Theses - Medicine
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Item Restricted New approaches and therapeutics for antimicrobial resistant Klebsiella: A comprehensive, multimodal approachZellmer, Caroline[Restricted]Item Embargo Cells of the Developing Human Heart and Great VesselsBayraktar, SemihThe human heart and the great vessels comprise various cell types that collectively support the heart’s homeostasis. Single-cell genomics has expanded our knowledge of the heart’s cellular heterogeneity. By gaining a deeper understanding of its cellular makeup, we can uncover how these cells converge to define cardiac identity and function and how perturbation to cellular identities can lead to diseases. This dissertation focuses on generating a high-resolution atlas of the first and second-trimester human heart and great vessels using single-cell genomics. The atlas contains 63 cell types with distinct identity, function, or location-specific signatures, such as the cardiomyocytes of different chambers and the vessel components specific to coronary or great vessels. Alongside molecular profiling, cellular communication mechanisms and gene regulatory networks are investigated, collectively aiming to advance *in vitro* models of cardiac cells. Chapter 1 introduces the topics covered throughout the thesis. After describing the materials and methods utilised in Chapter 2, an overview of the atlas is provided in Chapter 3. This is followed by a description of the individual cell types in subsequent chapters based on the macro anatomical structures they are associated with. Chapter 4 focuses on cardiomyocytes, which constitute the majority of the myocardium. In Chapter 5, the focus shifts to the vessels, encompassing the coronary and the great vessels. Chapter 6 explores the pericardium and endocardium as the two layers enveloping the myocardium. Ultimately, through the profiling obtained from the atlas, Chapter 7 proposes a framework to generate enhanced stem cell models to mimic the corresponding cells *in vivo*.Item Open Access The Epicardium as the Conductor of Cardiovascular Cells in Cardiac RegenerationRericha, PatrickStem cell therapy for cardiac repair is a field of regenerative cardiovascular medicine that is emerging as a promising therapeutic alternative to heart transplantation in patients suffering from chronic heart failure. Although promising, challenges to cell therapy for cardiac repair remain, including cell survival, maturation, graft size, revascularisation, and immunogenicity. It has recently been shown that co-transplantation of human embryonic stem cell (hESC)-derived epicardial cells (EPI) with hESC-derived cardiomyocytes (CM) improves cardiac repair processes with respect to engraftment, cell maturation, and graft and host vascularisation. One plausible explanation for the observed benefits of co-transplantation is paracrine-mediated effects of the epicardium towards CMs and endothelial cells (EC). While RNA sequencing data has been used to identify putative players in hESC-EPIs, little is known about their involvement in mediating hESC-CM maturation and angiogenesis. Here, the role of paracrine signalling in hESC-EPI-mediated promotion of hESC-CM maturation and angiogenesis is being investigated. To study the relative importance of secreted factors and extracellular vesicles (EVs) a protocol was established to split hESC-EPI conditioned medium into hESC-EPI-EVs and hESC-EPI-flowthrough, which is depleted in EVs. HESC-EPI-EVs failed to confirm the benefits on CM maturation observed in preliminary studies. Both fractions independently promoted tube formation in HUVECs *in vitro* and hESC-EPI-flowthrough additionally promoted proliferation and migration. Both fractions improved branching *in vivo* in chick yolk-sac membrane (YSM) assays. Characterisation of the hESC-EPI-EV-cargo on the protein and miRNA level lead to the identification of pro-angiogenic candidate factors that were screened *in vitro* and validated *in vivo*. Moreover, hESC-EPI-EVs reduced reactive oxygen species (ROS) in hESC-CMs and HUVECs *in vitro*. Viable mitochondria were transferred between hESC-EPIs and hESC-CMs and HUVECs, which was partly mediated by hESC-EPI-EVs. Identification of the paracrine signalling factors involved in the hESC-EPI-mediated promotion of CM maturation and revascularisation would make it possible to effectively address these challenges in stem cell therapy for cardiac repair, resulting in new therapeutic approaches.Item Embargo Domesticating the invader: how intronless genes escape HUSH silencingCohen-Gold, JonathanThe reverse flow of genetic information (the reverse transcription of RNA to DNA) allows retroviruses and retrotransposons to invade our genome. Together, retroelements make up more than 40% of the human genome. The HUSH epigenetic repressor complex defends the genome from retroelement invasion through the recognition and silencing of ‘intronless’ DNA, the hallmark of reverse transcription. Therefore, intronless cDNA provides the ‘abnormal molecular pattern’ which allows HUSH to distinguish foreign retroelements from host genes, making HUSH a part of the innate immune system. This presents a paradox: intronless genes (ILGs) comprise ~3-5% of the human protein-coding genome and are well expressed I.e. resistant to hush silencing despite themselves arising through retroelement invasion. I wanted to understand this paradox and determine how ILGs escape HUSH silencing. I initially assessed genome-wide RNA binding and transcriptional repression by HUSH to identify nucleic acid sequence determinants of HUSH-sensitivity. My findings suggest that ILGs evolve GC-rich sequence within their amino acid sequence constraints, through GC-biased codon usage and increased 5’UTR GC content. This enables their escape from HUSH despite the absence of introns, allowing beneficial retroelements to be domesticated. Decreasing GC content within an intronless coding sequence incrementally impaired recruitment of TREX, an essential mRNA export complex with a known GC binding RNA preference. This decreased GC content corresponded with reduced RNA export and increased HUSH repression. By tethering TREX components to intronless RNA, I could promote HUSH escape and this was most striking for ALYREF, an essential RNA- binding subunit of TREX which facilitates mRNA packaging. In contrast, depletion of TREX components (THOC1/2) induced HUSH-sensitivity of intronless reporters. As RNA binding by ALYREF and periphilin, the main RNA binding component of HUSH, is inversely proportional, I propose a model in which periphilin and ALYREF compete for nascent RNA binding. My results suggest this binding competition between periphilin and ALYREF determines the balance between HUSH-dependent silencing versus RNA nuclear export and gene expression.Item Embargo Mechanisms of Synthetic Lethality in Renal Cell CarcinomaBertlin, JamesClear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer, is characterised by biallelic inactivation of the von Hippel-Lindau (*VHL*) tumour suppressor gene, and inherited loss-of-heterozygosity of *VHL* underlies the eponymous hereditary cancer syndrome. The *VHL* protein product, pVHL, is the substrate recognition component of an E3 ubiquitin ligase complex responsible for targeting Hypoxia Inducible Factors (HIFs) for proteasomal degradation. Accumulation of