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Despite significant progress in the control and elimination of malaria over the past two decades, the global burden remains high. The COVID-19 pandemic has seen malaria cases and deaths increase markedly over 2019 resulting in 241 million malaria cases and 627,000 malaria deaths worldwide in 2020, an increase of 14 million cases and 69,000 deaths. Around 47,000 of these additional deaths were linked to pandemic-related disruptions in the provision of malaria prevention, diagnosis and treatment. The need for a highly efficacious childhood malaria vaccine has never felt more pertinent and in 2021, after 30 years of research and development, the World Health Organization recommended the first ever childhood vaccine against P. falciparum malaria, RTS,S/AS01E (RTS,S) for widespread use. The development of RTS,S, its deployment and continued evaluation has facilitated the synthesis of knowledge and data from across a wide range of different disciplines involved in malaria vaccine research. This depth of data has enhanced the development of mathematical modelling frameworks that combine immunological insights with epidemiological transmission models to address public health questions. These frameworks have formed a core part of the evaluation and policy recommendations surrounding RTS,S. The work presented in this thesis builds upon these modelling frameworks to provide insights into the potential impact of alternative RTS,S vaccination approaches. The two RTS,S approaches examined in this thesis are a delayed-fractional primary series and a seasonally targeted vaccination schedule, both of which have demonstrated promising efficacy in human challenge studies and field trials respectively. Drawing on data from the delayed-fractional RTS,S human challenge study I used a Bayesian framework to investigate immunological correlates of vaccine induced protection. I estimate that improvements to the quality, measured as antibody avidity, and not the quantity, measured as antibody titre, of the vaccine induced antibody response is critical to the increased efficacy against infection observed with this schedule. Next, I utilised data from seasonal malaria chemoprevention and seasonal RTS,S vaccination clinical trials to fit and validate an updated efficacy profile of the drug combination Sulfadoxine-pyrimethamine plus amodiaquine (SP+AQ) used for seasonal malaria chemoprevention using a Bayesian survival analysis framework. This approach enabled me to capture uncertainty in the protection provided by seasonal malaria chemoprevention over time. I then use this updated efficacy profile along with the existing RTS,S vaccine efficacy profile to replicate trial cohorts in a transmission model in order to validate the intervention models against clinical trial data. I found that the existing RTS,S model underestimated the protection provided by the seasonal vaccination schedule and explored several biologically motivated alterations to the model that brought results in line with those of the trial. These results combined with the trial reported antibody data suggest that efficacy improvements with this regime were not driven by increases in antibody quantity. Further model results suggest that when vaccination and chemoprevention were combined this resulted in potential synergistic interactions that enhanced the efficacy of SP+AQ in particular. This work resulted therefore in several updated versions of RTS,S and SP+AQ efficacy models that capture the current uncertainty in intervention effects. Finally extending these updated efficacy models from the validation exercise, I used a model of malaria transmission to investigate the long-term public health impact of novel RTS,S vaccination schedules compared to the original age-based RTS,S dosing schedule in seasonal settings. I considered the impact both in the presence and absence of seasonal malaria chemoprevention. I examined impact by degree of seasonality, transmission intensity and by wider health system and operational factors. RTS,S vaccination in seasonal malaria transmission settings could be a valuable additional tool to existing seasonal interventions, with seasonal delivery maximising impact relative to an age-based approach. Decisions surrounding deployment strategies of RTS,S in such settings will need to consider the local and regional variations in seasonality, current levels of other interventions and potential achievable RTS,S coverage. Open Access

Malaria epidemiology is characterised by extensive heterogeneity that manifests across a range of spatial and temporal scales. This heterogeneity is driven by a diversity of factors spanning the human host, the parasite, the mosquito vector and the environment. Together, variation in these factors lead to marked differences in the epidemiology of malaria across different settings; in where malaria is concentrated, how malaria is transmitted and who is most at-risk. These differences have material consequences for the impact of control interventions aimed at combatting the disease, underscoring the crucial need to better understand and quantify the factors underlying heterogeneity in malaria epidemiology and transmission dynamics. In this thesis, I use a combination of statistical and mathematical modelling to further our understanding of how variation in the epidemiological and entomological determinants of malaria transmission drives heterogeneity in dynamics across settings and explore the implications of this variation for control efforts. Accurate ascertainment of malaria infections represents a crucial component of malaria surveillance and control. Previous work has revealed the often-substantial prevalence of infections with parasite densities lower than the threshold of detection by microscopy (so called “submicroscopic” infections). The drivers of these infections remain uncertain, despite their established relevance to onwards transmission. In Chapter 2, I carry out a systematic literature review and meta-analysis exploring the prevalence of submicroscopic malaria infections and how this varies between settings. My results highlight extensive variation between settings, with much of this driven by a combination of both historical and current levels of transmission. Crucially, these results highlight significant variation in the prevalence of submicroscopic infections even across settings characterised by similar current levels of transmission, with implications for the utility of control efforts specifically targeting this infected sub-group depending on the context. Within communities, the distribution of malaria infections is frequently characterised by extensive spatial heterogeneity, which can make identification and treatment of infections challenging. In Chapter 3, using a regression-based approach, I characterise the fine-scale spatial clustering of malaria infections at the household level across a diverse range of sub-Saharan African settings through systematic analysis of 57 Demographic and Health Surveys spanning 23 countries. My results highlight that malaria infections cluster within households, and that the extent of this clustering becomes significantly more pronounced as transmission declines – a factor which will affect the comparative impact of household-targeting or whole-community based control strategies and result in their appropriateness depending closely on the levels of transmission characterising a setting. In addition to this spatial heterogeneity, malaria transmission dynamics are also frequently characterised by extensive temporal heterogeneity, a phenomenon underpinned by the (often annual) temporal fluctuations in the size of the mosquito populations responsible for transmission. Many questions remain surrounding the drivers of these dynamics however, questions that are rarely answerable from individual entomological studies (focussed on only a single location or species). In Chapter 4 I carry out a systematic literature review to collate anopheline mosquito time-series data from across India and develop a statistical framework capable of characterising the dominant temporal patterns in this dataset. The results demonstrate extensive diversity in the timing and extent of seasonality across mosquito species, but also show that this diversity can be clustered into a small number of “dynamical archetypes”, each shaped and driven by a largely unique set of environmental factors including rainfall, temperature, proximity to water bodies and patterns of land use. In Chapter 5, I apply this framework to time-series data from across South Asia and the Middle East for the highly efficient vector Anopheles stephensi, to better understand the factors shaping its seasonal dynamics and the likely impact of its recent establishment in the Horn of Africa. My results reveal significant differences in the extent of seasonality across Anopheles stephensi populations, with dynamics frequently differing between rural and urban settings, suggesting structural differences in how these environments shape patterns of vector abundance and potentially warranting different vector control strategies depending on predominant patterns of land-use. Integrating these seasonal profiles into a mathematical model of malaria transmission highlights the crucial need for an understanding of the timing of seasonal peaks in vector density if control interventions like IRS are to be most effectively deployed. Overall, the results presented here highlight some of the drivers influencing spatial and temporal heterogeneity in malaria epidemiology, quantifies how they contribute to the diverse malaria dynamics observed across different settings, and explores the implication of this variation for effective control of the disease. Open Access

Aryl Hydrocarbon Receptor (AHR) is a ligand activated transcription factor, which was initially characterised as an environmental sensor, that mediates toxic effects in response to harmful chemicals. Early research therefore focussed on exogenous ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which elicit a sustained activation of the AHR pathway, rather than a transient one associated with natural ligands. More recently, AHR has been implicated in obesity. The incidence of obesity has increased dramatically over recent decades, and carries a high burden to health care systems, along with increased prevalence of cardiovascular disease and cancer. Continued work surrounding obesity has shown that thermogenesis contributes to energy balance, and is now an attractive therapeutic target to attempt to reduce obesity rates. Findings show that AHR is not required for activation, nor regulation of thermogenesis through the characterisation of the global AHR knock out (KO) and adipocyte-specific AHR KO mouse models when exposed to cold. Both AHR KO and adipocyte-specific KO animals are able to maintain body temperature when exposed to acute cold challenge. No differences were observed in oxygen consumption nor energy expenditure. These mice were also able to maintain body temperature upon fasting prior to cold exposure. Endothelial cells (ECs) were also selected as an important cell-type to investigate AHR function due to the severe vascular defects of global AHR KO animals. Vascular disease is again associated with a high burden on healthcare, especially in the ageing population and in individuals who are obese. A human model system of human umbilical vein endothelial cells (HUVECs) was used to investigate novel functions of AHR in ECs in vitro. Bulk RNA sequencing revealed that AHR activation caused a downregulation of many cell cycle-related pathways. Functional data using EdU staining also verified that activation of AHR with various concentrations of FICZ for 6Hrs caused a transient accumulation of cells in G0/1, whilst S and G2/M remained unchanged when compared to DMSO control. Further protein expression analysis at a single cell resolution using Operetta indicated that these effects were mediated through E2F. In addition, AHR was knocked down in HUVECs, which saw a decrease in cells at G1/0 compared to control siKD cells, and an increase in S phase when stained with EdU. Cells reached confluency more rapidly in siAHR group, compared to siKD control group using Incucyte. Increases in E2F were also observed using the Operetta system. Open Access

