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Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are enteric pathogens that cause disease through intimate attachment and subversion of intestinal epithelial cells. This strategy depends on a type III secretion system (T3SS) to translocate effector proteins into host cells. EspJ is a T3SS effector protein that inhibits opsono-phagocytosis through an unidentified mechanism. This study aimed to determine the mechanism of action of EspJ through analysis of the protein itself and its effects on eukaryotic cell signalling. Bioinformatic analysis of EspJ revealed a predicted ADP-ribosyltransferase (ART) domain which mediates transfer of ADP-ribose from NAD on to a target protein. Through structural comparison with known ARTs, mutations were designed targeting predicted key ART domain residues. NMR analysis revealed that EspJ binds NAD and also that this interaction was disrupted by ART domain mutations. Mutation of the ART domain also disrupted inhibition of opsono-phagocytosis by EspJ. Further analysis of phagocytosis revealed that EspJ inhibits actin polymerisation and tyrosine phosphorylation during FcγR-mediated phagocytosis. During infection, EPEC and EHEC use the translocated effector Tir to initiate actin polymerisation pathways leading to the formation of actin rich pedestals on cultured cells. TirEPEC signalling is similar to that of the FcγR and so the impact of EspJ on pedestal formation was analysed. EspJ inhibited the tyrosine phosphorylation-dependent TirEPEC-mediated actin pedestal assembly but not the phosphorylation-independent TirEHEC signalling. Inhibition of EPEC pedestals correlated with reduced tyrosine phosphorylation and recruitment of the SH2-adaptor Nck. Importantly, an absence of Src family kinases (SFK) was observed in the presence of EspJ. EspJ inhibited FcγRIIa tyrosine phosphorylation suggesting that this effector acts at the initial stages of tyrosine kinase dependent actin polymerisation pathways preventing receptor phosphorylation by SFKs. Importantly, an intact ART domain was required for EspJ mediated inhibition of tyrosine phosphorylation and downstream actin polymerisation. Open Access

BACKGROUND: After Roux-en-Y gastric bypass bariatric surgery (RYGB) patients and animal models show a decreased preference for sweet/fatty foods. However, there are significant discrepancies in the findings of animal and human studies, and the mechanisms are not understood. AIMS: To investigate: 1. The effect of a high-fat (HFD) vs. low-fat (LFD) pre-operative maintenance diet on the ingestive behaviour of rats after RYGB 2. The effects of RYGB vs. gastric banding (BAND) surgery on food hedonics and brain reward systems in humans 3. The effects of RYGB on taste in humans METHODS: Study 1: Rats were fed either LFD or HFD before RYGB or sham surgery. Ingestive behaviour was assessed after surgery using food preference tests. Study 2: Brain reward responses to food were investigated using functional magnetic resonance imaging (fMRI), eating behaviour and metabolic phenotyping in body mass index (BMI) matched un-operated controls and patients after RYGB and BAND surgery. Study 3: The intensity and reward of sweet, fat and fat/sweet taste stimuli were assessed in patients undergoing RYGB using behavioural techniques. RESULTS: Study 1: Pre-operative maintenance diets with different fat contents, did not affect post-surgical weight loss or caloric intake. HFD-RYGB rats exhibited behaviour consistent with condition taste aversion to a familiar stimulus, compared to condition taste aversion to a novel stimulus in the LFD-RYGB rats. Study 2: Patients after RYGB had lower activation in brain reward systems to food and lower food hedonics than BAND patients and/or BMI-matched unoperated controls. Anorexigenic plasma gut hormones, plasma bile acids and dumping syndrome scores were higher in RYGB patients. Study 3: RYGB increased the intensity of fat/sweet taste solutions and reduced the reward value of fat/sweet stimuli. CONCLUSION: Pre-operative feeding may affect ingestive behaviour after RYGB. The mechanisms underlying the healthier food preferences after RYGB include the reduced reward value, and increased aversion to, high-calorie food and taste. Potential mediators are gut hormones, bile acids and altered gut nutrient sensing. Open Access

Regulation of gene expression is critical to govern distinct transcriptional programs for a cell type, lineage specification and developmental stage. Transcription is the first step in gene expression wherein RNA Polymerase II (RNAPII) transcribes protein-coding genes. Transcription is a highly coordinated process that involves a range of chromatin interactions including transcription machinery, chromatin remodellers and co-transcriptional RNA processing. Embryonic stem (ES) cells are pluripotent, self-renewing cells that can differentiate to give rise to all lineages making them an invaluable tool to study early development and in therapy. Genome-wide analysis in murine mES cells has identified 30% of known genes harbouring bivalent chromatin modifications along with repressive Polycomb complexes and a novel variant of RNAPII (modified as S5p+S7p-S2p-) with mechanistic implications in stem cell pluripotency, differentiation potential and lineage specification. To explore chromatin composition associated with different variants of RNAPII, I developed an unbiased method, ‘Proteome-ChIP’ (pChIP) wherein crosslinked chromatin is purified by immunoprecipitation followed by protein extraction and identification by Mass Spectrometry. Using an unbiased comprehensive experimental strategy and a novel systems biology approach, I qualitatively and quantitatively dissect the proteome composition and dependencies on RNAPII modifications during different stages of the transcription cycle. The work done in this thesis provides an invaluable resource of RNAPII chromatin interactions. We identify known and novel components of the co-transcriptional machinery, chromatin remodelling and RNA processing machinery. The work also uncovers novel processes associated with unusual RNAPII (S5p+S7p-S2p-) including DNA replication, Polycomb proteins and chromatin remodellers; many of these processes critical for stem cell viability and regulation. Extending the RNAPII-pChIP analysis on low complexity samples by Native-pChIP and Gradient-pChIP highlights the versatility of robustness of our method. The work described in this sheds light on regulatory chromatin processes specific to mES cells, which informs our understanding of stem cell biology and reprogramming.

The cohesion protein complex is critical for sister chromatid cohesion and proper chromosome segregation in cycling cells and has additional roles in gene regulation and chromatin organisation. Particularly, cohesin has been implicated in regulating genes critical for maintaining pluripotency in embryonic stem (ES) cells. Here I ask whether cohesin regulates gene expression during early stages of ES cell differentiation and the reprogramming of somatic cells towards pluripotency. In order to dissociate the role of cohesin in gene expression from cohesin’s essential functions during the cell cycle I have established genetic approaches to conditionally delete cohesion in ES cells and in somatic cells. By depleting cohesin in ES cells my results reinforce the suggested role of cohesin in pluripotency maintenance, but also show that cohesin removal preferentially leads to mesoderm and neuroectoderm differentiation. In addition, data presented here suggest that cohesin is required for the silencing of X-inactivating genes that occurs during the differentiation of female ES cells. To explore the role of cohesin in reprogramming I used experimental heterokaryons generated by fusing ES cells and somatic cells. In this system, the re-expression of ES-specific genes in somatic cell nuclei occurs in the absence of cell division, thereby obviating the need for cohesin for cell division-related functions. Interestingly, by depleting cohesion in ES cells I found that cohesin is not required for the ability of ES cells to induce reprogramming of somatic cells. Instead, cohesin depletion enhanced their reprogramming ability to re-activate the expression of pluripotency markers in somatic cells, possibly by a mechanism that involves the up-regulation of the reprogramming factor c-Myc. In contrast, somatic cells require cohesin to be reprogrammed. Cohesin-depleted somatic cells were unable to re-activate pluripoency markers and failed to silence lineage-specific somatic genes after fusion. Finally, ongoing experiments to establish a rapid and reversible system for proteolytic cleavage of cohesion in mammalian cells, will contribute to understand the mechanisms by which cohesin regulates gene expression in cellular differentiation and reprogramming. Open Access

Premature birth is a major issue for neurological health. Advances in medical care have led to a decrease in neonatal mortality; however, morbidity in preterm survivors remains substantial and neurocognitive issues, particularly involving cognition, language and behaviour are prevalent. Cerebral white matter injury is common in preterm-born infants and is associated with neurocognitive impairments. By identifying the pattern of connectivity changes in the brain following premature birth a more comprehensive understanding of the neurobiology underlying these deficits could be obtained. Whole-brain macrostructural connectivity was characterised in a group of preterm-born children, and the influence of age and prematurity was explored using a data-driven analysis of diffusion magnetic resonance imaging data. An age-adapted framework, combining anatomical and tissue segmentations with probabilistic diffusion tractography, was used to derive connectivity matrices, weighted by mean tract anisotropy: a measure of connective certainty and strength. In a sparsified, group-consistent connectivity matrix, a novel feature selection method comprising Lasso regression and stability selection was used to identify connections whose mean anisotropy was related to age at imaging or delivery. As hypothesised, older children were found to have greater connectivity in tracts involving frontal or temporal lobe structures. Increasing prematurity at birth was related to widespread, bilateral reductions in connectivity in all cortical lobes and several sub-cortical structures, consistent with previous histological and neuroimaging data. Results were robust to both increasing sparsity and the removal of subjects with serial imaging in the cohort. The pattern of white matter damage elicited in these results may be responsible for the neurocognitive impairments observed. This thesis presents a scalable, data-driven method, which could detect contrasting effects of development and prematurity in a sparse model of infant whole-brain structural connectivity. The approach can be used to evaluate connectivity in ever-increasing detail and undertake iterative discovery of the macroconnectome with increasing precision. Open Access

