers and later pathological manifestations is higher than that of other age classes, suggesting that survival of health insults in infancy or early childhood left teenagers more susceptible to fatal disease, particularly when their immune systems were vulnerable due to competing pubertal energy investments. Observed regional differences in skeletal marker rates suggest that this “double signal” may be more pronounced in populations with a high prevalence of TB. Perimortem trauma levels are equal to those of adults, though greater evidence of violence in the South and Southwest reflects the unstable social conditions in those areas. Regional, as well as temporal, trends were also identified in adolescent funerary preparations. Mid-nineteenth-century adolescents received preferential treatment, though general increases in mortuary elaboration overshadow this distinction in some later cemetery populations. Parental grief at the loss of near-adult offspring was expressed in the tendency to employ the metaphor of death as a journey when preparing adolescents for the grave, instead of the metaphor of sleep applied to younger children. Meanwhile, in frontier areas, independently living teenagers were often interred without familial involvement in the equivalent of paupers’ graves.

This thesis describes a large-scale forward genetic screen in zebrafish for host determinants of mycobacterial infection outcome that I participated in. Zebrafish larvae from families randomly mutagenized with N-ethyl-N-nitrosourea were infected with Mycobacterium marinum and monitored over time. Larvae with increased susceptibility to infection were identified by the presence of mycobacterial cording, a phenotypic proxy for failure of immunity. 279 unrelated families have so far been screened with isolation of phenotypic larvae from 31 families. Genetic mapping has been carried out on phenotype sorted larvae from 5 families to identify genomic regions harbouring causative mutations. Positional cloning has been completed on one such mutant, Guaguancó which was found to map to a nonsense mutation in the gene ubiquitously expressed transcript (uxt), a poorly characterised gene not previously associated with anti-mycobacterial immunity. Subsequent cellular characterisation demonstrated that uxt mutants manifest a normal immune response in the early stages of infection, but then show accelerated macrophage death resulting in granuloma failure. Pharmacological inhibition of cell death pathways associated with mycobacterial infection did not influence uxt mutant susceptibility to infection, suggesting that uxt deficiency results in a novel form of death in mycobacterium-infected macrophages.

Current therapy of tuberculosis (TB) has several limitations, such as risk of liver injury and intestinal dysbiosis due to frequent oral administration of antibiotics. Transdermal administration could be used to improve antibiotic delivery for treatment of Mycobacterium tuberculosis infection. Therefore, this thesis described a novel approach, using hydrogel-forming microneedle (MN) arrays to transdermally deliver TB drugs, namely rifampicin, isoniazid, pyrazinamide and ethambutol, which have different physicochemical properties. These drugs were individually prepared into three types of drug reservoirs, including lyophilised tablets, directly compressed tablets and poly(ethylene glycol) tablets. In the specific case of RIF, as the most hydrophobic drug compared to the other three, the inclusion complex between RIF and cyclodextrin was developed. Moreover, formulations of each drug reservoir type were optimised to achieve a rapidly dissolving tablet, and further integrated with hydrogel-forming MN arrays for in vitro permeation studies. Three types of hydrogel formulation were manufactured using different type of polymers and crosslinking processes. These MN arrays were then evaluated in terms of swelling ability, morphology and physical properties. Results of solute diffusion studies showed that drug permeation across the swollen hydrogel membrane was affected mostly by physiochemical properties and functional groups of each drug. In the in vitro studies, the amount of permeated drug through the hydrogel-forming MN arrays across the dermatomed neonatal porcine skin was affected by the drug solubility and reservoir design. The development of this transdermal TB drug regimen is still in the early stage, thus a further investigation to evaluate the effectivity, stability, safety, and efficacy prior to the clinical application will be required. For instance, in vivo pharmacokinetic studies must be done to determine the plasma concentration of the drug in the animal model. Besides, to ensure the efficacy of this system, pharmacodynamic studies must be also performed. On this basis, to reach clinical practice and ensure the benefit for TB treatment application, this novel approach should be tested in terms of the usability and acceptability to achieve the maximum impact of this work.