the HIF-2α isoform, in particular, drives renal tumourigenesis. *VHL* loss occurs at an early stage of ccRCC development, often several years before the clinical manifestation of disease, and so is observed throughout the entire mass of both primary and metastatic tumours. Therapeutic approaches which specifically target cells harbouring *VHL* mutations could therefore provide effective anti-tumour responses with minimal off-target effects. In this thesis, I undertake CRISPR/Cas9 screens in ccRCC cell lines deficient in *VHL*, and in paired *VHL*-reconstituted cells, aiming to identify genetic vulnerabilities of cells lacking functional pVHL. These reveal a synthetic lethal relationship between Core Binding Factor β (CBF-β) and pVHL. While CBF-β is critical for normal osteogenesis and haematopoiesis, little is known about its role in renal tissue. CBF-β classically stabilises the Runt related transcription factors (RUNX1-3) to improve their transcriptional efficiency. However, using a variety of cell biology techniques, I demonstrate that knockout of *CBFB* is lethal in cells lacking *VHL* through a mechanism which is independent of both direct CBF-β-RUNX binding and the high HIF activity of *VHL*-null cells. In addition, RNA sequencing and mass spectrometry reveal that CBF-β depletion causes a widespread upregulation of interferon (IFN)-stimulated genes (ISGs), although this does not contribute to the cell death phenotype. Rather than occurring through a canonical IFN response, CBF-β loss-induced ISG expression relies on the direct transcriptional activity of IFN Regulatory Factor 3 (IRF3) and its stimulation via STING. Together, these results reveal a novel requirement of *VHL*-mutant ccRCC cells for CBF-β, loss of which provokes cell death and increases ISG expression. Besides triggering direct tumour cell killing, depletion of CBF-β may present a strategy to reactivate anti-tumour immune surveillance in renal cancer.Item Embargo Identification and validation of new therapeutic targets for squamous cell lung cancerKottmann, DanielLung squamous cell carcinoma (LUSC) is a subtype of non-small cell lung cancer and constitutes around 25% of all lung cancer cases worldwide. Despite recent advancements in cancer therapeutics, no targeted therapies have been approved specifically for LUSC. The lineage- survival oncogene and transcription factor SOX2 is dysregulated by gene amplification in a large proportion of LUSC tumours and has been implicated as a key driver in multiple preclinical functional studies. As a transcription factor, SOX2 itself makes a challenging drug target. New therapeutic approaches are required for this disease, and this project identified and evaluated potential therapeutic targets for SOX2-driven LUSC. Multi-omics data was used to identify drug target candidates from the SOX2 interactome in LUSC, and ten target candidates were selected for *in vitro* validation. Targeting the PI3K/AKT/mTOR pathway showed benefits in SOX2-driven disease, and the transcription factor FOXM1 was highlighted as a promising target candidate in SOX2-driven LUSC tumours. Additionally, transcriptional data of LUSC tumours was used to identify differentially expressed genes in SOX2-high tumours, and identified TrkB (encoded by the *NTRK2* gene) as a potential drug target candidate. Expression of TrkB correlated with SOX2 expression in squamous tumours, showed a functional role in squamous cancers, and *NTRK2* was confirmed as a transcriptional target of the SOX2 transcription factor. Deregulation of TrkB expression and specific transcript variants showed an impact on cell fitness, survival, and downstream signalling. While TrkB.T1 was shown to be the predominant transcript variant of the *NTRK2* gene in LUSC and other squamous cancers, both TrkB.T1 and TrkB.FL variants had a functional role in oncogenic signalling. Targeting TrkB in patients with high SOX2 expression might therefore be a promising strategy for further evaluation in LUSC. Finally, this project characterised current *in vitro* models of LUSC and undertook early efforts to develop new models from human bronchial epithelial cells. Better models are needed to study the disease and to test new therapeutics. Ultimately, good *in vitro* models of LUSC remain a challenge and require future work. In summary, this project identified a set of drug target candidates in SOX2-driven LUSC and presented *in vitro* validation results to support the further development of selected targets. This work generated new insights into SOX2 pathobiology in LUSC and contributed to the development of new therapeutic approaches to treat this disease with clear unmet medical needs.Item Embargo Contribution of Causal Aquaporin-1 Mutations to the Pathobiology of Pulmonary Arterial HypertensionWood, JenniferPulmonary arterial hypertension (PAH) is a rare and debilitating disease characterised by elevated pulmonary arterial pressure and extensive vascular remodelling in the lungs, with a lack of effective curative options. The identification of genetic variations in genes encoding components of the bone morphogenetic protein pathway in individuals with heritable PAH has opened up promising avenues for therapeutics, with encouraging results from preliminary clinical trials targeting this pathway. To deepen the understanding of the molecular mechanisms underpinning this rare condition, detect additional causative genes, and identify novel potential therapeutic strategies, a comprehensive study involving whole-genome sequencing was conducted on a cohort of over 1000 PAH patients. Within this cohort, rare causative heterozygous variants in the Aquaporin-1 (AQP1) gene were identified among a small subgroup of PAH patients. AQP1 is a water channel protein critical for maintaining endothelial cell integrity and fluid homeostasis. The primary aim of this thesis is to provide insight into the potential pathobiological significance of these rare AQP1 variants in PAH. Computational structural analysis using Pymol was employed to gain preliminary insights into the mutational effects of the identified variants. Subsequently, mutant AQP1 proteins were overexpressed in Hek293T cells, then subjected to confocal microscopy and membrane biotinylation followed by biotin-based ELISAs, to investigate whether the mutant proteins are able to traffic to the cellular membrane. These studies suggested normal membrane trafficking for the Arg126Cys, Val176Glu, Leu181Phe, and Arg195Trp AQP1 mutants, with comparable levels to that of wild-type AQP1. In contrast, the Trp213Term mutant is predicted to display dysfunctional trafficking, although further validation regarding this nonsense mutant is required. In addition, water and solute permeability through the wild-type and mutant AQP1 proteins was assessed using stable transfection of MDCK cells and transient transfection of Hek293T cells, respectively, followed by calcein self-quenching assays. None of the PAH-causative AQP1 mutants exhibited permeability to glycerol or urea, suggesting a retained ability for strict solute exclusion. Interestingly, water permeability was significantly reduced in the Arg126Cys AQP1 mutant, near to or completely abolished in the Val176Glu, Arg195Trp, and Trp213Term AQP1 mutants, but unaltered in the Leu181Phe AQP1 mutant. Finally, due to the recurrent nature of the Arg195Trp mutation, identified