Background: Three billion people worldwide use solid fuels for cooking and heating their homes. The World Health Organization estimates that the resultant household air pollution causes 6.5 million deaths per year, predominantly in low- and middle-income countries. Despite causing half a million deaths per year from childhood pneumonia, the association between household air pollution and pneumonia in adults - a common cause of morbidity and mortality in sub-Saharan Africa - is not established. Studies of household air pollution are limited by difficulties in quantifying exposure levels, contributing to the relative scarcity of evidence. Addressing these methodological challenges would facilitate efforts to obtain the evidence required to reduce this health burden for the world's poorest populations. Focussing particularly in the sub-Saharan country of Malawi, this thesis aims to explore the challenges regarding exposure measurements in resource poor settings, and to provide evidence regarding the role of household air pollution in pneumonia in adults. Methods: The literature was systematically reviewed to establish the current evidence base for an association between household air pollution and pneumonia in adults. Two prospective studies examining the suitability of potential biomarkers of household air pollution exposure were undertaken: firstly, to establish the feasibility of using airway macrophage particulate load obtained from induced sputum samples as a biomarker; and secondly, to explore whether exhaled carbon monoxide is a suitable biomarker for use in field studies. Finally, a case-control study of HIV-positive and HIV-negative Malawian adults was undertaken to establish the role of household air pollution, and other potential risk factors, in pneumonia. Results: Eight studies regarding household air pollution and acute lower respiratory tract infection were identified, reporting conflicting data and with limited study quality. The two methods used to calculate airway macrophage particulate load were lengthy, complex and unreliable. Exhaled carbon monoxide tests were easy to use and acceptable to participants in Malawi, but were subsequently found to not correlate with measured air pollution exposures. 145 (117 HIV-positive; 28 HIV-negative) cases and 253 (169 HIV-positive; 84 HIV-negative) controls completed follow up in the case-control study. Household air pollution was not associated with pneumonia in HIV-positive (e.g. ambulatory particulate matter adjusted odds ratio [aOR] 1.00 [95% CI 1.00-1.01, p=0.141]) or HIV-negative (e.g. aOR 1.00 [95% CI 0.99-1.01, p=0.872]) participants. Chronic respiratory disease was associated with pneumonia in HIV-positive (aOR 28.07 [95% CI 9.29-84.83, p < 0.001]) and HIV-negative (aOR 104.27 [95% CI 12.86-852.35, p < 0.001]) participants. Conclusions: There is insufficient evidence in the existing literature to confirm an association between household air pollution and pneumonia in adults. Previous studies have been limited by methodological issues. To address these challenges, this thesis has added to the growing body of literature regarding biomarkers of household air pollution exposure. However, our finding suggest that neither airway macrophage particulate load nor exhaled carbon monoxide are well suited for use at scale in resource poor settings. This thesis reports the largest study of household air pollution and adult pneumonia to date, with detailed exposure and outcome assessments. Although the case-control study sample size was not met, we found no evidence for an association between household air pollution and pneumonia in Malawian adults; further studies should be conducted to ensure that future public health resources are appropriately targeted. Broader solutions, including tackling poverty, malnutrition and chronic respiratory disease, will likely be required to reduce the burden of pneumonia in resource poor settings.

Malaria is an infectious disease caused by Plasmodium parasites that are transmitted by the bite of female Anopheles mosquitoes. Successful acquisition and transmission of malaria parasites requires a female mosquito obtaining a blood meal from human hosts. The blood meal, which is rich in protein, is required for egg development. Most of the ingested protein is converted to lipid and stored in the fat body where vitellogenesis takes place. In this process, saturated fatty acids are converted to unsaturated fatty acids by the stearoyl-CoA desaturase (SCD1). Unsaturated fatty acids are also essential for maintaining cell membrane fluidity and other housekeeping functions. The main aim of this thesis was to functionally and phenotypically characterize the function of SCD1 during blood meal metabolism in the African mosquito vector Anopheles coluzzii. RNA interference (RNAi) silencing of the SCD1 gene and administration of a small molecule inhibitor of SCD1 had a significant impact on the survival of female mosquitoes following a blood meal. SCD1 knockdown (KD) caused a 100% mortality within 48 h after a human blood meal, while addition of the SCD1 small molecule inhibitor sterculic acid (SA) in the blood meal caused a 50% mortality within 72 h of blood meal. Microscopic analysis showed that SCD1 KD mosquitoes failed to develop eggs in response to the blood meal, while their thorax was filled with blood at 24 h post blood meal. These findings were highly consistent with electron microscopy data that showed increased plasma membrane rigidity and depletion of lipid droplets in the midgut epithelial cells. Transcriptional profiling using A. coluzzii oligonucleotide DNA microarrays showed that genes involved in protein, lipid and carbohydrate metabolism, as well as a large number of immunity genes were the most affected in blood-fed SCD1 KD versus control mosquitoes. Metabolomics profiling highlighted the biochemical framework by which the SCD1 KD phenotype is manifested after a blood meal, revealing increased amounts of saturated fatty acids and TCA cycle (and other interlinked pathway) intermediates in SCD1 KD and SA-treated mosquitoes. The data reported in this thesis reveal that silencing of SCD1 in female A. coluzzii mosquitoes leads to a metabolic syndrome primarily associated with the increase of saturated fatty acids and TCA cycle intermediates, which affects important biological functions leading to premature mosquito death. The accumulation of saturated fatty acids is also the likely cause of a potent immune response observed in the absence of infection, which resembles an auto-inflammatory reaction. These data provide important leads for the development of novel interventions aiming to block transmission of mosquito-borne diseases.