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide. Yet there is large geographical variation in incidence, with CRC often referred to as a ‘Westernised’ disease. A large body of both epidemiological and experimental evidence has linked the consumption of a diet high in fat and biological protein to increased CRC risk, whereas an inverse association has been demonstrated for high levels of fibre consumption. Moreover, it is theorised that diet’s impact on CRC risk is mediated through colonic microbial metabolism and the subsequent production of either pro- or anti-carcinogenic metabolites such as secondary bile acids and short chain fatty acids (SCFA), respectively. In the second and third chapters of this thesis, ultra high-performance mass-spectrometry (UPLC-MS) was used to investigate diet associated changes to faecal and urinary metabolites of participants in a Dietary Exchange Study. African American participants, who typically eat a high-fat, low-fibre, Western diet swapped diets for two weeks with rural African participants, who typically eat a high-fibre, low-fat diet, and vice versa. Previously published data demonstrated that Westernisation of the diet led to a striking increase in biomarkers of CRC risk in rural Africans, yet Africanisation of the diet led to decreased CRC biomarkers in African Americans. In Chapter 2, global profiling revealed six metabolites associated with changes to diet and CRC risk. Acylcarnitines increased in both faecal and urinary samples in response to the adoption of a Western diet and with further mechanistic evidence, could serve as biomarkers of diet associated increase in CRC risk. In Chapter 3, analysis of faecal samples using an UPLC-MS bile acid profiling method demonstrated increases in secondary bile acids associated with the consumption of a high-fat, low-fibre diet. However, in contrast to this trend, 3-ketocholanic acid (3-KCA) a derivative of carcinogenic lithocholic acid (LCA), increased with the consumption of a high-fibre, low-fat diet. This highlighted a potential LCA detoxification mechanism. In Chapter 4, this was investigated further. Firstly, both faecal microbiota and selected microbial species were shown to have the capacity to produce 3-KCA. This was a novel finding. Secondly, the carcinogenic potential of 3-KCA compared to that of LCA was investigated using a HCT116 cell line. 3-KCA was shown to be significantly less cytotoxic than LCA, and preliminary results also showed a trend towards reduced genotoxicity. Although further experiments, using additional cell lines and animal models, will be required to validate these results, these initial data imply that 3-KCA may be less carcinogenic than LCA. Taken together, these data shine a spotlight on the potential of synbiotic intervention, harnessing both probiotic species with 3-KCA producing capacity and the substrates which sustain them, in the detoxification of colonic LCA, and thereby in the reduction of CRC risk in high-risk populations. Accordingly, Chapter 5 set out to explore the potential of prebiotic intervention for CRC risk reduction in Alaska Native peoples, who suffer the highest incidence rates of CRC globally. Urinary and faecal samples from the first 20 participants of the ongoing ‘Fibre to reduce colon cancer in Alaska Native peoples’ study were analysed using Nuclear Magnetic Resonance (NMR) analysis. Although global faecal and urinary metabolic profiles were not significantly changed by the intervention, it remains to be seen whether this will remain the case when the remaining samples are analysed on study completion. Despite this, these data demonstrate the potential of NMR-based profiling in the non-invasive and accurate detection of CRC biomarkers. Collectively, the results presented in this thesis highlight how microbiota manipulation can potentially be harnessed to reduced CRC risk and how NMR and UPLC-MS can be used to assess associated metabolomic changes in a non-invasive manner. Open Access

Abnormal lipid metabolism is a common feature of cancers. In the clinic, elevated phosphocholine, an essential membrane lipid precursor, is revealed as a phenotype associated with malignant lipid metabolism. Overexpression of choline kinase alpha isoform (CHKA), is recognised as a predominant factor responsible for the phenotype, and, therefore, CHKA has become a novel therapeutic target for cancer. In the previous work by Trousil et al., a potent CHKA inhibitor ICL-CCIC-0019 was reported, which displayed high cellular activity but undesirable pharmacological properties to be ameliorated. In this thesis, CK146, an active analogous scaffold of ICL-CCIC-0019, bearing a reactive piperazine handle, is presented. This novel scaffold opens the further possibilities for structural elaboration of the classic pharmacophore by innovatively exploiting two advanced drug development strategies. In the first strategy, selective CHKA inhibition was attempted to be achieved by prodrug CK145, via the incorporation of an ε-(Ac)Lys motif into CK146. In the second strategy, a peptide ligand targeting prostate-specific membrane antigen (PSMA) receptor was embodied in CK146 to afford CK147, aimed to realise the targeted delivery to the malignant cells with high expression of PSMA receptors on cell surface. As expected, the precursor scaffold CK146 displayed high CHKA activity in kinase screening (CHKA activity as part of a 15 human lipid kinase screen: CK146: 69%, ICL-CCIC-0019: 53%) and good antiproliferative activity against four selected cancer cell lines (overall GI50 against HCT-116, A549, HepG2 and Caco-2: CK146: 2.5 ± 0.3 μM, ICL-CCIC-0019: 0.5 ± 0.02 μM). Proposed prodrug CHKA inhibitor (CK145) and PSMA-targeted CHKA inhibitor (CK147) were successfully synthesised. The pharmacological activity and pharmacokinetic profiles of the obtained compounds were evaluated in vitro. Although attempts to improve the pharmacological profiles of ICL-CCIC-0019 were not effective by these two modification strategies, important and informative structure-activity relationships were concluded and have been reported. Open Access

RNA helicases are the largest group of RNA interacting proteins in eukaryotes and are a vital mediator to the mRNA pathway. A key mediatory step in the mRNA pathway is the swift removal of mRNA possessing a premature termination codon (PTC). This is performed to prevent the formation of C-terminus truncated proteins which may have detrimental effects on the cell. The breakdown of PTC containing mRNA is performed by the nonsense mediated decay (NMD) pathway. Failures in this pathway can lead to many developmental issues and life-long conditions. One of the RNA helicases involved in this pathway is the DEAH-box 34 RNA helicase (DHX34). DHX34 has been shown to be key to the recruitment of SMG1 to UPF1, two key proteins in the NMD complex formation at the core of the PTC containing mRNA breakdown, and the subsequent phosphorylation of UPF1 to induce this mRNA decay. Although its role in the NMD pathway has been shown, and conditions have been linked to mutation in DHX34, its molecular mechanism and RNA remodelling activity are unknown. In this thesis, I use the optical tweezers single-molecule force-spectroscopy approach to investigate the interaction of DHX34 with RNA. I begin by designing and testing a novel assay to function within the optical tweezers and experimental requirements of this thesis. Next, I investigate DHX34’s ability to remodel duplex RNA through processive remodelling utilising a force-clamp assay. Here I demonstrate, through a lack of unwinding, that DHX34 is unlikely to be processive, but observe the remodelling of the hairpin beyond the immediate site of tension. Finally, I redesign the experimental approach to assess the remodelling activity of non-processive helicases using optical tweezers, achieved by introducing force-driven unfolding steps with the protein presence. This redesign, and the collected results, suggests that DHX34 preferentially binds ssRNA and most likely 5 maintains this through ATP turnover, and indicates an intermediate state to the currently recognised RNA helicase molecular mechanism pathway. Open Access

With advancements in sequencing technology there has been an unprecedented rise in human single nucleotide variant data in recent years. One of the key challenges within clinical genetics is distinguishing truly pathogenic from rare but benign variants. Many in silico tools have been developed with this aim but they often over predict pathogenicity particularly on novel variants. Here, I demonstrate how a framework designed to identify variants with functional equivalence by using information from variants in known related genes can help pathogenic variant interpretation. Using sequence alignments of human paralogues, known pathogenic variants within aligned positions can be used to transfer their annotations across to aligned variants. This Paralogue Annotation method is shown to be widely applicable exome-wide, with 71% of disease genes having at least one paralogue. As a classifier it performs more precisely than other contemporary variant predictors, having a precision of 94% or higher depending on the data. This however comes at the cost of limited sensitivity (17% and lower). But this is rescued when the framework was improved by altering the alignments to protein domains instead of whole gene sequences. The sensitivity was increased by 74% with a marginal 6% precision decrease. By expanding the framework to explore the usage of structural protein alignments instead of sequence alignments there is potential to further improve sensitivity, but current limited structural data means that predicted protein models must be relied on leading to further assumptions to be taken. In structural space, pathogenic variants across aligned models are statistically more likely to be closer together than benign and pathogenic variants. This framework can be used as a precise pathogenic variant classifier in sequence space, but overall, it can be used to search for functionally equivalent variants to variants of interest, which is a line of information not used by many. Open Access

The rapid rise in the prevalence of pathogenic bacteria that are resistant to front-line antibiotic drugs has necessitated a simultaneous upsurge in the use of “last-resort” antimicrobial agents. Colistin is one such antibiotic of last-resort that is increasingly used in the clinic as a salvage therapy to treat infections caused by multi-drug resistant Gram-negative microorganisms, including Pseudomonas aeruginosa and Escherichia coli. Unfortunately, despite its growing importance, colistin treatment is toxic, frequently fails, and resistance to the antibiotic is an intensifying threat. There is, therefore, a crucial requirement to augment the efficacy of colistin therapy, but efforts to do so are hampered by a lack of understanding about the drug’s mechanism of action. The work in this thesis initially uncovered a novel process by which colistin-susceptible P. aeruginosa cells survive exposure to the antibiotic, through the extracellular release of lipopolysaccharide (LPS) molecules that inactivate colistin in the external environment. In attempting to overcome this mode of drug tolerance by inhibiting LPS biosynthesis, critical insight into colistin’s bactericidal mechanism was obtained – namely, that the antibiotic kills Gram-negative bacteria by targeting LPS in the cytoplasmic membrane, not by interacting with membrane phospholipids, as previously thought. This finding in turn led to investigations about the site where colistin resistance, mediated by cationic chemical modifications to LPS, was conferred. It was shown that resistance to colistin in P. aeruginosa and E. coli, acquired through the harbouring of diverse plasmid-borne mobile colistin resistance (mcr) genes or chromosomal mutations, was in fact conferred at the cytoplasmic membrane, as opposed to the outer membrane of bacterial cells. Subsequent experiments revealed that intrinsic colistin resistance in Burkholderia cenocepacia was mediated at the outer membrane, and that strains of Enterobacter cloacae possess a unique inducible form of hetero-resistance to colistin. After characterising the mode of action of colistin and a number of potential causes of colistin treatment failure, new combination treatment strategies were designed to improve the effectiveness of colistin therapy. Using murepavadin, an inhibitor of the LPS transport system in pre-clinical development, to accumulate LPS in the cytoplasmic membrane proved to be particularly potent at amplifying the bactericidal activity of colistin in vitro against clinical isolates, in an in vivo murine lung infection model, and for overcoming colistin resistance. Furthermore, the capacity for colistin to permeabilise the outer membrane of colistin-resistant bacteria was exploited to re-sensitise Gram-negative pathogenic organisms to rifampicin, an antibiotic which normally cannot traverse the cell envelope. In summary, this work has identified how colistin works and why it often fails clinically, as well as providing urgently-needed novel solutions to enhance colistin efficacy and patient outcomes. Open Access