Tuberculosis caused by the Mycobacterium (M.) tuberculosis-complex (MTBC) of organisms remains one of the most prevalent and deadly infectious diseases of man and other animals. The mycobacteria responsible are a highly conserved group of pleomorphic acid-fast bacilli which cause chronic granulomatous infections. Tuberculous infections in humans and cattle often remain latent for prolonged periods of time before progressing to disease that has severe, negative consequences for the health and welfare of the infected host. Some of the organisms within the MTBC are highly specialised and limited to just a single or small number of host species whereas others, such as M. bovis, can infect a broad range of mammals including humans. Companion animals are susceptible to MTBC infections and understanding of the significance and frequency of these infections has grown in recent years. Cats and dogs share unrivalled proximity to their owners and therefore pose a small but real risk for the zoonotic transmission of tuberculous infections. Despite the high frequency of mycobacterial infections observed in companion animals, diagnostic tests to identify the commonly encountered mycobacterial species are lacking. The first aim of this work was therefore to improve on the currently available diagnostic test methodologies for companion animals. A diagnostic PCR assay was developed and applied to 380 histologically confirmed feline and eight canine mycobacteriosis samples. This novel assay specifically targeted the mycobacterial species most frequently identified by mycobacterial culture (M. bovis and M. microti) and was optimised for use with formalin-fixed tissue; a prerequisite for the safe handling of tuberculous tissue from companion animals by UK laboratories. The assay was suitable for both feline and canine tissue with a significantly quicker turnaround time, higher rate of test positive results and a significant increase in the proportion of M. microti diagnoses compared to culture results. Since evaluation of cytokines has shown diagnostic potential in other species, this project explored the potential of cytokine profiling in cats for the rapid and sensitive detection of mycobacteriosis. By evaluating serum/plasma from 116 naturally infected cats, this study demonstrated a consistent elevation in the cytokines associated with macrophage activation and antigenic stimulation compared to control cats. Sub-group analysis showed that elevations in PDGF-BB were specifically associated with M. microti infections whereas elevated TNF-α sensitively identified cats infected with M. bovis. Investigation of an unprecedented outbreak of M. bovis associated with the ingestion of a putatively contaminated raw food product led to the use of the interferon-γ release assay (IGRA) as a screening test for clinically healthy cats, a purpose for which it had not previously been evaluated. Nearly a third of clinically normal IGRA test-positive cats were subsequently found to have structural disease detected by diagnostic imaging. Though this raises questions regarding the specificity of the IGRA in clinically normal cats, it was an invaluable diagnostic tool to evaluate individual cats involved in the outbreak. The work on feline TB was complemented by similar investigations of canine TB. When an outbreak of M. bovis tuberculosis occurred in a kennel of 164 working Foxhounds, a testing strategy to successfully bring the outbreak under control and investigate the cause was developed. Collaborative work undertaken to screen at risk humans exposed to the hounds identified a latently infected person, highlighting the zoonotic risk posed by M. bovis infections in companion animals. Eight novel and existing diagnostic testing methodologies were evaluated for use in dogs of which a cell-based IGRA, and three serological tests comprising a novel comparative peptide ELISA, the Chembio DPP VetTB assay and the Idexx M. bovis Ab ELISA showed diagnostic potential for canine TB. Additional analysis employing a Bayesian latent class modelling approach revealed that the IGRA developed herein was as sensitive and specific as comparable tests in other species. The serological assays were shown to have markedly lower sensitivity than the IGRA but had higher specificities. All four tests had positive and negative predictive value estimates which indicate that these tests can be informative to clinicians who suspect cases of canine TB. A review of 1012 cases of canine TB highlighted an apparently lower incidence of infections in dogs compared to cats, but an increased severity of clinical signs when disease occurred. To investigate this discrepancy a protocol to derive macrophages from canine and feline bone marrow was developed. These cells acquired cell surface molecules indicative of a macrophage phenotype during ten days of culture with recombinant CSF-1. The response of primary macrophages and the DH82 canine histiocytic cell line was assessed following stimulation with LPS, infection with M. bovis Bacille Calmette Guerin, M. bovis AF2122/97 (the reference strain) or a clinical isolate of M. bovis. These investigations consistently revealed that DH82 cells do not accurately represent primary canine macrophage biology which was associated with altered morphology, lack of nitrite production and significantly reduced secretion of the pro-inflammatory cytokines IL-6 and TNF-α. Overall, the work presented in this thesis demonstrates novel advancement of the diagnostic methodologies for identifying cases of companion animal mycobacteriosis and in particular cases of TB. It further begins to explore the immunological basis for the clinical differences seen between species that may further contribute to novel testing and treatment strategies in the future.