Actinobacillus pleuropneumoniae (APP) is a bacterial pathogen that infects the porcine respiratory tract, causing the economically significant disease porcine pleuropneumonia. As yet, no highly efficacious or heterologous vaccine has been produced for its prevention. A better understanding of host-pathogen interactions, including the contribution of essential genes is important to elucidate novel strategies for the development of both vaccines and therapeutics. This study looked to identify the essential genes for growth of APP under numerous biologically important conditions using Transposon Directed Insertion Sequencing (TraDIS) and an individual mutant screen. The loss of diversity of an original 43,000 Tn mutant library, meant TraDIS was unsuccessful, however, a total of 16 putative essential genes were identified while screening mutants anaerobically. Using RNA-seq, changes in the APP transcriptome during in vitro and in vivo conditions were also elucidated. Findings illustrated that, compared to published in vivo (acute pig infection) transcriptome data, all conditions (growth in porcine serum, anaerobically in rich medium and in the Waxmoth Galleria mellonella) showed comparable gene expression levels, suggesting their potential as alternative models for the study of APP pathogenesis. Currently, no inducible gene expression system in APP has been published. Therefore, an anhydrotetracycline (aTc) inducible gene expression system was developed to verify predicted essential genes. After initial developmental issues, the aTc-inducible system proved functional and titratable in APP strain MIDG2331 and was used to verify atpB as essential for anaerobic growth. Genes identified as essential for anaerobic growth (atpB) or highly repressed in G. mellonella (lrp, MIDG2331_00346) were evaluated for their contribution to virulence and vaccine potential in G. mellonella. The atpB and lrp mutants were attenuated for virulence in G. mellonella, whereas deletion of a gene (MIDG2331_00346), encoding a hypothetical protein, had no effect on virulence. In addition, testing of the MIDG2331 lrp mutant suggested it may be a potential live- attenuated vaccine (LAV) candidate. Therefore, using these alternative models, coupled with the use of high-throughput methods, can provide suitable alternatives to the pig for the discovery of vaccine targets.

Opisthorchiasis is a zoonotic parasitic infection caused by human liver flukes. Opisthorchis viverrini (OV) is endemic in Southeast Asia along Mekong basin which estimated 9 million people are infected. Transmission to humans occurs through the consumption of uncooked fish; cyprinoid or white-scale freshwater fish containing infective stage metacercariae. The infection is mainly asymptomatic. Adult flukes could live up in the bile duct in the absence of treatment. Chronic infection is strongly related with bile duct cancer or cholangiocarcinoma. The International Agency for Research on Cancer has declared that Opisthorchis viverrini is a member of group 1 agent, carcinogenic to humans. Thailand has the highest incidence of cholangiocarcinoma in the world. However, opisthorchiasis is acknowledged as a neglected and underestimated disease on the global scale. In the present day, the epidemiological data on opisthorchiasis from OV infection in Thailand are considered outdated. An extensive cross-sectional study was undertaken from 1984 to 2001 under the National Control Program, but data on incidence is very limited. Moreover, infection dynamics are still poorly understood. This project aimed to comprehensively study the infection dynamics of OV infection using multiple tools in order to explore the infection in various aspects. Statistical models were developed to explore epidemiological data; prevalence, incidence and infection intensity, with risk factors for acquiring the infection. Infection dynamics will be described using mathematical modelling. The qualitative technique, by interviewing and group discussions, will be used to explain the cause of uncontrolled infection rate in bio-psycho-social aspect. Integrated study results will be used to develop community intervention strategies under the framework of the public health planning model. The overall outcome will be valuable for Thailand National Health Policy and epidemiological data will provide the basis for further rigorous academic research.

