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Antibiotic resistance (ABR) is a global problem impacting humans, animals, and environmental health, causing great economic loss and threatening food security. Whole genome sequencing (WGS) is a valuable tool when characterizing ABR bacteria from a one-health perspective, however, few studied have investigated evidence for direct sharing between one-health compartments. Studies by the OH-STAR project have used WGS to characterize Escherichia coli resistant to two critically important antibacterials (CIAs); third-generation cephalosporins (3GCs) and fluoroquinolones (FQs) from humans and cattle within a 50 x 50 km study area. Here we investigated 600 dogs in the same study region using WGS and phylogenetic analysis to characterize ABR E. coli in dogs and to identify evidence of sharing between dogs, humans and/or cattle. We also carried out risk factor analysis for excretion of CIA resistant E. coli in dogs. In addition, we characterized CIA resistant E. coli from zoo mammals in the study region. Faecal samples from 600 dogs and 167 samples from zoo mammals were processed to select ABR E. coli which were then subjected to WGS. We found evidence for sharing of CIA resistant E. coli between humans, cattle, and dogs in our study region, and identified "geographical associations", where dogs living in rural areas carried E. coli more similar to cattle than those living in urban areas. Risk factor analysis demonstrated a strong association between feeding raw meat and excretion of 3GC-R and FQ-R E. coli by dogs, confirming farm animal to dog transmission of these bacteria. We also found evidence for farm animal to human transmission of phylogroup A/B1 E. coli, accounting for 11% of human BSI within our study region in 2020, identified due to their carriage of farm animal specific ABR genes. Furthermore, we demonstrated the carriage of nitrofurantoin resistant E. coli by dogs, which may also be due to raw feeding, suggesting an unexpected farm animal reservoir for such E. coli. The zoo environment was a hotspot for evolution and circulation of CIA resistant E. coli, but there was no evidence of escape into the local community. These studies highlight the importance of ABR surveillance from a one-health perspective, which can be beneficial for the control of ABR bacterial prevalence.

Invasive bacteria are likely to encounter macrophages during their course of pathogenesis, especially if infection progresses to disease. The study of these interactions is crucial for our understanding of pathogenesis and the identification of therapeutic strategies to combat infection. In this thesis, I utilise a number of models to study and gain insight into these interactions. I demonstrate that hypervirulent strains of Klesbsiella pneumoniae (Kp) replicate within hepatic Kupffer cells (KCs) and confirm that replication is intracellular by utilising a modified gentamicin-protection assay on isolated murine KCs. I show that this replication precedes an influx of neutrophils which subsequently aggregate to form clusters that could develop into pyogenic liver abscesses characteristic of these Kp strains. Utilising an in vivo mouse model of invasive pneumococcal disease with infection occurring during the active or rest phase, I demonstrate that differential susceptibility of mice to Streptococcus pneumoniae infection is dependent on a diurnal phenotype within splenic marginal zone macrophages (MMMs). MMMs harbour a differential permissiveness to intracellular pneumococcal replication, with increased permissiveness during the active phase resulting in higher splenic bacterial burden, earlier and more pronounced release of intracellular bacteria into the systemic circulation, and higher levels of systemic pro-inflammatory cytokines. Finally, I utilise an ex vivo human spleen perfusion model to study the localisation of S. pneumoniae with human tissue-resident macrophages. I find that the majority of bacteria are associated with red pulp macrophages (RPMs), with an extremely low number localising with capillary sheath-associated macrophages (CSAMs). Interestingly, I also record an increase in the abundance of pneumococci within RPMs and perifollicular CSAMs, indicating potential replication within human splenic macrophages as observed within murine splenic macrophages. Taken together, my results emphasise the importance of the study of bacteria-macrophage interactions, especially for the identification of novel therapeutic interventions.

L. monocytogenes is a Gram-positive bacterium that is found ubiquitously throughout the world. It is a major foodborne intracellular pathogen and can cause a range of human diseases. It invades the cells of mammalian hosts, escapes the intracellular vacuole, replicates in the cytosol and spreads to neighbouring cells using actin-based motility. After breaching the intestinal barrier L. monocytogenes causes systemic infections in immunocompromised people. The intracellular lifecycle of L. monocytogenes is under the control of the regulator PrfA which regulates major virulence genes. It is widely characterised and well-studied. However, many studies on L. monocytogenes have used population level snapshot data which masks the kinetics of single cell interactions. Individual host cells and individual L. monocytogenes cells are heterogenous and infection outcomes depend on these heterogenous interactions. By designing a live cell imaging model, designed to specifically study single-cell interactions between bacteria and host cells, this study revealed novel host-pathogen interactions during infection. Successful replicative invasions are a rare event, only 9.1% (+-1.2%) of host cells are susceptible to infection and only 0.4% (+-0.05%) of the bacterial population can successfully form replicative invasions. L. monocytogenes forms aggregates in response to a proteinaceous host factor >10 kDa with the aggregates being PrfA regulated and ActA mediated. This is a novel strategy the bacteria use to increase invasion success rate through multiple invasion events. Aggregating bacteria were 3.5-fold more invasive than a non-aggregating mutant, formed 4-fold more replicative invasions in HeLa and 10-fold more in primary HUVEC cells. Additionally, in a competition assay the wildtype outcompeted the non-aggregating mutant by 10-fold. Aggregates were also shown to preferentially interact with and invade cells in the G2/M phase and this was due to InlB-Met interactions, as Met is upregulated during the G2/M phase. Host cells depleted of Met showed a 2.5-fold reduction in the number of associated bacteria. RNA-Seq analysis of L. monocytogenes treated with the causative host factor upregulated PrfA regulated virulence genes, and genes involved in iron uptake and glycerol metabolism. Analysis of gene expression between the wildtype and the non-aggregating mutant showed that aggregation downregulates genes involved in cationic peptide resistance. However, the process of aggregation did not upregulate the PrfA virulence regulon. Overall, this study has demonstrated the value of live imaging single cell interactions between human cells and pathogens and how this can uncover novel interactions that lead to a greater understanding of the biology of infection.

Iron deficiency and malaria are two major public health problems with largely overlapping epidemiology, and there is a complex relationship between iron and malaria. Iron deficiency can protect from malaria infection, whereas iron supplementation can increase the risk, and tissue iron loading can disrupt disease tolerance. Immune cells also need iron to support their function and iron deficiency can disrupt immunity. However, it is not known how tolerance or the immune response to malaria infection and vaccination is affected by iron deficiency. In this thesis, the effects of cellular iron deficiency on the host's response to malaria infection and vaccination were investigated. Recently mosquito transmitted P. chabaudi parasites were used to infect mice with a mutation in transferrin receptor 1 (TfrcY20H/Y20H mice), which causes decreased cellular iron uptake. We discovered that TfrcY20H/Y20H mice had higher P. chabaudi parasite burden, but comparable malaria induced anaemia. The disease phenotype of TfrcY20H/Y20H mice was also altered, as they had increased weight loss and exacerbated kidney damage, but attenuated liver damage compared to wild-type mice. In addition, disrupted cellular iron uptake had a range of inhibitory effects on the immune response to P. chabaudi. The expansion of key effector cells was reduced during the acute stage of infection and the germinal centre response was impaired in TfrcY20H/Y20H mice. Moreover, TfrcY20H/Y20H lymphocytes cultured in vitro had impaired proliferation and effector function, which could be completely rescued by iron supplementation. We also demonstrated that decreased cellular iron uptake weakened the immune response to blood-stage malaria vaccine RH5.1 and that mini-hepcidin induced hypoferremia disrupted the response to experimental liver-stage vaccine AdHu5-Py9. Hence, we have demonstrated for the first time that cellular iron deficiency has the potential to alter malaria disease tolerance and to impair the immune response to malaria infection and vaccination.

For over a century, it has been apparent that tumours behave somewhat like wounds that fail to heal. A number of studies have established several cellular and molecular parallels between wound healing and cancer, and a key one of these is inflammation, with both wounds and tumours triggering an inflammatory response that plays an essential role in the outcome of these tissue lesions. As a consequence, any potential therapy with the capacity to alter the wound/tumour inflammatory response is now considered clinically relevant. In this regard, infection is known to modulate the inflammatory response to both cancer and wound healing. Serendipitous findings extending back to those of Coley in the early 1900s, have shown cancer remissions occurring upon treatment with heat-killed bacteria, suggesting that infection can enhance, or prime, the host immune response to better recognise and eradicate cancers. During tissue repair, there is evidence that some aspects of the wound inflammatory response may be activated by microbial antigens, although, of course, if infection becomes overwhelming, this can lead to chronic, non-healing wounds. Whilst the longer term consequences of infection on cancer and wound healing have been partially explored, rather little is known about the precise cellular and molecular mechanisms by which bacteria-induced inflammation can influence these consequences, for example, how infection alters the behaviour of inflammatory cells and their cellular interactions with the tissue insult. Here I used the translucent larval zebrafish (Danio rerio), which has relatively recently become a powerful model of inflammatory-related human conditions, in combination with high-resolution confocal imaging and a novel bespoke automated cell tracking and cell-cell interaction software, to investigate how inflammatory cells behave and interact with repairing wounds and early cancer cells in sterile versus infection conditions. Using this approach, I showed that infection, delivered either systemically or locally, induced changes in the number and behaviour of inflammatory cells recruited to acute wounds and to pre-neoplastic cells, and these alterations led to a significant delay in wound healing, and to a reduction in the number of pre-neoplastic cells, which correlated with increased numbers of pro-inflammatory (tnfα-positive) macrophages. I expanded these studies into adult zebrafish where I observed that infection-induced inflammation inhibits the growth of established human-like melanomas. Finally, I tested a more therapeutically viable anti-cancer strategy by using a novel particle-based protocell system to selectively load macrophages and neutrophils, via their natural foreign body phagocytic abilities, with immunostimulatory, tumour-suppressing molecules (anti-miR223 or R848). I showed that anti-miR223-loaded protocells or R848-loaded protocells can effectively "reprogramme" macrophages (and neutrophils) towards a more pro-inflammatory, and thus anti-cancer state, in zebrafish (and cultured human macrophages), which led to a significant reduction in both larval pre-neoplastic burden and in the size and number of adult melanomas also.

Cystic fibrosis (CF) is a hereditary disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The lung disease is, however, also associated with dysregulation of the epithelial sodium channel (ENaC) which leads to dehydrated airways predisposing the individual to chronic cycles of infection and inflammation. ENaC is activated by trypsin-like channel activating proteases (TLPs), inhibition of which increases airway surface liquid volume and improves mucociliary function of primary human airway epithelial cells (hAECs) grown at air-liquid interface (ALI).

People with bronchiectasis and cystic fibrosis (CF) frequently suffer from recurrent infective pulmonary exacerbations (PEx) which lead to a progressive loss of lung function, reduced quality of life and increased morbidity and mortality. Determining how bacterial load and community composition changes with time may improve our understanding of the natural history of these chronic airway diseases. The aims of this thesis were to (i) to determine the psychometric properties of HRQol questionnaires in bronchiectasis, (ii) to measure the longitudinal change in bacterial load and community composition in patients with bronchiectasis when clinically stable and following antibiotic treatment of a PEx, (iii) to determine the antimicrobial susceptibility of P. aeruginosa and H. influenzae isolates cultured from sputum samples collected from patients with bronchiectasis and (iv) to determine changes in total bacterial and P. aeruginosa load following antibiotic treatment of a PEx in people with CF who received either microbiome directed orempirical antibiotic treatment. Results showed that all HRQol questionnaires had good internal consistency and short-term test-retest reliability in at least some domains. Responsiveness was reasonable with the meta-analysis showing a difference between the treatment and placebo effect. In people with bronchiectasis, no statistical difference was apparent in total bacterial, P. aeruginosa and H. influenzae load and microbiome composition during periods of clinical stability and following antibiotic treatment of a PEx. However, P. aeruginosa load was associated with lower lung function and H. influenzae load was associated with increased systemic inflammation .P. aeruginosa isolates were susceptible to tobramycin, ceftazidime and ciprofloxacin, while H. influenzae isolates were susceptible to ampicillin, ciprofloxacin and co-amoxiclav but resistant to azithromycin. In the CF study, no difference was observed in total bacterial and P. aeruginosa load between patients who received microbiome directed and patients who received empirical antibiotic treatment. Resilience in bacterial load and microbiome composition was observed in the lungs of patients with chronic airway diseases with no change observed during periods of clinical stability and following antibiotic treatment of a PEx.