AMP-activated protein kinase (AMPK) is a well characterised central regulator of energy homeostasis with many downstream substrates and potential therapeutic targets. Recently several kinases related to AMPK have been identified which are activated by the same upstream kinase, LKB1, and share significant sequence homology with the catalytic α subunit of AMPK. Previous studies have implicated NUAK1, one of these related kinases, in cell cycle regulation and other studies have identified various partner proteins but as yet there is no consensus model of its regulation or physiological role. This aim of this study was to investigate the regulation and interactions of NUAK1 using an in vitro system and also use an in vivo tissue specific mouse knockout system to investigate its function in two different tissues. The results of these studies reveal new insights into the regulation of NUAK1 in vitro and suggest that the kinase is capable of activation by autophosphorylation, in the absence of LKB1. NUAK1 is shown to interact with 14-3-3 proteins and evidence that this interaction may be important for the function of the kinase is presented. Cell based studies in mouse embryonic fibroblasts (MEFs) lacking NUAK1 show that it is involved in proliferation and there are suggestions of pathways involved in regulating this effect. NUAK1 liver-specific knockout mice were generated and extensive metabolic phenotyping analyses were carried out. In young animals deletion of NUAK1 in liver did not lead to any obvious changes in whole body metabolism. However, preliminary studies revealed a possible role for NUAK1 in metabolic regulation in older mice and in response to liver damage. Mice with heart-specific deletion of NUAK1 were generated and these mice were found to have a cardiac hypertrophy phenotype in response to angiotensin II treatment. Potential signalling pathways downstream of NUAK1 in the heart were then investigated in this model. Open Access

The ability to sense and initiate a response to situations of nitrogen-limitation is essential for bacterial survival. In extensively investigated organisms, the nitrogen-stress response consists of changes in intracellular metabolite levels, post-translational modification of proteins (such as metabolic enzymes) and a transcriptomic response mediated by a global response regulator. However, in mycobacteria the nitrogen stress response has not been comprehensively investigated. In this study mycobacterial nitrogen limiting conditions were optimised and the mechanism of GlnR activation investigated; M. smegmatis GlnR requires a highly conserved aspartate residue (D48), corresponding to a putative phosphorylation site, for function. In addition, a ChIP-seq approach combined with global expression analyses, permitted characterisation of the GlnR mediated global transcriptomic response stimulated during nitrogen. In M. smegmatis, 52 GlnR binding sites were identified, controlling the expression of at least 103 genes in response to nitrogen limitation. The majority of GlnR regulated genes were involved in nitrogen uptake and nitrogen scavenging. A consensus GlnR DNA binding motif was identified and AC-n9-AC DNA residues shown to be essential for GlnR:DNA binding. In M. tuberculosis 36 GlnR binding sites were identified in nitrogen limitation, however no consensus GlnR:DNA binding motif could be determined. Initial analysis suggests GlnR may be involved in a general stress response in M. tuberculosis, rather than mediating a nitrogen scavenging response as observed in M. smegmatis. This study provides the first global analysis of nitrogen limitation in mycobacteria and identifies GlnR as the main nitrogen response regulator. From this analysis it appears that the role of GlnR is different in M. tuberculosis compared to M. smegmatis, which may provide key insights into how pathogenic and non-pathogenic species survive nutrient limiting conditions. Open Access

Asthma and bacterial pneumonias are major causes of human mortality and morbidity throughout the world. To date many studies have investigated the possibility that bacteria exacerbate asthma but only a handful consider that asthma may cause bacterial infections. Recent clinical evidence suggests that bacterial infections cause serious complications in patients with asthma and that asthmatics show a 2-fold increased risk of invasive pneumococcal disease. This thesis examines the molecular mechanisms causing susceptibility of house dust mite (HDM) exposed lungs to bacterial infection. The main finding of this thesis is that HDM-induced allergic airways disease increases susceptibility to Streptococcus pneumoniae infection. Furthermore, the molecular pathways leading to the production of neutrophil chemoattractants in the lung are compromised and that despite the complexity of anti-bacterial pathways that are disrupted, the re-introduction of a single chemokine to the lungs with allergic airway disease enables clearance of S. pneumoniae that would otherwise prove fatal. However, a reduction in HDM-induced eosinophilia seen in ST2 deficient mice does not restore anti-bacterial immunity. This deficit in anti-bacterial immunity in HDM exposed lungs is associated with a change in resident alveolar macrophages into an alternatively activated phenotype, characterised by high mRNA and protein levels of RELMα, Ym1 and Arg1. These altered alveolar macrophages produce considerably less TNFα in response to Toll-Like Receptor (TLR) stimulation that is not a result of reduced TLR mRNA levels but due to an upregulation of TLR negative regulators particularly A20. A20 targets TRAF-6 that is found upstream of NF-κB activation. To prove a causal link naïve alveolar macrophages were transferred into allergic lungs prior to bacterial infection; such lungs handled the infection better confirming that alveolar macrophages are important in initiating the anti-bacterial response. Overall our findings highlight a change in specific innate immune pathways in the allergic lung that participate in susceptibility to bacterial pneumonia. Open Access

Osteoporosis is characterised by low bone mass, reduced bone mineral density and a deterioration of bone microarchitecture, resulting in increased susceptibility to fragility fractures. Oestrogen deficiency and thyrotoxicosis are established risk factors for osteoporosis. Oestrogen and thyroid hormone have opposing actions on adult bone and I hypothesise that accelerated bone loss at the menopause is due to unopposed actions of thyroid hormone on the skeleton. To test this hypothesis, I investigated the effect of altered thyroid status on the skeleton in adult sham-operated and ovariectomised wild type and thyroid hormone receptor (TR) α and β knockout mice (TRα0/0 and TRβ-/-). Skeletal phenotype analysis included determination of structural, densitometric, histomorphometric and biomechanical parameters. Skeletal phenotypes of euthyroid wild type mice were compared to hypothyroid and thyrotoxic wild type as well as TRα0/0and TRβ-/- mice. Bone mass was elevated in TRα0/0and reduced in TRβ-/- mice despite similar bone formation rates. These data suggest that the differences in bone mass are due to differing osteoclast activity. The skeletal phenotypes of TRα0/0 and TRβ-/- mice following manipulation of thyroid status were then described in detail. TRβ-/- and wild type mice rendered hypothyroid, had significantly lower bone formation than hypothyroid TRα0/0 mice suggesting that, in the absence of thyroid hormone, TRα may inhibit bone formation. Finally, the effect of oestrogen withdrawal in each of these groups of mice was also investigated. These studies demonstrate oestrogen deficiency bone loss was greater in hypothyroid compared to hyperthyroid mice, indicating that accelerated bone loss following oestrogen withdrawal cannot result from unopposed actions of thyroid hormones. Nevertheless, the studies contained in this thesis provide new insight regarding the roles of TRα and TRβ in bone maintenance and the effects of interactions between oestrogen and thyroid hormones on the regulation of bone mass. Open Access

COPD is currently the 4th most prevalent cause of death worldwide. Despite the global impact, there are no currently available treatments which impede disease progression. This lack of effective therapies is largely due to an inadequate understanding of the mechanisms which drive disease progression. Cigarette smoke (CS), the most important risk factor for COPD, is thought to initiate an inflammatory response in the lungs which becomes self-propagating and dysregulated. Chronically, this inflammatory response drives structural and functional changes. The mechanisms by which CS elicits this inflammatory response, however, remain unclear. Certain CS constituents are known to activate Transient Receptor Potential (TRP) ion channels. A number of TRP channels are actively expressed in the lung tissue or inflammatory cells, and a further few are also implicated in the generation of inflammation. Owing to these features, it was hypothesised that TRP channels A1, C6, M2, M8, V1 and V4 have a role in CS-induced airway inflammation and, consequently, the pathogenesis of COPD. To test this hypothesis, three murine models of induced airway inflammation were characterised: acute CS, sub-chronic CS and endotoxin (LPS). Lung-tissue TRP channel expression levels were measured in these models alongside human lung-parenchyma samples from non-smokers, smokers and emphysema patients. Mice deficient for specific TRP channels were profiled in the CS-model and the LPS-model to establish the role of TRP channels in the initiation of inflammation in disease and non-disease settings. TRPV1-/-, TRPV4-/- and TRPM8-/- mice exhibited significantly reduced levels of airway inflammation compared to wild-types after acute CS, but normal responses to the innate (LPS) challenge. This data suggests that modulation of TRP channels could represent a novel anti-inflammatory approach for combating smoke induced diseases like COPD without impacting on the normal, essential innate defence mechanisms. Open Access

The present thesis investigates the neural mechanisms supporting working memory (WM) guidance of visual attention, focusing on the role of the thalamus. Chapter 1 is a review of the relevant literature and sets-up the specific research aims. Chapters 2 and 3 explore the role of the thalamus on guidance of attention by WM contents. Stroke patients with focal-brain lesions performed a WM-guided search task. In valid conditions, the colour of the search target was pre-cued by the WM cue while on neutral conditions there was no cue prior to search. In invalid conditions, the WM cue specified the colour of a search distracter and the target appeared elsewhere. First, it was hypothesized that lesions to the thalamus could lead to deficits in attentional control (e.g. failing to separate irrelevant memory contents with relevant target information and leading to increased capture from WM-like distracters during invalid search conditions). An alternative hypothesis was that the thalamus may support the capture of attention by WM contents, hence thalamic patients would display little bias of attention from the WM contents, despite those contents are being maintained in memory. It was found that patients with focal-thalamic lesions especially in the ventrolateral nucleus, showed no search benefit from the valid cues on search as opposed to a control group of patients with lesions outside the thalamus and non-stroke patients. In the invalid condition, thalamic patients showed no capture by the irrelevant search item that matched the WM cue, whereas a group of healthy age-match controls exhibited the normal effect of capture by irrelevant contents held in WM. These observations suggest that lesions to the ventrolateral nucleus of the thalamus impair the capture of attention from WM contents. In Chapter 4, I aimed to establish the role of cortical structures that are known to be structurally connected with the ventrolateral nucleus of the thalamus (i.e. superior frontal gyrus) in WM guidance of attention. To do this, I investigated the effects of transcranial direct current stimulation (tDCS) of the dorsal frontal cortex in WM guidance of attention under distinct WM loads. I found that despite the effect of WM guidance of attention decreasing as WM load increased, frontal-tDCS modulated WM guidance in these conditions. We suggest that the dorsal frontal cortex forms part of a network alongside the thalamus in supporting WM guidance of attention. Finally, I conducted a functional Magnetic Resonance Imaging (fMRI) experiment (Chapter 5) with healthy volunteers to test the hypothesis that the thalamus plays a role in WM guidance when learning of abstract cue-target feature associations needs to take place for guidance of behaviour to emerge. I used four Japanese ideograms as WM cues, each associated with the colour surrounding the sought after target in the subsequent search display (valid trials). In the neutral condition, four different Japanese ideograms were presented that did not predict the colour of the target. Hence, for WM to guide attention the association between the abstract cue and the colour that surrounded the search target needed to be learned. I found that responses in the thalamus and the frontoparietal cortex displayed sensitivity to the predictiveness of the ideogram cues as WM guidance of attention emerged during learning. The findings reported in this thesis demonstrate the pivotal role of the thalamus in WM guidance of attention. Open Access