in 5 of 9 unrelated PAH patients with causal AQP1 variants, an Aqp1 R195W knock-in mouse model was generated to investigate its phenotypic effects *in vivo* and *ex vivo*. Surprisingly, neither heterozygous nor homozygous R195W mice displayed haemodynamic measurements suggestive of PH, either when exposed to normoxia or hypoxia. Nevertheless, the mutant mice exhibited significantly reduced urine osmolarity and signs of inflammation. Samples for future assessment of vascular remodelling through protein, mRNA, and immunohistochemistry analysis have been collected. In conclusion, this project has provided preliminary insights into the role of rare AQP1 variants in PAH and established crucial experimental protocols and reagents for further investigations. Ultimately, these findings may pave the way for the development of precision medicine approaches tailored to patients with specific AQP1 mutations, offering new hope for improved management and treatment outcomes in PAH.Item Embargo An unbiased proteomic approach to identifying cell surface markers in neutrophilsSubburayalu, Julien; Subburayalu, Julien [0000-0001-9243-0558]Introduction: Efferocytosis refers to the engulfment of dead cells by phagocytes such as neutrophils and macrophages and culminates in the beneficial removal of inflammatory cargo and auto-reactive material. While many of the mechanisms involved in efferocytosis have been described, the critically important ‘apoptotic-cell-associated molecular patterns’ have yet to be identified. Methods: Human whole blood samples were obtained from healthy volunteers. Neutrophils were isolated and subjected to a temperature shift to facilitate a wave of synchronised apoptosis. The plasma membrane proteome was assessed using tandem-mass-tag liquid chromatography-mass spectrometry (TMT-LC/MS) of apoptotic and time- and donor- matched non-apoptotic neutrophils, which allowed the identification of the plasma membrane proteins expressed or downregulated during apoptosis. In parallel, a method was developed to quantify efferocytosis. Briefly, apoptotic neutrophils or genome-modified neutrophil-like cells overexpressing the proteins identified to be up- regulated on apoptotic neutrophils, were fed to the monocytic cell line THP-1, which had been previously primed with phorbol-12-myristate-13-acetate (PMA) to acquire the functional properties of human macrophages. Efferocytosis was assessed by confocal microscopy and flow cytometry. Results: The late-stage apoptotic neutrophil plasma membrane profile identified several protein targets to be either up- or downregulated. The expression kinetics over time of ‘targets’ were then validated using flow cytometry. TIMD-4, a never previously described protein in (human) neutrophils, is actively up- regulated in preparation for apoptosis, and links to the phagocyte’s recognition and removal abilities of apoptotic cells, suggesting that TIMD-4 is a novel ‘eat me’ signal produced by apoptotic neutrophils. Conclusions: TMT-LC/MS can be successfully used to determine functionally relevant changes in the neutrophil plasma membrane, and has provided novel insights into the processes underlying efferocytosis of human neutrophils.Item Embargo Innate immunity in defence against infection and tissue repair of the urinary tractLoudon, KevinUrinary tract infections (UTI) are common and affect half of women and 5% of men during their lifetime. Most infections are caused by uropathogenic *Escherichia coli* (UPEC) and are limited to the lower urinary tract (cystitis), but ascending infection (pyelonephritis) can lead to fibrosis and the development of chronic kidney disease. A complex network of tissue-resident immune cells exists throughout the urinary tract that are vital for initiating and propagating local immune responses, however our understanding of their nature and function remains incomplete. In this thesis, we aim to understand two broad areas of tissue immunology: (i) the role of group 3 innate lymphoid cells (ILC3s) in bacterial cystitis and (ii) macrophage-derived amphiregulin (AREG) in acute pyelonephritis and infection-mediated scarring. Here, we identify type 17 immune response genes amongst the most up-regulated networks in the mouse bladder following UPEC challenge and identified ILC3s as a major source of these cytokines. Depletion of ILCs in Rag2-/- mice resulted in increased bladder bacterial load, identifying them as important players in bladder defence. Furthermore, we describe evidence of reciprocal cross talk between ILC3s and macrophages, with IL17 shaping bladder macrophage activation and polarisation. Together our findings reveal important insights into the orchestration and execution of type 17 immunity in bladder defence. AREG, is an epidermal growth factor receptor (EGFR) ligand that is expressed by a variety of activated immune cells, including macrophages and is a vital mediator of immune responses in infection and tissue repair. Using microarray analysis, we found AREG expression was higher in the renal medulla and augmented following UPEC stimulation. *In vivo, in vitro* and *in silico* experiments found renal macrophages to be a major source of AREG and mice globally deficient in *Areg* demonstrated increased susceptibility to pyelonephritis with impaired monocyte recruitment. Provisional findings in our haematopoietic- and myeloid-specific *Areg* knockout mice, also confirmed greater susceptibility to pyelonephritis. Furthermore, we describe accelerated repair of epithelial cells *in vitro* following the addition of recombinant AREG and conditioned medium from UPEC stimulated macrophages. Our data reveal a novel mechanism by which kidney MNPs interact with renal tubular cells to promote host-defence, organ integrity and repair in UTI. To study the role of AREG in infection-mediated kidney scarring we successfully developed a robust and reproducible *in vivo* murine model of chronic pyelonephritis. These experiments have found AREG expression to be highest in the medulla of kidneys with chronic pyelonephritis, and similarly enriched in medulla-derived macrophages. We found mice globally deficient in Areg were protected against kidney scarring and in 100% bone marrow chimeras, loss of AREG from the hematopoietic compartment offered greatest protection. Finally, using single-cell RNA sequencing we generate the first detailed atlas of the human kidney in chronic pyelonephritis and in conjunction with our murine data, offer insight into cellular mechanisms driving fibrosis, with the potential to inform strategies to reduce the development in CKD with patients with recurrent pyelonephritis.Item Embargo The Regulation of Cellular Iron MetabolismMartinelli, Anthony; Martinelli, Anthony [0000-0002-7285-7498]Iron is essential for life, but it presents a challenge to our cells because of its ability to drive free radical formation that damages proteins, lipids and DNA. While the consequences of iron excess or deficiency for disease are well described, it has only been recently recognised that iron-induced cell toxicity also forms part of a regulated cell death pathway, termed ferroptosis. This pathway is typically suppressed in tumours, but can be induced by cytotoxic T cells and contribute to anti-tumoural immunity. It is therefore essential to understand how cellular iron levels are controlled. In this thesis, I develop