Acute liver failure is a rare and devastating condition. In the Western world, paracetamol overdose is the leading cause of acute liver failure. Although there have been improvements in the management of patients with acute liver failure - most notably the acceptance of emergency liver transplantation as a therapeutic option - mortality rates remain high. The studies in this thesis focus on advancing our understanding of the short and long-term outcomes in patients with acute liver failure. In Scotland, all patients with acute liver failure are managed in the Scottish Liver Transplant Unit (SLTU). This thesis describes changes in the aetiology of acute liver failure, changes in the management of acute liver failure and changes in short-term outcomes in patients admitted to the SLTU over a 22-year period. In acute liver failure, the generally accepted paradigm is that if the patient survives to hospital discharge without requiring emergency liver transplantation, then they return to a level of health similar to that of the general population. The studies in this thesis disprove this theory, and demonstrate that spontaneous (transplant-free) survivors of acute liver failure have a significantly increased risk of death after discharge compared with the general population. Spontaneous survivors of acute liver failure also have a significantly increased risk of readmission to hospital. These observations challenge current thinking in the field of acute liver failure, and suggest that formal follow up strategies for these patients should be devised. It remains challenging for clinicians to determine prognosis in patients with acute liver failure, and to be confident in identifying which patients will survive without emergency liver transplantation. New prognostic scoring systems have been devised. Work in this thesis externally validates an American prognostic tool designed to predicted short-term transplant-free survival in a UK population of patients with acute liver failure. Although of some value in predicting prognosis, no single tool should be used in isolation to make clinical decisions about an individual patients' need for liver transplantation. Novel treatments, accurate prognostic scoring tools and formal follow up strategies are needed to improve short and long-term outcomes in patient surviving acute liver failure with and without emergency liver transplantation.

In recent years, e-cigarettes have been marketed as an alternative to cigarette smoking that is less damaging to health. However, there is a lack of scientific evidence to support this assertation that they are a safer alternative to cigarettes. This study aimed to evaluate the effect of e-cigarettes on key respiratory pathogens which included S. aureus (SA), P. aeruginosa (PA), H. influenzae (HI), S. pneumoniae (SP) and M. catarrhalis (MCAT). Results from this study have shown that neat e-liquids can reduce bacterial viability with particular flavours causing significant reductions in bacterial viability. It was found that e-liquids contained numerous chemical compounds some of which had antimicrobial properties. The greater the number of chemical compounds present in the e-liquid the greater the reduction in bacterial viability. E-liquid induced antimicrobial pressure affected HI, MCAT and SP isolates to a greater degree than PA and SA isolates. It was discovered that both vaped and neat e-liquids increased biofilm formation in PA and SA isolates whilst decreasing biofilm formation in HI, SP and MCAT isolates. The vaping of the e-liquids indicated that PGVG and flavours had significant effects on biofilm formation in bacteria. Additionally, e-cigarette vapour extract (ECVE) exposure modified the expression of two PA virulence factors, pyoverdine and pyocyanin. The chemical components of e-liquids components can act as environmental stressors resulting in modification of important bacterial virulence factors. Exposing respiratory bacteria to ECVE or cigarette smoke extract (CSE) before infection was found to result in significant increases and decreases in the release of IL-8 and IL-6 and activation of NF-κB in human cells. The degree of effect on cell inflammation appeared to be dependent on the bacterial isolate and whether it was exposed to ECVE or CSE. The exposure of respiratory isolates to ECVE or CSE can modulate the host immune response to infection. Overall, the study shows that e-cigarette exposure can induce changes in key respiratory pathogens associated with chronic lung diseases. These changes could impact host-bacterial interactions and may increase bacterial persistence and inflammatory potential.

The majority of antibiotics (approximately 80%) are prescribed by general practitioners (GPs) in the UK. Reducing inappropriate antibiotic prescribing is an important aim of antibiotic stewardship programmes (ASP) in order to maintain the efficacy of existing antibiotics for future generations. Analysis of longitudinal data from research databases in the UK including CPRD, SAIL Databank, and NHS digital open prescribing, can provide an insight into the effectiveness of current interventions, reports and guidelines aimed at reducing inappropriate prescribing. The aim of this thesis was to evaluate factors influencing antibiotic prescribing in primary care in the UK at patient- and practice level. Broadly, three research questions were considered: (1) To what extent does varying antibiotic prescribing for common infections influence infection-related complications? (2) Are patient characteristics and risk factors being used to inform antibiotic prescribing? (3) Are there any consistent antibiotic prescribing trends among English GPs and what factors influence this? Chapter 2 found the effect of physician preference antibiotic prescribing on the risk of infection-related complications among antibiotic users compared with non-antibiotics users. Chapter 3 demonstrates an inverse association between lower levels of practice level antibiotic prescribing for common infections and higher rates of infection-related complications by comparing high and low prescribing GP practices. In Chapter 4, this association was further explored and showed a large variability in absolute risk reductions for patients with an incidental common infection and an independence of predicted risk with GPs propensity to prescribe antibiotics. While Chapters 2-4 present the effect of antibiotic prescribing on infection-related complications for patients in primary care (RQ1/RQ2). These analyses do not provide insight into antibiotic changes through time and the potential of identifying GP practices for targeted antibiotic guidance. Thus, Chapter 5 explored the complex heterogeneity of antibiotic prescribing trends among English GPs and identified seven clusters with similar prescribing trends over time, which were not associated with deprivation scores (RQ3). Notable changes in prescribing trend were detected in early 2015, mid 2016, and late 2017. This thesis has the potential to improve understanding of the potential effects of reduced antibiotic prescribing in primary care and inform the implementation of more targeted identification of GPs and patients who will benefit most from a fundamental change in antibiotic prescribing behaviour.

Antimicrobial-resistance (AMR) is a global health concern. Data to support estimates of the AMR health burden are important to guide policy makers in developing appropriate interventions. The objective of this thesis is to improve the understanding of the epidemiology of AMR in low and middle-income countries (LMICs), where data is scarce. A prospective surveillance study on hospital-acquired infection (HAI) was conducted to estimate excess mortality attributable to AMR infection in a LMIC. Evaluating all types of infections and seven priority bacteria, I estimated that 16.0% of deaths involving bacterial infections were attributable to AMR. This is higher than is observed in high-income countries (HICs). One of the barriers in sharing AMR data in LMICs is a lack of expertise, time, and resources to analyse the data and write reports. I developed an open-access, offline, and easy-to-use application to fill in this gap. The tool was tested in seven hospitals in seven countries. AMR surveillance reports from those hospitals were shared on an open-access data repository platform and each has a digital object identifier that can be appropriately cited when re-used. It is known that low blood culture usage can bias the estimates of health burden of AMR. However, the magnitude of this effect is largely unknown. I performed a retrospective study with bootstrap sampling to illustrate the potential magnitude of biases in the estimates of AMR prevalence and incidence rates due to low blood culture utilisation rates. Finally, using a large retrospective data set, I applied a counterfactual framework to estimate the impact of delays in concordant antibiotic treatments. Attempts were made using observational data to emulate randomised controlled trials that are otherwise unethical to perform. This thesis describes work which generated data, provided evidence, and created a new tool for quantifying the health burden of AMR infections in LMICs.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host immune response to infection, characterised by oxidative stress and a 'cytokine storm'. The NLRP3 inflammasome is one of the first pathways activated in sepsis and leads to the activation of the pro-inflammatory cytokines IL-1β and IL-18, and gasdermin-D by caspase-1. Melatonin is a potent antioxidant and anti-inflammatory. 6-hydroxymelatonin (6OHM) is the main metabolite of melatonin and is apotent antioxidant and anti-inflammatory. The aim of this study was to investigate the effects of melatonin and 6OHM on the NLRP3 inflammasome in human immune cells, in conditions mimicking sepsis. For this purpose, the effects of melatonin and 6OHM were investigated in amacrophage model of inflammasome activation and in neutrophils extracted from the blood of healthy volunteers. The in vitro study showed that the NLRP3 inflammasome requires two signals to produce IL-1β, which have different roles in the activation process. In particular, K+ efflux appears to be essential for caspase-1 activation and, therefore, for the activation of the NLRP3 inflammasome. However, both signals are required for the maturation and release of IL-1β.Melatonin did not affect any of the inflammasome components or pyroptosis and reduced IL-1βlevels independently from NFκB. 6OHM reduced caspase-1 and pyroptosis, but not IL-1β levels, suggesting that caspase-1 is not the only enzyme capable of activating IL-1β. Melatonin and 6OHM inhibited NFκB activation independently from SIRT1 and may have pro-inflammatory properties. Sepsis is a life-threatening organ dysfunction caused by a dysregulated host immune response to infection, characterised by oxidative stress and a 'cytokine storm'. The NLRP3 inflammasome is one of the first pathways activated in sepsis and leads to the activation of the pro-inflammatory cytokines IL-1β and IL-18, and gasdermin-D by caspase-1. Melatonin is a potent antioxidant and anti-inflammatory. 6-hydroxymelatonin (6OHM) is the main metabolite of melatonin and is apotent antioxidant and anti-inflammatory. The aim of this study was to investigate the effects of melatonin and 6OHM on the NLRP3 inflammasome in human immune cells, in conditions mimicking sepsis. For this purpose, the effects of melatonin and 6OHM were investigated in amacrophage model of inflammasome activation and in neutrophils extracted from the blood of healthy volunteers. The in vitro study showed that the NLRP3 inflammasome requires two signals to produce IL-1β, which have different roles in the activation process. In particular, K+ efflux appears to be essential for caspase-1 activation and, therefore, for the activation of the NLRP3 inflammasome. However, both signals are required for the maturation and release of IL-1β.Melatonin did not affect any of the inflammasome components or pyroptosis and reduced IL-1βlevels independently from NFκB. 6OHM reduced caspase-1 and pyroptosis, but not IL-1β levels, suggesting that caspase-1 is not the only enzyme capable of activating IL-1β. Melatonin and 6OHM inhibited NFκB activation independently from SIRT1 and may have pro-inflammatory properties.

A chronic wound is any wound that does not progress through the normal stages of healing. These wounds can take months or even years to resolve and, in many cases, will never fully heal. Chronic wounds are highly prevalent, with up to 3% of people over 60 and as many as 5% of people over 80 suffering from a chronic wound. Patients with chronic wounds often suffer from chronic pain, limited mobility, social isolation, and poorer mental health. Not only are these wounds highly traumatic for patients, but they are also a significant burden for the NHS, with their treatment costing approximately £5.3 billion per annum. There are several reasons why a wound might fail to heal. The most common cause, however, is bacterial infection, and one of the most common causative organisms is Pseudomonas aeruginosa. When present in a wound, P. aeruginosa grows primarily in the form of a biofilm, a collection of bacterial cells encased in a self-produced polymer matrix. This matrix makes the bacteria highly resistant to antibiotic treatment, making wound infections very difficult, and often impossible, to eradicate. The formation of bacterial biofilms is governed by a process known as quorum sensing, in which bacteria produce and detect small signalling molecules known as autoinducers. This form of communication allows the cells to coordinate complex, population-wide, behaviours such as virulence factor expression, and biofilm formation. Several methods of disrupting quorum sensing, for example, via enzymatic degradation of the signalling molecules, have been proposed. One method competitive inhibition wherein a compound which competitively binds to the bacterial quorum sensing signalling receptors, effectively blocks the cell's ability to take part in quorum sensing. Many synthetic and natural compounds have been shown to be capable of such competitive inhibition but perhaps the most well studied of these are furanones. Furanones are one such family of naturally occurring chemicals which are structurally similar to the signalling molecules used by P. aeruginosa. This allows furanones to competitively bind to the quorum sensing signal receptor, thus, minimising biofilm formation by P. aeruginosa populations. This thesis aimed to develop a novel method of delivering furanones to chronically infected wounds. This work demonstrated that, due to their inherent instability, a minimally crosslinked polymer aerogel was the most appropriate system for the controlled delivery of these compounds. Further, this thesis has demonstrated that, whether they are applied early in the biofilm formation process or to mature biofilms, two furanones, 4-hydroxy-2,5-dimethyl-3(2H) furanone (HDMF) and sotolon, are capable of significantly reducing biofilm biomass in vitro. The final experimental chapter of this thesis aimed to develop a novel, clinically representative, in vitro model of chronic wound biofilm that could be used to assess furanone loaded aerogels as potential antibiofilm wound therapeutics. The developed model was validated by showing efficacy of clinically relevant wound dressings and, subsequently, was used to demonstrate that sotolon-loaded aerogels showed great potential for reducing biofilm in chronic wounds. This thesis concludes that, though complex to include in pharmaceutical formulations, naturally occurring furanones have excellent potential to be used as antibiofilm wound therapeutics. Furthermore, when incorporated into a minimally crosslinked poly (vinyl alcohol) aerogel material, these compounds retain their activity and can be delivered to clinically representative biofilms where they reduce biofilm biomass.