Tuberculosis (TB) accounts for over 1 million deaths each year, despite effective treatment regimens being available. Improving the treatment of TB will require new regimens, each of which will need to be put through expensive and lengthy clinical trials, with no guarantee of success. The ability to predict which of many novel regimens to progress through the clinical trial stages would be an important tool to TB research. as current models are constrained in their ability to reflect the whole spectrum of pathophysiology, particularly as there remains uncertainty around the events that occur. This thesis explores the use of computational techniques to model a pulmonary human TB infection. We introduce the first in silico model of TB occurring over the whole lung that incorporates both the environmental heterogeneity that is exhibited within different spatial regions of the organ, and the different bacterial dissemination routes, in order to understand how bacteria move during infection and why post-primary disease is typically localised towards the apices of the lung. Our results show that including environmental heterogeneity within the lung can have profound effects on the results of an infection, by creating a region towards the apex which is preferential for bacterial proliferation. We also present a further iteration of the model, whereby the environment is made more granular to better understand the regions which are afflicted during infection, and show how sensitivity analysis can determine the factors that contribute most to disease outcomes. We show that in order to simulate TB disease within a human lung with sufficient accuracy, better understanding of the dynamics is required. The model presented in this thesis is intended to provide insight into these complicated dynamics, and thus make progress towards an end goal of a virtual clinical trial, consisting of a heterogeneous population of synthetic virtual patients.

In the tuberculosis patients, Mycobacterium tuberculosis can stimulate production of hydrogen peroxide in the host as a result of immune response. The H2O2 accumulate in pulmonary cells, causing oxidative stress that could lead to the cancer. We select TB patients for this study which investigates the effects of quercetin as there is an increased incidence of latent TB among the migrant population in the past few years and TB can increase the risk of cancer. Sperm and lymphocytes were treated with DNA damage inducers and quercetin (10µM, 25µM and 100µM), the responses evaluated using the Comet and micronucleus techniques. The gene expressions of COX1, COX2, P53 and Bcl-2 and catalase protein expression were investigated using the qPCR and Western blot techniques. The results showed that a substantial reduction of DNA damage in lymphocytes from TB patients and sperm from healthy donors from * P ≤ 0.0283 to *** P≤0.001in the Comet assay. In the MNi assay, the effect of quercetin in lymphocytes was more significant in reduce DNA damage, whereas the DNA damage induced by a food mutagen was significant, from *p 0.0405 to ***p 0.001. The qPCR showed significance down-regulation of COX1 and Bcl-2 gene expression, rated between *p 0.045 and **p 0.0074. However, the catalase protein was up-regulated by the nano form of quercetin when using lymphocytes from TB patients and showed significant changes at *p 0.0236. In conclusion, the nano form was found to be more efficient at the reduction of DNA damage in the Comet and micronucleus assays. Also, it down-regulated COX1 and Bcl-2 and up-regulated the catalase proteins indicating a possible role for quercetin, in genoprotection to TB through its enzyme modulating effect.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is among the most successful human pathogens. Throughout their complex life cycle in the host – infection, growth, transmission – mycobacteria must continually resist host defences. A major mycobacterial survival mechanism lies in the unusual and complex cell envelope. The mycobacterial cell envelope provides a robust permeability barrier that helps the bacteria tolerate the variety of host insults it encounters. Many mycobacterial virulence factors are localised in the cell envelope. Here I have dissected the function of Erp (exported repetitive protein), a cell envelope associated, critical virulence factor, that is required for survival and growth specifically in host macrophages. I find that Erp appears to anchor the cell wall to the inner membrane, thereby preserving cell envelope ultrastructure. Localisation of Erp specifically to the cell wall is required to maintain the cell envelope as an impermeable barrier. Loss of Erp increases the permeability of the cell envelope, sensitising mycobacteria to both hydrophobic antibiotics and a range of antimicrobial molecules enriched in macrophages. Erp is mycobacterium-specific, and widely conserved in both saprophytic and pathogenic mycobacteria, suggesting that it co-evolved with the mycobacterial cell envelope to maintain a permeability barrier that was essential to counter ubiquitous environmental antimicrobial factors. Erp’s function then allowed mycobacteria to evolve into intracellular pathogens.