fluorescent iron reporters in cells and use them in genome-wide CRISPR/Cas9 mutagenesis screens, to map the cellular pathways of iron metabolism, identify novel regulators of iron levels, and determine their impact on the induction of ferroptosis. I first generated an iron reporter that is highly sensitive to changes in cellular iron abundance by endogenously tagging Iron Regulatory Protein 2 (IRP2) to the fluorescent protein Clover. I then undertook parallel genome-wide CRISPR/Cas9 mutagenesis screens in HeLa and A549 IRP2-Clover cells to identify genes that when mutagenised disrupt normal iron homeostasis. These screens have allowed me to map the main pathways involved in iron metabolism, as well as uncover genes without known links to iron homeostasis. SETD2, a histone methyltransferase, was a top hit in the screen, and provides the first example of a chromatin modifying enzyme as a mediator of iron levels. SETD2 depletion leads to an increase in the levels of the cargo receptor NCOA4, responsible for selective autophagy of the iron-storing protein ferritin, intracellular iron depletion and activation of the IRP2 response to promote iron uptake. SETD2 loss of function mutations occur in up to 28% of kidney cancers (ccRCCs). I go on to show that SETD2 loss in ccRCCs contributes to resistance to ferroptosis. Therefore, my work not only uncovers an unexpected link between SETD2 function and iron metabolism, but also explains how certain kidney cancers may evade anti-tumoural immunity via their resistance to ferroptosis.Item Embargo Blood Pressure, Arterial Stiffness and Cardiovascular Risk PredictionPavey, HollyThere is an important, unmet need to improve current cardiovascular (CV) disease risk prediction algorithms, allowing better stratification models for disease prevention and, ultimately, a personalised medicine approach. This is particularly important for ‘moderate risk’ individuals, where relatively few people will suffer a CV event, but where a large number of individuals are likely to be offered drug therapy. On the other hand, individuals below the treatment threshold will not be treated and many go on to have CV events. Indeed, the need for improved risk prediction has been highlighted by the initiation of the lower treatment threshold for hypertension from a systolic blood pressure (BP) of 140mmHg to 130mmHg in line with the US guidelines as recommended by the AHA/ACC. This would dramatically increase the number of adults requiring drug therapy. Aortic stiffness is an attractive novel biomarker, particularly for those with borderline hypertension, because it can be measured simply and non-invasively in large numbers of individuals, and shares a close association with BP. Stiffening of the aorta with ageing and disease occurs in almost all societies worldwide and indicates a deterioration of the ability of the large elastic arteries to ‘buffer’ the cyclical changes in BP resulting from intermittent ventricular ejection. Indeed, aortic stiffening appears to drive the development of systolic hypertension – by far the most common form of hypertension in older individuals and may, itself, provide a measure of end organ damage. However, the potential added value of aortic stiffness has not yet been examined in middle risk individuals or in those with borderline hypertension. It is also unclear whether BP is the main driver of arterial stiffness or whether other factors such as heart rate have an important role. This is an important question because ultimately aortic stiffness may provide a better measure of long term average BP than single clinic SBP readings or isolated 24-hr ambulatory measurements, which could improve CV risk prediction. In this thesis, an updated meta-analyses has been performed using data from 11 population based cohort studies consisting of 15,987 individuals at moderate CV risk, to assess the prognostic value of cfPWV beyond traditional CV risk factors. Novel risk scores including cfPWV measurements were derived and validated and compared to established CV risk scores used in clinical practice. These data showed that cfPWV was independently associated with CV risk after adjustment for established CV risk factors (age, sex, HDL, total cholesterol, smoking status, diabetes, antihypertensive medications) and that the addition of cfPWV to traditional CV risk factors significantly improved the ability of the model to discriminate between individuals who have an event and those who do not. Integrating the novel cfPWV risk scores into clinical practice in combination with the currently established risk models in a 2-stage screening programme and therefore additional screening with cfPWV measurements could reduce the risk of CV events by approximately 3% in the US and Europe, compared to the current guidelines. A subset of the studies with longitudinal data available contributed to longitudinal analyses allowing BP and heart rate trajectories to be modelled. When predicting CV events, the prognostic value of the SBP trajectory and cfPWV both acted independently, although the SBP trajectory was not an independent predictor of CV events. The results in this thesis suggest that a one off cfPWV measurement does not provide a good surrogate measure for long-term SBP measurements when predicting CV events. These analyses also suggested that current SBP, heart rate and age were the key predictors of current cfPWV, but that preceding trajectories of SBP and heart rate added a small amount of predictive value. Due to the COVID-19 pandemic occurring part way through this research project, an additional, unplanned project was performed looking at the effect of hypertension, absolute SBP and antihypertensive medications on the risk of severe COVID-19.Item Embargo Disease Surveillance and Implications on Public Health Decisions: Example of invasive Salmonellosis in sub-Saharan AfricaJeon, Hyon Jin*Salmonella enterica* is one of the most common blood stream infections in many parts of sub-Saharan Africa. An estimated 3.4 million people develop iNTS disease annually and the case fatality rate is as high as 20-25% in areas with limited resources. Around 11-20 million people are diagnosed with typhoid fever every year with 128,000 to 161,000 losing their lives to the disease. For typhoid fever, novel typhoid conjugate vaccines (TCV) are already developed and prequalified through the World Health Organization (WHO) while several vaccines against iNTS disease are currently undergoing clinical testing. Throughout the development process of new vaccines, different surveillance methods need to be deployed to allow an initial accurate assessment of disease burden, detailed knowledge on disease epidemiology, endemic areas for the target disease as well as the highest risk groups for infection. Post-vaccination, the impact of vaccines needs to be determined, which requires a different set of surveillance. Through this work, I investigate the use of disease surveillance for public health decision-making, using an existing bacterial surveillance infrastructure that was setup in the past 15 years in various African countries. This thesis investigates the epidemiology and transmission of invasive Salmonellosis, particularly iNTS disease, evaluates trends identified through the now over 10 year running surveillance, including potential relationships with other pathogens (i.e., *Plasmodium