Respiratory infections are of significant concern for individuals with chronic inflammatory lung diseases and there is an unmet need for novel antibiotics. Antimicrobial peptides possess antibiotic potential due to their antimicrobial and/or immunomodulatory properties. The project aim was to assess the therapeutic potential of several snake-derived AMPs and to evaluate the suitability of peptide delivery by nebulisation. In in vitro assays the peptides exhibited antimicrobial activity and/or anti-inflammatory effects with minimal cytotoxicity. We considered the impact of lung proteases on the peptides. Peptide incubation with CF sputum and protease inhibitors revealed the peptides were susceptible to proteolytic cleavage. Cleavage predictions highlighted neutrophil elastase (NE) as most likely responsible. All peptides were susceptible to cleavage by NE, and several peptides retained function following NE-incubation. Mass spectrometric analysis and cleavage predictions informed truncated derivative development, aiming to reduce production cost and potential immunogenicity. SnE1 and derivatives were tested as therapeutics for acute lung infection using a murine model of chronic lung disease. SnE1N treatment reduced lung bacterial load, whereas, SnE1 did not. SnE1 increased protein and IL6 concentration in the BAL fluid and reduced the number of immune cells, possibly due to cytotoxic effects, indicating increased inflammation; whereas, SnE1N did not. In nebulised delivery suitability testing, nebulisation did not alter peptide function. Sn1b and C2 were shown to be respirable in an adult spontaneously breathing model and all test peptides were found to be respirable in a mechanically ventilated breathing model. Assessment of nebulised droplet distributions suggested all test peptides would likely reach the lower airways. To conclude, AMPs remain an attractive candidate for antibiotic development. In this study, we found that SnE1N reduced the bacterial burden during acute lung infection in mice with chronic lung inflammation, warranting further investigation. Additionally, peptide delivery via nebulisation is likely feasible.

Neutrophils are major effectors of acute inflammation and microbial defence. Their infiltration and migration in injured tissue are critical for the inflammatory response. They are often observed navigating in a highly co-ordinated and directed manner leading to their aggregation at the target site of infection. This self-organised cell gathering is referred to as swarming. It is known that neutrophil swarming is driven by autocrine attractant production, notably of the lipid leukotriene B4 (LTB4). The decision to release attractants at the single-cell level is important and impacts the magnitude of the entire immune response. However, the precise mechanisms triggering this decision remain unclear. In this study, I employed in vivo imaging of zebrafish larvae to reveal the molecular processes that trigger the release of LTB4 and initiation of swarms. I developed a 2-Photon laser wound assay to elicit and visualise neutrophil swarming. A major limitation in previous studies of neutrophil swarms was the lack of tools to understand cell signalling dynamics during the response. To overcome this, I generated a new biosensor to probe for calcium levels in neutrophils as it correlates with the production of chemoattractants. Using this new tool, I revealed that neutrophils clustering at the target inflammatory site are experiencing sustained high calcium elevation. Using a new probe to follow the production dynamics of LTB4, I demonstrated that the rise of intracellular calcium promotes the biosynthesis of this key attractant. I further demonstrated that these calcium fluxes are triggered upon contact with necrotic tissue. This prompted me to interrogate the damage molecules driving these calcium signals in neutrophils. I discovered that the calcium fluxes were mediated by ATP that binds gated ion channels (P2X1) leading to rapid intracellular calcium uptake. Surprisingly, I found that live neutrophils can also trigger this calcium flux in other neutrophils upon mutual contact. Using chemical and genetic inhibition, I found that connexin-43 (Cx43) hemichannels, through their ability to release ATP, enable amplification of the calcium signal leading to chemoattractant production and subsequent neutrophil recruitment. I concluded that activation of LTB4 synthesis is a group decision reached via Cx43-dependent communication in pioneer clustering neutrophils. As Cx43 inhibition significantly reduced neutrophil aggregation at the target site, I investigated if this inhibition could have consequences for wound defence. For this, I developed a wound colonisation assay with Pseudomonas aeruginosa. Using this approach, I showed that Cx43 was crucial for the protection of wounds from opportunistic bacteria. Finally, I designed and generated an optogenetic tool to manipulate LTB4 biosynthesis dynamics in vivo. I demonstrated the effectiveness of this tool in immortalised cells and zebrafish larvae. The unique features of this tool make it very useful for a wide range of research applications on signalling dynamics. In conclusion, I have shown that by reinforcing damage signalling, Cx43 channels coordinate attractant biosynthesis in pioneer neutrophils. This generates an effective chemoattractant gradient source and promotes targeted aggregation and defence. This study, therefore, reveals a new mechanistic principle of collective behaviour that could be exploited in future pathological research.

During viral infections, the presence of viral-derived dsRNA triggers a translational shut-off response affecting translation of both host and viral-derived mRNAs. This response is mediated by the phosphorylation of Eukaryotic initiation factor 2α (eIF2α) by the double stranded RNA binding kinase PKR. However, the underlying mechanisms of this process are still not fully understood. Intriguingly, host antiviral genes escape this translational shut-off, suggesting a regulatory control mechanism that selectively allows the expression of essential self-defence genes. We have identified Interleukin enhancer binding factor 3 (ILF3) as an essential host factor required for efficient translation of the antiviral IFNB1 mRNA and a large subset of interferon-stimulated genes (ISGs) which are critical for establishing an effective antiviral state in the cell. By combining polysome profiling and high-throughput sequencing, we furthermore show that ILF3 is also required to trigger robust changes in transcription, alternative splicing and translation that arise from the activating the antiviral response. Our data suggests that ILF3-mediated regulation of RNA metabolism is necessary to establish a strong and vigorous cellular antiviral response.

Organ transplantation remains the best treatment for many patients with end organ disease. However, there is a discrepancy between organ supply and demand. Many donor organs are not used for transplantation because of risk attributes in the deceased donor ranging from infectious risks to specific risks associated with the cause of death. The aims of this thesis were to identify the number of deceased donors with risk attributes and then quantify the risks associated with using organs from such donors. This information was then used to provide evidence to the transplant community on which to base their decisions on using organs from such donors. This thesis used data in the UK Transplant Registry, which prospectively collects data on the clinical characteristics and follow-up of all donors and transplant recipients in the UK, and the Potential Donor Audit, which is a prospectively populated registry including all patients who die in UK critical care units of donation age. The main findings of this thesis were that there are a large number of deceased donors with risk attributes, in particular donors with increased risk behaviour for blood borne viral disease or with hepatitis C virus infection, whose organs are currently not used for transplantation, but that could safely be used. The thesis also describes that the transmission of meningitis/encephalitis from deceased donors to transplant recipients is a rare but serious complication of transplantation, but that transplantation of usually excellent organs from such donors should not be contraindicated. Different transplant centres display marked variations in practice in using organs from donors with different risk attributes, but centres that display greater risk appetite in using organs from higher risk donors do not have worse transplant outcomes compared to centres with lower risk appetites. Transplantation of organs from higher risk donors can result in excellent transplant outcomes. Wide variations in practice are seen across the UK, and to address this the UK aide memoire has been developed.

Preterm birth is the leading cause of neonatal mortality and morbidity. The uterine cervix is key in maintaining a healthy pregnancy. Excisional procedures for the treatment of cervical intraepithelial neoplasia (CIN) are associated with preterm birth (PTB), but underlying mechanisms are yet to be described. Intrauterine infection is involved in 40% of PTB and cervical damage is likely to facilitate ascending infection with vaginal bacteria. The aim of this thesis was to establish and characterise an in vitro and in vivo model of cervical damage to study its interplay with ascending infection. For this, the surfactant N-9 was used as a damage agent. N-9 was found to reduce the endocervical epithelial cells' viability and compromise their epithelial permeability in vitro. When vaginally administered in pregnant C57Bl/6 mice, N-9 disrupted the structural integrity of the cervical epithelium, caused an influx of neutrophils and resulted in increased cell proliferation in the basement membrane of the cervix. Similar where the findings in the vaginal epithelium. However, N-9-induced epithelial damage had no effect on timing of delivery or pup survival. Following vaginal infection with a luciferase-expressing Ureaplasma parvum, mice previously treated with N-9 exhibited higher rates of ascending infection, with increased bioluminescence signal in the upper reproductive tract. They also demonstrated higher bacterial titres in the amniotic fluid and higher bacterial product copy numbers in the reproductive tissues. This resulted in increased preterm birth rates among mice in this group compared to vehicle-treated controls. Infection with Ureaplasma parvum was characterised by an inflammatory response in the uterus, the placenta and the fetal membranes with increased expression of the proinflammatory cytokines TNFa, IL-1b, CXCL-1 and CXCL-2. This effect may be mediated by TLR2, the expression of which was also shown to increase accordingly. Overall, these findings suggest that cervical epithelial damage facilitates ascending infection. This is a potential mechanism explaining the higher PTB incidence among women treated for CIN. The robust model of cervical epithelial damage during pregnancy described in this thesis can be used to study the barrier function of the cervix and its interplay with ascending infection in future studies.

Human rhinoviruses (HRVs) are the most common causes for symptomatic respiratory infections, and have been linked to severe respiratory conditions in children, and in immunocompromised and elderly individuals. Currently there are no specific treatments or vaccination available for HRV infections and novel antiviral therapeutics are urgently required. Cathelicidins are a well-characterized family of Host Defence Peptides (HDP) with potent antibacterial, antiviral and immunomodulatory functions. This study investigates the antiviral activity of the human cathelicidin LL-37 against human rhinovirus together with the capacity of the peptide to modulate inflammation and host cell death in rhinovirus infection. We demonstrate that LL-37 has significant antiviral activity against HRV1B when the virus is exposed to peptide prior to cell infection, and when cells are infected prior to LL-37 treatment, indicating that LL-37 exerts its effects by directly targeting the virus and/or acting on host cells reducing their susceptibility to infection. Cathelicidin-mediated inflammatory pathway modulation was measured via quantification of IL-8, IL-6 and CCL5 gene expression and protein release. Our data indicates that LL-37 can significantly reduce pro-inflammatory gene expression and protein release induced by HRV1B infection in bronchial epithelial cells, when the peptide is exposed to HRV prior to infection. However, LL-37 was shown to increase HRV-induced inflammatory gene expression and protein release by bronchial epithelial cells, when cells were infected prior to LL-37 treatment. This data indicates that the cellular microenvironment and the context of cathelicidin exposure could determine the direction of cellular response to infection. We further demonstrate that LL-37 suppresses induction of apoptotic cell death in HRV-infected cells, which may represent a novel immunomodulatory role for LL-37 in the context of this infection. Taken collectively, these data suggest that cathelicidins represent an exciting therapeutic avenue for rhinovirus infections, via targeting of virus particles and modulation of host cell responses to infection.

Mycobacterium abscessus is an emerging pathogen with infections increasing worldwide, especially among Cystic Fibrosis (CF) patients. During my PhD, I studied key aspects of the biology of M. abscessus spp.; particularly, I studied host-pathogen interactions, antimicrobial resistance mechanisms, and genetic determinants of virulence. First, I performed phenotypic characterization of M. abscessus spp. clinical isolates obtained from CF patients classified according to subspecies and clustering. I found clustered isolates, representing probable transmission events, were phenotypically distinct from sporadic isolates and showed adaptation phenotypes associated with chronic lung infection, such as enhanced intracellular survival, increased antibiotic resistance, and metabolic adaptations to the host environment. Second, I assessed the role of an inserted element containing an active methyltransferase in M. a. massiliense. Infection experiments with an isolate containing the inserted element (BIR1049wt) and a knockout strain (BIR1049Δ1809078_1815649) showed decreased survival of BIR1049Δ1809078_1815649 within macrophages. RNAseq analysis showed a distinct gene expression pattern between both isolates, with a number of mycobacterial virulence factors upregulated in BIR1049wt. Third, I studied heritable non-mutational antibiotic resistance mechanisms in M. abscessus to linezolid and clofazimine. For both antibiotics, I found clonal isolates of M. abscessus spp. with varying susceptibilities and different gene expression patterns, suggesting transcriptional regulation of antibiotic resistance. Mutation- mediated resistance to clofazimine was also found due to mutations in two transcriptional regulators predicted to regulate efflux pumps. Last, I evaluated the potential of repurposing a kinase inhibitor (compound H) in clinical trials for the treatment of cancer and CF, to treat M. abscessus infection. I found compound H enhanced killing of intracellular M. abscessus in macrophages through stimulation of autophagy and lysosomal function. I further studied over 60 chemical analogues of compound H in order to find a more active and specific compound for M. abscessus infection.

