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Long-lasting activation of inflammation leads to chronic conditions and is particularly common in autoimmune diseases. The causes of this self-activation in those conditions are still unknown, but these diseases are often also associated with an increase in oxidative stress. In fact, reactive oxygen species (ROS) are released during the inflammatory response and can cause oxidative damage, which in turn can lead to maintenance of inflammation. However, ROS are not only toxic species but can also act as signalling molecules to regulate immune responses, for instance via thiol modification. Thiols present in the cysteine residues of protein are among the most sensitive targets of ROS. They can undergo many redox changes, including glutathionylation or disulphide-linked dimerisation, all of which alter the protein and thus its function, localisation and secretion. This “redox regulation” regulates many cellular processes such as apoptosis, cell development and differentiation, homoeostasis and the immune response. In this project, we hypothesise that changes in thiol oxidation affect the inflammatory response and two different approaches have been set up to track redox changes in inflammatory conditions. Firstly, the role of endogenous glutathione (GSH), the main thiol antioxidant, was investigated. For this purpose, we used RAW cells, a mouse macrophage cell line. Cells were depleted of endogenous GSH and then stimulated with a standard inflammatory stimulus, bacterial lipopolysaccharide (LPS). A microarray analysis was then performed to identify changes in the gene expression profile. Results indicated that endogenous GSH does not decrease the inflammatory response but, on the contrary, favours the host antiviral response as its depletion results in an impaired LPS-induced increase in gene expression of genes in the interferon pathway, including oas2, mx2 and irf9. The biological significance of these results was later confirmed in cells infected with influenza A, showing that the antiviral response elicited by LPS was inhibited by GSH depletion. The second approach of this work was the use of a pegylated maleimide (MalPEG – 10kDa) to determine the redox state of three “redoxkines”, protein thiol/disulphide oxidoreductases with inflammatory properties: Trx, Prx1 and Prx2. MalPEG covalently binds to free thiols causing a mobility shift that can be detected by Western blot, leading to differences in the migration of oxidised and reduced proteins. After LPS stimulation, clear changes in the redox state were detected both intracellularly and in secreted proteins. To identify potential membrane targets of redoxkines, we set up a technique to identify proteins with redox-sensitive exofacial thiols on the cell surface. The results of this work show that activation of inflammatory pathway in macrophages brings about a number of redox changes in protein thiols, some of which may be related to GSH signalling, which are important in the regulation of both inflammation and host defence.

Head and Neck squamous cell carcinoma is the 6th most common cancer in the first world and oral squamous cell carcinoma the 10th. It comprises 2-3% of all new malignancies diagnosed in the United Kingdom every year. Despite decades of research, the life expectancy of this disease remains the same as it was 30 years ago and its incidence has been rising over the last 10 years. 85% of lingual cancers are found on the tongue’s lateral border. It is known that these cancers spread early to regional lymph nodes. A metastatic cervical lymph node is the single most important factor in the prognosis of lingual cancer, with its presence indicating a 50% reduction in survival rates. Therefore finding predictive factors within these cancers that could foresee whether it will spread early would improve diagnosis, treatment and potentially survival.

Lung cancer is the leading cause of death by cancer worldwide. There is an urgent need to expand the range of molecular targets and therapies, as well as biomarkers to diagnose disease, stratify high-risk patients and predict response to therapy. The importance of epigenetic transcriptional silencing as a mechanism of tumour suppressor gene inactivation is well recognized. DNA methylation is the dominant process necessary for this epigenetic inactivation and represents a valuable source of new biomarkers and therapeutic targets. The objective of this project was to seek de novo, candidate lung cancer tumour suppressor genes that are subject to transcriptional silencing by means of promoter methylation. Selected candidates would have their potential as prognostic biomarkers in non-small cell lung cancer characterized and any functional consequences of their silencing investigated. A genome-wide, microarray-based screen involving methylation and gene expression analysis in a panel of cell lines, incorporating treatment with the demethylating agent 5-aza-2′-deoxycytidine, was completed. From this, the TGF-β accessory receptor Endoglin and the transcription factor ZNF655 were identified and confirmed as novel genes subject to methylation-dependent silencing in lung cancer. Methylation of both genes was detected in primary lung cancer tissues. There was a strong trend towards shorter survival for patients with methylated Endoglin in two separate cohorts. Functional experiments demonstrated that silencing of Endoglin leads to increased invasion and epithelial to mesenchymal transition (EMT) in 2 NSCLC cell lines in vitro. In summary, I have validated the use of a high-throughput microarray screening strategy to identify novel epigenetically silenced tumour suppressor genes. I have identified Endoglin and ZNF655 as common targets of epigenetic silencing in lung cancer and have shown a functional role for Endoglin silencing in lung cancer progression.