falciparum* malaria and COVID-19), and eventually disaggregates the use of surveillance and obtained data for public health decision making to use TCV and upcoming iNTS vaccines in endemic settings. The research presented here relies mainly on data collected through Severe Typhoid in Africa program conducted from 2016 to 2019, as well as the Typhoid Fever Surveillance in Africa Program (TSAP) from 2011-2014 for long-term trends determination. I report incidence of iNTS disease estimated from 2016 to 2019 in five sub-Saharan African countries, with the focus on young children considering the potential iNTS vaccine introduction in these settings. iNTS remains one of the major febrile illnesses in sSA, and the biggest burden lies on the young children under the age of two. I further investigated the severity of the iNTS disease and AMR profiles in different countries, and assessed the association between iNTS disease and *P*. falciparum malaria, one of the known risk factors to iNTS disease. In addition, I used phylogenetic analysis to investigate the different *S*. Typhi strains circulating in Madagascar for the past ten years, and participated in updating the pan-African *S*. Typhimurium phylogenetic tree. Findings from these work can have significant potential impact on public health as they show how such surveillance data can help make the informed decision. Lastly, with the Covid-19 pandemic which started during my PhD work, I evaluated how COVID-19 had an impact on the surveillance activities and severity of disease in Madagascar. Overall, I demonstrated how disease surveillance can make an impact on the public health decision making with an example of the invasive Salmonellosis surveillance in sSA. Data collected from surveillance not only show the burden of the disease and the extend of AMR, but also guide the way for the policy makers to make an informed decision on the public health matters including vaccination. Well-established, sustainable surveillance system can monitor the impact of interventions such as vaccination and detect other new pathogens and outbreak in real-time as it was witnessed in our study sites during the pandemic.Item Embargo Deep learning of regulatory sequence variation in Pulmonary Arterial HypertensionTilly, Tobias; Tilly, Tobias [0000-0002-6762-5342]Pulmonary arterial hypertension (PAH) is a rare and fatal lung disease. To date, in only a third of idiopathic patients, the cause can be attributed to rare genetic variation in the protein-coding space. The sequencing of 13,343 whole genomes by the NIHR BioResource for Translational Research – Rare Diseases (NBR), including 1,216 PAH samples, provides an unprecedented opportunity to estimate the contribution of regulatory genome variation to the development of PAH. This work aims to determine whether sequence-based predictions of epigenetic features can be used to narrow down the possible regions of interest and allow aggregation of variants into functional groups for association testing. A convolutional neural network (CNN) has been trained using publicly available data sets to predict epigenetic features from DNA sequences. The model was tested against known enhancer regions and its accurate performance was verified; two approaches were developed for the evaluation of the epigenetic features. Firstly, an epigenetic importance score supplies general information about the availability of epigenetic profiles within a region to explore the non-coding space. Secondly, a regulation score combines the predicted features into activating and repressing subsets for more detailed analyses to gauge the regulatory impact of variants. Based on the regulatory impact and other common variant annotations, variants were filtered and aggregated for over-representation analysis, comparing cases with controls. These scores were used in an outlier analysis using the Fisher's exact test and a sweep across the landscape of 1,135 PAH gene-associated enhancers, using SKAT-O. After p-value adjustment, over 80 regions were found significant. The statistical analysis revealed likely disease-causing sequence variation in ENG enhancers, as well as strong associations in ACVRL1 and KLK1 enhancers. Here, I present an extended search into enhancer networks associated with PAH, unlocking the non-coding space for genomic medicine.Item Embargo Development of a Lentivirus-mediated Gene Therapy Targeting HIV-1 RNA to Eliminate Infected CellsBuckingham, AmandaOver 38.4 million people worldwide are living with HIV-1, the etiological agent of acquired immunodeficiency syndrome and the cause of over 40.1 million deaths. The latent HIV-1 reservoir is the major roadblock to cure and necessitates lifelong antiretroviral therapy (ART), which is vulnerable to drug resistance. I contributed towards the development of a novel therapeutic strategy that aims to eliminate cells actively expressing HIV-1 and those harbouring HIV-1 reactivated from latency. This approach hijacks the currently unexploited HIV-1 alternative RNA splicing process to functionalize a defective cell suicide enzyme (*HSVtk*ΔAUG1) through targeted RNA *trans*-splicing with the HIV-1 D4 splice site. In-frame *HSVtk* translation is initiated from the start codon of the HIV-1 *tat1* donor exon in chimeric mRNA. HSVtk activates the prodrug ganciclovir (GCV), with cytotoxic metabolite GCV-triphosphate (TP) disrupting DNA synthesis to selectively kill HIV-1-expressing cells. Following proof-of-principle transfection studies, therapeutic constructs were engineered for lentivirus-mediated delivery to HIV-1-infected cells in vitro. I first optimized production of a panel of VSV-G-pseudotyped D4-targeting lentivectors for high infectious titre and low transfer plasmid carryover. I confirmed trans-splicing between HIV-1 *tat1* and *HSVtk*ΔAUG1 RNA in Jurkat T cells co-transduced with HIV-1NL4-3ΔE and therapeutic vectors. However, I found that translation of catalytically active polypeptides from internal AUGs in *HSVtk*ΔAUG1 caused dose-dependent cytotoxicity with GCV in uninfected cells. Modification of internal AUGs in D4 opt 2 effectively mitigated this major off-target effect. Based on the MTT assay, D4 opt 2 in combination with GCV reduced the viability of HIV-1NL4-3ΔE-expressing Jurkat T cells by approximately 50% with no impact on uninfected cells. For improved therapeutic potential, I replaced the promoter in D4 opt 2 with that for the human *EF1α* gene, resulting in 4.4-times more RNA payload. EF1α-driven D4 opt 2 with GCV reduced the viability of HEK293T cells expressing full-length HIV-1NL4-3 by up to 70%. I developed a panel of lentivectors delivering opt 2 with low (ScrambleV2), moderate (ScrambleV1), or high (D4) affinity predicted in silico for target HIV-1 pre-mRNA and found that the strength of interaction between opt 2 and HIV-1 correlated with the propensity for *trans*-splicing and elimination of HIV-1-expressing cells by the opt 2 cell suicide system *in vitro*. Having demonstrated that this HIV-1-targeted cell suicide system could eliminate cells actively expressing HIV-1, I next investigated its potential against latent HIV-1 in J- Lat 10.6 cells. I found LRAs exerted class and dose-dependent effects on viability which could affect the outcome of shock and kill. Despite their superior reactivation potential, NF-κB agonists made J-Lat 10.6 cells more