The innate immune system is the first line of defence against infection. It is comprised of physicochemical barriers and a variety of cell types including macrophages and dendritic cells. Pathogens express specific pathogen associated molecular patterns (PAMP) which are recognised by pattern recognition receptors (PRR) on macrophages to initiate an innate immune response. Gram-negative bacteria such as Salmonella enterica serovar Typhimurium express a range of bacterial PAMPs recognised by Toll-like receptors (TLRs) including lipopolysaccharides (LPS) recognised by TLR-4 and lipoproteins by TLR-2. The activation of TLRs results in activation of nuclear factor κB (NF-κB) to drive transcription of mRNA coding for pro-inflammatory proteins such as tumor necrosis factor α (TNF-α) and pro-interleukin (IL) 1β. Myeloid cells also possess intracellular PRRs including the nucleotide-binding domain and leucine-rich repeat (NLR) family. NLR family CARD domain- containing protein 4 (NLRC4) and NLR family pyrin domain-containing protein 3 (NLRP3) are the main NLRs engaged in recognising S. Typhimurium infection, leading to formation of the inflammasome. The inflammasome is a macromolecular complex assembled in the cytoplasm, and usually contains a NLR, the structural protein apoptosis-associated speck-like protein containing a CARD (ASC) and effector enzymes such as cysteine-dependent aspartate-directed protease (caspase) -1 and caspase-8. This structure is responsible for processing the cytokines pro- IL-1β and pro-IL-18 to their mature form and is involved in triggering a pro-inflammatory process of cell death termed pyroptosis. The formation of the inflammasome therefore results in cell death and secretion of proinflammatory cytokines which play important roles in controlling infections. Inflammasome activity must be tightly coordinated, as its dysregulation is associated with a variety of auto-inflammatory and auto-immune diseases. The signalling events leading to inflammasome assembly are poorly understood and the molecules involved in fine-tuning its activity are only beginning to be discovered. The aim of this thesis was to discover new molecules involved in inflammasome activation and/or in keeping its activity in check. To achieve this goal, I performed S. Typhimurium infection assays in primary bone marrow derived macrophages (BMDM) derived from C57BL/6 mice wild type (WT) and compared the resulting cellular viability, intracellular bacteria counts and IL-1β production to that of BMDMs derived from C57BL/6 mice lacking proteins involved with, or suspected to be involved with, innate immune activity. Amongst the proteins I studied, caspase recruitment domain 9 (CARD9) inhibited inflammasome-mediated IL-1β production. Multiple independent genome-wide association studies link this protein to inflammatory pathologies such as Crohn's disease, but its role in canonical inflammasomes was largely unexplored. To investigate how CARD9 inhibits inflammasome-mediated IL-1β production I have conducted assays in WT and Card9-/- BMDMs, including stimulation of specific NLRs with their purified ligands, infection with bacterial strains deficient in NLRC4 activation, and infection assays in presence of pharmacological inhibitors. By employing these approaches, I observed that CARD9 has a negative role on NLRP3-dependent IL-1β production. Specifically, in response to activation of the NLRP3 by Salmonella infection, CARD9 negatively regulates pro-IL-1β transcription, and decreases IL-1β processing by inhibiting spleen tyrosine kinase (SYK)-mediated NLRP3 activation and represses caspase-8 activity in the inflammasome. CARD9 expression is suppressed in the course of S. Typhimurium infection which may act as a mechanism to increase IL-1β production during the infection. In conclusion, I have established a connection between CARD9 and IL-1β production by the canonical NLRP3 inflammasome and elucidated some of the mechanisms involved in this process. I have also found evidence that other proteins are likely to be involved in inflammasome regulation and the elucidation of their roles will be addressed in future studies.

Human cytomegalovirus (HCMV) is a widespread human herpesvirus pathogen and prototypical member of the β-herpesvirus subfamily. Like all herpesviruses, the virus establishes a lifelong latent infection following host exposure, which has the potential to reactivate periodically and contribute to recurrent disease processes. In individuals with weak or compromised immune systems, such reactivation can lead to profound pathology. Understanding how latent infections are maintained is important for uncovering how HCMV causes disease. The study of viral genes that are expressed during latent infection grants insight into how latency is regulated and how it could be therapeutically targeted. To that end, this project has sought to evaluate the functional significance of one such viral gene termed LUNA in the context of latency. In models of experimental latent infection based on primary myeloid cells, levels of viral gene transcription were found to be significantly reduced following infection with LUNA deletion mutant viruses, consistent with corresponding observable changes in post-translational histone modifications over the viral promoters of latency-associated genes. Additionally, using luciferase reporter systems, latency-associated viral gene promoters became activated in response to the expression of wild-type LUNA. Together, these findings argue for a role of LUNA in regulating viral gene expression during latent HCMV infection. One possible mechanism by which LUNA may fulfil its role is by targeting cellular ND10 structures, known intrinsic inhibitors of herpesvirus gene expression, for disruption. In support of this, latently infected cells were found to be devoid of ND10, a phenotype that was recapitulated by the direct expression of wild-type LUNA. Furthermore, mutation studies confirmed the identification of a novel deSUMOylase activity encoded by LUNA that was responsible for mediating ND10 disruption. Use of a catalytically inactive LUNA mutant in transcriptional analyses of latent infection also generated similar results as with the LUNA deletion viruses. Overall, these data support the hypothesis that LUNA serves as an important regulator of viral gene expression during latency, which is likely linked to its ability to target ND10 structures for disruption, thus raising the possibility that inhibition of deSUMOylation may serve as a novel therapeutic strategy to target latent HCMV infection.

Systemic inflammation is thought to be an important driver in chronic neurodegeneration. During systemic infection, the inflammatory status of the periphery is communicated to the brain and conserved sickness behaviours initiated. However, in the context of dementia the same inflammatory stimulus might trigger delirium. Delirium is a severe, transient neuropsychiatric condition characterised by altered levels of arousal, inattention, cognitive deficits and psychoses. Delirium and systemic inflammation exacerbate the trajectory of pre-existing dementia, and are associated with increased risk of future dementia. Accumulating experimental studies suggest microglia are “primed” by chronic neurodegeneration, such that a subsequent inflammatory insult – central or systemic – induces an increased inflammatory response which manifests as exaggerated sickness behaviours. To date there have been no studies of microglial priming in the context of pure tau pathology, without amyloid pathology, and none investigating acute sickness behaviour in such a model. The overarching aim of this thesis is to address this gap in the literature and further our understanding of the interactions between systemic inflammation, neuroinflammation and neurodegeneration in the context of tauopathy. The P301S mouse over-expresses human mutant tau protein under the Thy1.2 promoter. It develops hyperphosphorylated and insoluble tau accumulations and progressive neuronal loss. Consequently, P301S mice develop progressive hind limb paralysis. This study identified the horizontal bar task, a test of motor control and coordination, conducted at weekly intervals from 8-22 weeks of age, as a non-invasive measure of disease progression. In addition, a detailed temporal profile of pathological hallmarks at 8, 9, 10, 11, 12, 16 and 20 weeks of age was determined. Key results presented here demonstrate progressive, superficial neuronal loss in the cortex of P301S mice, with associated astrogliosis and surprisingly this occurs in the absence of apparent cortical microgliosis. In stark contrast, there is progressive microgliosis in the spinal cord of P301S mice. On this background, lipopolysaccharide (LPS), a chemical moiety found on the outer surface of gram-negative bacteria, was used to mimic a systemic bacterial infection. P301S mice and C57BL/6 control mice were injected, at 10 or 16 weeks of age, intraperitoneally with 500 μg/kg LPS or saline and were monitored in the following hours and weeks. Acutely, P301S mice showed signs of an exaggerated, longer lasting sickness response. Importantly, exaggerated acute symptoms extended beyond those typically associated with sickness behaviour; LPS induced an exaggerated acute impairment of horizontal bar performance in P301S mice and not C57BL/6 mice – a function which is known to be impaired in P301S mice later in disease. Impairments were age-dependent in terms of timing of injection. These data suggest an interaction between acute infection and existing CNS vulnerability leading to acute neurological dysfunction that is not a feature observed in sickness in a normal animal. LPS-injected P301S mice also showed, again age-dependent, increased rate of decline in motor performance compared with controls. There was no evidence of microglial priming in P301S mice. LPS caused an acute increase in AT8-positive phospho-tau however this did not persist until end stage. At 22 weeks of age there was significant disease-associated cortical neuronal loss in the vehicle-injected P301S mice, and additional superficial cortical neuronal loss in LPS-injected P301S mice and control mice. There was significant IBA1-positive microgliosis in the spinal cord of P301S mice at end stage which was further increased in LPS-injected P301S mice. Taken together these data indicate a clear and clinically relevant interaction between systemic inflammation and tau-associated neuropathology with acute and long-term functional consequences. In the absence of evidence of microglial priming, future work will explore potential mechanisms.

Bacteria are enclosed by a peptidoglycan sacculus, an exoskeleton-like polymer composed of glycan strands cross-linked by short peptides. The sacculus surrounds the cell in a closed bag-like structure and forms the main structural component of the bacterial cell wall. As bacteria grow and divide, cell wall remodelling by peptidoglycan hydrolases results in the release of peptidoglycan fragments from the sacculus. In Gram-negative bacteria, these fragments are efficiently trapped and recycled. Gram-positive bacteria however shed large quantities of peptidoglycan fragments into the environment. For nearly five decades, Gram-positive bacteria were thus assumed not to recycle peptidoglycan and this process has remained enigmatic until recently. In this thesis, the occurrence and physiological role of peptidoglycan recycling in the Gram-positive pathogen Staphylococcus aureus was investigated. S. aureus is an important pathogen, and is becoming increasingly resistant to many antibiotics. Through bioinformatic and experimental means it was determined that S. aureus may potentially recycle components of peptidoglycan and novel peptidoglycan recycling components were identified and characterised. Though disruption of putative peptidoglycan recycling in S. aureus appears not affect growth or gross morphology of this bacterium, potential roles for peptidoglycan recycling in cell wall homeostasis and in virulence were identified. This is to my knowledge the first demonstration of a potential role of peptidoglycan recycling in either of these aspects of bacterial physiology in any Gram-positive bacterium. This is an important step forward in understanding the basic biology of Gram-positive bacteria, and in understanding the mechanisms of virulence in S. aureus. Future study of this process in S. aureus and other Gram-positive bacteria promises to reveal yet further facets of this process and its functions, potentially leading to the identification of novel therapeutic approaches to combat infections.

Prostate cancer is the second most common cancer in men. Studies now indicate a strong association of the disease with chronic bacterial infection, which may ultimately induce oncogenesis. It has been shown that P. acnes is the predominant organism isolated from prostatectomy samples and is significantly associated with organ inflammation. Further evidence suggests that P. acnes is able to chronically infect the prostate gland and in vitro experiments clearly indicate its potential to drive oncogenic changes. This thesis investigates the oncogenic potential of specific prostate-derived P. acnes phylogenetic types, with the ultimate aim of developing risk stratification approaches for the development of the disease. In vitro infection models were used to compare the effects of different phylogenetic types of P. acnes on prostate epithelial cells. Soft agar assays and quantitative PCR (qPCR) were used to investigate the molecular expression profiles associated with endothelial-mesenchymal transition (EMT) and inflammation during bacterial infection. Cells infected with phylotype IA1 were able to form colonies in soft agar assays, indicating cellular transformation. Additionally, qPCR results showed a decrease in the expression of the EMT marker E-cadherin. These findings were supported by immunostaining experiments. Furthermore, qPCR expression analysis revealed that phylotype IA1 infection induces changes suggestive of apoptosis resistance, increased proliferation and androgen-independent growth, all characteristics of advanced treatment-resistant cancer. In contrast, cells infected with type III strain exhibited expression changes consistent with anti-cancer behaviour, including increased apoptosis and androgen-dependent signalling. These findings highlight the strain-specific response to infection and suggest the attractive possibility that infection with P. acnes may be a modifiable risk factor for prostate cancer. Since some phylotypes of P. acnes may be oncogenic, while others are cancer-protective, the development of a screening test to identify the presence of infection may therefore prove valuable to stratify patients at risk of oncogenesis.

