The increasing emergence of resistance to commonly used therapies has placed a huge strain on the prevention and control of malaria; therefore, there is an urgent need to develop novel antimalarial agents. The aim of this research was to design and synthesise a library of potent antimalarial compounds, with desirable pharmacokinetic profiles, in order to identify a drug candidate suitable for preclinical development. This research was divided into two main sections: x The synthesis of compounds deigned to inhibit IspD, a novel target in antimalarial drug discovery x The late stage development of a series of endoperoxide-based antimalarials, which are derived from the structure of artemisinin A library of benzisothiazolinone compounds was generated to target the IspD enzyme. Many of these compounds displayed low micromolar inhibitory activity against both enzymatic and phenotypic assays in vitro and an investigation into structure-activity relationships around the core of these benzisothiazolinones was also conducted. The most potent compound to emerge, a CH2 linked benzisoselenazolone, had an IC50 of 0.17 μM against PfIspD and 5.54 μM against Pf3D7. These compounds represent a novel class of IspD inhibitor, which have the potential for further development as antimalarial agents. A number of 1,2,4,5-tetraoxane analogues were also prepared in order to develop an antimalarial agent suitable for a single-dose cure. The most potent analogue, N205, had an IC50 of 1.3 nM and an average mouse survival of 26.3 days (66% cure rate) following a single dose. A less than optimal stability profile for N205 led to the further development of another potent tetraoxane analogue, E209. Optimisation of the synthetic pathway led to the generation of E209 in a series of five high-yielding steps that are suitable for large-scale production. E209 represents the first 1,2,4,5-tetraoxane that is comparable, in terms of both efficacy and PK/PD profiles, to OZ439, and is a candidate for pre-clinical development.

Antivenom is the only effective treatment for snakebite and comprise immunoglobulins obtained from venom-immunised horses or sheep. Globally, more than 45 manufacturers make over 120 snake antivenoms; it is a regulatory requirement that the venom-neutralising efficacy of all antivenoms are assessed preclinically. The World Health Organisation (WHO) recommended preclinical tests of efficacy are the median lethal venom dose (LD50) and median effective antivenom dose (ED50) assays performed in mice. They result in substantial pain and suffering to the mice with death/survival as their metric. With NC3R-funding, we sought to apply the ‘Refine, Reduce and Replace’ principles of animal experimentation to these murine assays. Pain is a near-universal symptom of snake envenoming, and one of our objectives was to identify an effective analgesic that could be utilised without invalidating the assay results. The Mouse Grimace Scale and Activity scores were used to measure pain. We examined the effects of two opioid analgesics, buprenorphine and morphine, in a range of venom LD50 and ED50 assays. Both were effective at reducing pain scores, but death rates were higher in those which had received buprenorphine, hence morphine is preferable. We demonstrate that each venom exhibits a distinct set of lesions, the severity of which appears time and dose dependent, and that the observed murine pathological lesions show significant similarities to those reported in envenomed human victims. Applying the 3R principles, we have used pathological observations, in combination with ante mortem observations, to establish more humane end-points, consequently reducing the duration of LD50 and ED50 assays from 24 to 6 hours. In addition, we have implemented a ‘dose-staging’ element into experimental design in which one dose is given and the next dose(s) selected based on the results of the previous dose, reducing total mice required. To reduce the numbers of assays, and therefore mice, we have shown an excellent correlation between in vitro binding assays, cytotoxicity neutralisation assays and in vivo ED50 using antivenoms derived from the same pool of donor animals. Comparison of the results of in vitro binding assays between 35 different venom/antivenom combinations showed a poor correlation overall, but the correlation improved when each of five venoms were considered separately. The possibility of replacing the in vivo LD50 and ED50 tests, using a cell-based neutralisation assay was investigated using two cell lines from diverse tissues of origin, namely VERO epithelial-type cells and neural SH SY5Y. All venoms studied produced a cytopathic effect in both cell lines, with the VERO cells being more sensitive to viper venoms and SH SY5Y cells to the effects of elapid venoms, when both cell lines were grown in co-culture. However, variability of results made optimisation of a neutralisation assay inadequate for use as an alternative to in vivo tests.