difficult to eliminate. In contrast, the 26S proteasome inhibitor bortezomib (BTZ), known to sensitise HIV-1-infected cells to death, enhanced shock and kill. I discovered that the nucleoside analogue DNA methyltransferase inhibitor decitabine (DAC) is an adjuvant of the HIV-1-targeted cell suicide system, with DAC-TP and GCV-TP known to synergise. A ≥65% reduction in J-Lat 10.6 viability was achieved when cells were stimulated with BTZ, DAC, or a TNFα/DAC ‘shocktail’ prior to treatment with EF1α-driven D4 opt 2 and GCV. My work supports further development of our HIV-1-targeted cell suicide system against cells actively expressing HIV-1 and those chronically infected when combined with appropriate LRAs.Item Open Access Novel Approaches to the Emergency Management of HyperkalaemiaHumphrey, Toby; Humphrey, Toby [0000-0002-1084-3701]Hyperkalaemia is the term used to describe raised potassium level in the blood and, if severe, can constitute a medical emergency. Prevalence estimates have historically varied greatly depending on the population investigated with reports ranging from 1-10% in patients admitted to hospital. Management of severe hyperkalaemia (serum potassium >6.5 mmol/L) is typically undertaken in hospital with the use of intravenous insulin and dextrose as first line treatment, which requires regular monitoring, often requires repeat dosing and can expose patients to additional risk, morbidity and a prolonged length of hospital stay. Thus, there is an unmet need to develop a treatment regimen that both acts quickly and specifically enough to be of use in the acute setting but also which is safe and well-tolerated by patients. This thesis investigates the epidemiology and management of hyperkalaemia and explores clinical outcomes associated with hyperkalaemia with a view to enhancing the design of hyperkalaemia management trials by identifying clinically meaningful endpoints. This thesis was undertaken during the COVID-19 pandemic, which caused wide-spread disruption to non-COVID related research, and as a result also includes my work on the design and execution of the COVID-19 prophylaxis trial PROTECT-V. To improve understanding of the prevalence of hyperkalaemia we conducted the first systematic review (chapter two) of all reported observational studies describing the prevalence and/or incidence of hyperkalaemia. This work has provided a comprehensive published review on the prevalence and incidence of hyperkalaemia across a large number of relevant sub-groups and clarifies our understanding of risk-factors for developing hyperkalaemia. Pooled mean hyperkalaemia prevalence was 1.3% amongst the general population, 8.7% amongst adult inpatients and up to 20.7% in patients requiring renal replacement therapy. Another sub-group at risk of hyperkalaemia are users of medications that inhibit the renin-angiotensin-aldosterone system (RAASi) which are commonly prescribed as a cornerstone therapy to patients with chronic kidney disease or heart failure who already have an increased risk of hyperkalaemia. The prevalence of hyperkalaemia in RAASi users was 5.8% which rises to 12.2% amongst hospital inpatients taking RAASi. Hyperkalaemia can cause stoppages in RAASi therapy and the work described in chapter 3 which resulted in a publication, explores the adverse clinical impact of these stoppages in a large UK primary care dataset that was linked to secondary care records. The risk of hospitalisation, cardiac arrhythmia, heart failure hospitalisation and cardiac arrest were all higher amongst patients who suffered interruptions or cessation of their RAASi therapy. The fourth chapter reports the largest, published UK observational study to date of the emergency management of hyperkalaemia. Our work highlights that insulin dextrose treatment failed in over one third of patients and hypoglycaemia occurred in one in five patients. This work was used to provide clinically meaningful trial endpoints and guide the design of the HELP-K trial that is described in chapter 5. This trial aimed to provide contemporary evidence for the use of novel potassium-binding medications in the emergency setting to try and reduce the risk of treatment failure with insulin dextrose and to limit the associated morbidity of hypoglycaemia and prolonged length of hospital stay reported in chapter 4. Whilst there remains much work to be done to improve the evidence base for emergency hyperkalaemia management the work contained within this thesis helps improve the epidemiological understanding of hyperkalaemia in the real-world setting with contemporary knowledge that, I hope, will be useful to health-care providers, health policy makers and patients.Item Embargo An Investigation into the Effects of Macrophage Polarisation on Interleukin-1 ExpressionKrzyzanska, DominikaMacrophages are innate immune cells characterised by functional heterogeneity and plasticity. The prevailing dogma divides macrophages into two opposing subtypes: the pro-inflammatory M1 and anti-inflammatory M2 macrophages. Macrophages are also the main cell type that produces two powerful cytokines that drive chronic inflammation and adaptive immunity - interleukin (IL)-1α and IL-1β. Although the role of M1 macrophages as drivers of inflammation has been extensively studied, unravelling the function of M2s in inflammation still requires further investigation. The work presented in this thesis challenges the dogmatic division of M1/M2 cells as pro/anti-inflammatory, respectively. We demonstrate that M2 macrophages produced substantially more IL-1α and IL-1β following LPS treatment, compared to M0 or M1 macrophages, and we have validated these findings in vivo. This may have important implications for diseases with heightened M2 polarisation, where they could contribute to previously unrecognised pro-inflammatory, IL-1-driven phenomena. In addition, we identified novel mechanisms of IL-1 regulation in macrophages. We show that HECT E3 ligase inhibition led to dose-dependent stabilisation of IL-1α, and we also identified AREL1 and TRIP12 as prospective candidates that target IL-1α for degradation. These findings suggest that a reduced rate of IL-1 degradation resulting from defects in E3 ligase expression and/or activity might have detrimental effects in inflammatory diseases. Further investigations to identify the cause of amplified IL-1 in M2s guided us to explore TBK1 signalling, which revealed that TBK1 inhibition significantly decreased IL-1α in M2s, but increased IL-1β in M0 and M1 macrophages. We also observed that this subtype-specific effect on IL-1 was not dependent on type I interferon or IRF3 signalling. Thus, understanding the subtype and context-specific effects of TBK1 inhibition may have important implications in, for example, TBK1-driven diseases. Finally, we used mRNA sequencing to examine transcriptional changes potentially underlying IL-1α/β expression in LPS-treated M1/M2 macrophages, particularly to identify the molecular mechanism behind the heightened IL-1 expression in M2s. The analysis revealed that LPS-treated M2 macrophages retained the characteristic M2 polarisation markers and importantly, identified IGF-1 signalling as a candidate regulator of IL-1. We show that LPS-treated M2 macrophages secreted high levels of IGF-1 and that IGF-1R inhibition reduced IL-1α/β expression in M2s. Therefore, targeting IL-1 