Hip replacement surgery has experienced a dramatic evolution in recent years supported by the latest developments in many areas of technology and surgical procedures. Unfortunately complications that follow hip replacement surgery remains the most challenging dilemma faced both by the patients and medical experts. The thesis presents a novel approach to segment the prosthesis of a THR surgical process by using an Active Contour Model (ACM) that is initiated via an automatically detected seed point within the enarthrosis region of the prosthesis. The circular area is detected via the use of a Fast, Randomized Circle Detection Algorithm. Experimental results are provided to compare the performance of the proposed ACM based approach to popular thresholding based approaches. Further an approach to automatically detect the Obturator Foramen using an ACM approach is also presented. Based on analysis of how medical experts carry out the detection of loosening and subsidence of a prosthesis and the presence of infections around the prosthesis area, this thesis presents novel computational analysis concepts to identify the key feature points of the prosthesis that are required to detect all of the above three types of complications. Initially key points along the prosthesis boundary are determined by measuring the curvature on the surface of the prosthesis. By traversing the edge pixels, starting from one end of the boundary of a detected prosthesis, the curvature values are determined and effectively used to determine key points of the prosthesis surface and their relative positioning. After the key-points are detected, pixel value gradients across the boundary of the prosthesis are determined along the boundary of the prosthesis to determine the presence of subsidence, loosening and infections. Experimental results and analysis are presented to show that the presence of subsidence is determined by the identification of dark pixels around the convex bend closest to the stem area of the prosthesis and away from it. The presence of loosening is determined by the additional presence of dark regions just outside the two straight line edges of the stem area of the prosthesis. The presence of infections is represented by the determination of dark areas around the tip of the stem of the prosthesis. All three complications are thus determined by a single process where the detailed analysis defer. The experimental results presented show the effectiveness of all proposed approaches which are also compared and validated against the ground truth recorded manually with expert user input.

In this thesis I develop and implement “antibody landscapes”, a method to profile immunity against a pathogen as a function of antigenic differences between a range of strains. Theoretically applicable to any antigenically variable pathogen and measurement of immunity, the work here focusses on antibody-mediated immunity against the A/H3N2 influenza subtype. Applying the methodology to study annual serum samples from individuals monitored for influenza infection over a period of six years, patterns of influenza immunity were found to be remarkably distinct and maintained almost unchanged over time in the absence of influenza exposure. Upon infection, the initial response is strikingly antigenically broad, including responses against viruses far beyond the extent of cross-reactivity observed after a primary infection. Analysis of two vaccination cohorts, one receiving an antigenically advanced vaccine strain and one a more typical vaccine strain choice, revealed many of the same patterns of response as seen with infection. Antigenically advanced vaccination generated greater responses against later strains but surprisingly, due to equivalent boosting of prior immunity, this came at no cost to responses generated against contemporary or older strains. Exploring in more detail the development of immunity over time, analysis of a cohort of children demonstrated that - in contrast to adults with diverse exposure histories - antibody responses to a first infections were remarkably similar in pattern and magnitude. Interestingly, for second infections, although post-infection antibody titres against circulating strains were comparable to those after first infections, overall cross-reactivity of the response against future antigenic variants appeared to be diminished. The findings here underline the significant role prior-immunity plays in affecting the response to new exposures and the importance of understanding it. An important conclusion is that by failing to account for it, current approaches to influenza vaccine strain selection may be suboptimal and pre-emptive vaccine strain updates may improve overall vaccine efficacy where immunity to current strains already exists in the population. Building on the work presented here should help to optimise strain choice and vaccine efficacy even further.

Infections are a common co-morbidity in Alzheimer's disease (AD), and evidence suggests that infections can exacerbate neuroinflammation and increase cognitive decline in AD patients. In AD, immune changes are observed both in the central nervous system (CNS) and in the rest of the body. However, only a few studies have investigated immune responses to infection in AD. Here, two extensively studied infections, Toxoplasma gondii (T. gondii) and Trichuris muris (T. muris) were used to investigate infection in AD. T. gondii is a protozoan parasite which is common globally, including in the developed world where AD cases are increasing dramatically. Infection with T. gondii starts in the gut, before becoming systemic and then infecting the CNS, where the parasite forms a chronic cyst infection. In contrast, T. muris is a nematode parasite, which remains localised to the gut. Notably, T. gondii is known to alter neuroinflammation and behaviour. T. gondii forms cysts preferentially in the areas of the brain commonly affected by AD, such as the hippocampus, which therefore makes it an interesting model to study co-morbidity. AD is often associated with advanced age. As we age, our immune system declines, and an important unanswered question is whether age impacts on the immune response to infection. This is of particular significance when considering chronic infections such as T. gondii, which require immune surveillance to prevent parasite recrudescence. Therefore, the aim of this thesis was to investigate infection in AD by determining: whether the immune response to an infection is altered in AD; whether the immune response to an infection in AD differs with age; what the effects of infection are on neuroinflammation, pathology and behaviour in AD; what are the effects of chronic infection with T. gondii. Immune responses to infection were altered in both the 3xTg-AD and the APP PS1 mouse models of AD, including increased inflammation and weight loss in AD mice following infection. Although older (eleven to twelve-month-old) 3xTg-AD mice showed some alterations in cytokine responses following infection, overall there were no major difference compared to younger (five to six-month-old) animals. Additionally, infection was found to alter neuroinflammation in both 3xTg-AD and APP PS1 mice, though differently. In 3xTg-AD mice, microglia activation increased following infection with T. gondii and T. muris, showing that infection did not need to be in the brain to alter neuroinflammation. In APP PS1 mice, a decrease in microglia activation occurred after infection with T. gondii, which was accompanied by an increase in IL-1alpha production and increased amyloid beta levels in APP PS1 mice following infection. However, no changes were found in behaviour following infection with T. gondii or T. muris in AD mouse models. Finally, chronic T. gondii infection was investigated in the TgF344-AD rat, which was established as a suitable AD model with both amyloid and tau pathology in which to study chronic infection. This work adds to a growing body of literature to suggest that infections are detrimental to AD patients, and that future measures to decrease morbidity could focus on further study of infections in AD, and the development of strategies to better prevent infections in AD patients.

Background Antibiotic resistance is an important societal health issue. The greatest risk factor for developing a resistant infection is antibiotic use. Almost 75% of all antibiotics in the UK are prescribed in the community, and mostly for acute respiratory tract infections (RTIs). Yet, the majority of RTIs are self-limiting, viral and do not need antibiotic treatment, especially in young children. While the effects of antibiotic-resistant infections have been widely studied in hospitals (e.g. the MRSA 'superbug'), we know less about how antibiotic-resistant infections affect people in the community, even though this is where most antibiotics are prescribed. There is also widespread public misconception about antibiotic use and resistance despite several high-profile, multimillion antibiotic awareness campaigns. This is important to address because consultation behaviour and expectations for antibiotics are a significant determinant of antibiotic use in the community. Methods Three studies were conducted for this thesis. First, a systematic review and meta-analysis to assess the evidence of the impact of antibiotic resistance for patients with common infections in the community; second, a retrospective analysis of routinely collected primary care data to examine the relationship between antibiotic exposure and antibiotic 'response failure' in preschool children presenting with acute RTIs; third, a qualitative interview study to explore parents' perceptions and understanding of antibiotic use and resistance when they consider consulting in the community with their preschool child who has a respiratory tract infection. Results Antibiotic resistance significantly impacts on patients' illness burden for common infections in the community. Patients who presented in community health care settings with antibiotic-resistant E. coli urinary tract infections and S. pneumoniae respiratory tract infections were more likely to experience delays in recovery after antibiotic treatment. From routinely collected primary data (2009-2016), preschool children receiving two or more antibiotic courses in the previous year for acute RTIs had greater likelihood of antibiotic 'response failure' to treatment for subsequent acute RTIs compared to children that had received no previous antibiotics. When interviewing parents of young children, most parents were quite reticent about antibiotics for their children. However, very few considered antibiotic resistance as a possible harm of antibiotics. Parents thought their families were at low risk of antibiotic resistance because their families were 'low users' of antibiotics and did not perceive any association between their individual consumption of antibiotics and the development and spread of antibiotic-resistant bacteria in the community. They wanted future antibiotic awareness campaigns to have a universal message relevant to their families that fit into their daily lives. Conclusions The findings challenge the perception that antibiotic prescribing and resistance in the community poses little or no additional risk to patients in the community, or is remote from everyday prescribing decisions. Rapid diagnostic tests and other prognostic tools need to be promoted and evaluated to better identify patients who might need an antibiotic, and reduce the risk antibiotic response failures. Clinicians and parents should exercise caution about whether further antibiotics for acute RTIs are likely to be beneficial in those children who have received two or more antibiotic prescriptions for acute RTIs during the previous 12 months. Incorporating this into clinical practice guidelines and decision-support systems will help clinicians and parents consider a non-antibiotic strategy for acute RTIs. Future guidelines, campaigns and interventions around antibiotic resistance should tailor initiatives to outcomes that patients and clinicians in the community can relate to and slot into their daily lives. More research is needed to evaluate the impact of other common infections in primary care, and determine the relative contribution of antibiotic resistance to patients not responding to antibiotic treatment for common infections.

Heart failure and infection are recognised complications of cardiac pacing. Right ventricular (RV) pacing is established to have detrimental effects on myocardial function and is associated with a broad spectrum of left ventricular dysfunction, from mild pacing-induced ventricular dysfunction (PIVD) to more severe manifestations such as pacing-induced cardiomyopathy (PICMP) and heart failure in patients with preserved myocardial function at the index implantation. In view of this consideration, there is a clear need for a strategy to identify a priori patients who are likely to develop PIVD with identification of pre-symptomatic LV dysfunction which can be targeted by upgrade to biventricular pacing to prevent progression of LV dysfunction. Infection is another recognised complication of cardiac implantable electronic device (CIED) implantation. Early diagnosis of infection is essential in order to avoid spread of infection and late-infective complications. In this thesis we examined the clinical utility of two different non-invasive imaging modalities for (i) the detection of pacing-induced ventricular dysfunction (PIVD) and (ii) the early diagnosis of CIED infection. In a prospective observational study of pacemaker recipients with high-grade atrioventricular block and preserved baseline left ventricular ejection fraction (LVEF >55%), one-month global longitudinal strain (GLS) was significantly reduced in patients who would subsequently develop PIVD and PICMP. Further analysis indicated that a one month GLS of ≤14.5 had high predictive accuracy for identifying patients who would subsequently develop PIVD at 12 months and in a multivariate model one-month GLS ≤ 14.5 was identified as an independent predictor of PIVD and PICMP. In a second study we examined the diagnostic utility of 18F-fluorodeoxyglucose positron emission tomography with computed tomography ( 18F-FDG PET/CT) in the early diagnosis of CIED-generator pocket infection (CIED-GPI) in patients where, at initial presentation, it was unclear if their symptoms were due to infective or non-infective pocket complications. 18F-FDG uptake in the region of the generator pocket was expressed as a semi-quantitative ratio (SQR, defined as the maximum count rate around the CIED divided by the mean count rate between normal right and left lung parenchyma). 18 F-FDG activity was significantly higher in cases that required extraction compared to those that did not and controls with high diagnostic accuracy to differentiate infected from non-infected devices demonstrated. The studies undertaken in this thesis demonstrate the clinical potential for two novel imaging modalities in the diagnosis of pacemaker complications. Firstly, that one-month, but not pre-implant GLS has high diagnostic accuracy for predicting the development of PIVD and that 18F-FDG PET/CT has high diagnostic accuracy in the early diagnosis of CIED-GPI, whereby 18F-FDG PET/CT was shown to be better than clinical judgement in differentiating infected from non-infected devices at initial presentation.