Chiggers are the tiny six-legged larval stage of mites in the family Trombiculidae. These mites, particularly the genus Leptotrombidium, act as important vectors of Orientia tsutsugamushi, the causative agent of scrub typhus disease in the Asia-Pacific region (including Thailand). Although the medical impact of these mites has been recognized in the country due to the increasing incidence of the disease in humans, knowledge of the ecology and epidemiological role of these mites is still very limited to date. A systematic review of mite-associated bacteria was conducted from 193 publications (1964 - January 2015) providing a reference database of bacteria found in mites of agricultural, veterinary and medical importance. Approximately 150 bacterial species were reported from 143 mite species with Cardinium, Wolbachia and Orientia as the dominant genera. Nationwide field sampling of small mammals from 13 locations in Thailand revealed a high diversity of chigger mites. From approximately 16,000 mites isolated from 18 host species examined (1,574 individual animals), 38 chigger species were found including three species new to science (i.e., Trombiculindus kosapani n. sp., Helenicula naresuani n. sp. and Walchia chavali n. sp.) and 10 new records for the first time in the country. Brief taxonomic information for the morphological identification of chiggers is provided. A combination of autofluorescent and brightfield microscopy was demonstrated to be a novel approach to study both the morphology and DNA profile of the same individual chigger. Most chigger species showed low host specificity. The diversity of chiggers on hosts was influenced by host intrinsic (i.e., host phylogeny and maturity) and extrinsic factors (i.e., habitat and geographical location). Chigger species richness and host-chigger network connectance were found to be interrelated variables explaining human scrub typhus incidence in Thailand. Chigger-associated bacteria were investigated for the first time using an Illumina MiSeq 16S rRNA amplicon sequencing approach. DNA of O. tsutsugamushi was detected in the chigger population as expected. In addition to O. tsutsugamushi, Borrelia and Mycobacterium were identified aspotential pathogens of human and animals. Potential symbiotic bacteria of arthropods; e.g., Candidatus Cardinium, Pseudonocardia, Rickettsiella and Wolbachia were also discovered for the first time in chiggers. An important technical limitation was that chigger DNA starting quantity (individual specimens versus pooled mites) was found to have a significant impact on the apparent microbiome profile. These outcomes from the studies of chigger taxonomy and the ecology of host-chigger interactions, as well as the composition of the microbiome in chiggers, are of key importance to the chigger research field, providing essential information for disease epidemiology with vector control implications.

A. fumigatus is a ubiquitous fungus that causes invasive pulmonary aspergillosis (IPA). IPA is one of the most life-threatening infections in immune compromised patients. Corticosteroids are commonly prescribed as immunosuppressive agents for transplant patients, and IPA mostly occurs in the setting of potent transplant maintaining immune suppressive therapy. Corticosteroid immune suppression represents a major risk factor for IPA, as it inhibits the killing capacity of alveolar macrophages and results in fungal growth that recruits non-sterilizing PMNs to the site of infection that leads to tissue damage. This PhD aimed to characterize effects of the sphingosine-1-phosphate (S1P) pathway inhibitors SKI-II and FTY720 for their utility in attenuating the characteristics of IPA immunopathology, such as inflammation and fungal development. Firstly, it was demonstrated that A. fumigatus-mediated cytokines and chemokines were down regulated in vitro in the presence of inhibitors, and direct sphingosine kinase (SphK) inhibition resulted in a phagocytic defect but not S1P1 receptor antagonism. Inflammatory responses and fungal burden were then evaluated in immunocompetent mice with FTY720, and it was shown that cytokine and chemokine responses were inhibited without compromising fungal clearance. Further to this, FTY720 was evaluated for its efficacy in the hydrocortisone model of aspergillosis. Both pro-inflammatory mediators and fungal burden were reduced, and increased survival was observed. Secondly, the antifungal properties of both SKI-II and FTY720 were investigated. SKI-II was shown to have limited activity with germination kinetics but inhibited hyphal extension, whereas FTY720 was demonstrated to inhibit fungal germination and hyphal growth significantly. Genetic deletions in A. fumigatus of SphK and gprD putative genes were generated, corresponding to mammalian sphingosine kinase 1 and S1P1. Phenotypic examination showed that mutants were sensitive to cell wall stress and had altered germination kinetics, and ∆gprD was less immunogenic. Overall these findings in this thesis indicate the S1P1 inhibitor FTY720 has antifungal activity in vitro against A. fumigatus with therapeutic effects in vivo in the HC model of IPA and could be developed as an antifungal therapeutic.