Whilst cough is a defensive reflex, in respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) the cough reflex may become exaggerated and unproductive, leading to presentation of chronic cough as a troublesome symptom. Chronic cough is the most common reason that patients seek ambulatory care in the UK, and cough medications are one of the largest segments of the global over-the-counter drugs market, yet there are no cough medications (prescription or OTC) available that are both efficacious AND safe, indicating a large unmet clinical need. β-adrenergic receptor (β-AR) agonists, methylxanthines (theophylline) and fatty acid amide hydrogenase inhibitors (FAAHi) are peripherally acting drugs with a proven safety profile in humans for other uses, but are not currently recognised as anti-tussive medications. This thesis aimed to examine whether these drugs possess anti-tussive activity, and if so, the mechanisms by which they inhibit cough. These drugs were all effective at blocking the in vivo cough response to tussive stimuli in naïve guinea pigs, and in addition β-AR agonists and theophylline inhibited tussive stimulus evoked cough in guinea pigs previously exposed to cigarette-smoke, which display a clinically relevant enhanced cough phenotype. In vitro and in vivo assays of guinea pig vagus nerve/neuron activation were used to show that these compounds inhibit depolarisation of airway-innervating peripheral sensory nerves. Pharmacological and genetic tools were used to investigate the receptors and signalling pathways activated by these drugs. Whilst β-AR agonists, theophylline and FAAHi had different mechanisms of action, a common component was that all three classes of compound act on various potassium channels, thereby reducing the excitability of sensory neurons. This work suggests that drugs that inhibit peripheral sensory nerve activity have the potential to be efficacious as anti-tussives. Specifically the compounds tested have improved clinical side-effect profiles over currently used anti-tussives, and therefore have potential as therapies for chronic cough. Open Access

DNA methylation is an epigenetic modification important in many cellular processes such as maintenance of genome stability, establishment and maintenance of imprinting, transposon silencing, chromatin remodelling and control of gene expression. It is therefore important to understand how this modification is established and erased. We set out to develop a sensitive, liquid–chromatography mass–spectrometry method to measure global levels of DNA methylation (5mdC), as well as hydroxymethylation (5hmdC), a potential intermediate of DNA demethylation. With the new Agilent 6490 QQQ LC–MS we were able to detect as little as 50 amol of 5mdC and 5hmdC. We used this method to quantify levels of DNA methylation from DNA extracted from only 100 cells, allowing us to compare DNA methylation levels in early zygote development. Given that the evidence for DNA demethylation in early zygotes comes from methods using either antibody staining or bisulfite sequencing, this is the first direct demonstration that global DNA demethylation occurs in zygotes. Myoblast differentiation is a well-known model of DNA demethylation, where locus-specific DNA demethylation at the promoters and enhancer elements of myogenic regulatory factors, such as myogenin and MyoD, induce muscle specification and differentiation. It has been proposed, however, that the DNA demethylation process involves a large proportion of the genome and that it occurs in the absence of replication, indicating an active process. Therefore, in the second part of this work I set out to further investigate the global scale of epigenetic events associated with myoblast differentiation. Whilst some of the myoblast differentiation experiments showed a marked wave of DNA demethylation (up to 51%) others did not show any changes in DNA methylation level, showing that myoblast differentiation and DNA demethylation are not co-dependent. Addition of a DNA demethylating agent, 5-Aza-3’-deoxycytidine, to the growth medium of differentiating myoblast enhanced the differentiation process. On the other hand, inhibition of Poly (ADP- ribose) polymerase 1 activity, which has been shown to be mechanistically involved in DNA demethylation, inhibited the differentiation process. My work documents the kinetics of 5- v methylcytosine abundance during the myoblast differentiation together with the expression dynamics of factors previously linked to the DNA demethylation process. Open Access

Gene therapy is a promising approach for the treatment of cancer. While most progress in gene therapy has been achieved using eukaryotic viral vectors, they have had limited success in systemic gene therapy applications due to undesired uptake in non-targeted tissues, insertional mutagenesis, and immunogenicity. These shortcomings have driven the development of gene delivery vectors derived from alternative sources. The bacteriophage-derived Adeno-associated virus-phage (AAVP) is a novel hybrid prokaryotic-eukaryotic viral vector consisting of a mammalian transgene cassette flanked by ITRs from AAV serotype 2 inserted into an intergenomic region of an M13 bacteriophage. Anti-tumour therapy following systemic administration has previously been demonstrated in several in vivo cancer models, with tumour specificity achieved through display of an αv integrin-targeting ligand on the viral capsid. However, despite the apparent success of this novel vector in several cancer models, high titres of AAVP are required for transduction of mammalian cells. The mechanisms of entry and intracellular trafficking of the targeted AAVP vector were therefore investigated in order to identify key barriers to transduction. Using a combination of flow cytometry, confocal, and electron microscopy techniques, together with pharmacological agents, RNAi and dominant negative mutants, it was demonstrated that receptor-mediated endocytosis of the AAVP vector is both dynamin and clathrin-dependent. Following endocytosis, the majority of AAVP particles are sequestered by the endosomal-lysosomal degradative pathway, which presents a major obstacle to gene delivery by the vector. The feasibility of improving AAVP-mediated gene transfer through endosomal/lysosomal disruption was demonstrated using the lysosomotropic agent chloroquine. Subsequently, novel multifunctional AAVP vectors bearing endosomal-escape peptides were generated. These novel vectors demonstrated improved transgene expression and retained integrin-targeting capabilities of the RGD peptide. These studies will prove invaluable for the future development of AAVP. Furthermore, the findings presented in this thesis underline the importance of such studies for the improvement of gene therapy vectors.

Molecular imaging is an ideal platform for non-invasive detection and assessment of cancer. In recent years, the targeted imaging of CXCR4, a chemokine receptor that has been associated with tumour metastasis, has become an area of intensive research. CXCR4 is a GPCR whose interaction with its natural ligand SDF-1α is essential for embryo development and haematopoiesis. Over-expression of this receptor is associated with aggressive types of cancer and potentially metastatic tumours. Several classes of CXCR4 inhibitors have been reported in the literature, including TN14003-, FC131- and IT1t-derivatives. The aim of the research presented herein was to develop CXCR4-targeted probes for positron emission tomography. To this end, we designed and synthesised 20 new potential tracers belonging to those three classes of CXCR4 inhibitors, for labelling with fluorine-18 or gallium-68. In vitro and in vivo evaluation of these radiotracer candidates allowed for further understanding of the structure-activity relationship and pharmacokinetic properties of these types of compounds. In particular, our gallium-68-labelled tracer based on the structure of TN14003 displayed the essential features for the identification of CXCR4-expressing tumours in a clinical setting. Open Access

The mammalian CKS family consists of two highly conserved proteins, CKS1 and CKS2. Both are able to bind to cyclin dependent kinases 1 and 2 (CDK1 and CDK2) with high affinity and were suggested to modulate CDK activity and thus cell cycle control. CKS proteins have also been reported to facilitate the ubiquitination of cyclin A by the anaphase promoting complex (APC). Additionally CKS1 has been shown to have an important role in the recognition of the cyclin dependent kinase inhibitor p27Kip1 by the SCFSkp2 (Skp1-Cul1-F-box-protein) ubiquitin ligase. Elevated expression of both CKS proteins is often found in a variety of tumours and is correlated with poor prognosis. This might indicate an involvement of CKS proteins in the process of transformation from normal to cancer cells. Previously it was shown that down-regulation of CKS proteins in cancer derived cell lines results in a loss of their high proliferative capacity. I surmise, that a possible oncogenic effect of CKS proteins might be due to the deregulation of replication. To study the role of CKS proteins as oncogenes, different stages of cancer development were modelled in the human diploid fibroblast cell line, IMR90. Over-expression of CKS proteins was combined with defined genetic events known to be associated with transformation such as activation of telomerase (hTERT) and blockage of the p53 and RB pathways. Over-expression of CKS variants in IMR90 cells led to checkpoint activation and growth arrest reminiscent of oncogenic stress. Down-regulation of the checkpoint proteins p53 and RB however could alleviate the growth arrest and led to a slight growth advantage of the CKS over-expressing fibroblasts. Analyses to understand the molecular mechanisms behind the checkpoint activation in primary IMR90 were performed addressing, in particular, the involvement of an altered CDK activity.

Thiol-disulphide reactions are critical to the structure and function of von Willebrand factor (VWF). Intracellular multimerisation is catalysed by intrinsic thiol isomerase activity of CGLC motifs in its propeptide. Conserved CG(L/I)C motifs were identified in the D3, D4 and C1 domains which may possess thiol isomerase activity and contribute to intracellular synthesis of VWF or to extracellular thiol-disulphide exchange. Nine cysteine residues are unpaired in a proportion of plasma VWF molecules and are hypothesised to participate in disulphide-mediated self-association. This thesis aimed to study VWF self-association, the participating individual cysteines and the intrinsic isomerase activity of VWF. To study the unpaired cysteines, point mutants and C domain deletion mutants (where most of these cysteines are located) were made. To study the role of the conserved CG(L/I)C motifs, two complementary types of mutant were made: replacement of D2 domain (known isomerase activity) with either the D3 or D4 domain; and insertion of glycine between vicinal cysteines (known to disrupt isomerase function). Mutants were expressed in HEK293T cells. Mutants which were retained intracellularly were studied by measuring lysate pro-VWF and by EndoH digestion. Secreted mutants were studied by quantifying expression, static collagen binding activity and free thiol content. Mutant VWF function was assessed by measuring its ability to capture platelets when perfused over collagen under shear stress. The lateral self-association of VWF was studied under both static and flow conditions using plasma-derived and recombinant VWF. The results demonstrate that the individual cysteine residues and the C domains of VWF play a crucial role in the folding of VWF during synthesis. Furthermore, the CGLC motif in D3 plays a critical role in the synthesis of VWF. However, extracellular self-association of VWF does not appear to play a significant role in the capture of platelets to collagen under physiological flow conditions.