The work described in this thesis is focused on the application of fragment-based approaches for two essential mycobacterial target proteins, fumarate hydratase (fumarase) from Mycobacterium tuberculosis (Mtb) and tRNA (m1G37) methyltransferase (TrmD) from Mycobacterium abscessus (Mab). With Mtb fumarase a high-throughput screening (HTS) hit was used to design a small library of fragments in a deconstruction-reconstruction approach. These fragments were screened using a range of both biochemical and biophysical methods. The resultant fragments showed evidence of weak protein binding. As an alternative strategy, derivatives of the HTS hit were synthesised and screened by a biochemical assay, which identified nanomolar inhibitors of this enzyme. In addition, X-ray crystallography was also carried out with a range of these compounds. Selected compounds were subsequently screened by collaborators at the NIH against Mtb. With the enzyme TrmD from Mab, the fragment hits identified were used as the basis of a fragment-merging approach to develop potent inhibitors, guided by structural biology. In the implementation of this approach, synthesis and biophysical techniques were extensively utilised, including both differential scanning fluorimetry and isothermal titration calorimetry. This approach led to the development of novel inhibitors with low nanomolar affinity. Select compounds were screened by collaborators against both Mab and Mtb in vitro. In light of encouraging activity against Mtb, the TrmD homolog in Mtb was expressed and screened against select compounds to demonstrate the broader applicability of the lead series.

Tuberculosis affects millions of people worldwide every year. The current treatment for TB is divided into a regimen of both first- and second-line drugs, where first-line treatments are more tolerated and require shorter treatment lengths. With rising levels of resistance, alternative treatment regimes are urgently needed to fight this disease. Ethionamide, a second-line drug is administered as a prodrug which is activated in vivo by the enzyme EthA, which is in turn regulated by EthR. The disruption of the action of EthR could lead to novel therapeutics which could enhance the efficacy of ethionamide, and raise it to a first-line treatment. The work reported in this thesis examines the elaboration of three chemical scaffolds using fragment-based approaches to develop novel inhibitors capable of disrupting the EthR-DNA interaction. The first scaffold, 5-(furan-2-yl)isoxazole was investigated by fragment-merging approaches and produced compounds with the best of these having a KD of 7.4 uM. The second scaffold, an aryl sulfone was elaborated using fragment-merging strategies. This led to several modifications of the fragment, leading to several variants with KDs around 20 uM. With both of these series the affinity could not be improved below 10 uM and due to the synthetic complexity a further scaffold was prioritised. The third scaffold was explored was a 4-(4-(trifluoromethyl)phenyl)piperazine using fragmentgrowing from the NH of the piperazine to probe deeper into the EthR binding pocket. In addition to this, SAR around the 4-(trifluoromethyl)phenyl group was assessed to explore the interactions with EthR. These modifications led to compounds with nanomolar IC50s. A range of compounds were then screened by REMAssay to determine the boosting effect on ethionamide, and this identified compounds with up to 30 times boosting in the ethionamide MIC. The final chapter examines a concept where compounds were designed to exploit the dimeric nature of EthR by linking two chemical warheads with a flexible linker. These compounds are examined using mass spectrometry to investigate the stoichiometry of the interaction to provide insight into the binding of these extended compounds and exploring an alternative strategy to inhibit EthR. The work in this thesis demonstrated the successful use of fragment-based approaches for development of novel EthR inhibitors which showed significant ethionamide boosting effects.

Background: Phagocytes have diverse roles including the ingestion and clearance of microbes, foreign particulate matter and dead cells. The study of patients with phagocyte dysfunction, either because of young age or inborn errors of immunity, could help design novel immunomodulatory treatments. However, such insights have been limited by a lack of access to the relevant clinical samples. Methods: Phagocytes from three groups of infants and children with clinical features of phagocyte dysfunction, obtained by novel sample collection methods, were studied by in vitro functional and transcriptomic testing. Results: Analysis of the clinical phenotype combined with functional testing of patient phagocytes demonstrated: i) primary alveolar macrophages isolated from healthy infants exhibited poorer control of Mycobacterium tuberculosis replication, as well as reduced expression of genes involved in lysosomal function and mycobactericidal activity, as well as genes encoding chemokines, which may explain the observed clinical vulnerability of infants to severe tuberculosis; ii) impaired control of mycobacterial replication by monocyte-derived macrophages of children with deletion of genes involved in cholesterol metabolism (LIPA and CH25H) is associated with clinical susceptibility to infection caused by Bacillus Calmette-Guérin vaccination; and iii) neutrophils from children with glucose-6-phosphatase catalytic subunit 3 deficiency exhibited increased inflammatory responses, which drives inflammatory bowel disease and can be corrected by haematopoietic stem cell transplant. Conclusion: Age-dependent and genetic factors make clinically important contributions to phagocyte immunity.