Vietnam has a high burden of viral hepatitis. This thesis strives to advance its elimination, addressing important gaps in the literature that I hope will contribute to treatment guidelines and health policy, both in Vietnam and internationally. Firstly, to define the hepatitis epidemic in Vietnam, I assimilate all published seroprevalence data since 1990 to estimate pooled prevalence of HBV, HCV and HDV in high and low risk populations. I show that although blood safety has improved, and HDV is largely confined to high-risk populations, a renewed focus on birth dose HBV vaccination and targeted HCV screening and treatment of people who inject drugs, is urgently required to meet elimination targets. The next chapters address HCV therapy, namely predictive factors for selecting individuals who could be treated for shorter duration, treatment failure in relation to rare HCV subtypes, and the clinical importance of resistance mutations. I describe a prospective clinical trial evaluating the efficacy of shortened sofosbuvir and daclatasvir therapy, based on early virological response: firstly, in genotype 1 or 6-infected individuals with mild disease (chapter 3) and then in genotype 6-infected individuals with advanced liver fibrosis (chapter 4). I show that shortened therapy, with retreatment if needed, can reduce antiviral use while maintaining high cure rates, but that day 2 virologic response alone is not an adequate predictor of cure. I demonstrate that a high frequency of putative NS5A inhibitor resistance mutations in genotype 6 infection does not impact cure rates, negating the need for costly genotyping in Vietnam. In my final data chapter, I explore an innovative means of decentralising HCV care. In two independent study populations from Vietnam and the UK, I show that an increase in routinely taken alanine transaminase after HCV therapy is a reliable screen for treatment failure that could substantially reduce reliance on nucleic acid testing in remote and resource-limited settings. Open Access

The extrinsic incubation period (EIP) of malaria is the time required for Plasmodium parasites to undergo sexual reproduction and asexual replication within the mosquito (sporogony). The EIP is long relative to mosquito life expectancy meaning most mosquitoes do not live long enough to transmit malaria and small changes in mosquito survival strongly affect malaria transmission. The EIP is typically estimated in a population of experimentally infected mosquitoes, which are dissected on different days post infection for the presence of infectious, salivary gland sporozoites. I developed a novel mathematical model to estimate the EIP distribution from these data (Chapter 2). This model accounted for heterogeneity in mosquito infection and infection-dependent differences in mosquito survival, which could affect the EIP estimated using historical methods. Existing malaria transmission models that incorporate temperature-dependent changes in the EIP mostly use an existing degree-day model, which was parameterised using the time for the first Anopheles maculipennis mosquitoes (a species complex native to Europe) to be dissected with sporozoites. I therefore estimated the effects of constant temperature on the EIP distribution among Anopheles gambiae sensu stricto mosquitoes (a dominant Plasmodium falciparum vector) (Chapter 3). These EIP estimates were similar to the degree day model except at low temperatures. Theoretically, accounting for the complete EIP distribution marginally affects malaria transmission compared to assuming that all mosquitoes have the mean EIP. If vector control and mosquito mortality are high, however, accounting for heterogeneity in the EIP is important because mosquitoes with a short EIP become more important for transmission (Chapter 4). In the wild, temperature is not constant. I therefore developed a model to predict changes in the EIP of mosquitoes exposed to fine scale diurnal and seasonal fluctuations in temperature using the constant-temperature EIP relationship (Chapter 5). Model performance was assessed using laboratory data from mosquitoes that were exposed to a range of different temperature profiles. Incorporating fluctuations in temperature improved the model performance. Accounting for changes in the mosquito age distribution due to seasonality in mosquito emergence is theoretically important when measuring sporozoite prevalence (Chapter 6). I incorporated seasonality in mosquito emergence and temperature-dependent changes in the EIP into a mechanistic malaria transmission model and assessed the model’s predictive accuracy using seven years of monthly sporozoite prevalence samples from a holoendemic area of Senegal. Seasonality in mosquito emergence caused a small increase in predictive accuracy, and temperature-dependent changes in the EIP had a very small effect if mosquito emergence was allowed to vary and no effect if mosquito emergence was assumed to be constant (Chapter 7). Open Access

Adult T-cell leukaemia/lymphoma (ATL) is an aggressive T-cell malignancy that occurs in around 5% of carriers of the human T cell leukaemia virus type 1 (HTLV-1). Identifying the carriers at an early stage has been difficult and current methods, such as use of proviral load (PVL), lack specificity. In this thesis, a flow-cytometry based approach, using T-cell receptor subunit quantification to identify dominant clones and to calculate an oligoclonality index (OCI), was able to identify carriers with a five-fold increased risk of transformation compared to high proviral load alone. ATL driver mutations can be present many years prior to disease in subjects who subsequently transform. In order to identify whether low frequency mutations associated with oncogenesis were also present in HTLV-1 carriers with high OCI and unknown outcomes, samples were subsequently flow-sorted into populations representing dominant clones, infected polyclonal cells and Other CD4+ cells. Eight out of nine high-OCI subjects (89%) had a mutation detected in a gene frequently mutated in ATL, at a variant allele fraction of greater than 0.1, compared to no subject with a low-OCI. Three subjects had a mutation in a dominant clone in the TCR/NFκB pathway; all three of these subjects subsequently transformed to ATL. Differential expression analysis demonstrated similarity between dominant clones and previous patterns of gene expression seen in ATL, that were distinct from differential expression seen in Infected Polyclonal cell populations. There were some patterns of gene expression unique to clones with mutations in the TCR/NFκB pathway, which may represent late change. In this work, I have demonstrated that High-OCI HTLV-1 carriers have mutational and transcriptional profiles resembling those seen in ATL, and these may reflect differing stages of transformation. These differences could be exploited for therapeutic gain and earlier treatment of HTLV-1 carriers at the highest risk of transformation. Open Access

The bone microenvironment (BM) is highly heterogeneous and is altered significantly during our lifespan. Blood vessels in the bone provide specialised niches that support processes such as osteogenesis, haematopoiesis and adipogenesis. The function of the female hormone, oestrogen has been attributed to bone remodelling during puberty, pregnancy, and menopause. However, the role that oestrogen signalling plays in the bone endothelium and its subsequent changes in the bone microenvironment have not been well understood. The results in this thesis show that systemic oestrogen administration promotes bone angiogenesis and osteogenesis and inhibits adipogenesis. These micro-environmental changes correspond to the changes observed with a physiological oestrogen surge during pregnancy and its depletion during menopause and ageing. Blood vessel physiology during pregnancy, menopause and ageing is regulated by oestrogen signalling via oestrogen receptor alpha (ERα) and G protein-coupled oestrogen receptor 1 (Gper1) but is not dependent on oestrogen receptor beta (ERβ), characterised using endothelial-specific transgenic mouse models. Interestingly, gene expression analysis of young bone endothelial cells (EC) revealed minor sex differences regarding endothelial cell metabolism. Metabolic analyses show increased active mitochondria in adult males compared to females; and demonstrate the role of oestrogen in reducing mitochondrial function. Additionally, glycolytic and mitochondrial stress tests indicate that oestrogen promotes a glycolysis- and oxidative phosphorylation-independent energy metabolism without mitochondrial perturbation. Further characterisation of EC metabolic requirements led to the discovery of fatty acid β-oxidation (FAO) being the primary energy source, more so promoted by oestrogen, drawing FAs from BM adipocyte lipolysis. Oestrogen-deplete conditions such as menopause and ageing lead to the accumulation of ferroptosis-induced lipid peroxides (LPO) due to impaired lipid metabolism when angiogenic type H vessels are lost, with an increase in intracellular ferrous ions. Finally, the novel use of the antioxidant Liproxstatin-1 (Lip-1) closely mimics oestrogenic action in alleviating harmful reactive oxygen species (ROS) and inhibiting LPO generation, by restoring angiogenic type H vessels and stimulating bone formation in aged mice. The findings strongly suggest the use of Lip-1 as a potential therapeutic agent in treating post-menopausal osteoporosis as an alternative to hormone replacement therapy. Open Access

Plasmodium falciparum malaria is a preventable but deadly parasitic disease, leading to an estimated 409,000 deaths per year, 95% of which occur in the African region. Despite overall reductions in the risk of malaria mortality in the past two decades, progress in further improvements has stagnated since 2016. Timely access to effective first-line artemisinin combination therapies (ACT) remains one of the most important components of case management and successful malaria control interventions. The effect of delay to treatment is difficult to study but important to quantify and include in estimates of malaria morbidity and mortality. This thesis aims to explore the coverage and effectiveness of antimalarials in the community, quantify the impact of delay to treatment on severe malaria and mortality, and evaluate current interventions that reduce treatment delays. Using nationally representative household surveys conducted in Sub-Saharan African communities, I estimate the coverage of different types of antimalarials and rates of potential parasite clearance following self-reported antimalarial treatment. I also develop and calibrate an individual-based model to the household survey data to explore antimalarial effectiveness in the community. Factors affecting antimalarial effectiveness, such as duration of treatment and type of provider are also explored. Further, I conduct a systematic review and individual participant meta-analysis to quantify the impact of prompt treatment on reducing severe malaria and different severe malaria phenotypes. Using these, I model the relationship between age and presentation with severe malaria symptoms and subsequently estimate the burden of severe malaria phenotypes and malaria mortality across sub-Saharan Africa using a model of malaria transmission, developed by Imperial College London. Finally, I estimate the impact of prompt treatment in terms of severe malaria cases and deaths averted using baseline and endline data from the Rapid Access Expansion (RAcE) programme, following implementation of integrated Community Case Management (iCCM) interventions in high-burden countries of Sub-Saharan Africa. This thesis explores several key aspects relevant to progression to different severe malaria phenotypes and mortality and demonstrates the importance of prompt and effective first-line ACT treatment, good treatment adherence, adequate provision of community health care services, as well as regulation and provision of high-quality antimalarials. It also highlights differences in both the fatality and the impact of delay to treatment for different severe malaria phenotypes. Understanding the distribution of these phenotypes and their contribution to malaria mortality is important in informing malaria control interventions. Open Access