Introduction Epilepsy is a heterogeneous collection of neurological diseases characterised clinically by recurrent seizures. Pre-clinical models implicate derangements in ligand-gated receptor-mediated neurotransmission in seizure generation and termination. In this thesis, the author quantified activated N-methyl D-aspartate- and opioid peptide receptor availability in adults with focal epilepsy. Methods This thesis consists of three positron emission tomography (PET) studies of adults with focal epilepsy, using [18F]GE-179 (activated NMDA receptors) and [11C]diprenorphine (DPN; opioid receptors) radioligands. A novel resolution-recovery technique, Structural Functional Synergistic – Resolution Recovery (SFS-RR), was applied to pre-existing paired [11C]DPN PET datasets acquired from adults with temporal lobe epilepsy (TLE). Activated NMDA receptor availability was quantified in adults with frequent interictal epileptiform discharges (IEDs), by regional compartmental modelling and model-free voxelwise analyses. Statistical parametric mapping was used to identify significant differences in volumes-of-distribution (VT) between populations. Results [18F]GE-179 had good brain extraction with a relatively homogeneous distribution and moderately-paced kinetics in grey matter. The two brain compartments, four rate-constants model best described the radioligand’s kinetics in grey matter. Global increases in [18F]GE-179 VT were seen for seven of 11 participants with frequent IEDs. Focal increases in [18F]GE-179 VT of up to nearly 24% were also identified for three of the 11 participants. A post-ictal increase in [11C]DPN VT was identified in the ipsilateral parahippocampal gyrus. Discussion This first-in-man evaluation of [18F]GE-179 evidenced several properties that are desirable in PET radioligands, but the specificity of binding requires further characterisation. The results suggest focal increases in activated NMDA receptor availability in participants with refractory focal epilepsy, and also post-ictal increases in opioid peptide availability in the parahippocampal gyrus in TLE. Both findings may have pathophysiological relevance, and illustrate the potential of quantitative ligand PET with advanced post-processing to investigate changes in inhibitory and excitatory receptor systems in the epilepsies in vivo. Open Access

Metabolomics is a technology which allows us to probe a wide array of interactions between metabolites. These interactions can be revealed by statistical correlations between metabolite levels that may arise via a range of mechanisms. To measure metabolite levels, two main techniques are used: Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR). For the measurement of correlation structure high analytical reproducibility of the assays is required. While NMR has previously been shown to be reproducible, LC-MS, has not been similarly assessed. To assess the reproducibility of LC-MS for urinary metabolomics, a multi-laboratory study was devised. We find that the technology is highly reproducible, both within and between laboratories with CVs of < 17%, < 5s drift and under 10% ppm between labs. In LC-MS, ionisation of a single compound can lead to multiple charged species such as isotopologues, adducts etc. These multiple signals have a high mutual correlation and we show that this allows them to be identified with high sensitivity and specificity. The inferred statistical interactions between different metabolites can also be affected by analytical errors. An algorithm was designed to remove statistical metabolite links that could have been caused by the analytical technique. Using this method, a higher confidence can be placed on the remaining interactions, suggesting that they are potential biological interactions. Finally, most biological interactions are dynamic in nature, leading to correlations through time between metabolite levels. To explore these dynamic links, two temporal approaches were developed. These methods are designed to discover temporal correlations between metabolites and to test whether they vary between bio- logical conditions. We successfully demonstrate the methods in both LC-MS and NMR datasets. Overall, this thesis shows that correlation structure in metabolic profiling data is reliable, can be successfully filtered to improve quality and can be interrogated to reveal a new kind of dynamic metabolic biomarker. Open Access

Exposure to pollution and active or passive smoking have been associated with a worsened asthma severity and a reduced response to treatment. These poorly controlled asthmatics are responsible for the majority of the economic burden of the disease but how pollution and/or cigarette smoke (CS) impacts on the disease is not well understood. The aim of this thesis was to develop a murine model of allergic asthma where CS exposure results in a change in model phenotype and the sensitivity of the response to pharmacological intervention. Two preclinical models of allergic asthma were utilised: the ovalbumin (OVA) model which had previously been established in-house, and the house dust mite (HDM) model which I developed in this thesis. As topical HDM exposure is known to cause innate inflammation I developed an allergic model where HDM challenge resulted in inflammation only in the mice which had been previously sensitised to HDM. The allergic inflammation in this model was accompanied by allergic airway hyper responsiveness, however the LAR was not observed in this model. CS exposure did not have a dramatic impact on the cellular inflammation in either the OVA- or the HDM-driven model, nor did it impact upon the anti-inflammatory effects of oral steroid treatment with the exception of the addition of a steroid-insensitive neutrophil population. However CS exposure attenuated the AHR observed in the OVA and the HDM models. Finally cigarette smoke exposure not only enhanced the OVA-induced LAR but also rendered this response completely insensitive to oral steroid treatment. Further investigation into the effects of CS in these two models may provide clues as to the mechanisms behind the effect of smoking on asthma in the clinic. The CS-enhanced LAR model could be invaluable in understanding the clinical phenotype of treatment resistance in smoking asthmatics. Open Access

Ageing in the absence of disease is associated with reduced cognitive function. Cognitive function depends on the integrated operation of large-scale distributed brain networks. In this thesis I use structural and functional magnetic resonance imaging to test the hypothesis that age related cognitive decline arises from disruption to the integrated operation of large-scale networks, and the disrupted operation of these networks is associated with structural disconnection to white matter tracts. There are five studies presented in this thesis. The first study demonstrates that from middle-age onwards there are reduced grey and white matter volumes and increased damage to widespread regions of white matter, suggesting disconnection across many neuronal networks. In the second study, analysis of thalamo-cortical connectivity reveals a reduced volume of thalamo-frontal projections with age that follows an allometric scaling law and predicts a decline in executive function. Next, I show that disruption in functional and structural connectivity within large-scale networks that occupy regions within the frontal lobe predict age–related differences in cognition. Reduced functional connectivity at the level of the frontal lobe within these networks is associated with micro-structural damage to widespread white matter pathways, suggesting that white matter damage in remote networks can disrupt interconnectivity within other networks. I then investigate the effect of age-related white matter damage on large-scale network function and behaviour using a response inhibition task. I demonstrate that older adults fail to deactivate the Default Mode Network during response inhibition. Failure to deactivate the DMN is associated with impaired inhibitory performance. Regulation of the DMN is predicted by the structural integrity within a remote brain network, the Salience Network. This work identifies DMN dysfunction as an underlying factor in cognitive deficits associated with increasing age, and confirms that white matter damage interrupts the interaction between large-scale networks in the ageing brain. Open Access

Introduction Epilepsy, defined as the recurrence of unprovoked seizures, is one of the commonest serious conditions in neurology. The World Health Organisation (WHO) estimates a prevalence of 50 million people worldwide, with a temporal lobe focus being the commonest cause of complex partial seizures. Patients with temporal lobe epilepsy (TLE) often have reduced GABAA receptors at their seizure focus, and poor memory performance. Blockade of GABAA receptors containing alpha5 subunits is promnestic. Animal models have also demonstrated alterations in cannabinoid type 1 (CB1) receptor availability in response to seizures. The studies presented in this thesis were designed to first determine the reliability of measurement with two novel PET tracers for the expression of alpha5 subunits of GABAA receptors ([11C]Ro15 4513) and CB1 receptors ([11C]MePPEP). These were then used in human TLE to try and elucidate mechanisms of memory impairment and minimally invasive characterisation of the seizure focus. Methods Adult healthy volunteers underwent paired scans with [11C]Ro15 4513 (GABAA alpha5 receptor partial inverse agonist) and [11C]MePPEP (CB1 receptor mixed inverse agonist and antagonist). Test–retest variability was characterised for both radiotracers with quantification in regions spanning high and low receptor concentrations by regional compartmental modelling and a variety of regional and voxel-wise model-free analyses (spectral analysis and variants). Semiquantification with modified standard uptake values (mSUVs), in widespread clinical use, was also explored. In the clinical studies, healthy volunteers were compared with TLE patients using Statistical Parametric Mapping software. Paired post-ictal and interictal studies were obtained with [11C]MePPEP to determine changes in response to seizures. Single PET scans were obtained with [11C]Ro15 4513 to determine changes in relationship to memory impairments. Results [11C]Ro15 4513 could be reliably quantified with voxel-wise spectral analysis, and the simplified reference tissue model, but not mSUVs or compartmental models. For [11C]MePPEP, voxel-wise spectral analysis, a one tissue compartment model and simple mSUVs were the most reliable methods in controls, while preserving between-region differences. CB1 availability in TLE was higher, at the group level, in the ipsilateral temporal lobe in post- ictal scans than in controls, and negatively correlated with time since last seizure. In individual patients, however, focal increases were not consistently found in the epileptogenic temporal lobe. [11C]Ro15 4513 scans could be obtained in 12 patients but have not yet been fully analysed. Discussion I demonstrated that two novel PET tracers can be reliably quantified, using a much larger cohort and a much greater variety of methods than available in the literature in the case of [11C]MePPEP, and performing such an analysis for the first time for [11C]Ro15 4513. This laid the foundation for the clinical study of CB1 receptor availability in TLE patients. The hypothesis of an upregulation of this inhibitory G-protein coupled receptor type in response to single spontaneous seizures could be confirmed, but the method was not so far useful in individual patients. [11C]Ro15 4513 PET holds promise for the investigation of the mechanisms of memory impairment in TLE. Open Access