Whole genome sequencing (WGS) is an attractive approach for mycobacteria diagnosis and epidemiological studies. It provides the potential for a rapid method that produces detailed information and could theoretically be used as a routine tool in clinical settings. This thesis focuses on the benefits and challenges involved in transforming molecular approaches into practical clinical mycobacteriology in general, and in particular WGS, as well as examining how it might be implemented. We first set out to improve the quantification of viable mycobacteria cells in vitro and make the molecular bacterial load assay (MBLA) sensitive enough to use in future clinical trials that monitor treatment response. The results showed the assay is rapid and accurate in its detection and count of viable bacteria. WGS was tested with different types of mycobacteria species to address different epidemiological questions. WGS not only provides a higher resolution result than traditional epidemiological methods but it can rapidly identify an outbreak, thus simplifying the investigation and reducing the cost. WGS accurately identified the sources of TB recurrence and could therefore have a potential role in determining the endpoints for clinical trials. Rapid genotyping of species in this way has been demonstrated in our studies. In addition, WGS has the ability to, in most circumstances, predict TB drug resistance. This could also prove very beneficial from a clinical standpoint. We used different approaches in our studies; for example, single nucleotide polymorphism threshold methods and the creation of a putative outbreak reference genome, which can be used in future outbreak investigations. WGS is a cost-effective, high-resolution method with a short turnaround. This makes it potentially usable as a routine tool in clinical settings and reference laboratories. Future studies are needed to improve the mycobacterial genome sequencing procedure, analysis and bioinformatics in order to implement WGS in clinical practice.

Tuberculosis is a disease that kills more people every year than any other infectious disease and is caused by the human pathogen, Mycobacterium tuberculosis (Mtb). This disease can be treated by a standard six month course of four antimicrobial drugs that have been in use since the 1960s. However, the rise of multi-drug resistant and extensively drug-resistant strains of TB has complicated the efforts to eradicate the disease. Therefore, there is a critical need for the development of new anti-TB drugs with a novel mechanism of action that can speed up treatment duration and help avoid resistance. The discovery of twenty genes encoding cytochrome P450 enzymes in the Mtb H37Rv genome sequence has pointed to the significance of these enzymes in the physiology and pathogenicity of this bacterium. Consequently, the characterisation of these Mtb P450 enzymes may define their physiological roles of which can be a novel anti-tubercular drug target. To date, the characterisations of selected Mtb P450 enzymes have highlighted their diverse and unexpected roles in the metabolism of cholesterol and lipids and the production of secondary metabolites. Biochemical and biophysical studies of these enzymes provided knowledge of their active site properties that may be exploited for drug discovery. Therefore, with the prospect of defining novel functions and identifying novel drug targets, characterisations of the remaining orphan Mtb P450s is of interest. M. tuberculosis CYP141A1 and CYP143A1 are orphan enzymes with unknown physiological function in Mtb which is characterised in this study through use of various spectroscopic and biophysical techniques. Interestingly, CYP141A1 can be expressed in form of which 54 amino acids (Del54CYP141A1) are deleted from the N-terminus. Although Del54CYP141A1 still retain spectroscopic characteristics, this form of P450 cannot be crystallized. Optimisation of full-length CYP141A1 buffer composition resulted to the formation of reproducible crystals and determination of CYP141A1 structure. Spectroscopic and structural characterisations presented in this thesis revealed many characteristics of CYP141A1 and CYP143A1 are comparable to previous Mtb P450s reported to date. CYP141A1 and CYP143A1 active site consist of b-type heme iron ligated by cysteine residue and a water molecule at its proximal and distal face, respectively. Both enzymes bind tightly to azole antifungal drugs highlighting their potential as a drug target. In addition, fragment-based screening applied to CYP141A1 and CYP143A1 provided the starting point for the development of potent, isoform-specific inhibitors for both orphan Mtb P450 enzymes. The first crystal structure of CYP141A1 and identification of new fragment binders of CYP141A1 and CYP143A1 are presented in this thesis. Overall, this research remains significant in providing new knowledge on the spectroscopic and structural properties of the M. tuberculosis P450s CYP141A1 and CYP143A1.