Tc-99m-MIBI is a substrate for ATP-binding cassette (ABC) transporter proteins, including P-glycoprotein (P-gp), and has been used to image their expression, mainly in cancer. Imaging P-gp expression may have a role in renal transplantation and investigating drug toxicity. The aim of the project was to develop a functional imaging assay for ABC-transporters using Tc-99m-MIBI.

Background - Traditional audit methods are limited in their ability to provide short feedback loop to identify underperforming surgical units in time for them to respond appropriately. Moreover, case mix and other confounding factors limit the usefulness of crude mortality figures. More advanced industrial methods such as cumulative sum method (CUSUM) have therefore become of interest to surgeons. Hypothesis – Continuous monitoring of outcome in aortic aneurysm surgery using CUSUM technique (with optimisation using fractional polynomial mathematical models) can be applied, and do provide significant higher and more accurate detection rate of outliers when compared to traditional audit methods. Methods – Using anonymised records from National Vascular Database (NVD), three monitoring systems were applied in real time: Cumulative mortality (reflecting traditional audit process), funnel plot, and CUSUM (SPRT). VBOHM risk score was used to adjust for case-mix. Outliers were detected using different detection levels (h) and odds ratios (OR) with variable mortality rates (p). Performance of the three monitoring models was compared using direct alarm signals, sensitivity and specificity analysis, receiver operating curve (ROC), and average run length (ARL). Choosing control limits to maximise efficiency was approximated using direct simulation, Markov chain, and fractional polynomial techniques. Results –In-hospital mortality following elective Abdominal Aortic Aneurysm (AAA) repair between 1995 and 2011 in 140 centers were monitored. Compared to traditional audit methods, CUSUM has significant sensitivity to the outlier status of each vascular unit, with average number of CUSUM alerts of 0.89 when there is no outlier status, rising up to 23 alerts when there is an outlier status. Maximising the sensitivity and specificity of detecting outliers by CUSUM technique (also called incontrol ARL) while minimising false alarms (also called out-of-control ARL) was achieved using different range of values for control limits (h) and odds ratios (OR). For best CUSUM performance, values of OR=3, p=3, and h=1.25 has been shown to detect outliers correctly in 53% of case, and reject correctly in 59% of cases. This corresponds with CUSUM sensitivity of 80% and specificity of 80%. CUSUM has a positive predictive value of 78% and negative predictive values of 82%, with accuracy reaching 80%. Fractional polynomial technique and CUSUM simulation behavior were shown to correlate well (R > 0.88, p < 0.05) to the real-time NVD data analysis. Conclusion - To the best of our knowledge, our results demonstrated for the first time that using CUSUM (SPRT) model is both feasible and beneficial when used to comprehensively analyze a national dataset, validating the performance of all contributing Units, triggering alarm signals where necessary, and testing the sensitivity and specificity of each detection method with different decision making thresholds.

Everyday life is pervaded by prospects presenting risks and potential rewards, and the ability to decide effectively is critical for survival. Existing literature implicates integrated action of multiple neocortical and subcortical areas, yet the mechanisms underlying risky decision-making remain debated. Here, the processing of elementary prospects, bearing variable potential losses, gains and associated outcome probabilities was investigated through multiple modalities. Autonomic monitoring indicated very limited involvement of peripheral arousal in economic parameter representation, qualifying the somatic marker hypothesis. Electroencephalography demonstrated marked dissociation between long- latency cortical potentials, tracking potential gains and value, and alpha-band arousal response to potential losses and amount magnitude. Univariate functional MRI revealed a differential cortical representation of potential losses and gains, action of the mesial prefrontal cortex as decisional comparator and involvement of the insula in encoding outcome uncertainty. Subsequent graph-based network analysis demonstrated that the medial and anterior lateral prefrontal cortices harbour key value-integrating hubs, characterized by large numbers of effective connections and high topological centrality. Predicated on the apparent lack of striatal involvement indicated by functional MRI, a behavioural experiment was conducted on patients with basal ganglia degeneration, confirming normal decisional performance. Exploring the role of the lateral prefrontal cortex in regulating risk propensity and representation of gains and losses, a direct-current neuromodulation experiment was performed: though no changes in decisional pattern were observed, enhancing right hemisphere activity boosted confidence, echoing the biases observed in pathological gamblers. These results highlight that value determination proceeds through a distributed neocortical representation of specific economic parameters, particularly losses, feeding into densely-interconnected integrative hubs in the mesial and anterior lateral prefrontal cortex. Future work will need to confirm the effect of real vs. virtual financial endowment, explore the generalization to prospects presenting different combinations of options and investigate relevant patient populations.