Intra-tumour biological heterogeneity is a characteristic shared by all cancers and is thought to contribute to treatment failure. Within-lesion spatial heterogeneity can be qualitatively visualised in Positron Emission Tomography (PET) imaging. Quantifying the variability of the biological processes and the complexity of the signal being measured in PET oncology is essential. The aim of this thesis was to develop and validate intensity- and spatial-based metrics to quantitatively account for the complexity of radiotracer uptake and to annotate intra-tumour PET heterogeneity. Texture analysis was employed to characterise the in vivo tumour heterogeneity of cell proliferation in breast tumours using 18F-fluorothymidine (18F-FLT) PET. The repeatability of the feature measurements was assessed in patients who had two PET scans prior to therapy. Associations between features at baseline and clinical response measured after three cycles of chemotherapy were explored. Associations between feature changes at one week after the start of chemotherapy and clinical response were also explored. Furthermore, the influence of analysis parameters and imaging protocols were studied. A subset of textural features produced reliable measurements and were associated with treatment response. A technique based on multifractal analysis was also developed for characterising the space-filling properties of an object of interest in PET imaging. The derived spatial index was further combined with intensity metrics and the technique was shown to correct for partial volume effects. The method was illustrated on mathematical objects, validated on test-retest 18F-FLT PET clinical data and applied to realistic PET simulations. This work contributes to the demonstration that intensity- and spatial-based image analysis methods can supplement existing methods in PET quantification studies. These techniques provide some improvements on existing methods to derive classical quantitative PET indices and permit extraction of additional information to further characterise patient populations in the clinical setting and in relation to therapy. Open Access

Toxoplasma gondii affects a range of intermediate and secondary host species, including humans, with cats (Felidae) being the definitive host. The mechanism of action of T. gondii in its natural rat host can provide profound insights as to evolutionary trajectory of parasite manipulation. I used a range of non-invasive behavioural and physiological assays to assess the impact of infection on different aspects of innate rat behaviour. One key unique focus was an examination of the potential effect(s) of novel genetically modified parasite lines that overexpress tyrosine hydroxylase (TgTH) in comparison to wildtype T. gondii. The aim here was to test the hypothesis that TgTH, through its subsequent impact on neuromodulator levels, plays a significant mechanistic role in host behaviour modification. Consistent with previous studies, there was a clear effect of T. gondii on host behaviour, with effects seen in generalised anxiety and feline response tests. However contrary to predictions, there was little or no significant differences between wildtype and TgTH overexpressor infected rats in feline response tests, implying that TgTH does not play a major role in feline or olfactory related predation response. The implications of the role of TgTH in generalised anxiety and activity levels were more mixed. This suggests that other mechanisms of action, probably multiple, are involved. This work increases our understanding of the potential mechanisms this parasite has evolved to attain specific manipulation of the rodent intermediate host to increase transmission success to the feline definitive host. The results thus have both evolutionary implications for parasite-altered behaviour in intermediate host species, and applied implications in terms of the potential impact on health and behaviour of all infected hosts, including humans. Open Access

Objectives: to assess the value of quantitative vascular imaging by power Doppler ultrasound (PDUS) as a tool that can be used to stratify patient risk of joint damage in early seropositive RA while still biologic-naive but on synthetic DMARD treatment. Methods: 85 patients with seropositive RA

Dengue is a vector-borne disease found across much of the world, with an increasing number of cases annually. This thesis explores the dynamics of dengue infection within an individual, and the possible impact of this at a population level. I use mathematical modelling and statistical analysis, tightly coupled with data, as a way of tying together the important components and processes during infection. I model the virus and immune dynamics, capturing the differences between individuals, disease severity and primary/secondary disease (with a focus on hypothesised secondary mechanisms). Within the immune dynamics I concentrate on antibody, looking at the role of antibody in limiting infection. Within this framework I also consider the impact on these dynamics of an antiviral. The final section of this thesis brings together this closer consideration of virus dynamics and considers their impact at a population level. Using data from biting experiments I am able to characterise the “infectivity” of an individual over time, how this varies between individuals and groups (as above), and how this compares to previous transmission modelling assumptions. In terms of control I look at how this “infectivity” is altered by antivirals and by wolbachia infected mosquitoes.

Considerable development in the field of nanotechnology is increasingly yielding novel applications of nanoparticles. The unique properties of nanoparticles in particular their high aspect ratio (length : width ratio), however could pose potential risks to the user. A high throughput genetic screening platform, RISCI (robotic single cDNA investigation), was previously established for the systematic evaluation of single gene activities. Here, RISCI was utilised to identify pro-apoptotic genes as well as genes involved in the positive and negative regulation of silica nanoparticle-induced cell death. This project describes the further development of the screening platform by harnessing its capability to screen a cDNA library comprising approximately 30,000 full length, completely annotated, and sequenced human genes for novel regulators of apoptosis. It integrates an extensive skill sets and is broadly organised into three major phases: Setup, Screen and Analysis. The integration of a pro-apoptosis treatment to screen for inhibitors and sensitizers is a novel aspect of the current experimental setup, along with the low redundancy library. The extensive setup phase focused on technical aspects. The cDNA library, acquired as plasmid DNA, was transformed into a bacterial host for replication and subsequent DNA isolation. A new high-throughput process was developed encompassing the production of competent bacteria and a heat shock transformation protocol, which was subsequently transferred onto the robotic platform. In parallel, the software controlling the robots was redeveloped to allow for execution of user-defined protocols while novel transfection protocols were adapted for automation. The screen identified 699 apoptosis inducers, 1,141 inhibitors and 626 sensitizers. Bioinformatics analysis revealed that the inducers were highly enriched for cell death associated terms, while the inhibitors were strongly associated with cancer profiles. Both inducers and sensitizers were predominantly achieving the functional effect on the protein level, but inhibitors were mainly transcription based. Enriched metal response genes also suggest that the silica nanoparticles were causing their toxicity through reactive oxygen species generation. Intriguingly, the screen identified many noncoding sequences as being functionally capable of regulating apoptosis. These noncoding candidates are capable of regulating the protein coding counterparts identified from the screen. The truly interesting part of the project outcome remains those unknown candidates that were implicated in apoptosis regulation for the first time. Dissemination of the consolidated candidate list would help accelerate the experimental validation of these candidates and aid other researchers in deriving novel hypotheses when the candidates are placed in their research context. [For supplementary files please contact author].

The work is focussed on the HIV epidemic among key populations in Latin America, a region that has received limited international attention due to the concentrated nature of its epidemic. It faces important challenges in prevention and risks missing the opportunity to control the epidemic. Despite the evidence available showing the disproportionate burden of infection among key populations, principally men who have sex with men (MSM) and to a lesser extent injecting drug users (IDU) and female sex workers (FSW), a small percentage of countries’ HIV prevention spending is allocated towards prevention interventions among these groups. In this thesis, we first estimate the distribution of new infections by type of exposure in the Dominican Republic, Mexico and Peru and find that MSM account for a large share of new infections in the three countries (over 50% in Mexico and Peru and 30% in Dominican Republic). This highlights that this population should be prioritised in prevention programmes. We then turn to interventions for MSM, and investigate whether pre-exposure prophylaxis (PrEP) to prevent HIV acquisition among MSM in Lima, Peru represents a cost-effective intervention at the population level. We find PrEP to be a potentially cost-effective intervention although unaffordable at a large scale. We conclude it should be considered as an additional tool within a combination prevention approach among this population. Following these, we seek to understand the dynamics, causes and consequences of risk behaviours and HIV infection in another key population that has rarely been prioritised by public health programmes: prison inmates. We use data from the largest prison in Peru and find that HIV prevalence is 4 times higher than national prevalence and inmates engage in riskier sexual behaviours than in the general population. Nevertheless, there are indications of risk behaviours and HIV prevalence reducing in recent years. To help direct prevention efforts in this setting, we characterise the risk profile of inmates who engaged in sex with FSW, other men and in unprotected sex with casual partners and identify inmates who have sex with men as being at heightened risk of infection (due to higher levels of drug use and unprotected sex). We use those data to develop a mathematical model that represents the HIV epidemic in the prison. Contrary to expectations, we find that incidence is likely to be less than 1% per year within the prison and that the dynamics of HIV in prisons are principally driven by inmates infected before their incarceration. We further estimate that since the beginning of the epidemic, at least 5% of HIV cases identified in Lima have passed through the prison, suggesting that the routine screening system in place at prisons could make a substantial contribution to controlling the HIV epidemic across the city. Finally, we formulate a conceptual framework of HIV risk among prison inmates in Latin America focussing on the social and underlying determinants of risk and develop a structured questionnaire to test the hypotheses proposed through the framework and obtain information relevant to the design of interventions within prisons and at the community level. Together this work draws on social and quantitative science to provide new insights into key populations in Latin America that should contribute to stronger and responsive HIV prevention programmes, to the benefit of all those that remain at risk of HIV in the region.

Genetics and epigenetics are widely expected to revolutionise our understanding of health and disease. However any attempt to extract relevant information from noisy data requires a combination of modelling and statistical techniques. Given the number of genes and the complexity involved in the genome, sophisticated methods will be needed to properly capture the information that is contained. Many mechanisms and variables can affect and control the expression of a gene. In this thesis, it is specifically spatially coherent variations in transcription which are investigated. Several different areas were examined, producing a broad set of results. Important findings include the demonstration of spatial coherence as the result of epigenetic effects, the creation and validation of a technique to detect spatial coherence, and the extension of spatial modelling to epigenetic data. Other important results include the detection of spatial coherence variation due to confounding variables (PMI and neuronal concentration) and the development of new spatial modelling techniques. The results indicate that spatial modelling provides a useful approach to investigating unusual and unknown aspects of epigenetic and transcriptional regulation.