signalling in inflammatory diseases characterised by high levels of IGF-1 might be an important therapeutic strategy. To conclude, the work presented in this thesis enhances our understanding of IL-1α and IL-1β regulation in macrophages and should enable revaluation of the roles of macrophages in health and disease.Item Open Access Clustering of immune-mediated diseases using genomic dataNicholls, Katherine; Nicholls, Katherine [0000-0002-4070-1317]Studying immune-mediated diseases (IMD) yields insights into the active immune system. In this thesis I cluster patients with IMD based on RNA-seq data using two extensions to clustering, and also cluster the diseases themselves using GWAS summary statistics. I conducted an extensive study of biclustering methods and found that a Bayesian biclustering method called SSLB had best performance on simulated datasets and recovered biologically relevant biclusters in a knockout mouse dataset and a sorted blood cell dataset. Through the study I developed tools for processing and analysing biclustering results. I applied this knowledge to perform biclustering, with SSLB, of a sorted blood cell dataset containing patients with six immune-mediated diseases. Amongst the biclusters was a bicluster recovering the genes that escape X-inactivation and a bicluster capturing type 1 interferon response, enriched for samples from patients with systemic lupus erythematosus. I found that whilst it is an advantage that biclustering has sufficient complexity to describe the immune system comprehensively, this also means that the results of the biclustering are themselves complex and thus interpretation can be a real challenge. In order to study RNA-seq data when summarised using key immune gene signatures, I developed DPMUnc, an extension to a Dirichlet process Bayesian clustering model which allows the uncertainty associated with the data points to be taken into account. I was then able to cluster patients based on their average gene expression across gene signatures, for example finding the expected enrichment of lupus patients in a cluster with high expression of interferon genes. I also clustered the immune-mediated diseases themselves using GWAS summary statistics. DPMUnc separated autoimmune from autoinflammatory diseases and isolated other subgroups such as the EGPA subtypes and multiple sclerosis. This thesis thus reveals an interesting trade-off between the complexity of the data and the utility of the results. Biclustering is sufficiently complex for the heterogeneity of the data but the results are difficult to interpret. In contrast, although some information is lost by summarising into key gene signatures, it allows for more concrete analysis.Item Embargo Understanding and predicting pulmonary exacerbations in Cystic Fibrosis using Machine LearningSutcliffe, DamianCystic Fibrosis (CF) is the most prevalent, life-limiting, multi-system, genetic disorder, affecting over 100,000 individuals globally. The condition is caused by mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene, which corrupts the instructions for making the corresponding CFTR protein. In the lungs, the defective protein leads to a reduced depth to, and acidification of, the airway surface liquid, a sticky and viscous outer mucous layer, and impaired mucociliary clearance. This in turn leads to airway obstruction and chronic bacterial infection. Recurring, sudden, clinical deteriorations in respiratory symptoms - termed acute pulmonary exacerbations (APEs) - cause cumulative damage to the lungs, and are the most significant driver of mortality and morbidity in CF. However, despite this, currently little is known about their pathophysiology, nor what triggers them. The ability to accurately predict impending APEs would permit earlier treatment, reduce inflammatory lung damage, and directly benefit life expectancy. The average time to treatment varies from a few days to around a month, and so there is a significant opportunity to reduce the delay from current levels. Achieving this outcome was the driving force behind my research. First, I developed an unsupervised machine learning model (the Alignment Model) that, for the first time, was able to generate a characteristic profile of the changes in physiology and symptoms during an APE, and to define an accurate start point for exacerbations. Of particular interest was the existence of a partial interim recovery approximately 10 days after the start of the APE. By extending the model I was also able to identify three distinct classes of APE - one closely resembled the global profile, another showed declines in symptoms before FEV1, and finally one that showed signs of repeated exacerbation and a steeper decline. Second, I used the inferred exacerbation start dates from the Alignment Model to categorise the full set of study days into stable vs unstable (APE episodes). Using this training data, I developed a supervised ML model (the Predictive Classifier) that was able to predict the onset of APEs with 83.6% reliability, and on average 9.5 days earlier than current clinical practice. For this reseach, I leveraged the SmartCareCF study home monitoring data-set, and importantly, I was also able to quantify that including physiological measures in the data collection process resulted in a 51% improvement over self-reported symptom scores alone. Given the already time-consuming treatment regimen for people with CF, this is a key justification of the value of providing these additional measurements. I observed that the performance of the Predictive Classifier was negatively affected by increasing amounts of missing input data. In order to use the predictive algorithm in a clinical setting, it is critical to understand the extent to which any missing input data might have affected the predictions, and, therefore, whether any given prediction can be trusted (is safe) or not. Consequently, in my next area of research, I generated a synthetic data-set that represented the sensitivity of performance of the Predictive Classifier to the amount and pattern of missing data points. I then used this to train a separate ML model (the Safety Classifier) that was able to determine whether the APE predictions were safe or not. It achieved a PR-AUC of 89.7% and an ROC-AUC of 91.2%. Additionally, I was able to use the Safety Classifier iteratively to determine the minimum amount of data that was required to guarantee a safe APE prediction, which could be used in future studies to optimise the data collection requirement. Finally, I was able to apply my research to a new ongoing adult CF home-monitoring study (Project Breathe). The results for both the Alignment Model and the Predictive Classifier were consistent with my earlier findings. However there were three significant environmental factors that should be noted: i) The study period coincided with the broader rollout of triple modulator therapy, which I was able to show reduces the frequency of APE’s by 75%; ii) Also there was an extended period of covid-19 enforced isolation, which reduced cross-infection risk; iii) The data collection compliance was significantly lower - for the five most important measures, it was less than half that of SmartCareCF. Despite the overall data-set being nearly five times the size, there were relatively fewer usable APE events - both in absolute count (55) as