No description available.

Tuberculosis kills more people than any other infection. Individuals harboring latent Mycobacterium tuberculosis infection (LTBI) carry a lifetime risk of reactivation to active disease and a better understanding of LTBI is a research priority. Studies to investigate dormant cells of M. tuberculosis when subjected to nutrient limitation, using in vitro and in vivo models to simulate LTBI, are the subject of this thesis. In vitro, extended culture with fatty acids (oleic acid) as the carbon source, leading to non-replicating persistence (NRP) was used to compare bacilli in different growth phases. Transcriptomic analyses revealed that NRP bacilli down-regulated genes involved in lipid biosynthesis, aerobic respiration, TCA (tricarboxylic acid) cycle metabolism and ATP synthases, while genes associated with 3-oxidation of lipids were induced, when compared to exponential phase (EXP) bacilli. The gene expression profiles of bacilli in vitro were compared with those from bacilli isolated from intracellular and in vivo models and the data indicated that lipid metabolism and nutrient depletion may be important during LTBI in humans. Guinea pig models of active and latent M. tuberculosis infection were established and methods of bacterial RNA extraction from infected lung were investigated, with the aim of characterising in vivo derived bacilli using transcriptomics. Technical challenges in obtaining sufficient RNA for transcriptomics could not be overcome using the available technology. The infectivity in guinea pigs, of bacilli from different growth phases generated in vitro was investigated to determine the relationship between the transcriptome and infectivity. Compared with EXP or stationary phase (STAT), NRP bacilli showed a delay in establishing infection but similar bacterial loads and pathology were observed at later time points. In summary, using an in vitro nutrient depletion model, growth phase-specific gene expression signatures and infectivity characteristics of M. tuberculosis were defined which contribute to the understanding of bacilli during latent infection.

Treatment of tuberculosis requires months of antimycobacterial therapy. This tolerance to antibiotics displayed by Mycobacterium tuberculosis could be attributed to biofilm formation. Biofilms are the cause of many chronic infections. The aim of this thesis was to apply laboratory methods for the culture of Mycobacterium smegmatis and M. tuberculosis H37Rv biofilms and to further characterise these bacterial phenotypes in terms of their physiology, gene expression and drug susceptibility The Modified Robbins Device (MRD) and the Constant Depth Film Fermenter (CDFF) laboratory models were applied alongside the previously established well-plate pellicle model. Antibiotic efficacy studies of M. tuberculosis pellicles identified drug-tolerant bacteria. These pellicle biofilms exhibited tolerance to rifampicin and isoniazid many times above the planktonic minimum inhibitory concentration (MIC). CDFF biofilms were tolerant to a planktonic MIC of isoniazid. CDFF and pellicle biofilms of M. tuberculosis and pellicle biofilms of M. smegmatis were investigated in terms of their gene expression using microarrays to determine the underpinning molecular mechanisms behind biofilm formation Biofilms of both mycobacterial species upregulated lipid metabolism and biosynthesis. M. tuberculosis biofilms upregulated genes involved in the type seven secretion system (T7SS) and genes which code for PE/PPE proteins. T7SS is known to interact with some PE/PPE proteins, many of which are cell-surface associated. These gene expression profiles suggested significant restructuring of the cell wall and provides a genetic basis for extra-cellular matrix formation. Also, low levels of metabolic activity were identified within these biofilms using fluorescent staining with viability dyes and flow cytometry. Overall, this thesis provides a controlled method for mycobacterial biofilm formation using the CDFF and confirms that M. tuberculosis H37Rv biofilms comprise antibiotic tolerant cells.