Heterochromatin is implicated in the negative regulation of gene expression. To understand the effects of heterochromatin on RNA polymerase-II (RNAPII) mediated transcription, this study focused on the FXN gene where abnormal silencing induced by expanded (GAA)n repeats causes Friedreich's ataxia (FRDA), an incurable neurological disorder. Here, the silenced FXN locus was found to be modified by the heterochromatic histone marks H3K9me3, H3K27me3 and bound by HP1β. This pathological heterochromatinisation was partially reversed by the histone deacetlyase inhibitor nicotinamide, which upregulated FXN expression to potentially therapeutic levels in vitro, ex vivo and in vivo. In addition, RNAPII was shown to be stalled in the first exon of the FXN gene and degraded by the proteasome in FRDA but not in healthy cells. This was linked to increased proteasome binding at the -silenced FXN locus in a pattern reminiscent of the heterochromatin marks. Importantly, proteasome inhibition restored stalled RNAPII levels in FRDA and upregulated FXN to potentially therapeutic levels in vitro. Moreover, experiments with healthy human cells and wild-type mouse thymus revealed enriched levels of proteasome binding in other heterochromatic regions (e.g. pericentromeric repeats and SINEs) which were also de-repressed by proteasome inhibition; suggesting that the effect seen on the pathological FXN locus represented a specific example of a more generalised phenomenon. Overall, this introduces a novel mechanism whereby heterochromatin might be maintained in a silent state. In this thesis, heterochromatin effects were also investigated in relation to sexually dimorphic gene expression. Microarray analyses revealed hundreds of autosomal genes sensitive to sex chromosome-complement rather than gender. HP1β-repressed genes and SINE elements were over-represented within this gene group suggesting a potential link between heterochromatin, proteasome-dependent silencing and sexually dimorphic gene expression. The results reveal a novel layer in the regulation of sexually dimorphic genes with implications for understanding sex-bias in physiology and disease.

This thesis describes investigations into exogenous factors that influence fate-choices during human pluripotent stem cell (PSC) differentiation in vitro. I describe novel growth factor environments and small molecule regimes that provide patterning signals enabling directed differentiation of human PSCs towards biomedically relevant neurons. I provide evidence that the TGFβ growth factor Activin can promote ventral telencephalic differentiation. Small adjustments in Activin administration can produce an over-representation of forebrain derived medium spiny neurons and cortical interneurons, with significance in Huntington’s disease and epilepsy respectively. Additionally, I manipulate the earliest growth factor environments of PSC differentiation to lead to broad changes in rostro-caudal patterning. Inhibiting FGF signalling leads to a midbrain-like phenotype that can be further differentiated towards a ventral midbrain dopaminergic fate, a cell type that degenerates in Parkinson’s disease. Our novel manipulations and differentiation protocols provide insights into early events in human development, which would otherwise be impossible to study. This logic can also be applied to investigate diseased states during human development and ageing via use of disease-specific cell lines. The mature neurons produced may provide a tool that can be applied to large-scale drug screening assays and toxicology testing as well as having the potential for cell based therapies in future years.

Epithelial ovarian cancer is the commonest cause of death from gynaecological malignancy. Platinum based chemotherapy remains the cornerstone of first-line therapy for ovarian cancer, however relapse is common and acquired resistance is frequently observed on subsequent lines of platinum based treatment. Because activation of the PI3K/AKT pathway has been shown to play a role in acquired platinum resistance phenotype of ovarian cancer, combination of platinum with AKT pathway inhibitors holds promise for resensitising tumours to platinum. The aims of this thesis were i) to evaluate the effect of pharmacological AKT inhibitors AKT on the resensitisation of clinically-derived platinum resistant ovarian cancer cells to platinum, and ii) establish molecular imaging approaches for monitoring resensitisation to platinum in vivo combined with biochemical profiling of pathway activity. Treatment of platinum sensitive PEO1 ovarian cancer cells with cisplatin induced growth inhibition and apoptosis. The AKT inhibitor, API-2, resensitised paired platinum resistant PEO4 cells to cisplatin. These in vitro studies established 1h pre-treatment time point with API-2 followed by cisplatin as the most appropriate schedule for further studies. Resensitisation to platinum appeared to be a pathway effect rather than a specific effect of API-2. In order to establish the utility of PET imaging for therapy response, a bilateral tumour xenograft model comprising of platinum sensitive PEO1 and the platinum resistant cell line PEO4 was developed. Both [18F]fluorothymidine ([18F]FLT)-PET, which measures proliferation, and [18F]fluorodeoxyglucose ([18F]FDG)-PET, which measures glucose metabolism detected responses to cisplatin alone in PEO1 tumours and the combination of cisplatin and API-2 in both PEO1 and PEO4 tumours. Correlative reduction in phosphorylated PRAS40, a direct downstream marker of AKT activity indicated that in vivo changes in imaging variables of the combination treatment resulted from AKT inhibition. The imaging changes were also linked to histological reductions in Ki67 labelling index. AKT Biochemical profiling of tumours obtained after imaging confirmed that the changes in tumour [18F]FDG and [18F]FLT uptake were due, at least in part, to reductions in the expression of glucose transporter Glut-1 and hexokinase activity, as well as decreases in TK1 expression, respectively. These studies demonstrated that [18F]FDG-and [18F]FLT-PET hold promise for clinical evaluation of platinum resensitisation in patients with platinum resistant ovarian cancer. Given also that combination treatment with cisplatin and API-2 induced apoptosis in PEO4 cells in vitro, further studies were conducted with the aim of establishing [18F]ICMT11 PET, a specific cleaved caspase-3/7 radiotracer, for monitoring re-sensitisation to platinum in PEO4 tumours. [18F]ICMT11 uptake detected by PET increased after combination treatment but not with cisplatin alone. Parallel increases in TUNEL and cleaved caspase-3 staining by immunohistochemistry were observed consistent with the PET outcome. Thus, [18F]ICMT11 PET could also find utility in monitoring early responses to platinum therapy in combination with AKT inhibition. In addition to the 3 imaging agents described above, analysis of baseline levels of integrin αvβ3/5 expression, an index of angiogenesis, as well as myo-inositol uptake were investigated as potential discriminators of platinum resistant phenotype. These preliminary experiments were inconclusive. In summary PET imaging of proliferation, glucose metabolism and apoptosis were shown to be promising techniques for early detection of resensitisation to platinum therapy in platinum resistant ovarian cancer and warrant further investigation.

This thesis presents an investigation of bilingualism as a motor learning skill, with success ultimately measured in terms of strength of a foreign accent, in contrast to the many studies of bilingualism in terms of linguistic competence. My research used functional magnetic resonance (fMRI) imaging to investigate feedforward (motor) and feedback (auditory and somatosensory) systems involved in the production of foreign speech and how these systems are modulated by proficiency levels. I investigated the function of the frontal operculum and the temporo-parietal junction (TPJ) – planum temporale (posterior auditory association cortex) and parietal operculum (somatosensory association cortex) – during speech. The frontal operculum, strongly lateralised to the left, has been associated with speech since Broca performed his classic post mortem lesion-deficit analysis. Interest in the TPJ has arisen because of recent publications proposing the posterior half of the left planum temporale (± adjacent parietal operculum) as a ‘sensorimotor interface’ for speech production. My research compared activity within the frontal operculum and the TPJ during overt and covert speech. A second fMRI study examined retrospective proficiency based on existing language skills in people with English as a foreign language who were scanned during speech production in their native language and in English. A third fMRI study manipulated proficiency by training monolingual native English participants in the production of foreign speech sounds, with scanning pre- and post-training. This allowed measures of changes in activity (indicating rapid plasticity) following a short period of behavioural training in articulating novel foreign speech sounds. Training effects were observed predominantly in the striatum, and further analyses indicated that striatal activity in vocal learning is modulated by proficiency.

The bone marrow serves as a reservoir for leukocytes and stem cells, from where cells can be mobilised into the circulation and can be recruited to sites of inflammation. Mobilisation of cells out of the bone marrow is dependent on their migration across the bone marrow sinusoidal endothelium, which is thought to be structurally and functionally different to endothelial cells from other vascular beds. In order to characterise the bone marrow endothelium and to study the molecular mechanisms involved in the mobilisation of cells, a protocol to isolate bone marrow endothelial cells and to grow them in vitro was developed. The bone marrow contains a number of distinct progenitor cell populations, including endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs). Whether these populations of stem cells are recruited from the bone marrow to the lungs was investigated in two contrasting models of lung disease: the house dust mite (HDM) model of allergic airways disease and the bleomycin model of pulmonary fibrosis. In the HDM model increased recruitment of EPCs to the inflamed lungs was associated with increased peribronchial angiogenesis, and reduced EPC numbers in the bone marrow. Blocking VEGF inhibited EPC recruitment to the inflamed lungs and reduced the associated peribronchial angiogenesis. In this model, no recruitment of MSCs to the inflamed lungs was observed. However, in the bleomycin model, a significant elevation in MSC numbers was observed in the circulation, lung tissue and BAL fluid. Experiments to block the recruitment of MSCs to the lungs in response to bleomycin injury were performed, along with investigations into the recruitment of exogenously administered MSCs to the injured lungs. A population of MSCs residing in the naïve lungs was identified, which are phenotypically similar to bone marrow MSCs, but can be distinguished by their size and expression of specific cell surface antigens.

This thesis explores the roles of the gut hormones Peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) in the modulation of human brain reward pathways utilising functional magnetic resonance imaging (fMRI). PYY and GLP-1 are co-released post-prandially and both have been shown to reduce appetite and inhibit food intake when administered to humans. They have the potential to be developed into anti-obesity therapies, with the expectation that low-dose combination therapy may provide more effective weight loss and limited side effects. There are currently no safe and effective medications available to treat obesity, and yet this global health crisis continues unabated. In this context, a study of the mechanisms by which gut hormones exert their anorectic effects, may guide the rational development of new drugs. To date, the effects of GLP-1 alone and the ways in which PYY and GLP-1 combine to modulate brain activity in humans are unknown. This thesis contains a set of functional MRI experiments designed to determine these effects in healthy, fasted, normal-weight human subjects. Results are compared with the changes in brain activation patterns observed physiologically following a meal. For the first time in humans, I have demonstrated that, in conjunction with a comparable effect on lunchtime energy intake, combined infusion of PYY3-36 and GLP-17-36amide to fasted subjects results in a similar modulation of brain activity as observed following a large breakfast. This supports the proposal that these hormones are major physiological mediators of satiety in humans. Both the fed state and the administration of anorectic gut hormones to fasted subjects, reduces activation in multiple brain reward regions in response to visual food-cues. This confirms that circulating gut hormones modulate the hedonic processing of food. The lack of any obvious differential activation pattern between PYY3-36 and GLP-17- 36amide raises the possibility that they act at corticolimbic structures via a final common pathway.