The persistence of biofilms in hospital settings are associated with Healthcare Associated Infections (HCAI), causing increased morbidity, mortality and healthcare costs. The resistance of biofilms against commonly used hospital disinfectants has been well reported. Metal and metal oxide nanoparticles (NP) such as silver (Ag), copper (Cu), zinc oxide (ZnO) and copper oxide (CuO) exhibit antimicrobial properties against various pathogens. Methods: Biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus in a Centre for Disease Control (CDC) biofilm reactor and a 96 well plate was compared. A three stage approach including Minimum Biofilm Reduction Concentration (MBRC), R2 values and log(10) reductions was used to assess the efficacy of Ag and ZnO NPs both alone and in combination against P. aeruginosa and S. aureus biofilms. Atomic Absorption Spectroscopy (AAS), Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) was used to further assess the antimicrobial ability of the metal and metal oxide NPs. The prevention of P. aeruginosa and S. aureus adherence on Ag and ZnO thin film coating on silicon (Si) surfaces was also investigated, as well as icaC, ebpS and fnbB gene expression in S. aureus biofilms. Results: The CDC biofilm reactor demonstrated to be the most effective method for P. aeruginosa and S. aureus biofilm production in comparison to 96 well plates, with lower standard errors of the mean (SE) and higher replicability. Individual MBRC of ZnO and Ag NPs in suspension were 256 and 50 µg/ml for P. aeruginosa and 16 and 50 µg/ml for S. aureus respectively. The concentrations in combination were reduced by at least a half, with concentrations of 32/25 µg/ml of ZnO/Ag NPs in suspension resulting in a significant (p ≤0.05) reduction of 3.77 log(10) against P. aeruginosa biofilms and 8/12 µg/ml of ZnO/Ag NPs in suspension resulted in a 3.91 log(10) (p ≤0.05) against S. aureus biofilms. Both combinations showed an additive effect. Time point analysis confirmed that a 24 hour treatment is vital for any significant (p ≤0.05) antimicrobial activity. AAS data suggested that the Ag+ ions quenched Zn2+ ions, therefore the antimicrobial efficacy of the combination is mainly due to Ag+ ions. Damage of the biofilms from Ag and ZnO NPs was observed in the SEM imaging and energy dispersive X-ray (EDX) analysis confirmed the adherence of Zn and Ag within the biofilms. CLSM imaging showed dead (red) cells of P. aeruginosa and S. aureus biofilms throughout the depth of the biofilm. P. aeruginosa formation was reduced by 1.41 log(10) and 1.43 log(10) on Ag and ZnO thin film coatings respectively. For S. aureus, a reduction of 1.82 log(10) and 1.65 log(10) was obtained for Ag and ZnO coating respectively. Only low levels of ribonucleic acid (RNA) were achieved so no further gene analysis could occur. Conclusion: Reductions of ≥3 log(10) were observed for P. aeruginosa and S. aureus biofilm treatment with ZnO/Ag NP suspensions. It can be concluded that the ZnO/Ag NP suspensions had greater antimicrobial activity than Ag and ZnO coated surfaces owing to large concentrations of Ag+ and Zn2+ ions acting upon the biofilms. The slower release of ions from coated surfaces suggest an inadequate concentration of ions in the media, which are therefore unable to prevent biofilm formation as rapidly as NP suspensions, however provide a sustained release of ions over time. The results from this investigation propose that Ag and ZnO NPs in suspension could be a potential alternative to disinfectants for use in nosocomial environments against P. aeruginosa and S. aureus biofilms.

Intestinal dwelling parasites live in close association with the complex microbiota that inhabit our intestinal tracts. The intestinal helminth, Trichuris muris, depends on these bacteria for egg hatching and successful establishment of infection within the epithelium of the caecum and colon. Infection causes significant alterations to the host intestinal microbiota, including a decrease in bacterial diversity and shifts in proportions of certain bacterial groups. This is accompanied by a decrease in Foxp3+ regulatory T cells and changes to the metabolic potential of the host microbiota, consequently impacting host health. However, the factor(s) driving these changes and the existence and role of its own intestinal microbiota is unknown. Infection of C57BL/6 and immunodeficient SCID mice with a high dose (~ 200 embryonated eggs) and a low dose (~ 20 embryonated eggs) of T. muris was used to determine the impact of worm burden and the adaptive immune system on the host intestinal microbiota, in comparison to naïve controls. Microbiota analysis was performed by 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) and Illumina sequencing. This revealed that infection-induced microbiota changes were dose dependent and high level infection caused an increase in the Bacteroidaceae and Enterobacteriaceae families, independently of the host adaptive immune system. Development of a surface sterilisation protocol enabled the internal T. muris microbiota to be analysed by 16S rRNA gene DGGE and fluorescence in situ hybridisation (FISH). The resulting data indicated that T. muris requires its own diverse intestinal microbiota that is derived from, but distinct to, that of its host. A core microbiota is selected and maintained by the parasite regardless of the surrounding host microbiota. The parasite microbiota is important for its fitness, shown in vitro using an antibiotic motility assay and in vivo using germ free (GF) mice. Furthermore, infection with T. muris causes a significant reduction in caecal butyrate concentrations and consequently a decrease in the expression of butyrate transporters in caecal tissue. Interestingly, the T. muris microbiota is able to produce the short-chain fatty acid (SCFA) butyrate, which the parasite is unable to make itself yet secretes into its local environment. Together these strategies promote the long term survival of T. muris within the intestinal niche, adding a new level of complexity to the interaction between the pathogen, the host and their respective microbiotas that underpins successful chronic nematode infection.

Nontyphoidal Salmonella (NTS) causes invasive, and frequently fatal, disease in African children. The burden of disease secondary to NTS reflects inadequacy of Salmonella-control strategies in Africa, with expanding antibiotic resistance, and no licensed anti-NTS vaccine. The delivery of improved interventions to prevent, diagnose, and treat invasive NTS (iNTS) infection, will be facilitated by an improved understanding of the biological determinants of susceptibility to iNTS, including host genetic factors. To identify host genetic determinants of iNTS disease, we performed a GWAS and replication analysis of NTS bacteraemia in African children. This analysis identified and validated a common genetic variant in STAT4 associated with increased iNTS risk. To characterise the function of the NTS-associated STAT4 variant, we utilised a genotype-selectable bioresource of healthy European adults and samples from African children with iNTS disease. In these experiments, the risk genotype at STAT4 is associated with reduced STAT4 RNA expression in stimulated leukocytes, and reduced IFNγ production in both ex vivo stimulated natural killer cells and in the serum of African children with acute NTS bacteraemia. To validate genetic variation suggestively associated with NTS bacteraemia in the GWAS, NTS-associated loci with evidence of regulatory function were prioritised for functional characterisation. Using in vitro models of intracellular Salmonella infection and RNA interference, I characterise the role of a candidate NTS-susceptibility determinant, EVI5L, in Salmonella infections. Finally, applying a pathway enrichment analysis to the NTS bacteraemia GWAS demonstrated that NTS-associated genetic variation in African children is enriched for methionine salvage enzymes. I further investigate the potential for host-pathogen interaction in this pathway, generating and characterising Salmonella mutants deficient in methionine metabolism. Taken together, these data represent the first unbiased assessment of genetic susceptibility to iNTS disease in unselected populations. These results have important implications for the design of Salmonella-control strategies for use in Africa.

In recent years, a new threat to barley crops has emerged and gained substantial attention due to its rising economic importance. Relatively little is known about the infection strategy and development of Ramularia leaf spot (RLS) disease on barley. Therefore the overall aim of this project was to increase the understanding of fundamental biology and life cycle of the causal agent, R. collo-cygni. Chapter 2 describes the horizontal transmission of the fungus on barley. Both field and transgenic R. collo-cygni isolates expressing GFP and dsRed fluorescent reporter markers were utilised to visualise the infection progression in living host tissues by various light and confocal microscopy. The existence of a previously unknown structure called stomatopodium (infection peg), involved in the penetration of stomata, was demonstrated. The fungus initially exhibited symptomless epiphytic growth, extending above epidermis and connecting the hyphal aggregates inside substomatal cavities and subsequent initial sporulation. However, during the transition into symptomatic phase, the organised intercellular growth of hyphae into the mesophyll was observed. This hyphal network was involved in the production of asexual spores demonstrating that the raprture of epidermal layer was responsible for local necrosis observed for RLS. In addition to barley, several other speculated R. collo-cygni hosts have been used to verify their compatibility to the pathogen. In chapter 3, a whole plant inoculation assay was developed to investigate the mode of the fungal seed-borne transmission by using GFP expressing strain of the fungus. It is shown here for the first time that the vertical transmission is systemic, involving symptomless colonisation of embryo and closely resembled the mode of dissemination observed for Neotyphodium species, mutualistic fungal endosymbionts on grasses. The impact of fungal infection on seed germination ability was also examined that revealed no significant difference between clean, moderate and high levels of R. collo-cygni DNA. Chapter 4 represents an attempt to discover and analyse the sexual development in R. collo-cygni. As a first step to understand the sexual reproduction cycle in this apparently asexual species, the genetic structure of the mating system was characterised by using PCR-based techniques which demonstrated the heterothallic nature of the fungus. The defined population of R. collo-cygni field isolates was then screened for the presence of the discovered mating type idiomorphs (mat) to determine the frequencies of the mating types in the defined R. collo-cygni populations. The segregation ratio of mat1 and mat2 close to 1:1 indicated a frequent sexual reproduction. In order to verify the existence of functional sexual stage in R. collo-cygni, potential sexual development was induced using the potentially compatible isolates and a comprehensive analysis was undertaken by correlative use of light-, confocal- and low temperature scanning electron microscopy. Two types of multicellular bodies were observed and described. First was the speculated Asteromella stage (male donor) that carries spore-like spermatia. The second structure initially resembled sclerotia that in only a few instances developed into perythecium/ pseudothecium that appeared to carry the sexual spores, ascospores enveloped in asci. Chapter 5 demonstrates the role of rubellin toxin in symptom development by using autofluorescence phenomenon. The structure of putative molecular machinery involved in rubellin biosynthesis was addressed by using bioinformatics approaches and the complete R. collo-cygni genome sequence. A gene cluster encompassing several components of other known secondary metabolite biosynthesis pathways, such as that of dothistromin and aflatoxin, was found and putative protein function of the genes is hypothesised.

Introduction: Emerging pathogens (EP) in cystic fibrosis (CF) include organisms that have infected individuals with CF for many years e.g. Burkholderia multivorans and Mycobacterium abscessus and more recently identified potential pathogens in CF e.g. Pneumocystis jirovecii and Pandoraea spp. The clinical implications of infection with these organisms are emerging but much remains unknown. Current evidence suggests that infection with some EP is associated with a worse prognosis. This thesis aimed to investigate the epidemiology, prevalence and clinical impact of EP in adults with CF.Methods: (1) The prevalence of P. jirovecii was determined in adults attending Manchester Adult Cystic Fibrosis Centre (MACFC) who were clinically stable versus those experiencing an acute pulmonary exacerbation (PEx). (2) The prevalence of M. abscessus at MACFC was determined, isolates of M. abscessus were strain typed, and cross infection risk was assessed. The clinical impact of Gram-negative EP was assessed by: (3) assessing their prevalence and determining if any patients attending MACFC harboured identical strains and had opportunities for cross infection to occur, and by (4) following these patients longitudinally and comparing outcome with age, gender and FEV1 matched Pseudomonas aeruginosa infected controls. Results: (1) P. jirovecii was detected via sputum PCR in 10 (4.4%) of 226 samples tested from 111 patients. P. jirovecii was more likely to be detected in samples taken from an acute pulmonary exacerbation compared with samples taken from stable patient visits (7 (9.2%) of 76 exacerbations samples versus 3 (2%) of 150 stable visit samples, p = 0.033). (2) Prevalence of M. abscessus was stable at ≤3.6% from 2010 to 2015. 21 patients (91.3%) with a positive culture for M. abscessus since 2010 were infected with M. abscessus subsp abscessus. 2 clusters of 7 and 6 patients harboured strains with identical variable number tandem repeat profiles and some of these patients had opportunities for cross infection to occur. 28.6% of patients developed M. abscessus pulmonary disease, 38.1% were persistently culture positive with no related pulmonary disease, and 33.3% spontaneously cleared M. abscessus from their sputum. (3) Prevalence of Gram-negative EP ranged from 1.9% (Ralstonia spp.) to 6.2% (B. multivorans). Small numbers of patients shared strains of B. multivorans; Stenotrophomonas S. maltophilia and Achromobacter; Ralstonia and Pandoraea species. Epidemiological connections consistent with possible cross infection were found in patients infected with Pandoraea and Ralstonia species. (4) Patients with B. multivorans; S. maltophilia; Ralstonia spp. and Pandoraea spp. had higher antibiotic requirements than P. aeruginosa infected matched controls. B. multivorans; Achromobacter spp.; Ralstonia spp. and Pandoraea spp patients had median FEV1 (% predicted) values ≥10% (absolute) lower than the overall median FEV1.Conclusion: Prevalence of all EP investigated at MACFC was low. P. jirovecii was approximately 5 times more likely to be detected in patients with acute PEx compared with stable patients suggesting it may be a cause of PEx. Results suggest that some patients attending MACFC may have acquired infection with M. abscessus subsp abscessus, Pandoraea spp. or Ralstonia spp. through cross infection. Patient numbers are too small to establish this with certainty and a common environmental source is possible. Gram-negative EP other than Achromobacter spp. were associated with higher acute antibiotic requirements than P. aeruginosa matched controls suggesting these EP are associated with an increased risk of PEx. The fact that many Gram-negative EP were associated with lower median lung function may indicate that these EP cause accelerated lung function decline or that patients with more advanced disease are at most risk of acquiring EP.