Hospital associated infections result in longer patient stays, increased treatment costs and morbidity and mortality. Novel technologies utilising UV light, hydrogen peroxide vapour and ozone have been designed to provide high-level environmental decontamination, in an attempt to prevent patient acquisition of nosocomial pathogens. However due to safety concerns, these technologies are only suitable for intermittent terminal cleaning, and surfaces can become quickly re-contaminated. Recently developed systems which utilise antimicrobial 405 nm violet-blue visible light have been successfully used for continuous decontamination of surfaces and air, in the presence of patients. This study investigated some fundamental questions surrounding the use of antimicrobial 405 nm light for clinical decontamination. Initial investigations studied the antiviral efficacy of 405 nm light on a model for the nosocomial pathogen, Norovirus. Studies showed positive antiviral effects in suspension and on clinically-relevant surfaces when virions were exposed in minimal media (DPBS), however this inactivation efficacy was significantly improved (85% lower dose) when the virus was suspended in organically-rich, biologically-relevant media (such as saliva and blood plasma). This enhanced inactivation is likely due to photoexcitation of the suspending media, and was demonstrated using fluorescence spectrophotometry, with excitation peaks seen for all suspending media except minimal media. A systematised review also compared 405 nm light inactivation of viruses with other clinical pathogens (bacteria, fungi), and found that viruses (exposed in minimal media) are more resilient structures, requiring higher doses for equivalent reductions, likely due to differing inactivation mechanisms. Further studies also investigated the potential for proliferating and non-proliferating bacteria, Staphylococcus aureus, to become tolerant to 405 nm light. Results demonstrated that exposure to 405 nm light during cultivation resulted in higher dose requirements for complete inactivation and increased stress tolerance, however the process was unlikely to be selective. Results also demonstrated that repeated sub-lethal exposure of antibiotic sensitive and resistant vegetative cells did not give rise to tolerance or alter antibiotic susceptibility. This study has provided significant fundamental information about antimicrobial violet-blue light. The results demonstrate proof-of-concept evidence of the virucidal efficacy of 405 nm light, as well as demonstrating that bacterial tolerance is unlikely. These results further support the clinical use of antimicrobial 405 nm light for continuous environmental decontamination, with implementation likely to aid infection control and reduce hospital associated infections.

Many Gram-negative bacterial pathogens such as Shigella and adhesive invasive Escherichia coli (AIEC) cause infections characteristic of hyperinflammation. These infections require antimicrobial therapy. However, due to the widespread emergence of multiple drug-resistant strains, alternative strategies must be sought to combating infectious diseases. It has been shown that natural compounds such as propolin D are able to control Shigella growth inside host cells. Geraniol is another natural product which has a chemical structure similar to the side chain of propolin D, which possesses properties potentially useful for antimicrobial therapy. qPCR analysis revealed that propolin D caused extensive bacterial envelopE stress, as indicated by a changed expression of key bacterial genes involved in stress responses. Propolin D also enhanced the autophagy activity of the host cells; the intracellular growth of S. sonnei was significantly reduced in wild type HEK293 cells but not changed in ATG5 knockout cells. Propolin D was unable to enhance septin cage as intracellular S. sonnei formed actin tails in the presence of propolin D; septin cage would restrict formation of actin tails. Geraniol has been shown to target the major virulence regulator, DsbA, which is vital for Shigella’s survival in the reducing host cell cytosol. Geraniol and geranylxvacetate inhibited DsbA function in vitro; wild type DsbA efficiently reduced fluorescently labelled Di-E-GSSG whereas a mutant protein, DsbA33G, was less potent in this in vitro assay. By supplementing acidic and nutrient-poor medium with geraniol the growth of S. sonnei and AIEC strains was severely inhibited. Geraniol was effective in protecting of Galleria. mellonella larva from S. sonnei and AIEC infection. The Galleria mellonella larvae were highly tolerant to geraniol – indicating the great potential of geraniol for future in vivo and clinical studies. In light of previous reports that geraniol synergistically works with antibiotics and induces IL-10 from macrophages, it was concluded that geraniol holds great potential in treating Shigella and AIEC infections.

The objective of this thesis was to investigate various aspects of the epidemiology of human papillomavirus (HPV). Prospective studies were performed on a total of 334 patients attending to genitourinary medicine departments in London. The patients had either clinical indications for genital biopsy, urethral gonorrhoea, anogenital warts or a current partner with anogenital warts. Biopsy, scrape, swab and blood samples were taken. In addition, a new method, the "Superglue (SG) technique" was devised. HPV types 6, 11, 16, 18, 31 and 33 were identified in the specimens by DNA extraction followed by the polymerase chain reaction, Southern blotting and hybridisation. HPV DNA was detected in 31.8 to 96.6% of biopsies with histological evidence of wart virus infection, 83.3% of biopsies with neoplasia and 18.2% of those with other histological changes. HPV DNA was detected in 31.3 to 61.1% of urethral loop specimens, 18.0 to 24.8% of urethral swab specimens, 6.3 to 12.5% of urine specimens, 37.5% of vaginal washes and 21.4% of rectal swab specimens. Fifty percent of samples obtained with the SG technique containing human cells had detectable HPV DNA. HPV types 6 and 11 tended to be found in biopsies from lesions clinically associated with HPV infection and in the urethras of men with warts seen near the urinary meatus. HPV types 16 and 18 tended to be found in specimens obtained by techniques other than the biopsy method and from clinically atypical lesions. Infections with multiple HPV types were common. Different sites from one patient sometimes had HPV types in common.