The eukaryotic core promoter is an essential regulatory part of the genome. Its sequence dictates the transcriptional output from any specific gene and it can alter the output in response to external and internal stimuli. The transcriptional start site (TSS), where RNA polymerases bind and initiate transcription, is at the base of the core promoter, and dynamic mapping of initiation sites can reveal how the cell utilises alternative core promoter features. I first map core promoters in Drosophila development by using a low-input, single-nucleotide resolution mapping protocol for identification of TSSs, SLIC-CAGE. The maternal and zygotic genomes are mostly guided by distinct codes of initiation, with maternal genes being dependant on nucleosome positioning signals and zygotic genes on precisely-positioned motifs. Despite specific initiation rules used by the maternal and zygotic genomes, I find no evidence for overlapping codes used in the same promoter, a feature that has been demonstrated to be conserved in vertebrate development. However, I also demonstrate that there is evidence of alternative use of YC and YR initiation in promoters harbouring both. One of the most striking features of core promoters revealed by accurate TSS mapping is the two modes of initiation. Sharp initiation is characterised by a single TSS usage, while broad initiation refers to multiple, closely-spaced TSSs. Much less is known about how transcripts arising from broad initiation differ in their downstream processing. I demonstrate that individual TSSs have distinct translational properties by combining SLIC-CAGE with polysome fractionated RNA. This is particularly striking in YC/YR dual-initiation promoters, which might explain their high prevalence in the genome. Finally, I demonstrate differential translational dynamics of zygotic RNAs initiated at specific core promoter motifs in the zygote, as well as RNAs expressed in both maternal and zygotic genomes. Again, YC and YR initiation in the zygotic genome show distinct properties, with high expression but low translation of YC promoters in the zygote, suggesting that the high transcription compensates for the inefficient translation. Open Access

The association between alcohol consumption and cardiometabolic disease risk has been described as a J or U-shaped curve attributed to a combination of harmful and beneficial effects varying with volume of intake. Diet is an established risk factor for cardiometabolic disease and related traits However, few studies examining this association adequately control for residual confounding by dietary intake. Therefore, the aim of this thesis was to investigate the relationship between alcohol consumption and markers of cardiometabolic health independent of dietary intake. Cross sectional analyses were conducted using data from the Airwave Health Monitoring Study – a British occupational cohort (n = 9,581). Alcohol consumption behaviour was determined from questionnaire and 7-day diet record data. Diet quality was determined by measuring adherence to the Dietary Approaches to Stop Hypertension (DASH) diet using data from the 7-day diet records. Markers of cardiometabolic health included: adiposity (body mass index and waist circumference), blood pressure, cholesterol, HbA1c and C-reactive protein. All analyses were replicated in an independent UK cohort using UK Biobank data (n =146,888). As part of this thesis, genetic analyses were conducted to investigate whether genes implicated in the regulation of HDL-c may facilitate some of the cardioprotective effects attributed to alcohol intake. Heavy alcohol consumption is associated with a deterioration in cardiometabolic health. The risk of cardiometabolic disease as indicated by a cardiometabolic risk score was lowest amongst moderate drinkers and highest amongst never, and heavy drinkers. Increasing alcohol intake was associated with a deterioration in diet quality and had an additive effect of total energy intake. Diet quality did not modify the relationship between moderate alcohol intake and cardiometabolic risk. Alcohol intake did not alter the effect of genes implicated in the regulation of HDL-c. These findings were validated in a sub-sample of the UK Biobank cohort. To conclude, the findings from this thesis show that alcohol consumption plays a key role in determining diet quality and cardiometabolic risk. Specifically, risk of developing obesity. Open Access

Pancreatic ductal adenocarcinoma (PDAC) is the 7th leading cause of cancer related death. Deregulation of the apoptotic pathway, including upregulation of inhibitor of apoptosis proteins (IAPs), occurs at an early stage in PDAC resulting in aggressive tumours which are often resistant to therapy. Currently, use of IAP antagonists in clinical trials aims to sensitise cancer cells to apoptosis. However, these therapies show a lack of efficacy in a large proportion of the patient population. Therefore, elucidating the molecular mechanisms associated with resistance to IAP antagonists could optimise treatment strategies for PDAC. In this study, three different screening methodologies are used to identify novel resistance drivers to IAP antagonists. Firstly, a genome-wide CRISPR-Cas9 screen was conducted to identify 1025 potential genetic contributors (p

Major depressive disorder (MDD) is a debilitating psychiatric disorder characterised by a complex underlying biology and poor response to pharmacological antidepressant strategies. Given the heterogeneity of MDD and the diverse range of available treatment options, there is an increasing desire to develop and implement precision medicine approaches to tailor existing treatment strategies to the biological system of the individual. In this thesis, high-resolution omics data (connectomics [fMRI], metabolomics [1H NMR] and immunomics [inflammatory cytokines]) collected from the Canadian Biomarker Integration Network in Depression (CAN-BIND) study has been integrated to facilitate the deep phenotyping of MDD. In addition, this approach has been used to predict the treatment response to two common antidepressant drugs, monotherapy with the selective serotonin reuptake inhibitor (SSRI) escitalopram (10-20 mg) or combination therapy with escitalopram and the dopaminergic antipsychotic aripiprazole (2-10 mg). This approach identified a multi-modal panel of sex-specific biomarkers of MDD and treatment response, highlighting a strong immunometabolic component in depressed males, but not females. Unsupervised clustering methods indicated the superiority of biological (neuroimaging) over symptom-based (clinical questionnaires) data for the stratification of patients into MDD subtypes with differential response to treatment. More importantly, a set of multi-modal, sex-specific biomarkers were identified that predicted treatment response with escitalopram monotherapy (84.7% accuracy) or aripiprazole augmentation (88.5% accuracy). In addition to highlighting potential new aspects of the biology of MDD (e.g. relevance of lipoprotein size and density for their relation to depression), this work is one of the first attempts to apply systems biology approaches to high-resolution biological data from a large clinical trial to predict later treatment outcome. With the validation of the findings presented in this thesis in independent cohorts, and with further development of omics technologies, leading to cheaper and high-throughput screening of the patient population, pre-dose biomarkers have the potential to achieve personalised treatment. Each year, escitalopram and aripiprazole are prescribed to an estimated 26 million and 7 million individuals respectively, and over one third of them do not respond. Thus, being able to predict response to antidepressant medication from baseline biomarkers has enormous clinical and socioeconomic benefits. Open Access

Nitrogen is an essential nutrient for bacterial survival and growth, being a constituent of almost all cellular building blocks, including nucleotides, amino acids, and cell wall components. The adaptive response to nitrogen starvation in Escherichia coli has been well characterised at the transcriptional level, but relatively little beyond that. Moreover, the role of post-transcriptional regulation of gene expression is being increasingly recognised as a vital facet of bacterial adaptive responses to stress. Therefore, the ubiquitous post-transcriptional regulatory protein Hfq was chosen as a surrogate to study the post-transcriptional basis of the adaptive response to nitrogen starvation. Here, we demonstrate a requirement of Hfq for optimal survival, and maintenance of cellular metabolism, during long-term nitrogen starvation. Using single-molecule tracking photoactivatable localisation microscopy (SM-PALM), we present a novel property of Hfq in long-term nitrogen starved bacteria whereby it forms a single, distinct focus. The Hfq foci form gradually as nitrogen starvation persists, correlating with Hfq’s role in regulating cellular metabolism. Notably, the Hfq foci rapidly disperse when bacteria are replenished with nitrogen, thus cementing the idea that the Hfq foci play a role in the adaptive response to nitrogen starvation. Moreover, we establish that the above described Hfq foci contains the components of the RNA degradosome – namely RNase E, PNPase and RhlB. This discovery suggests that the Hfq foci/RNA degradosome structure in nitrogen starved bacteria may have a role in managing the fate of RNA in stressed bacteria functioning akin to analogous liquid-liquid phase separated (LLPS) structures described in stressed eukaryotic cells. Hence, we term the Hfq foci/RNA degradosome structure the H-body. Our investigation reveals that H-bodies share several features of LLPS structures. Although many details of the mechanistic basis and prevalence of H-bodies yet remain elusive, their discovery most likely heralds a new facet of post transcriptional gene regulation in bacteria. Open Access

The rise of antibiotic treatment failure represents a serious global health problem. Along with antibiotic resistance, antibiotic tolerance and persistence make up the three phenomena underlying the ability of bacteria to survive treatment with bactericidal antibiotics. Primarily distinguished by their penetrance within a bacterial population, antibiotic tolerance and persistence are superficially similar phenomena, with both relying on low antibiotic uptake or target activity due to slow or arrested bacterial growth. As such, it is widely assumed that similar mechanisms underpin the ability of persistent and tolerant bacteria to survive antibiotic exposure. However, to date, little evidence directly supports mechanistic equivalence between these two forms of antibiotic recalcitrance. In this thesis, I compare the molecular mechanisms enabling the survival of antibiotic tolerant and persistent Salmonella Typhimurium during macrophage infection. First, I identify purA as a multidrug antibiotic tolerant Salmonella mutant. Using this mutant, I then directly compare the physiological states adopted by antibiotic tolerant Salmonella and wild type persisters during macrophage infection. Specifically, I show that tolerant Salmonella adopt a near-dormant state, undergoing minimal DNA damage during macrophage infection. In contrast, persisters retain an active but vulnerable physiological state that is prone to the accumulation of double-stranded DNA (dsDNA) breaks during macrophage infection. In view of these initial observations, I next explore the dependence of Salmonella persisters on various DNA repair pathways for survival during macrophage infection, uncovering that these bacteria rely extensively on RecA-mediated DNA repair to overcome their inherent vulnerability to the intramacrophage environment. Finally, I compare the relative ability of tolerant and persistent bacteria to initiate infection relapse after cessation of antibiotic treatment. This revealed that, despite their vulnerability, persisters retain the ability to initiate infection relapse as long as they are protected by RecA-mediated DNA repair. Overall, this work highlights the importance of RecA-mediated DNA repair for wild type Salmonella persisters to survive within the host during antibiotic treatment and to initiate infection relapse following drug withdrawal. Open Access

Height and weight are indicators of healthy versus unhealthy nutrition. Body-mass index (BMI) measures weight gain beyond what is expected from height gain. Having short stature or too little weight for one’s height, represented by low BMI, as well as having excessive weight for one’s height, represented by high BMI, increases the risk of morbidity and mortality. My thesis aimed to provide summary statistics that characterise the distributions of height and BMI and their interrelationship. Data were collated via the NCD Risk Factor Collaboration (NCD-RisC) network, from population-based studies that had anthropometrics measurements from 1985 to 2019 for a total of 2,896 studies with 187 million participants. Of these, 1,282 surveys provided over 11 million participants aged 20 to 79 years, with 1,021 surveys of over 1.4 million women aged 40-49 years and 815 surveys of over 870,000 men aged 40-49 years. I used a hierarchical Bayesian model to estimate mean, variance and skewness of height and BMI, as well as their correlation coefficient; I also used a regression model to estimate the contribution of the change in mean BMI to the change of prevalence in underweight and obesity. In 2019 versus 1985, mean and variance of both the height and BMI distributions increased in most countries and sexes; skewness of the height distribution remained around zero for both women and men in most countries, while skewness of the BMI distribution, although it was a positive number for both sexes, it decreased in women from most countries and increased in men from all countries. Changes in the prevalence of underweight and total obesity, and to a lesser extent severe obesity, were largely driven by shifts in the distribution of BMI, with smaller contributions from changes in the shape of the distribution. The correlation coefficient between height and BMI did not change significantly from zero for most countries and sexes. Considering that the height gain was not proportional to the BMI increase, segments of the two distributions were affected heterogeneously so policy makers and health practitioners need to tackle the double burden of malnutrition. Open Access