Cytomegalovirus (CMV)-based vectors are a promising vaccine platform with an ability to induce high levels of durable, immediate, effector memory T (TEM) cell responses against their heterologous encoded pathogen target antigen. The primary focus of this thesis is centered on a hypothesis that targeting of essential CMV tegument proteins by using regulatable protein-destabilization is suitable as a conditional vaccine attenuation strategy. CMVs are generally benign, however in individuals whose immune systems are immature or weakened, CMV can be a significant pathogen causing substantial morbidity and mortality. Previous studies have shown that replication-defective versions of CMV made by conventional permanent deletion of essential genes are not compromised in terms of their immunogenicity. However, such non-conditional attenuation requirs parallel development of complementing cell lines, which is technically difficult, expensive and not suitable for use in many situations and environments, in particular low and middle-income countries (LMICs). Recently, protein destabilizing domain (DD) technology has been developed that has the potential to be brought against this issue. DDs are conditionally unstable protein domains that can provide regulatable degradation of a desired protein by genetic fusion to the targeted protein. Addition of a small-molecule binding ligand stabilizes the DD, thereby increasing levels of the targeted protein in a rapid and dose-dependent manner. Using the murine CMV (MCMV) system, we hypothesized that fusion of a DD to essential tegument proteins of MCMV would be capable of regulating the stability of the tegument proteins resulting in generation of conditionally-attenuated MCMV vectors. Part one of this thesis details construction and in vitro characterization of recombinant MCMVs using this DD-based destabilization strategy, and identifies this approach as able to provide a means for production of CMV-based vaccines that differ in their levels of attenuation based on the specific tegument protein targeted. The second part of the thesis is concerned with initial development of CMV as a vaccine vector against pandemic influenza A (IA) virus infection. IA virus is a respiratory pathogen that despite the availability of vaccines continues to have an enormous impact on population health and world economy. Standard seasonal (epidemic) IA vaccines provide only ‘homosubtypic’ immunity. The omnipresent potential for emergence of pandemic IA subtypes, for which the human population possesses no immunity, makes IA a major global health concern. The hypothesis being tested in these studies is that targeting of more conserved IA virus proteins with CD8+ TEM cell-based immunity by using CMV-based vectors as a quintessential inducer of such ‘effector’ memory responses, will provide the desired heterotypic immunity capable of preventing pandemic IA. This initial study determined the capacity of a MCMV-based vaccine expressing the conserved IA proteins nucleoprotein (NP), polymerase (PA) and non-structural protein 2 (NS2) to induce immunity in mice as a strategy to prevent pandemic IA emergence.