CD8+ T cells form a crucial arm of the adaptive immune system and act as sentinels against infection and cancer. The role of autophagy, a major lysosomal degradation pathway, in T cell biology is currently limited. Here, we show that autophagy is required for naïve CD8+ T cell homeostasis. When the essential autophagy gene Atg7 is deleted in the T cell lineage (T-Atg7-/- mice), mice develop lymphopaenia leading to increased CD8+ T cell homeostatic proliferation. When CD8+ T cells encounter antigen, they undergo an initial expansion before contracting, leaving behind a population of long-lived memory cells that provide long-lasting immunity. How CD8+ T cells utilise autophagy when responding to infection is currently unknown. We demonstrate that autophagy is dispensable for CD8+ T cell expansion, but loss of Atg7 results in the failure to establish CD8+ T cell memory to influenza and MCMV infection. Interestingly, CD8+ T cell memory becomes defective in old age, although the cellular mechanism for this is largely unknown. Here, aged mice were shown to exhibit defective CD8+ T cell expansion and memory formation to immunisation. Autophagy levels were diminished in antigen-specific CD8+ T cells from aged mice and the CD8+ T cell response of old mice to vaccination could be rejuvenated in an autophagy-dependent manner using the compound spermidine. These results suggest that targeting of the autophagy pathway might be beneficial as a route to effective T cell modulation and enhanced immunity to infection and vaccination.

Infection after trauma complicates the patients clinical course. Infection leads to longer critical care and hospital stays, has been associated with increased mortality rates and places considerable cost pressures on health economies. The predictors of infection after severe injury are not known, and the effects on outcomes other than mortality are under-reported. The overall objective of this research was to characterise factors predictive of infection in severely injured patients admitted to critical care. A prospective cohort study of 271 patients investigated admission factors predictive of the development of infection. A second study of 280 patients evaluated post-injury immune cell changes and the association with infection. Thirdly the relationship between early coagulopathy and infections was investigated in 158 patients. Finally a study of 385 patients examined the use of Tranexamic Acid (TXA) and its association with infection and other outcomes. Infection was a significant burden for severely injured patients. Admission hypoperfusion was the only early characteristic associated with the development of infection, and a dose dependent relationship was observed between severity of shock and increased percentage of infection (p<0.01). Lymphopenia prolonged to day four post injury was strongly predictive infection (OR 0.10, CI 0.02-0.48, p<0.01). At 24 hours, the anticoagulant Protein C was lower in those with infection (Infection: 70.2 iu/dL vs. No infection: 83.3 iu/dL p=0.02), and increased fibrinolysis was also associated with infectious complications (Infection: 6156 μg/L vs. No infection: 3324 μg/L p=0.03). There was a trend to a beneficial relationship between TXA and infection, and it was independently associated with reduced organ failure (OR 0.27, CI: 0.10 – 0.73, p=0.01) and mortality (OR 0.16 CI 0.03 - 0.86, p=0.03). In severely injured patients, admission shock, prolonged lymphopenia and early coagulation dysfunction post severe injury were independent predictors of infection. Timely modulation of these responses after trauma may help to reduce the burden of infection.

Foot-and-mouth disease virus (FMDV) is a highly infectious virus affecting cloven-hoofed animals. The most prominent of its clinical signs is the development of vesicular lesions on the feet and in or around the mouth, which are a consequence of extensive FMDV-induced epithelial cell death. Currently, there is no certain biological knowledge on why extensive epithelial cell death occurs in some FMDV-infected tissues, but not in others. Using the epithelial tissues of tongue and dorsal soft palate as examples of a tissue where lesions occur and one that does not visibly exhibit FMDV-induced cell death, this work aims to identify the potential drivers of epithelial cell death and survival. A partial differential equation (PDE) model informed by experimental data on epithelial structure, is used to test epithelium thickness and cell layer structure as potential determinants. A second PDE model investigates FMDV-interferon (IFN) dynamics and their impact on the levels of cell death and survival, while an experimental study is undertaken to provide data for model validation. The work carried out casts light on the important role of a variety of factors including FMDV replication, IFN production and release, and IFN antiviral action.

Immune activation is generally acknowledged to be costly. These costs are frequently assumed to result from trade-offs arising due to the reallocation of resources from other life-history traits to be invested in immunity. Here, I investigated the energetic basis of the costs associated with immune activation in Drosophila melanogaster. I found that immune activation significantly reduced fly fecundity (45%) and also caused a decline in metabolic rate (6%) but had no effect on body weight. To understand the factors behind reduced fecundity and metabolic rate I measured feeding and found that food intake was reduced by almost 31% in immune-challenged D. melanogaster. These findings suggest that fecundity costs of immune activation result not from the commonly accepted resource reallocation hypothesis but probably because resource acquisition is impaired during immune responses. The individuals of any animal population generally vary greatly in their ability to resist infectious disease. This variation arises due to both environmental heterogeneity and genetic diversity. Genetic variation in disease susceptibility has generally been considered to lie in the nuclear genome. Here, for the first time, I explored the influence of mitochondrial genetic (mtDNA) variation on disease susceptibility. I crossed 22 mitochondrial haplotypes onto a single nuclear genome and also studied epistasis interactions between mitochondrial and nuclear genomes (mitonuclear epistasis) by crossing five haplotypes onto five different genetic backgrounds. I found that fly susceptibility to Serratia marcescens was influenced significantly by mtDNA allelic variation. Furthermore, the effect of mitonuclear epistasis on fly susceptibility to S. marcescens was twice as great as the individual effects of either mitochondrial or nuclear genome. However, susceptibility to Beauveria bassiana was not affected by mtDNA allelic variation. These findings suggest the mitochondrial genome may play an important role in host-parasite coevolution. The Mother’s Curse hypothesis suggests that sex-specific selection due to maternal mitochondrial inheritance means that mitochondria are poorly adapted to function in males, resulting in impaired male fitness. Mother’s Curse effects have previously only been studied for two phenotypic traits (sperm-infertility and ageing) and their generality for broader life-history has not been explored. I investigated the impact of mtDNA allelic variation on 10 phenotypic traits and tested whether the patterns of phenotypic variation in males and females conformed to the expectations of the Mother’s Curse hypothesis. I found that seven of the 10 traits were significantly influenced by mtDNA allelic variation. However, there was no evidence that the effects of this variation differed between males and females. I therefore concluded that Mother’s Curse is unlikely to be a general phenomenon, nor to provide a general explanation for sexual dimorphism in life-history traits. Overall, this thesis explored the impacts of immunity costs, mitochondrial genetic variation, mitonuclear epistasis and sex-specific mitochondrial selection on D. melanogaster life-history.

The core of the dissertation lies in developing two novel systems bioengineering approaches, a synthetic Escherichia coli killer-prey microecology, and a combined infection-inflammation NET-array system, to investigate the role of the mechanochemical complexity of the microenvironment in driving the microbe-microbe and host-microbe interactions, respectively. Herein, the first part of the dissertation includes designing and engineering a synthetic E. coli killer-prey microecological system where we quantified the quorum-sensing mediated interactions between the engineered killer and prey E. coli bacterial strains plated on nutrient-rich media. In this work, we developed the plate assay followed by plasmid sequencing and computational modeling that emphasizes the concept of the constant evolution of species or acquired resistance in the prey E. coli, in the vicinity of the killer strain. We designed the microecological system such that the killer cells (dotted at the center of the plate) constitutively produce and secrete AHL quorum-sensing molecules into the microenvironment. AHL then diffuses into the prey cells (spread throughout the plate) and upregulates the expression of a protein that lyses the prey. Through time-lapse imaging on petri plates automated using a scanner, we recorded the "kill wave" that originates outside the killer colony and travels outward as the prey dies. We found that the prey population density surrounding the killer decreased in comparison to other locations on the plate far from the killer. However, some of the prey colonies evolve to be resistant to the effects of AHL secreted by the killer. These prey colonies resistant to the killer were then selected and confirmed by plasmid sequencing. Using this empirical data, we developed the first ecological model emphasizing the concept of the constant evolution of species, where the survival of the prey species is dependent on the location (distance from the killer) or the evolution of resistance. The importance of this work lies in the context of the evolution of antibiotic-resistant bacterial strains and in understanding the communication between the microbial consortia, such as in the gut microbiome. Further, the second part of the dissertation includes quantifying the interactions between immune cells (primary healthy human neutrophils) and motile Pseudomonas aeruginosa bacteria in an inflammation-rich microenvironment. Neutrophils, being the first responding immune cells to infection, defend by deploying various defense mechanisms either by phagocytosing and killing the pathogen intracellularly or through a suicidal mechanism of releasing their DNA to the extracellular space in the form of Neutrophil Extracellular Traps (NETs) to trap the invading pathogens. Although the release of NETs is originally considered a protective mechanism, it is shown to increase the inflammation levels in the host if unchecked, ultimately resulting in end-organ damage (especially lung and kidney damage), as with the severe cases of sepsis and COVID-19. In our work, we developed a combined infection-inflammation NET-array system integrated with a live imaging assay to quantify the spatiotemporal dynamics of NET release in response to P. aeruginosa infection in an inflammatory milieu at a single-cell resolution. Importantly, we found increased NET release to P. aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but not leukotriene B4 (LTB4), compared to the infection alone. Our device platform is unique in that the nanoliter well-assisted individual neutrophil trapping enables us to quantify NET release with single-cell precision. Besides, incorporating confined side loops in the device helped us study the role of mechanical confinement on NET release, showing reduced NET release from neutrophils confined in the side loops compared to the relatively wider chambers of our microsystem. In summary, our work emphasizes the importance of studying the heterogeneity of NET release in host defense and inflammation. In the future, our system can be used for screening novel neutrophil-based immunotherapies and serve as a valuable research tool in precision medicine.

Recent advances in sequencing technology and computing power mean that we are in an unprecedented position to analyse large viral sequence datasets using state-of-the-art methods, with the aim of better understanding pathogen evolution and epidemiology. This thesis concerns the evolutionary analysis of rapidly evolving RNA viruses, with a focus on avian influenza and the use of Bayesian methodologies which account for uncertainty in the evolutionary process. As avian influenza viruses present an epidemiological and economic threat on a global scale, knowledge of how they are circulating and evolving is of substantial public health importance. In the first part of this thesis I consider avian influenza viruses of haemagglutinin (HA) subtype H7 which, along with H5, is the only subtype for which highly pathogenic influenza has been found. I conduct a comprehensive phylogenetic analysis of available H7 HA sequences to reveal global evolutionary relationships, which can help to target influenza surveillance in birds and facilitate the early detection of potential pandemic strains. I provide evidence for the continued distinction between American and Eurasian sequences, and suggest that the most likely route for the introduction of highly pathogenic H5N1 avian influenza to North America would be through the smuggling of caged birds. I proceed to apply novel methods to better understand the evolution of avian influenza. Firstly, I use an extension of stochastic mutational mapping methods to estimate the dN/dS ratio of H7 HA on different neuraminidase (NA) subtype backgrounds. I find dN/dS to be higher on the N2 NA background than on N1, N3 or N7 NA backgrounds, due to differences in selective pressure. Secondly, I investigate reassortment, which generates novel influenza strains and precedes human influenza pandemics. The rate at which reassortment occurs has been difficult to assess, and I take a novel approach to quantifying reassortment across phylogenies using discrete trait mapping methods. I also use discrete trait mapping to investigate inter-subtype recombination in early HIV-1 in Kinshasa, the epicentre of the HIV-1 group M epidemic. In the final section of the thesis, I describe a method whereby epidemiological parameters may be inferred from viral sequence data isolated from different infected individuals in a population. To conclude, I discuss the findings of this thesis in the context of other evolutionary and epidemiological studies, suggest future directions for avian influenza research and highlight scenarios in which the methods described in this thesis might find further application.