India harbours the world’s largest burden of tuberculosis, with approximately 27% of the estimated global burden. Although considerable measures have been taken since the roll-out of the End TB strategy, in 2015, to increase case detection and improve treatment quality, the overall quality of care remains poor and estimates on incidence remain highly uncertain, because of the poor notification rates, especially from the private healthcare sector, where nearly half of all individuals seek care. In 2017, India’s National Strategic Plan for Tuberculosis Elimination brought forward extremely ambitious targets of an 80% reduction in incidence by 2025. The first step of the plan proposes a scale-up of case-detection through active case-finding, in order to increase case notification rates and reduce the diagnostic and treatment initiation delays, often associated with tuberculosis care. The evidence for the value of active case-finding interventions for tuberculosis, however, is mixed and highlights a substantial geographical imbalance of studies. A recent systematic review has encouraged the implementation of targeted active case-finding interventions, suggesting these may have an important effect on the prevalence of disease. Despite this, there remains some scepticism around these interventions, which can be costly and difficult to implement on a large scale, and because of the diverse number of factors that can affect their impact. This thesis aims to investigate the value of routine active case-finding interventions in high-burden settings, implemented using available tools for screening and diagnosis. Epidemiological data is used in the development of the mathematical models used in the analyses, and costing information is included to allow an estimation of the financial implications of this undertaking. Open Access

Transposable elements (TEs) are discrete DNA sequences that multiply and move within their host genomes. These elements are widespread across eukaryotic species and occupy over 50% of the human genome, however their abundance and diversity vary widely between species. The factors shaping the differences in TE content are poorly understood. Investigating the evolution of TEs has its difficulties, because their sequences diversify rapidly and TEs are often transferred through non-conventional means such as horizontal gene transfer. I developed methods for the visualisation and analysis of TE content across a multitude of genomes. Furthermore, I used these techniques to investigate epigenetic mechanisms and their potential role in the evolution of TE abundance and diversity amongst species. First, I constructed a sequence similarity network (SSN) to study the sequence evolution of Tc1/mariner elements across focal nematode species. With this method I was able to identify an unknown connection between two TE families and an associated convergent acquisition of a domain from a protein-coding gene. Second, I developed a weighted bipartite network to investigate how TE content across species is shaped by epigenetic silencing mechanisms. I show that the presence of PIWI-interacting RNAs (piRNAs) is associated with differences in network topology after controlling for phylogenetic effects, indicating higher tolerance of TEs in species with piRNAs. Additionally my analysis of single cell RNA sequencing data from Caenorhabditis elegans embryogenesis gives evidence that some TE families are differentially expressed during C. elegans embryonic development and show cell type specific expression patterns. Together this thesis demonstrates how network-based approaches can be used to identify hitherto unknown properties of TE evolution across species. It gives an insight into the factors responsible for TE diversity, including epigenetic mechanisms and the beneficial effects of TEs for their host genomes. Open Access

The age of -omics has set a precedent for precision medicine, yet one of the most prevalent challenges is multi-omics data integration, with no platform yet that can efficiently integrate clinical and multi-omic data. This thesis uses several integrative data strategies to deconvolute complex multisystemic disease states, utilising clinical, cytokine profiling, metagenomic and metabonomic data to gain greater depth and understanding. High-throughput metabolic profiling methods have been employed for in-depth systemic analysis of SARS-CoV-2 infection, Primary Biliary Cholangitis and paediatric burn injury. Correlation analysis and derived clustering methods were utilised for data integration, following multivariate analyses and reveal disease state specific signatures. Similarly, different biofluids (plasma, urine, faeces) contributed specific complementary information. Several systemic biomarkers were identified for each disease, each with hallmarks of organ dysfunction. This work highlights the value of data integration in heavily confounded data and disease. Open Access

Chronic low-grade inflammation is linked to a multitude of chronic diseases and conditions. However, chronic low-grade inflammation has not been fully characterised, as there remains conflicting and inconclusive evidence from epidemiological studies. My thesis aimed to explore chronic inflammation using a multi-omic approach. Data from the UK Biobank and Airwave cohort was used. Within this thesis I report genome-wide association on C-reactive protein (CRP), a marker of systemic inflammation, in UK Biobank participants (N = 427,367) and the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium (total N= 575,531). I identify 266 independent loci, of which 211 were not previously reported. Gene-set analysis highlighted 42 gene sets associated with CRP levels (p ≤ 3.2 x 10-6) and tissue expression analysis indicated a strong association of CRP related genes to the liver and whole blood gene expression. Phenome-wide association identified 27 clinical outcomes associated with genetically determined CRP, including five phenotypes of the respiratory system, nine of the circulatory system, three musculoskeletal, three sense organs, one mental disorder, five endocrine/metabolic, and lastly one genitourinary. Mendelian randomisation analyses of chronic diseases supported a causal association with several outcomes, such as schizophrenia (beta (β) = -0.074, 95%, p = 7.83 x 10-6), chronic airway obstruction (β=0.330, p=7.94 x 10-4) and breast cancer (β = 0.044, p = 2.02 x 10-5). Metabolome-wide causal association MR analyses tested CRP associated genetic variants with 123 NMR platform measured metabolites and identified five with convincing evidence, including citrate (β=-0.13, p=1.6 x 10-5) and phenylalanine (β=0.13, p=1.3 x 10-5). In combination these results provides insight into the genomic, phenotypic, metabolomic profile, and functional properties of CRP proxied systemic chronic low-grade inflammation. Results provide novel findings, which can be investigated further to understand the underlying molecular pathways of disease afflicted by systemic chronic low-grade inflammation. As CRP proxied chronic inflammation is modifiable, this may be of interest for therapeutic intervention. Open Access

Immunity and metabolism are closely intertwined. Immune responses are resource-demanding and metabolic changes are therefore required to the supply the immune system with the required energy and raw materials. In pathological infections where the immune response becomes dysregulated, these metabolic changes can contribute to pathology. Work using the model organism Drosophila melanogaster has been productive in uncovering how the immune system regulates metabolism, but details of actual metabolic changes that occur during infection, and their functions, are poorly known. This project sought to uncover the metabolic changes that occur during the Drosophila innate immune response to infection and understand the consequences of these changes. By combining mass spectrometry-based metabolomic analysis with the genetic analysis of Drosophila, I found that the immune response to systemic bacterial infection caused an increase in nitrogenous waste metabolism. Further investigations suggested that this increase in nitrogenous waste is due to an increase in the upregulation of endogenous protein degradation via an ESCRT-III mediated process. Expression of CHMP2B, encoding a core ESCRT-III component, was indicated to be required in the fat body for optimal translation of antimicrobial peptide transcripts. Together these data suggest a model in which the fly upregulates endogenous protein degradation in order to rapidly provide in-demand amino acids for immune effector production, with catabolism of the resulting excess amino acids causing an in increase in nitrogen waste metabolism. This project has thus revealed a profound metabolic change induced by a successful immune response in Drosophila. As the cost of this change is the degradation of the host’s own protein, this a probable mechanism of metabolic pathology in the context of a dysregulated immune response. Studying the etiology of such metabolic pathology will contribute to our understanding of conditions underpinned by metabolic dysregulation, such as sepsis and infection-induced cachexia. Open Access

During evolution, variation is created by mutational processes in an organism's genome, and then these variants remain or are purged as a result of selective pressures and drift. Mutation rates are biased at many scales across the genome, and the selective pressure acting on the generated variation as a result of mutations also varies greatly. One well studied example of mutational heterogeneity at single-base resolution concerns methylated cytosines, which mutate at an approximately 10-fold higher rate than their unmethylated counterpart. In Chapter 1, I demonstrate that cytosine methylation not only affects mutation risk at the methylated base but also exerts mutational effects on its neighbouring nucleotides. In human, bases surrounding the methylated cytosine accrue fewer SNPs than bases in the vicinity of unmethylated sites. In plants, on the other hand, I demonstrate that the opposite is true. I show that this difference is not driven by differential sampling of sequence or chromatin contexts, and is instead likely due to alternative lesion processing between the species. Regarding selective heterogeneity, studies in the past have largely focused on differential conservation of genetic elements between species, an approach ill-equiped to capture selection against gaining a novel deleterious function. In Chapter 2, I develop and validate a novel approach to interrogate selection against deleterious gain-of-function motifs in protein coding sequences, which is based on derived allele frequency distributions. I show how derived allele frequencies can be used to detect selection against particular amino acid motifs genome-wide. This thesis elucidates forces biasing the creation and persistence of genetic variation and highlights the entwined nature of mutational bias, selective pressures, and the technical challenges involved. Open Access