A successful cancer gene therapy requires systemic gene delivery vectors capable of efficient and selective gene transfer to tumour sites. Two main approaches have been undertaken for targeted cancer gene therapy: i) restriction of transgene expression in tumour cells by using tumour specific promoters and ii) selective delivery of vectors of gene therapy to tumours by ligand targeting. Amongst many characterised tumour specific promoters, the stress-inducible promoter of the Glucose-regulated protein 78 (Grp78) gene holds great potential to improve the practice of cancer gene therapy as its activity is highly induced under conditions often found in the tumour microenvironment such as acidic pH, glucose deprivation and chronic anoxia. However, the clinical application of the Grp78 promoter in cancer gene therapy has been limited by lack of delivery strategies via the systemic routes. In 2006, an efficient tumour targeted vector, adeno-associated virus/phage termed AAV/Phage or AAVP, was reported. In this AAVP vector, the M13 bacteriophage (phage) was used as a vehicle to target αv integrin receptors, which are selectively overexpressed in tumours, and to deliver the AAV transgene cassette driving transgene expression from a cytomegalovirus (CMV) promoter. Systemic administration of the targeted AAVP viral phage vector has been shown to successfully mediate therapeutic transgene expression in several preclinical tumour models. Thus, the AAVP particle provides a suitable vector candidate for double targeting of cancer after systemic administration. Herein, I combined ligand-directed targeting of AAVP vector with transcriptional targeting using the Grp78 promoter in one single platform. In the first half of the thesis I show that the double-targeted vector provides sustained and long-term transgene expression in transduced tumour cells in vitro. Moreover, compared to the conventional phage carrying the CMV promoter, the new hybrid phage provided a significant tumour killing both in vitro and in vivo using the HSVtk gene and ganciclovir suicide therapy. More importantly in chapter 5, I describe a novel mechanism of Grp78 promoter activation by HSVtk and ganciclovir suicide therapy. In chapter 6, in addition to the vector safety, I show the superior tumour transduction in vivo after systemic administration of double-targeted AAVP vector over the original AAVP carrying the CMV promoter. In Chapter 7, I demonstrate strong dose dependent chemotherapeutic induction of the Grp78 promoter in the context of AAVP vector. Together, all data presented in this thesis validate the great potential of the Grp78 promoter in targeted systemic cancer gene therapy and report the efficacy of the double-targeted AAVP vector which proves useful for translation into clinical applications.

Tuberculosis (TB) continues to be a public health concern in high-income countries, such as the UK, with disease notifications primarily, and disproportionately, occurring in foreign-born immigrants who have a higher TB incidence than the local-born population. Underlying this epidemiology is the synergy of migration from high TB burden regions and the reactivation of imported latent TB infection (LTBI) in the initial years after migration which has refocused attention on immigrant screening for TB. In addition, amongst foreign-born individuals, TB disease primarily manifests as an extrapulmonary phenotype although the relative importance of demographic, host, mycobacterial lineage and environmental factors remains unclear. International, and national (UK-specific), surveys of the current systems in place for immigrant screening were undertaken. Using empirical UK immigrant screening data obtained from a multi-centre assessment of single-step IGRA testing and a prospective, community-based evaluation of different screening modalities, estimates for LTBI prevalence, yields for screening and the relative performance of different screening methods were computed. Decision-analysis models were constructed, with the data obtained from these empirical studies, to assess the cost-effectiveness of screening for LTBI at different incidence thresholds with different screening tools with and without port-of-arrival chest radiography to enable specific policy recommendations to be made. To assess demographic, mycobacterial lineage and environmental factors associated with specific clinical phenotypes, data from three independent Northern hemisphere TB databases (Birmingham, UK; US CDC and London, UK) were analysed. High-income countries prioritised screening for active TB but LTBI screening, paradoxically, was limited and those countries that did screen for LTBI varied significantly in whom they targeted and how they screened. Similar patterns were seen in the UK with heterogeneity particularly evident in the incidence threshold at which screening was instigated and the screening tools used. Moreover, screening for LTBI was inversely related to TB burden in the responding area. Data from the empirical studies of immigrant screening revealed that LTBI prevalence varied from 17-30% (depending on screening tool) and that prevalence was independently associated with increasing TB incidence in country of origin. The proportion of latently infected individuals identified (with LTBI) decreased as the TB incidence screening threshold increased. Health economic analyses revealed that the most cost-effective screening model would be to dispense with port-of-arrival chest radiographic screening and screen for LTBI at an intermediate screening threshold (250 or 150/100000) using single-step IGRA testing (with the QuantiFERON Gold in-tube). Data on the association of demographic and mycobacterial lineage factors revealed that, on multivariate analysis, extrapulmonary disease was most strongly associated (and more strongly than lineage) with non-White ethnicity. Mycobacterial lineages were geo-ethnically restricted and certain lineages (Euro-American and East African Indian) were preferentially associated with pulmonary disease whilst others, such as the East Asian lineage, were associated with extrapulmonary disease. In a separate assessment of demographic and environmental factors, local-born Whites had a significantly lower proportion of extrapulmonary disease compared to local-born Non-Whites; both groups had a significantly lower proportion of extrapulmonary disease as compared to foreign-born Non-Whites. In foreign-born Non-Whites, the proportion of cases that were extrapulmonary significantly increased as time elapsed between migration and the development of active TB before stabilising. In the US dataset, states with higher levels of sunshine had significantly lower levels of extrapulmonary TB. In the London cohort, individuals with purely extrapulmonary tuberculosis (14.1 nmol/L) had significantly lower mean vitamin D levels compared to individuals with pulmonary tuberculosis only (25.8 nmol/L)(p=0.0003). Severe vitamin D deficiency (

Highly active antiretroviral therapy (HAART) improves morbidity and mortality in HIV-1-infected patients yet reversal of immune dysfunction remains incomplete. The study objective was to determine the impact of immunomodulatory agents on T-cell function and phenotype in treated, chronic HIV-1 infection. Recombinant human growth hormone (rhGH) was administered daily to patients at a pharmacological dose for 12 weeks. Proliferative CD4 and IFN-γ-producing CD8 HIV- 1-specific T-cell responses were induced, however these responses declined with less frequent dosing. In a further study, patients that received a lower, physiological dose of rhGH for 40 weeks demonstrated increased IFN-γ ELISpot responses to pools of both MHC Class I and MHC Class II restricted HIV-1 Gag peptides at week 40. T-cell phenotypes showed a trend toward a reduction in the expression of activation markers. A randomised, open-label, phase I clinical trial investigated the effects of therapeutic immunisation, interleukin (IL)-2, granulocyte macrophage colony stimulating factor (GM-CSF) and rhGH in HIV-1+ subjects on HAART. Immunisation plus IL-2, GM-CSF and rhGH administration resulted in increased IFN-γ, IL-2 and perforin production in response to peptide pools of Gag, elevated CD4 T-cell counts and improved CD4/CD8 T-cell ratios at week 48. Subjects that received cytokine and hormone therapy (with or without immunisation) showed reduced immune activation and senescence, and for all patients, exhaustion markers were downregulated by week 48. Distinct Gag- and Nef- specific responses were not found to correlate with particular immunophenotypes. Further analysis of T-cell phenotypes from healthy controls showed immune defects to persist in HIV-1 infection despite HAART, and notwithstanding that, duration of HAART positively correlated with CD4 T-cell count. Together, the data presented here demonstrate the beneficial effects of immunotherapy in treated chronic HIV-1 infection and illustrate the potential to reverse T-cell dysfunction, furthermore highlighting the necessity for the induction and maintenance of HIV-1-specific immune responses.

This study focuses on integrating and applying computational techniques for modelling quantitative traits and complex diseases, such as hypertension and diabetes, using the rat model system and translating the findings to humans. Complex disease traits are heritable, highly polygenic, and influenced by environmental factors. Human studies, like Genome Wide Association Studies (GWAS), have identified many genetic determinants underlying these traits but have provided little information about the functional effects of these variants and mechanisms regulating the disease. This study takes a systems-level approach for looking at the genetic regulation of complex traits in the rat by analysing multiple phenotypes, genomewide genetic variation and gene expression data in multiple tissues. I integrated these multi-modality datasets in the BXH/HXB rat Recombinant Inbred (RI) lines, an established model of the human metabolic syndrome, to identify candidate genes, pathways and networks associated with complex disease phenotypes. I evaluated methods for Expression Quantitative Trait Locus (eQTL) analysis and used sparse Bayesian regression approaches to map eQTLs in the RI lines, delineating a new, large eQTL data resource for the rat genetic community. I have also developed and applied signal processing and time series analysis methods to physiological traits to extract more detailed indices of blood pressure, and integrated these with genetic, expression and eQTL data to inform on the regulation of these traits. Then, using publicly available data, I used comparative genomics approaches to elucidate a set of genes and pathways that can play a role in human diseases. This study has provided a valuable resource for future work in the rat, by means of new eQTLs in multiple tissues, and physiological time series phenotypes and approaches. This has enabled an integrative analysis of these data to give new insights into the regulation of complex traits in rats and humans.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurological disorder caused by the selective degeneration of upper and lower motor neurons, for which there are currently no effective treatments. 10% of ALS cases are familial, of which 15-20% are caused by mutations in the copper/zinc superoxide dismutase gene (SOD1). The primary triggers for motor neuron degeneration in ALS are unknown, but research in patients and SOD1 models has revealed several mechanisms which may contribute. These include: oxidative stress, mitochondrial abnormalities, inflammation and protein aggregation. This study focused on the pre-clinical testing of two-potential therapeutics for ALS in the SOD1G93A murine model of the disease; metformin, an anti type II diabetes drug which has been shown to have anti-inflammatory and anti-oxidant properties and the ability to bring about mitochondrial biogenesis, and trehalose, a chemical chaperone and enhancer of autophagy, which has been shown to reduce protein misfolding and aggregation. We performed an initial study in which oral metformin administration from 35 days increased the survival of functional motor units in the hindlimbs of male and female SOD1G93A mice at 100 days. Consequently we performed a dose-response survival study in SOD1G93A mice with longitudinal monitoring of weight and neurological score. Surprisingly, metformin had no effect in males and brought about a dose-dependent negative effect on the onset of neurological symptoms and on disease progression in females. We hypothesise this negative effect may have resulted from a metformin-induced reduction in oestrogen production. Oral trehalose administration was tested using the same survival study format. Although trehalose treatment brought about a dose-dependent delay in weight-loss in males, it had no effect on the onset or progression of neurological symptoms or on survival in male or female mice. We conclude that neither metformin nor trehalose represent strong candidates for clinical trial in ALS patients when administered orally.