My work has involved the investigation of physical and physiological issues related to submarine escape and rescue, including animal experiments using goats, human experiments,engineering and equipment based trials and also the development of a number of mathematical models for the prediction of equipment performance and physiological aspects of decompression. In addition to my published papers I have authored or made substantial contribution to over sixty reports funded by the United Kingdom Ministry of Defence (UKMoD). Experiments in which I made significant contribution in terms of original conception, design, data acquisition and analysis have provided evidence for the efficacy of surface oxygen as a treatment for decompression illness following submarine tower escape and demonstrated that breathing raised partial pressures of carbon dioxide in a distressed submarine then switching to diving quality air in the escape tower is not likely to prevent successful escape. The submitted work demonstrates an advance in our knowledge of the risks of decompression illness and a substantially improved level of confidence in the predictions of mathematical models of decompression illness for submarine tower escape. These are the first semiempirical probabilistic models of decompression illness to combine animal and human decompression illness data using body mass as a scaling factor. Provision of more detailed information on risks of decompression illness and likely outcomes has allowed me to argue for a change in escape policy which has been accepted by the UK Royal Navy (RN). My suggestions for optimising tower escape are to be tested on board UK submarines in the future, prior to possible adoption by the RN. The iso-risk curves I developed for probability of decompression illness and predicted probability of survival following submarine escape have been included as advice in the latest update to ATP-57, the NATO submarine search and rescue manual.

Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis (ZVL) in the Mediterranean region, with the domestic dog as the main reservoir host. Phlebotomus (Larroussius) ariasi is the principal vector in cooler, forested ecotopes in southwest Europe, which suggests that it might be subject to environmental and geographical isolation. However, the population genetics of P. ariasi had been little studied before this thesis, which investigated how the population differentiation of this vector might affect its ability to spread northwards, or persist in the Mediterranean region, in response to climate and habitat change. Thirty-six spatio-temporal populations of P. ariasi were molecularly characterized across its range, predominantly from southwest France but including geographical outgroups from Spain, Portugal and North Africa. Phylogenetic and population genetic assessments were made based on five DNA sequences: mitochondrial cytochrome b, nuclear elongation factor-la and apyrase, plus two anonymous nuclear loci, AAm20 and AAm24. The results demonstrated the absence of cryptic sibling species of P. ariasi and the selective neutrality of each locus. Mitochondrial DNA revealed a historical phylogeographic structure, which was consistent with Pleistocene climate change driving multiple haplogroup divergences within glacial refuges and phalanx-like population expansions in interglacial periods. Nuclear loci mostly showed isolation by distance, but some supported restricted gene flow between the Pyrenees and the Massif Central, France, as indicated by cytochrome b. A glacial refuge may have existed north of the Pyrenees. The genetic diversity observed in the northeast Pyrenees, France, permitted an assessment of the effects of broadleaf forest fragmentation on the differentiation of P. ariasi. No conclusive evidence was found to support contemporary genetic substructuring or impoverishment associated with a recent increase in forest fragmentation. The salivary peptide apyrase revealed a geographical pattern of polymorphism consistent with the other selectively neutral loci. A range of selection tests indicated that apyrase was not evolving under positive directional or balancing selection and, therefore, a genetic arms race with the mammalian host and/or Leishmania parasite was not supported. The approach taken provides a proof of principle for helping to assess apyrase and other salivary peptides as vaccine candidates against leishmaniasis.

New diagnostics and treatments for malaria have renewed hope in the developing world as they promise relief from the debilitating effects of this illness. Accompanying these interventions are a growing number of economic evaluations assessing their efficiency. To ensure the relevance of economic evaluations to decision making purposes it is imperative that they use best available computational and statistical approaches. This thesis initially discusses the necessary requirements for economic evaluations to ensure they provide appropriate decision recommendations. This is followed by four evaluations of malaria diagnostics and treatments using methods new to the context of malaria. The first study expands the range of factors included in the evaluation of diagnostic tests, addressing compromised adherence to test results and societal costs associated with antimalarial use. The second analysis demonstrates how models can be designed as decision support tools allowing stakeholders to enter local data along with other parameter estimates, priorities and values. Both Bayesian and deterministic models are presented for comparison. The third analysis demonstrates the use of multilevel models for economic evaluations based on multi-centre trials. The chapter compares the results of a multilevel model evaluating treatments for severe malaria with those obtained in a standard analysis. The fourth study uses a Markov model to evaluate the efficiency of Home Management of Malaria programmes. The use of a Markov model addresses the restricted portrayal of malaria infection and illness that has characterised many previous evaluations. In addition to contributing to a better understanding of the cost-effectiveness of the latest malaria treatments and diagnostic tests, this thesis seeks to bridge the growing gap between recent methodological advances in the field of economic evaluation, and the relative paucity of evaluations producing practical and effective policy recommendations for areas of the world where the burden of malaria and other diseases is heaviest.