Pancreatic β-cells in the islets of Langerhans regulate glucose homeostasis by secreting insulin in response to elevated levels of glucose in the circulation and impairment of their function can contribute to the development of type 2 diabetes (T2D). MicroRNAs (miRNAs) are short non-coding RNAs that silence gene expression post-transcriptionally and play a vital role in the development and function of endocrine cells. MiRNAs fine-tune the expression of β-cell genes to regulate glucose homeostasis whilst their own expression is altered in T2D. MiR-125b-5p (miR-125b) is a highly conserved miRNA that is ubiquitously expressed and targets many genes in a cell-specific manner. Its role is well characterized in diseases such as cancer, but in β-cells remains unclear. Islet miR-125b expression is upregulated by glucose through the energy sensor, AMP-activated protein kinase (AMPK), and positively correlates with body mass index (BMI) in human islet donors. Thus, we hypothesized that miR-125b may contribute to the deleterious effects of hyperglycaemia in β-cells and aimed to determine the function of miR-125b in these cells. Here, I show that CRISPR/Cas9 mediated knockout of miR-125b in a human β-cell line (EndoCβ-H1) enhances glucose-stimulated insulin secretion (GSIS) and results in shorter, more fragmented mitochondria, whilst overexpression of miR-125b in a mouse insulinoma cell line (MIN6) results in reduced insulin content, the appearance of enlarged lysosomes and protects cells against cytokine-induced β-cell death. Importantly, overexpression of miR-125b in β-cells of mice causes impairments in glucose tolerance due to defects in insulin secretion, attributed to the strong reduction in mature insulin granule content and crystallization and abnormally enlarged lysosomes. Furthermore, miR-125b has a profound impact on the expression of genes involved in lysosomal and mitochondrial function in β-cells. In summary, my study has shown that miR-125b is a negative regulator of insulin secretion and a potential fine-tuner of mitochondrial and lysosomal metabolism in β-cells. My findings provide new insights into the role of miR-125b in β-cell function and point towards the inhibition of this miRNA in these cells as a potential therapeutic approach for the treatment of T2D. Open Access

Polycomb repressive complex 2 (PRC2) has a well characterised role in maintaining gene silencing and is essential for normal development, tissue homeostasis and is frequently dysregulated in cancer. The classical role of PRC2 is to catalyse post-translational modification of histone tails, with tri-methylation of Histone 3 lysine 27 strongly associated with gene silencing. Recent studies have highlighted additional non-classical roles for PRC2 including methylation of non-histone substrates, direct transcriptional activation and association with cell signalling cascades. Here I show that loss of core PRC2 components results in reduced Wnt signalling in embryonic stem cells (ESCs). Mouse ESCs that lack Suz12 show virtually undetectable canonical Wnt signalling. ESCs lacking Suz12 fail to differentiate. Intriguingly, efficient downregulation of pluripotency genes, but failure of induction of lineage specific genes is observed. In addition to the contribution to canonical Wnt signalling, beta-catenin also complexes with E-cadherin at cell surface adherens junctions. Importantly, altered E-cadherin localisation is observed in PRC2 null ES cells. E-cadherin localisation is normalised at cell-cell contacts between PRC2 mutant and wild type ES cells when grown together. Normalisation of E-cadherin localisation occurs concomitant to reestablishment of canonical Wnt signalling, and rescue of neuronal differentiation. The dual residency protein Afadin, a constituent of the intracellular adherens junction complex is also shown to have altered subcellular localisation in the absence of Jarid2 or Suz12. Taken together these results suggests a potential non-classical role for PRC2 in regulation of the adherens junctions, which in turn modifies the contribution of cadherin associated beta-catenin to Wnt signalling, and differentiation. Open Access

Crescentic glomerulonephritis (GN) caused by deposition of immune complexes within the glomerular capillary wall is a common cause of rapidly progressive glomerulonephritis and an important cause of end-stage renal failure. Current treatment with generalised immunosuppression is suboptimal in terms of efficacy, and is associated with significant side effects and long-term toxicity. Monocytes and macrophages are important in mediating crescentic GN, but little work has been done to phenotype the subpopulations involved and determine their respective contributions to glomerular inflammation. I developed a live glomerular imaging model using confocal microscopy in order to monitor intravascular monocyte subset behaviour during nephrotoxic nephritis (NTN) in a novel WKYhCD68- GFP monocyte/macrophage reporter rat strain. Additionally, I used flow cytometry and gene expression analysis to analyse ex vivo the glomerular leukocyte infiltrate during NTN. Using live glomerular imaging, I showed that non-classical monocytes surveyed the glomerular endothelium via the b2 integrin LFA-1 in the steady state. During NTN, nonclassical monocytes were recruited first, but subsequent recruitment and retention of classical monocytes was associated with glomerular damage and the onset of proteinuria. Importantly, monocytes recruited to the glomerular vasculature during NTN did not undergo transendothelial migration. This finding suggests that inflammation in crescentic GN caused by immune complex formation within the glomerulus is predominantly intravascular, driven by dynamic interactions between intravascular blood monocytes and the endothelium. Glomerular endothelial cells and non-classical monocytes overexpressed a distinct chemokine axis, which may orchestrate inflammatory myeloid cell recruitment and expression of damage mediators. Lewis rats, that are inherently resistant to experimental GN, demonstrated reduced recruitment of classical monocytes during NTN, suggesting a role for CD16 in mediating glomerular damage. In conclusion, my data suggest that monocyte subsets with distinct phenotypes and effector functions may be important in driving inflammation in experimental crescentic GN caused by immune complex deposition within the glomerulus. LFA-1 dependent endothelial surveillance by non-classical monocytes may detect immune complexes through CD16, orchestrating the inflammatory response through intravascular retention of classical monocytes, which results in glomerular damage and proteinuria. Open Access

The liver is crucial for maintenance of metabolic homeostasis in eukaryotes. Dysregulation of metabolism in liver leads to obesity, type 2 diabetes, non-alcoholic fatty liver disease and cardiovascular disease. Liver disease alone costs approximately $157 billion per year, a huge burden on healthcare services and society globally. Pathways including glycolysis, lipogenesis and bile acid metabolism are frequently dysregulated and are subject to regulatory changes at the transcriptional, post-translational and signalling levels. However, alternative pre-mRNA splicing, a major contributor to both transcriptome composition and proteomic diversity has not been widely investigated in the context of hepatic metabolic disorders. This thesis demonstrates that there is a consistent mis-regulation of splicing factors and splicing events in liver under the influence of obesogenic diets (high fat, HFD and high fructose, FD). The motif of the splicing factor RBFOX2 is highly enriched surrounding alternatively spliced exons in healthy livers (control diet, CD) during feeding and fasting cycles, and between AS exons of obese livers (HFD vs CD and FD vs CD). Enhanced individual nucleotide crosslinking immunoprecipitation (eiCLIP) of RBFOX2 in primary hepatocytes showed that RBFOX2 targets are involved in cholesterol and lipid trafficking, homeostasis and regulation. Generation of a liver-specific RBFOX2 knockout mouse model (LΔRbfox2) showed that in high fructose loading, LΔRbfox2 mice have disrupted cholesterol and phospholipid metabolism. In response to increased lipogenesis from fructose consumption RBFOX2 regulates a network of targets. Use of splice switching oligos (SSOs) enabled subtle manipulation of RBFOX2 splicing targets to regulate lipid metabolic pathways. This may enhance and inform therapeutic strategies to combat metabolic dysregulation and liver disease. Open Access

With the fast development of biological techniques, high-throughput omics data is available, which also raises many big problems in other subjects like mathematics: how to deal with such big data so that meaningful biological insights can be found. Among the various omics data, I mainly work on Matrix-Assisted Laser Desorption Ionization Mass Spectrometry and Single Cell RNA Sequencing data. Although there already exist many mature analysis pipelines and machine learning algorithms for analysing these kinds of omics data, there remains improvement space. In this thesis, I firstly describe the relevant challenges of omics data analysis in detail. Then I explain popular analysis pipelines and algorithms which are frequently utilized during the analysis. Finally, I illustrate the modified analysis pipeline, math models and corresponding results. Open Access

Introduction Empirical evidence isolating the independent effect of armed conflict on health is scant, has methodological limitations, and neglects a growing burden of non-communicable diseases (NCDs). This thesis aims to evaluate the impact of armed conflict on civilian morbidity and mortality globally. Methods Two systematic reviews assessed the existing evidence on conflict and NCDs (Chapters 4 & 5). Longitudinal, fixed effects panel regression models used data from the Uppsala Conflict Data Program and Global Burden of Disease to assess relationships between four armed conflict specifications and health outcomes. Outcomes included all-cause and cause-specific civilian mortality (Chapter 6), and maternal and child mortality and health service coverage (Chapter 7). Models were adjusted for ten confounders, lagged by between one and ten years, and were age- and -sex stratified. Multiple sensitivity analyses assessed model robustness. Results Systematic review findings found positive but inconsistent associations between conflict and NCDs, although two thirds of studies had low methodological quality. Empirical research chapters included 193 countries between 1990 and 2017 and found that more intense conflicts (wars) drove associations. Wars were associated with an increase in civilian mortality by 81.5 per 100,000 population (95% CI 14.3-148.8), predicting 29.4 million (95% CI 22.1-36.6) deaths globally over the study period, two thirds of which were from communicable, maternal, neonatal, and nutritional diseases and 20% of which were from NCDs. Males appeared more affected than females, particularly for deaths from injuries, and point estimates were largest for children under 5 years and adults over 69 years. Wars were positively associated with all maternal and child health indicators, predicting 14.1 million (95% CI 11.5-16.7) infant deaths. Sensitivity analyses demonstrated robustness of findings. Discussion This thesis comprehensively and robustly contributes to understanding the detrimental impact of conflict on civilian morbidity and mortality globally, including on NCDs and across the life course. Open Access

Tolerance mechanisms are vital for maintaining immune homeostasis in the intestinal system. The intestinal system must remain primed against pathogens whilst nutrients are absorbed without an inappropriate inflammatory response. Regulatory T-cells (Treg) are a key cell type for maintaining tolerance, primarily acting to suppress inflammatory T-cell responses. This mechanism is exploited by helminth parasites, including Heligmosomoides polygyrus (H. polygyrus), in order to suppress the anti-helminth T-cell response and establish chronic infection. H. polygyrus infection therefore provides a useful model for studying the dynamics of T-cell regulation in vivo. The current understanding of T-cell and Treg dynamics in vivo is limited. This thesis uses the novel Timer Of Cell Kinetics and Activity (Tocky) reporter system developed by the Ono Lab to overcome this knowledge-gap. Tocky reveals whether a gene of interest is actively transcribed or has previously been transcribed in real-time. Here, Foxp3-Tocky mice are used to report transcription of the key Treg gene Foxp3, and Nr4a3-Tocky mice are used to reveal T-cell receptor signalling through transcription of the Nr4a3 gene. This thesis identifies that during early primary H. polygyrus infection, a proportion of infection-reactive T-cells appear to induce and maintain Foxp3 transcription. Further analyses, including transcriptomic analysis, support that activated T-cells are a source of Treg in this context. Within secondary infection, where mice exhibit resistance to re-infection, Foxp3 transcription within Treg appears to be very similar to that in primary infection. However, the Treg pool is offset by the memory T-cell pool. In conclusion, I demonstrate that a subset of T-cells activated in H. polygyrus infection up-regulate Foxp3 transcription, potentially as a mechanism to suppress their own anti-parasite T-cell response. In secondary infection, Foxp3 transcription is similarly initiated and maintained, however, due to disproportionate expansion of the memory T-cell population, the Treg response is ineffective. Open Access