This thesis addresses the hypothesis that multiple sclerosis (MS) patients have an imbalance in the frequencies of natural killer cell subsets, with fewer immunoregulatory and more proinflammatory NK cells compared to healthy controls. Ten NK cell subsets were defined according to expression of CD56, CD8, CD27 and CD57. Subset frequencies were compared by performing multiparameter flow cytometry on PBMCs from healthy controls and patients with clinically isolated syndrome (CIS) or relapsing-remitting MS. CD56dim CD8+ CD57+ CD27- NK cells had a higher frequency in untreated relapsing-remitting MS patients compared to healthy controls and their frequency was decreased in patients on interferon-β treatment. CD56bright NK cells, particularly the CD8- CD27- subset, had a significantly lower frequency in untreated MS and CIS patients. CD8- CD57- CD27- subsets of both CD56dim and CD56bright NK cells were expanded in interferon-β-treated patients in remission but not relapse. Characteristics of two CD56dim subsets displaying an apparent reciprocal relationship (CD8+ CD57+ CD27- and CD8- CD57- CD27-) were compared by qRT-PCR, flow cytometry and killing assays. CD56dim CD8+ CD57+ CD27- NK cells were more cytotoxic and expressed higher levels of transcripts encoding IFNγ, T-bet and other factors associated with IFNγ signalling. This is consistent with the hypothesis that there is a shift towards a more pro-inflammatory NK cell phenotype in MS patients, which is reversed by interferon-β treatment. In order to determine whether these NK cells exert their effects mainly in the blood or in the central nervous system (CNS), mononuclear cells were extracted from the CNS of Line 7 mice, which develop spontaneous autoimmune demyelination due to expression of a human MS-associated HLA/TCR combination. NK cells were detected in spleen, but not CNS, of these mice, suggesting that their role in this particular disease model is restricted to regulation of other leukocytes outside the CNS.

Background Email is a popular and commonly used method of communication, though not widely used in the healthcare setting. In England, policy has pushed for the introduction of email as a method of consultation in general practice. However, there is very little evidence available on its use in England and professional bodies have taken a neutral or negative stance to its use. The aim of this study was to explore the potential for email as a method of consultation in English general practice, and describe the key factors that may influence its normalisation. Methods A mixed methods approach was used. A systematic review was carried out to identify the extent of the experimental evidence base. An interview study was carried out with patient and professional (GPs, practice managers) participants who had used email for consultation, to establish their experiences and opinions, and to identify barriers and facilitators to its use. Normalisation process theory was used in interpreting the interview study results, so that the factors influencing normalisation might be identified. Results The review found that the existing evidence base for email consultation is poor and as such it is not possible to draw conclusions as to the effect of email as an intervention. The interview study identified that email consultation largely arose as a result of patient demand, and it brought advantages for both groups. However there were problems associated with it and these related largely to the uncertainties surrounding its use. Conclusions Email consultation is not a normalised method of consultation in English general practice despite the positive factors that patients and professionals associate with its use. It lacks a regulatory framework, which makes it difficult to use. Formal guidance on how to approach its use is required, along with methodologically rigorous research upon which policymakers and practitioners can base their decision making.

GABAergic drugs, such as benzodiazepines, are widely used in clinical practice yet their immune side effects are poorly understood. Preliminary studies have suggested that immune cells express GABAA receptors indicating that they may be controlled by GABA signaling. Herein parallel preclinical, translational and epidemiological approaches are described to help understand the importance of GABAA immunomodulation. The hypothesis is that GABA signaling acts to reduce responsiveness to a pathogen and thus that GABAergic drugs will increase susceptibility to infection. To inform on the clinical importance of this work, data from a subgroup analysis of the Maximizing Efficacy of Targeted Sedation and Reducing Neurological Dysfunction (MENDS) trial (where the relative effects of lorazepam, and dexmedetomidine were compared) are described in septic and non‐septic patients. Consistent with the hypothesis, avoidance of lorazepam sedation decreased mortality by 70% in septic patients but did not affect outcome in non‐septic patients. As preclinical data suggests that benzodiazepines increase mortality at subsedative doses we next conducted a population‐based cohort and nested case‐control design analysis of The Health Improvement Network (THIN), a comprehensive UK general practice database. Benzodiazepines exposure increased the incidence of community acquired pneumonia (CAP) and both 30‐day and long‐term mortality from CAP. Based on these significant accumulating data of the harm of exposure to benzodiazepines during an infection, animal studies were conducted to understand (i) the biological plausibility of our findings and (ii) the mechanism of the effect. In a series of mouse studies the prototypical benzodiazepine, diazepam, increased mortality from Streptococcus pneumoniae through potentiation of GABAA signaling. The increased mortality was associated with increased pathogen load and a delayed cytokine response to the infection. However cellular recruitment was not affected, indicating that local mechanisms were perturbed. Immune cell profiling revealed that alveolar macrophage and monocytes abundantly expressed subunits of the GABAA receptor, compatible with benzodiazepine sensitivity. Ex vivo studies showed that GABAA receptor activation decreased cytokine responses, phagocytosis and bacterial killing by alveolar macrophage likely via inducing an intracellular acidosis. Finally based on the immune cell profile of GABAA receptors we predicted that benzodiazepines that do not target the α1 GABAA subunit would lack the immune suppression observed by nonselective drugs. In accordance with this hypothesis we show that these selective benzodiazepines do not provoke intracellular acidosis, affect cytokine release or bacterial killing of macrophage ex vivo. In vivo the selective benzodiazepine did not increase mortality from infection or increase pathogen load.

Cough is under the control of sensory afferents which innervate the airways via the vagus nerve. Cough is an important protective reflex that clears the airway, but can become exacerbated and deleterious when associated with airways diseases, in which there is enhanced release of inflammatory mediators and a decrease in lung pH. These mediators sensitise airway afferents and could be driving enhanced cough associated with inflammation. Transient Receptor Potential (TRP) ion channels are associated with several disease pathologies. TRPV1 has an established role in cough, and is implicated in the aetiology of chronic cough; and TRPA1 is a promising new target. Involvement of these ion channels in the tussive reflex is awaiting comprehensive investigation. I have therefore explored the role of TRPA1 and TRPV1 in tussive responses to the endogenous irritants prostaglandin E2 (PGE2), bradykinin (BK) and low pH. To do this I have used selective antagonists and genetically modified mice in models of human, guinea pig and mouse vagal sensory nerve depolarisation; conscious guinea pig cough; and guinea pig primary ganglia cell imaging. TRPA1 and TRPV1 were shown to mediate PGE2 and BK-induced nerve depolarisation, cough, and activation of ganglia cells. In contrast, low pH-induced nerve depolarisation and ganglia cell activation was mediated via TRPV1 or Acid Sensing Ion Channels (ASICs); whereas, cough was partially attenuated with TRPA1 or TRPV1 antagonists. In summary, I have identified that TRPA1 and TRPV1 mediate PGE2 and BK-induced cough; and provided evidence that low pH-induced sensory nerve activation is mediated via TRPV1 and ASICs, but a role for TRPA1 is still unclear. These are exciting findings which add to our understanding of the mechanisms that drive the cough reflex in the healthy state; builds a base for investigating cough hypersensitivity in disease; and could help to guide the development of novel efficacious anti-tussive therapies.

Clinical and epidemiological studies were conducted to identify factors related to the wide range of serum-insulin values encountered in man. A reliable modification to the double antibody immunoassay for insulin was established. Serum-insulin levels were log-normally distributed. Lean healthy subjects showed a ten fold range of insulin values. "Insulin resistance" was identified in consistently high insulin secretors, who showed correspondingly higher blood-sugar values. Normoglycaemic and borderline diabetic patients with vascular disease or obesity had elevated serumrinsulin levels, but more severe diabetes was associated with a delayed and inadequate response to oral glucose. The one hour sample after 50 g. glucose identified most clearly the "insulin resistance" of normal subjects as well as the impaired insulin response of diabetics. In population studies, the independent relationships of various factors with the "one hour" serum-insulin levels were delineated. Blood-sugar levels showed the strongest association with serum-insulin. The latter rose steeply with blood-sugar levels up to 180 mg/100 ml., but beyond this a progressive decline of insulin values occurred. Other major factors associated positively and independently with elevated serum-insulin levels were age, obesity, hyperuricaemia, and hypokalaemia. The insulin response to the standard glucose load varied inversely with height. Females had consistently higher levels than males. Fasting serum-insulin levels were closely correlated with serum triglycerides both in normal subjects and those with coronary heart disease. Presumably insulin may have a physiological role in2 promoting hepatic triglyceride synthesis. Measurement of serum-insulin provided useful insight into possible mechanisms of disease; but was of limited use in discriminating diseased individuals. In the stages of diabetes mellitus, borderline hyperglycaemia was related to "insulin resistance", which may, in part, reflect varying vascular permeability. More severe hyperglycaemia was associated with a progressive impairment of insulin response, of undetermined cause, although hyperosmolality may contribute to this. Hyperinsulinism is possibly a "pre-diabetic" phenomenon. In subjects with vascular disease in the population, the association with elevated serum-insulin levels was confirmed, particularly in the younger subjects with coronary heart disease and in hypertensives generally. Serum-insulin may usefully characterize the metabolic status of population samples, for healthy and active subjects showed low levels that did not rise until late age; whereas obese, hyperglycaemic, or vascular disease subjects had prematurely elevated levels, and an accelerated rise with age. The findings are compatible with the hypothesis that insulin has an aetiological role in the early genesis of atherosclerosis. Open Access