The red squirrel (Sciurus vulgaris) is native to Eurasia, but in the UK its survival is being threatened by the non-native grey squirrel (Sciurus carolinensis). Since its introduction to the UK from the USA the grey squirrel has increased its range at the expense of the red squirrel. Although competition for resources clearly plays a role in this replacement, an infectious viral disease, caused by squirrelpox virus (SQPV) and hosted apparently asymptomatically by the grey squirrels, has now been recognised as a major contributing factor. Little is known about the pathogenesis of infection in grey squirrels in comparison to red squirrels, but understanding this is essential to determining how the virus spreads within and between the red and grey squirrels. The aims of this thesis were to investigate the course of SQPV infection in red and grey squirrels and possible routes of virus transmission. Specifically, for the first time, a novel Real Time PCR (qPCR) assay and immunohistochemistry were used to investigate the presence of SQPV in various tissues from naturally infected red squirrels and experimentally infected grey squirrels. In diseased red squirrels SQPV DNA was found in several tissues with the highest amounts being found in skin samples. This reflects the multiple lesions that were easily visible on the red squirrel carcasses. There was no indication of systemic disease although the viral DNA was detected, at lower levels, in other internal organs. Grey squirrels were experimentally infected with SQPV isolated from naturally-infected red squirrels with fatal clinical disease. In contrast to SQPV-infected red squirrels no clinical lesions, other than mild scab formation at the site of inoculation, were found in the grey squirrels post-infection. No gross pathological changes indicative of systemic infection were observed and these findings were reflected in the qPCR and histopathology results. Viral DNA was only detected by qPCR in samples from the site of inoculation (scarified skin) and at lower concentrations in other skin tissues such as digital and eyelid skin. In addition, histopathology and immunohistochemistry examination revealed evidence of infection characterized by ballooning degeneration of keratinocytes, and acanthosis and spongiosis of the epidermis. These skin lesions were self limiting and minor compared to the infected red squirrel skin samples. The molecular variation in the virus isolated over time from different parts of the UK was also investigated. Seven SQPV isolates (4 from Scotland and 3 from England) were tested and results indicated that there are no significant changes in the amino acid sequence of any of the three genes examined apart from one amino acid change (one base change) in one gene. All Scottish isolates examined showed this change in comparison to English isolates. The results in this thesis show that there is a mild pathology associated with SQPV infection in grey squirrels. Scabs form at the site of infection but are less proliferative than in infected red squirrels, though they may still serve to contaminate the environment with virus leading to further outbreaks of disease. In contrast it seems likely that the proliferative lesions suffered by red squirrels and the greater amounts of virus that this leads to are likely to be more significant to the epidemiology of disease in localised outbreaks.

Shigella flexneri attacks cells of the intestinal tract, causing over 1 million deaths annually from bacterial dysentery. A type III secretion system (T3SS) initiates the host-pathogen interaction and transports virulence factors directly into host cells via a needle complex (NC) comprising an extracellular needle and membrane-spanning basal body. Rings formed by the single-pass membrane proteins MxiG and MxiJ are arranged concentrically within the inner membrane ring (IMR) of the NC. The Neterminal domain of MxiG (MxiG-N) is the predominant IMR cytoplasmic structure, however it was structurally and functionally uncharacterised. Determination of the solution structure of MxiG-N in this study revealed it to be a forkhead associated (FHA) domain, although subsequent analyses of conserved residues suggested it does not have the canonical role in cell-signalling via phospho-threonine recognition. Subsequent positioning of the structure in the electron microscopy (EM) density for the S. flexneri NC supported models with 24-fold symmetry in the IMR. Both MxiG and MxiJ also have significant periplasmic domains, which were purified to homogeneity in this study, facilitating preliminary characterisation of their structures and intermolecular interactions. In addition, the entire IMR within the context of intact basal bodies was isolated and visualised in vitro by EM. The essential function of MxiG-N could be to localise the putative cytoplasmic ring (Cering) at the base of the T3SS. Although absolutely required for secretion, the Csring component, Spa33, was structurally uncharacterised. The crystal structure of the Cvterminal domain of Spa33 (Spa33-C) was determined in this study, showing an intertwined dimer that aligned with homologous structures and exhibited a novel interaction with the N-terminus of the ATPase regulator, MxiN. Subsequently, Spa33-C was identified as an altemative translation product of spa33 that formed a 2: 1 complex with Spa33 in vitro. This complex oligomerised further, demonstrating for the first time that Spa33 has the propensity to form the ordered, high molecular weight assemblies that would be required for C-ring formation in S. flexneri.

The BacA protein plays a key role in the symbiosis of Sinorhizobium meliloti with the leguminous plant alfalfa (Medicago sativa) and is proposed to be the transmembrane subunit of an ATP‐binding cassette (ABC) transporter. BacA homologues are also present in Brucella species, enteric bacteria (known as SbmA) and Mycobacterium tuberculosis. The S. meliloti‐alfalfa symbiosis, although beneficial to both partners, can also be viewed as a chronic infection. The M. tuberculosis BacA protein is important for M. tuberculosis for the maintenance of a chronic infection. In order to study the M. tuberculosis BacA protein a codon optimised M. tuberculosis bacA gene was synthesised and cloned into vectors for expression in an S. meliloti ΔbacA mutant. Evidence is presented that M. tuberculosis BacA sensitises an S. meliloti ΔbacA mutant towards the glycopeptide bleomycin and the truncated proline rich peptide Bac71‐16, and further that a functional ATPase domain is essential to perform BacA mediated peptide transport. The M. tuberculosis BacA protein protected an S. meliloti ΔbacA mutant from being killed by host defensins. In addition, it was determined that M. tuberculosis BacA‐mediated protection of the legume symbiont S. meliloti against legume defensins as well as mouse alveolar lavages and human ‐defensin 2 is dependent on an ATPase domain which is present in the M. tuberculosis BacA protein. M. tuberculosis encounters ‐defensins during mammalian infections in the host’s lungs and my data show that BacA is likely to be important in conferring immunity to these peptides. The mechanism of persistent infection by M. tuberculosis is therefore very reminiscent of the Sinorhizobium ‐ legume interaction. Also Salmonella enterica is able to cause asymptomatic infections and about 5% of these develop a chronic carrier state and are able to spread the pathogen. In enteric bacterial species SbmA is in close proximity to the putative lipoprotein YaiW. In this study it was determined that YaiW is exposed on the cell surface and that it is involved in the protection of E. coli and potentially Salmonella spp. against a cysteine rich host peptide. YaiW is potentially involved in swarming motility. It was also determined that an alfalfa plant infection model can distinguish between Salmonella strains forming acute and chronic infections. Staphylococcus aureus is one of the major hospital acquired and opportunistic pathogens. Asymptomatic carriers, infected with multi‐drug resistant strains (MRSA) pose a threat to immunocompromised individuals. Here, novel compounds and ways from a variety of sources have been tested for their potential antimicrobial activity against a range of multi‐drug resistant clinical S. aureus isolates. This project significantly advanced the molecular understanding of asymptomatic bacteria‐host infections and helped to understand and establish novel ways to treat infections with multi‐drug resistant clinical pathogens.

Recently, cholesterol metabolism has been shown to modulate the infection of several viruses and there is growing evidence that inflammatory response to infection also modulates lipid metabolism. However little is known about the role of inflammatory processes in modulating lipid metabolism and their consequences for the viral infection. This study investigates host-lipid viral interaction pathways using mouse cytomegalovirus, a large double-stranded DNA genome, which represents one of the few models for a natural infection of its natural host. In this study, transcriptomic and lipidomic profiling of macrophages shows that there is a specific coordinated regulation of the sterol pathways upon viral infection or treatment with IFNγ or β (but not TNFα, IL1β or IL6) resulting in the decrease of free cellular cholesterol. Furthermore, we show that pharmacological and RNAi inhibition of the sterol pathway augments protection against infection in vitro and in vivo and we identified that the prenylation branch of the sterol metabolic network was involved in the protective response. Finally, we show that genetic knock out of IFNβ results in a partial reduction while genetic knock out of Ifnar1 completely abolishes the reduction of the sterol biosynthetic activity upon infection. Overall these results support a role for part of the sterol metabolic network in protective immunity and show that type 1 IFN signalling is both necessary and sufficient for reducing the sterol metabolic network upon infection; thereby linking the sterol pathway with IFN defence responses.

Clostridium difficile is a major problem as the aetiological agent of antibiotic associated diarrhoea. The mechanism by which the bacterium colonises the gut is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The aims of this study were to further define roles for selected surface proteins using a knockout approach, to evaluate the feasibility of surface protein based immunotherapeutics and to obtain structural information using X-ray crystallography. Mutants of cell wall-binding domain (PFam04122) containing proteins CD1036, CD2735, CD2784, Cwp66, CD2791, Cwp84, CD2795 and the flagella cap (FliD) were created. Mutants were characterised with regard to growth, sporulation, toxin production, adhesion in vitro, and, for the Cwp84 mutant, using the in vivo hamster model. The surface-located cysteine protease, Cwp84, was found to play a key role in maturation of the C. difficile S-layer, yet the Cwp84 mutant still caused disease with a similar pathology to the wildtype. Culture supernatant levels of toxin A were increased in CD2735, Cwp66, CD2791, CD2795 and particularly in Cwp84 and FliD 24 hr cultures, while CD2735, Cwp66, CD2791, CD2795 mutants also showed reduced adherence to Caco-2 cells compared to the wild-type. Passively administered immunotherapy, generated to low pH surface protein extracts of the C. difficile R20291 strain, did not protect hamsters from challenge with the cognate strain. Structural studies were undertaken on the surface proteins CD2791, Cwp66 and CD2767. Crystallisation conditions were identified for a recombinant N-terminal domain of CD2767 and an X-ray data set collected to 2 Å, although the structure was not solved by molecular replacement. Together these results further our knowledge of C. difficile surface proteins, although further work is required to identify which surface proteins play key roles in vivo during infection.

Industrialization and advances in technology over the last half-millennium have led to dramatic changes in human society. The diet of our ancestors, once rich in plant matter of extraordinary chemical diversity, has been replaced with a heavily processed diet. Similarly, the rise of urban centers has resulted in an environment devoid of many microbial and chemical exposures around which our ancestors evolved. This rapid shift in human society is sharply correlated with two facets of modern human health: an extensive restructuring of the human gut microbiome and a dramatic increase in the incidence of chronic inflammatory diseases and infection. The gut microbiome is an incredibly complex ecosystem of microorganisms residing in the chemically complex milieu that is the gut. Diet has emerged as instrumental in driving the composition and dynamics of the gut microbiome, and in the development of diverse human diseases. While the impact of dietary carbohydrates (fiber) on the gut microbiome and human health is well characterized, there is a dearth of information as to how plant small molecules (phytochemicals) and other xenobiotics, many of which are thought to be bioactive, interact with or are transformed by the gut microbiome to affect human health. The dynamic interplay between multiple biotic and abiotic factors in this ecosystem makes deconvoluting molecular mechanism challenging using next-generational community profiling tools that profile community structure alone. This research aims to synergize bottoms-up and top-down approaches to define the landscape of phytochemical metabolism by the gut microbiome as a lever on human health. Chapter 2 describes the characterization of phytochemical glycoside metabolism across diverse gut bacteria, discovery of the genetic and molecular basis for differential glycoside catabolism across both gut bacteria and phytochemical substrates and the identification of a phytochemical-specific bacterial catabolic enzyme, and identification of two phytochemical-derived microbial metabolites with novel bioactivities: protection from experimental colitis and selective antagonism of C. difficile. Chapter 3 describes the identification of a novel small intestinal factor that regulates oral tolerance via modulation of both the microbiome and immune components of the gut, discovery of a dietary microbial tryptophan catabolic pathway that generates food allergy-protective Treg-reactive metabolites, and therapeutic intervention against this factor protecting against loss of tolerance in both early life and adulthood. Together, these findings demonstrate novel microbiome-diet axes wherein gut bacterial catabolism of dietary xenobiotics generates chemically-diverse metabolites that regulate the development of both local (inflammatory bowel disease; enteric C. difficile infection) and global (food allergy) disease processes. Furthermore, these findings emphasize the importance of reducing both the host and microbial components of the gut microbiome to their most basic, tractable parts—microbes, genes, and molecules to mechanistically identify tractable elements for therapeutic intervention.