Background: Tuberculosis (TB), the deadliest infection worldwide, causes severe tissue destruction associated with excess inflammation. This process is driven by matrix metalloproteinase (MMP) enzymes regulated by the innate immune response. Patients also experience profound metabolic changes, such as weight loss. TB-infected macrophages show the Warburg effect, a metabolic shift from oxidative phosphorylation to aerobic glycolysis. However, the relationship between innate inflammation and cellular metabolism in TB is unclear. Materials/methods: Primary monocyte-derived macrophages (MDMs) or normal human bronchial epithelial cells (NHBEs) were incubated with specific metabolic inhibitors or transfected with siRNA. Glycolysis was blocked with the hexokinase (HK) inhibitor, 2-deoxyglucose (2DG). Cells were then directly infected with live, virulent Mycobacterium tuberculosis (M.tb) H37Rv or stimulated with TB cytokine networks. Protein secretion and expression, gene expression and functional tissue damage were measured by ELISA, luminex, zymography, Western blot, real-time PCR and DQ collagen assay. HK2 immunohistochemistry was performed in samples from a murine TB model and TB patients. Results: HK2 was highly expressed in murine and human tissue sites of TB inflammation. In vitro, 2DG downregulated gene expression and secretion of MMP-1 and the proinflammatory cytokine IL-1b; expression of the hypoxic transcription factor HIF-1a; functional matrix degradation and intracellular bacillary growth. The metabolic regulator AMPK upregulated MMP-1 secretion in TB, while MMP-1 was downregulated by the Pi3-kinase-Akt-mTORC1 signal transduction pathway. Conclusions: MMP-1, HIF-1a and pro-inflammatory cytokines are modulated by glycolysis, AMPK and the Pi3-kinase-Akt-mTORC1 pathway in TB. Immunometabolic regulation of tissue destruction in TB might provide new avenues for host-directed therapies. Open Access

Taenia solium taeniasis/cysticercosis is a neglected zoonotic disease caused by the Taenia solium pork tapeworm, responsible for a significant global public health and economic burden. Infection of the central nervous system with T. solium cysts, leading to neurocysticercosis, is the single most important risk factor for acquired (infectious) epilepsy worldwide. Economic consequences pertain not only to the human health sector, by also to the animal sector, resulting from pig infection in the food-value chain. The 2012 World Health Organization (WHO), first, roadmap and the London Declaration on Neglected Tropical Diseases (NTDs) called for a validated strategy towards T. solium control and elimination by 2015, and for interventions to be scaled up in selected countries by 2020. These targets have not yet been met. More recently, the second, post-2020 NTD WHO roadmap (published in April, 2020) reformulated the milestones for T. solium, as hyperendemic settings/countries having adopted intensified control by 2030. Mathematical transmission models can support efforts aimed to setting, achieving, and evaluating the proposed 2030 goals. Although a number of transmission dynamics and control frameworks for T. solium taeniasis/cysticercosis already exist, refinement of some of these will be required for the purposes of increasing their usefulness to inform public health policy and practice. Taenia solium has a complex multi-host transmission cycle, including the adult tapeworm stage in the human definitive host, larval stages in the intermediate porcine host (with humans also acting as accidental intermediate hosts), and contamination of the environment with eggs/proglottids. Various T. solium models have been published, capturing the transmission cycle to varying degrees of complexity. Therefore, this thesis first reviews the current state of the T. solium transmission modelling field, including assessing models for the wider Taeniidae family of tapeworms to identify important gaps and understand how these may be addressed. Characterising incidence patterns, using Force-of-infection (FoI) catalytic models fitted to age-(sero)prevalence data for porcine cysticercosis, human taeniasis and human cysticercosis, was identified as an important gap and, therefore, comprises a central component of this thesis. FoI estimates thus produced are applicable to parameterise T. solium transmission models, while reviewing age-(sero)prevalence profiles globally enables exploration of whether immunity-driven age-resistance, or exposure heterogeneity mechanisms operate in the human and porcine hosts. Putative T. solium taeniasis/cysticercosis endemicity levels are also presented, with the FoI estimates obtained being placed within the different proposed levels to investigate patterns between incidence parameters and (sero)prevalence. A deterministic, compartmental T. solium transmission model (EPICYST) is refined (by including, among other features, age structure in human and pig populations) with the aim of assessing the impact of the minimum pig slaughter age, an important setting-specific variable, on the outcome of simulated pig- and human-directed, age-targeted interventions. The resulting model outputs are presented in the context of the post-2020 WHO NTD goals (“intensified control in hyperendemic settings/countries”), by reporting, for a generalised hyperendemic setting, the number of years required, and the levels of intervention coverage necessary to achieve three distinct porcine cysticercosis operational prevalence thresholds proposed (ranging from lenient (

T he human-restricted, typhoidal Salmonella Paratyphi A (S. Paratyphi A) and S. Typhi are the major c auses of enteric (typhoid) fever and are endemic in regions with poor sanitation. Despite the recent i ncreased rate of S. Paratyphi A isolation from patients in Asia, its pathogenesis remains largely u nknown. Asymptomatic chronic carriage in the gallbladder is encountered in about 5% of patients a nd is facilitated by efficient immune evasion. In this study, we have shown that bile changes the e xpression of > 5% of genes in S. Paratyphi A, including both bile tolerance and virulence-associated g enes. S. Paratyphi A and S. Typhi may differentially regulate certain metabolic pathways in response t o bile. Furthermore, a clinical S. Paratyphi A isolate appears to exhibit distinct regulatory mechanisms. A s inflammasomes have been shown to play a key role in Salmonella infection, we also investigated t heir role following infection of macrophages with S. Paratyphi A, using S. Typhi and S. Typhimurium a s controls. This work demonstrates that S. Paratyphi A and S. Typhi induce pyroptosis, which is lower t han that triggered by S. Typhimurium. While the pathway activated during S. Typhi infection remains u nclear, S. Paratyphi A-triggered pyroptosis occurs via activation of caspase-1, caspase-4, caspase-8 a nd NLRP3. Both S. Paratyphi A and S. Typhi require their SPI-1 injectisome to enable inflammasome a ctivation. However, while the Vi antigen of S. Typhi is dispensable for limiting pyroptosis, the S.Paratyphi A FepE-mediated synthesis of very long O-antigen chains impairs macrophage cell death a nd a ΔfepE mutant elicited enhanced inflammasome activation. Very long O-antigen chains can also a ct as an inflammasome dampening mechanism in S. Typhimurium, but reduced fepE expression i ndicates that this strategy is not exploited by this pathogen. Therefore, this work points towards distinct mechanisms of virulence by S. Paratyphi A, highlighting the need for a systematic c haracterisation of its molecular pathogenesis. Open Access

Lung cancer stands as one of the deadliest diseases, responsible for the most cancer related deaths worldwide. The UK 5-year survival rate of non-small-cell lung cancer (NSCLC), the predominant subtype of lung cancer, stands at 9.5%, highlighting an unmet need for therapeutic intervention. A key issue is the lack of efficacy current chemotherapy regimens have in the clinic. These therapies often suffer from poor tumour targeting, resulting in dissemination throughout the body and inadequate concentrations in the tumour. This causes deleterious side effects contributing to a reduced patient quality of life and ultimately survival. Nanomedicine may serve to overcome the current therapeutic hurdles in treating NSCLC; the use of nanoparticles (NPs) for the delivery of drugs can improve drug targeting to tumours, increasing efficacy and attenuating off-target side effects. NPs can be used to deliver multiple drugs and be made from varying materials such as gold (AuNPs) or polymers. Furthermore, the discovery of oncogenic mutations in genes like EGFR present druggable targets in patients harbouring the appropriate mutations. This can also be taken advantage of using NPs to more directly target tumours and increase therapeutic response. Therefore, the aim of this thesis was to develop novel NP formulations comprised of a chemically modified variant of the tyrosine kinase inhibitor afatinib and gold (Afb-AuNPs) or in combination with vinorelbine as a polymeric dual chemotherapy formulation (Dual-NPs). Drug-bearing NPs were synthesised using a combination of organic chemistry and hydrophobic ion pairing, after which the NPs were extensively characterised to discern their physicochemical properties. We then sought to investigate the in vitro efficacy of NPs. Cell viability studies revealed Afb-AuNPs and Dual-NPs were significantly cytotoxic to various NSCLC cell lines and comparatively nontoxic to noncancerous cells. Moreover, NP formulations were found to significantly inhibit proliferation of A549, H226 and PC-9 cells 3 compared to clinical formulations as determined by electric cell-substrate impedance sensing. The mechanism of uptake in cancer cells was elucidated using fluorescent NPs as a model system and quantified using confocal microscopy. Finally, the in vivo activity of biocompatibility of Dual-NPs was investigated in a physiologically relevant murine model of NSCLC. Taken together, these results highlight the therapeutic potential for NP formulations of chemotherapy. Open Access

Amyloids are characterised by their innate capacity to aggregate into insoluble fibrils, which are commonly recognised for their cytotoxicity and association to neurodegenerative diseases. Their unique physicochemical properties are exploited by bacteria for various functional roles, including the formation of extracellular matrix that is linked to biofilm construction and antimicrobial resistance. The curli fimbriae of Escherichia coli was the first functional bacterial amyloid (FuBA) to be discovered. Two operons encode a curli secretion system that is comprised of several distinct proteins including curli-forming subunits, chaperones, amyloid inhibitors, and an outer-membrane (OM) transporter. Amyloid secretion systems enable FuBA fibril formation, whilst minimising their cytotoxicity to the host-cell. A functional amyloid in Pseudomonas (Fap) operon was recently identified in the Pseudomonas genus encoding a novel FuBA secretion system. Unlike the curli system, detailed insight into the Fap system is lacking. The curli and Fap secretion systems export biochemically similar amyloid forming subunits; however, their FuBA secretion systems are genetically distinct. The biophysical studies of this thesis sought to provide further insight into the structure and function of FapF, a uniquely structured OM transporter, as well as FapD, a periplasm-residing protein that is predicted to serve a proteolytic and chaperoning role within the Fap system. Previous reports demonstrate the evolutionary co-conservation of FapF and FapD, suggesting their functional co- dependence. In this thesis, a combination of biophysical techniques, including nuclear magnetic resonance (NMR) spectroscopy, are used to demonstrate the presence of a unique, asymmetric, parallel trimeric coiled-coil domain within the periplasmic N- terminus of FapF. Furthermore, a transient interaction between FapF and FapD was identified under solution conditions. Protocols that enable the near-native study of the FapF OM domain by solution-state NMR spectroscopy were also optimised. These studies pave the way for future research to enhance our mechanistic understanding of FuBA secretion systems, with aim to increase our capacity to modulate FuBA formation. Open Access