well as in overall proportion (1%) - and so this reduces my confidence somewhat in the generalisability of the results. Additionally, the Safety Classifier showed that only 10% of the overall Project Breathe study days would be determined as safe to make an APE prediction which was approximately five times lower than for SmartCareCF. A material increase in data collection would be required to be able to use the algorithms in a clinical setting. Overall I was able to meet my research objectives, demonstrating there is a signal in the home measurement data that can be used to identify APEs, building an ML model that could reliably and accurately predict the onset of APEs over a week earlier than current clinical practice, and developing a framework to ensure these APE predictions are safe in the context of missing input data. I am excited to be taking the results of my research into a clinical trial later this year.Item Open Access The molecular epidemiology of Salmonella Typhi and applications for policyCarey, MeganSalmonella Typhi (S. Typhi) is a Gram-negative bacterium and the etiologic agent of typhoid fever. High rates of typhoid were historically associated with urban slums in South Asia with poor sanitation, but recent multicentre surveillance studies have demonstrated that typhoid is also a major problem of urban and rural areas in sub-Saharan Africa. Typhoid necessitates antimicrobial therapy; however, antimicrobial resistance (AMR) poses a serious threat to the effective clinical management of typhoid, particularly in South Asia, where resistance to all oral antimicrobials used to treat typhoid has been reported. New typhoid conjugate vaccines (TCVs) are highly efficacious and two have been prequalified by the World Health Organization (WHO); a small number of countries have introduced TCVs into their national immunization programs. However, many countries lack primary surveillance data to inform decision-making for TCV introduction. Here, aiming to aggregate and analyse globally representative whole genome sequencing (WGS) data to inform public health action, I conducted a genomic investigation of the global distribution and transmission dynamics of AMR S. Typhi. With colleagues from Pakistan and India, I conducted phylogenetic analyses of the first molecularly confirmed azithromycin-resistant S. Typhi isolated in Pakistan and India and put them into context with contemporaneous azithromycin-resistant isolates. I found that single point mutations in acrB (efflux pump) were emerging independently in these settings, potentially associated with selective pressure. AMR in S. Typhi is associated with the H58 lineage, which arose recently before becoming globally dominant within a relatively short time. Given this lineage’s association with AMR, I sought to investigate how, when, and where it emerged. Working with collaborators from the United Kingdom Health Security Agency (UKHSA), we performed phylogenetic and phylodynamic analyses using S. Typhi from returning travellers to the UK between 1980 and 1995. This dataset, which contained the earliest described H58 S. Typhi, indicates that the prototype H58 organisms were MDR, and that they emerged spontaneously in India in 1987 and became radially distributed throughout South Asia. These early organisms were associated with a single long branch and possessed mutations associated with increased bile tolerance, suggesting that the first H58 organism was generated during chronic carriage. The subsequent increased use of fluoroquinolones led to several independent mutations in gyrA, leading to decreased fluoroquinolone susceptibility. The apparent ability of H58 to acquire and maintain AMR genes continues to pose a threat, suggesting that TCVs should be deployed across South Asia to minimise the potential emergence of new drug-resistant variants. I worked with a broad range of collaborators to establish the Global Typhoid Genomics Consortium (GTGC) to encourage sharing of S. Typhi genomic data and standardised metadata, and to enable analysis and visualisation of these data in context to support public health decision-making. Here, I engaged with groups sequencing S. Typhi genomes to analyse and draft a global update paper; the final dataset contained >13,000 sequences. This work provides an updated overview of AMR and genotype distribution and illustrates key international transmission events, with the aim of informing TCV introduction decision-making and treatment guidelines. To create a translational link between laboratory scientists, genomics experts, clinicians, and policy makers, I outlined use cases for WGS in surveillance, diagnostic development, clinical management, and informing vaccine introduction as well as typhoid control. I hope to advance this work by fostering broader participation in the GTGC, and by encouraging funding support for the generation and analysis of S. Typhi WGS data. I also intend to create greater visibility for, and advocate for the value of, such data in conversations with country decision-makers and other policymakers via the WHO.Item Open Access FAMIN in dendritic cells biochemically restrains T cell primingRamshorn, KatharinaFAMIN is a purine nucleoside enzyme that was first identified in genome-wide association studies (GWAS) as increasing risk for Crohn’s disease (CD). Defects in the enzyme that result in loss of catalytic activity are the only known monogenic cause of Still’s disease, a severe autoinflammatory condition that predisposes for the risk of developing macrophage activation syndrome (MAS). MAS can be triggered by common viral infections and result in hyperactivation of T lymphocytes. The work presented in this thesis builds on the discovery that FAMIN-sufficient mice are protected against influenza A infection, with dendritic cell (DC)- dependent excessive activation of CD8+ T cell responses in their FAMIN-deficient counterparts. Loss of FAMIN activity in DCs was shown to result in increased priming of both CD4+ and CD8+ T cells both in vitro and in vivo, resulting in enhanced antigen-specific cytotoxicity, IFNg secretion, and T cell expansion. Here, we discovered that FAMIN controls the pace of membrane trafficking and DC antigen processing to restrain T cell priming via a cytosolic NADH/ NAD+-dependent mechanism. We describe wide-reaching metabolic defects resulting from loss of FAMIN activity, including in the core pathways of glycolysis and TCA cycle activity, as well as upstream changes in glucose, fatty acid and amino acid metabolism. Notably, depressed rates of glycolysis and oxidative phosphorylation DCs lacking FAMIN function were not responsible for increased T cell priming capacity. Instead, we find that FAMIN balances flux through adenine-guanine nucleotide interconversion cycles to maintain the cytosolic NADH/ NAD+ ratio. We found that FAMIN additionally regulates T cell priming by DCs directly through the enzyme’s purine nucleoside phosphorylase (PNP) activity, converting exogenous hypoxanthine to inosine that acts on the T cell adenosine 2A receptor (A2AR). In summary, FAMIN is a multifunctional purine nucleoside enzyme enabling flux through purine interconversion pathways highly integrated with cytoplasmic and mitochondrial metabolism. In DCs, FAMIN acts as a biochemical checkpoint to restrain T cell priming and prevent autoimmunity and autoinflammation.