Existing estimates of the incidence of infectious intestinal disease (IID) caused by norovirus in the community are based on electron microscopy or reverse transcription- polymerase chain reaction (RT-PCR). Neither method accurately represents norovirus disease burden: electron microscopy has poor diagnostic sensitivity, RT-PCR has poor diagnostic specificity. In this thesis, for the first time, viral load measurements were used to identify cases of norovirus-associated IID, to update the existing, electron microscopy-based estimates of norovirus disease burden in the community in England and to conduct a comprehensive analysis of risk factors for sporadic norovirus-associated IID. The prevalence and characteristics of asymptomatic norovirus infection in England were also described. Data for this work were taken from the Study of Infectious Intestinal Disease faecal specimen archive, which had been subject to semi-quantitative real- time RT-PCR norovirus testing. Finally, routine surveillance data and time-series regression modelling were used to produce an additional and independent estimate of the incidence of general practice consultations for norovirus-associated IID in England and Wales. Using viral load for norovirus diagnosis, the age-adjusted community incidence of norovirus-associated IID in England was 4.5 per 100 person-years (95% credibility interval: 3.8,5.2), equating to 2 million episodes per year between 1993 and 1996. Amongst children aged less than five years, the community incidence was 21.4 per 100 person-years (95% credibility interval: 15.9,27.7) and the incidence of consultations to general practitioners for norovirus-associated IID was 3.2 per 100 person-years (95% credibility interval: 2.6,3.8), with 100 000 children visiting their GP for norovirus- associated IID each year. The main risk factor for sporadic, community-acquired norovirus-associated IID was contact with a person with IID symptoms. This result indicates that reduction of person- to-person transmission would substantially decrease the burden of norovirus- associated IID in the community in England, e. g. through good hand hygiene and appropriate cleaning of environmental surfaces.

The Toll/IL-l Receptor (TIR) domain is an essential signalling module in eukaryotic innate immune signalling pathways. Homotypic interaction between TIR domains allows the formation of a signalling platform in which molecules are able to interact and activate each other to initiate an immune signalling cascade. Proteins containing TIR domains have also been discovered in bacteria. Studies have subsequently shown that these proteins are able to modulate mammalian immune signalling pathways dependent on TIR interactions and that this forms an evasion strategy for bacterial pathogens. In this study a bioinformatic search for proteins containing TIR domains was carried out across unicellular organisms, including bacteria. TIR domain proteins (Tdps) from highly pathogenic bacteria were down-selected for investigation. After an initial screen of their activity, a Tdp from Yersinia pestis, the causative agent of plague, was down-selected for further investigation. The bioinformatic analysis found a high representation of Tdps in bacteria generally classified as non-pathogens, and that TIR domains are promiscuous in their co-occurrence with other domains. This analysis also showed that they are not necessarily conserved between strains and species. These findings question the universal role of Tdps in the pathogenic evasion of a host immune response and suggest they may have other functions. Initial screening of down-selected Tdps showed that they were able to modulate immune signalling pathways in vitro, but studies with a Tdp-deficient mutant of Y. pestis did not demonstrate a role for this protein in the virulence of Y. pestis in a mouse model. However, this Tdp-deficient mutant did display two characteristics in vitro: an increased autoaggregation phenotype when compared to wild-type Y. pestis and an inability to survive as well as wild-type bacteria in conditions of high salinity. These fmdings indicate that TIR domain proteins may have other roles in bacterial physiology unrelated to immune evasion.