The Structural Maintenance of Chromosomes (SMC) complex family forms an essential part of the genome’s organisational machinery in all domains of life. In eukaryotes condensin is one such complex that mediates the packaging of DNA for segregation during cell division. This pentameric ATPase is comprised of two elongated Smc proteins (Smc2 and Smc4), a kleisin type protein (Brn1) and two HEAT repeat proteins (Ycg1 and Ycs4). Despite the important role of condensin in such fundamental cell processes its overall structure, and consequently its molecular mechanism, are currently unknown. This study describes the processes leading to production of the first atomic resolution model of the Saccharomyces cerevisiae condensin complex in the ATP free apo state by cryo electronmicroscopy. This model demonstrates that several features such as a folded conformation in the Smc protein arms are common to multiple members of the SMC complex family. Furthermore, previously described structural features such as the location of the Ycs4 subunit in contact with the Smc protein head domains, and a discontinuity in the arms of the Smc proteins called the joint, are also described as part of the whole complex. Finally, the position of the Ycg1 subunit is also described as sitting flexibly under the Smc head domains, anchored by the Brn1 subunit to the rest of the complex. Also described in this study is preliminary work to further characterise the biochemical properties of the condensin complex in the context of chromatin using the Lumix C-trap optical tweezer system. Condensins’ ability to compact DNA in real time and to translocate on a fixed DNA substrate in the C-trap system are demonstrated, as is ongoing work to chromatinise the same DNA substrate in situ. This chromatinisation involves the purification of recombinant S. cerevisiae histone octamers and histone chaperone Nap1 which have been successfully applied to DNA within the C-trap to create a chromatin substrate suitable for future experimentation. Open Access

Background: The streptococcal cell surface M protein is arguably the most studied virulence factor of S. pyogenes and is postulated to be essential for S. pyogenes survival in the human host through mechanisms which culminate in evasion of opsonophagocytic killing. Despite the central role ascribed to M protein in combatting bacterial clearance, we identified selection and emergence of M protein defective S. pyogenes which was related to a premature stop codon and secretion during experimental infection. We therefore set out to determine the impact of M protein loss and the release of extracellular M protein on streptococcal disease. Methods: Isogenic strains with mutations in emm1 were constructed using standard techniques. Adhesion to host factors was measured using solid phase adherence assays, and bacterial survival and uptake were assessed in human whole blood and neutrophil phagocytosis assays. Bacterial survival and dissemination was investigated using in vivo models of intramuscular and intranasal infections, including coinfections to determine competitive advantage of strains. The prevalence of M protein mutations in whole genome sequences was analysed and the effect of vaccination using M1 on bacterial clearance was assessed. Results: M1 protein mutants were unable to bind fibrinogen, fibronectin and collagen, and were vulnerable to immune killing and neutrophil uptake in comparison to the WT parent strain in vitro. Surprisingly, and in contrast to in vitro clearance studies, M protein mutations did not affect bacterial survival 3 h after intramuscular infection of mice. The strain shedding truncated M protein was recovered in greater numbers than the parent strain expressing full length anchored M protein from certain niches 24 hours post infection, such as lymph nodes. This advantage was confirmed using coinfection experiments. In contrast, M protein mutants were unable to establish intranasal infection in mice. While no other truncating mutations were found in M protein when analysing whole genome sequences, it was shown that all M1 S. pyogenes tested secrete soluble M protein during normal culture. Conclusion: M protein is not required for survival of S. pyogenes during invasive infection, and some M protein mutations seem to provide an advantage in the lymphatic niche. Expression of soluble M protein is an important characteristic of all S. pyogenes strains and may affect immune evasion and pathogenesis. Open Access

Background: Our understanding of psychedelic drug action has rapidly developed over recent years. Yet despite impressive progress, fundamental questions remain. Determining post-psychedelic long-term brain effects and neurobiological mechanisms underlying positive psychological change would hold significant value – not just for the field of psychedelic science, but for the wider fields of neuroscience and mental health. Moreover, serotonin 2A receptors – the key site of action for psychedelic drugs – are widely expressed across the vascular system in addition to their expression in the brain. This complicates our understanding of the neurophysiological correlates of the human psychedelic brain state which largely stems from indirect measures of neuronal activity by extraction of blood-flow related parameters. Aim: To address the knowledge gaps and further our understanding, this thesis investigated (i) post-psilocybin changes in pathological beliefs in patients with treatment-resistant depression (TRD), (ii) acute and long-term effects of psilocybin in healthy humans, and (iii) psychedelic vasoactivity in mice. Methods: STUDY 1 investigated changes in pessimism in TRD patients versus controls via an objective measure of cognitive biases. STUDY 2 measured the acute and long-term effects of psilocybin in healthy humans via multimodal neuroimaging and a broad repertoire of psychological and behavioural assessments. STUDY 3 characterised the behavioural and vascular effects of 25CN-NBOH in mice via behavioural pharmacology with the head twitch response (HTR) as a behavioural read-out and pulse oximetry, respectively. Results: STUDY 1: Psilocybin treatment remediated pessimism in TRD patients one week post-dosing. STUDY 2: Brain signal complexity was related to insightfulness experienced during the peak intensity of a high-dose psilocybin experience. Psychological insight modulated positive long-term outcomes and persistent brain effects correlated with positive long-term psychological changes four weeks after a high-dose psilocybin experience. STUDY 3: 25CN-NBOH induced a (i) dose- and time-dependent, ketanserin-sensitive increase in HTRs, which was subject to short-interval and subchronic tolerance, and (ii) temperature-dependant increase in heart rate, decrease in breath rate, and increase in arterial blood flow. Conclusions: This thesis revealed post-psilocybin treatment changes in pathological beliefs in TRD patients. In healthy participants, the present findings identify insight as a mediator of long-term positive outcomes, provide a potential neural correlate of acute insight during a high-dose psilocybin experience, and show persistent neural changes that correlate with well-being outcomes long after that experience. The vasoactivity intrinsic to psychedelic drug action revealed here in mice provides considerations for blood-flow based neuroimaging techniques to measure acute psychedelic brain states. Further, using optogenetics to image cortical activities in awake behaving mice might take our mechanistic understanding of the underlying processes from basic receptor pharmacology to systemic circuit dynamics. In conclusion, this thesis presents novel results that considerably broaden the field’s knowledge, provide inspiration for future work and potentially impact wider society and mental health care. Open Access

The principal role of Von Willebrand Factor (VWF) is platelet capture at sites of vascular injury, via interaction of its A1 domain with platelet glycoprotein Ibα (GPIbα). The GPIbα binding site overlaps with a binding site for heparin, the functional significance, and physiological ligand, of which remains unknown. Previous studies have demonstrated heparin inhibition of VWF-dependent platelet binding, a secondary effect distinct from heparin’s anticoagulant role. Using a physiologically representative flow assay, this secondary effect has been demonstrated, with unfractionated heparin (UFH) exerting the greatest inhibition. This antithrombotic action of heparin is a potential mechanism for unpredictable bleeding complications during cardiopulmonary bypass surgery (CPB) when high concentrations of UFH are needed to maintain the extracorporeal circuit. A prospective cohort study to determine the changes in VWF antigen activity and function, occurring pre- and post-UFH in CPB demonstrates a significant rise in the VWF antigen (VWF:Ag) level by the end of surgery, with a relative fall in VWF collagen binding. The VWF Ristocetin Cofactor (VWF:RCo) fell but not significantly following UFH, with recovery seen in the absolute VWF:RCo after UFH reversal with protamine sulphate (PS) but not in the VWF:Ag/VWF:RCo. Both heparin and PS sulphate affect many components of haemostasis and these off-target effects, alongside the consumptive and dilutional coagulopathy during CPB make prediction of bleeding difficult. Recombinant VWF heparin binding site variants demonstrate impaired platelet capture on a collagen surface, suggesting a greater degree of overlap between the two binding sites or that the heparin-binding site of VWF has a role in the GPIbα interaction, identifying a potential physiological ligand. Heparan sulphate (HS) within the endothelial glycocalyx has been suggested as the physiological ligand for the VWF heparin-binding site. Removal of HS from the glycocalyx of human umbilical vein endothelial cells is a promising model to investigate this further. Open Access

Working memory impairment after traumatic brain injury (TBI) is common and can lead to long-term disability. Predicting which patients are likely to develop impairments and providing effective treatments is currently challenging. This is in part due to the heterogeneity in damage acquired across the population. Diffusion tensor imaging (DTI) provides a means to quantify white matter damage associated with working memory impairment after TBI. I therefore investigated whether the development of a diagnostic pipeline using DTI could sensitively detect axonal injury in individual patients and provide insight into the impairments observed. Next, I applied a graph theoretical approach to determine whether distinct patterns of white matter damage were associated with working memory impairment and whether these measures could predict impairment in subacute TBI. Finally, I examined whether the application of machine learning to neuroimaging data could predict the effect of methylphenidate on working memory in patients and whether multimodal imaging improved prediction models. I found that almost a third of patients with no visible MRI damage had evidence of axonal injury when using my diagnostic pipeline and that patients diagnosed with impairment had significantly poorer cognitive and functional outcomes. I found that patterns of white matter damage associated with alterations to the structural topology of the working memory network were related to working memory impairments after TBI and that network measures could accurately predict working memory outcomes in a novel, subacute TBI population. Finally, I demonstrated that the prediction of treatment effects on working memory performance could be achieved, but only when multimodal imaging was used. These findings provide mechanistic insights into the development of working memory impairment after TBI and a diagnostic pipeline that can account for heterogeneity in the population. This work also identifies clinically useful prognostic markers, and provides evidence to support the development of more tailored treatments after TBI. Open Access