Background: Chronic Kidney Disease (CKD) is common and is associated with an increased cardiovascular morbidity and mortality which is not completely explained by traditional risk factors. Non-traditional risk factors include arterial stiffening and calcification. Several calcification regulatory proteins (CaRP) are implicated in arterial stiffening. Fetuin-A may be important in inhibiting mineralisation via physicochemical interaction with calcium and phosphate, and can form circulating CalciProtein Particles (CPP) in pro-calcific states. Osteoprotegerin (OPG) & Receptor Activator of Nuclear Factor K-B Ligand (RANKL) control bone resorption and are also expressed in the vasculature. They may be important in vascular calcification. Fibroblast growth factor 23 (FGF-23) promotes urinary phosphate excretion and is also thought to be implicated in vascular disease. This study tests the hypotheses that these CaRP are associated with aortic stiffening in pre-dialysis CKD, and that CaRP and/or arterial stiffening are associated with outcomes. Method: 200 CKD stage 3 & 4 subjects were enrolled in this study. Subjects underwent annual measurement of aortic stiffness with carotid-femoral pulse wave velocity (C-F PWV) and CaRP were measured. Outcomes measures were C-F PWV and high sensitivity troponin T (hs-cTnT), a recently introduced biomarker of adverse cardiovascular outcome assessed at baseline, rate of change of C-F PWV, rate of change of renal function and survival. Major Results: OPG and CPP were independently associated with arterial stiffness. OPG, RANKL and FGF-23 were associated with hs-cTnT, independently of other recognised risk factors. Neither CaRP nor arterial stiffness were associated with rate of change of renal function. OPG:RANKL was associated with adverse survival in addition to hs-cTnT. C-F PWV increased across the follow up period and was independently associated with the combined end point of renal and patient survival in a time dependent manner. Conclusion: This study provides epidemiological evidence of the association between CaRP and a variety of cardiovascular measurements and outcomes including arterial stiffness, stiffening and survival. Arterial stiffness was associated with the combined outcome of death or progression of renal disease. Evidence of an association between arterial stiffness and either progression of renal disease or survival alone could not be proven.

Inflammation is a consequence of the activation of innate immunity and represents an important component of several pathological conditions, including not only the complication of infections but also sterile and autoimmune diseases. An early event in inflammation is represented by the production of proinflammatory cytokines and both their production and action have often been associated to oxidative stress. The redox status of the cell is therefore a key regulator of inflammation and glutathionylation (formation of mixed disulphides between cysteine residues of proteins and glutathione) is considered an important mechanism of this regulation. While most of the studies in the past focused on glutathionylation of intracellular proteins and transcription factors, the main goal of this project was to verify whether glutathionylated proteins are released by inflammatory cells and if these have a biological role. Using redox proteomics, we identified several proteins in the supernatants from Raw 264.7 cells (murine macrophages) stimulated with bacterial lipopolysaccharide (LPS). Among the identified proteins, we focused our attention on Peroxiredoxin 2 (Prx2), an antioxidant enzyme involved in cells protection against oxidative stress by removing H2O2. Released Prx2 was also detected in supernatant from human peripheral blood mononuclear cells (PBMC) and human macrophages. Prx2 levels were also increased in the serum of LPS-treated mice. We could confirm that Prx2 is released in the glutathionylated form. Moreover it was observed that the intracellular level of glutathione affects Prx2 release suggesting a role for glutathionylation in the mechanism of its release. The second part of the project was to verify whether released glutathionylated proteins may act as mediators of inflammation. To this purpose, the possible inflammatory role of released Prx2 was studied. The results showed that extracellular Prx2 induced an increase of TNF-α production in Raw 264.7 cells and in human macrophages. In conclusion, Prx2 is released during inflammation in a redox-dependent manner, in addition to its well-known intracellular role as enzyme, Prx2, in its released form, can also play a role in inflammatory response.

Background: Asthma affects approximately 300 million people worldwide ', 5.2 million of these people live in the UK2, 1.1 million of these are children" Asthma is one of the most common chronic diseases and is the fourth leading cause of morbidity worldwide, and there is no indication of a decline in prevalence. It is hypothesised that a range of gene- environmental interactions may influence the susceptibility, severity and medication response of asthma in children and young adults". Methods: To explore these issues, two studies have been established to create datasets that will describe the phenotypic and genotypic characteristics of children with asthma in the paediatric population across Sussex and Scotland. This thesis is the output of doctoral research using data from these studies (BREATHE and PAGES) that aims to explore the interactions between variants of six genes implicated in airway remodelling and relevant environmental factors and their influence on the severity of asthma in children and young adults. This thesis is divided by analysis of individual variants. The thesis included one variant of Chitinase 3-Like-1, two variants of Matrix metalloproteinase 9, two variants of Matrix metalloproteinase 12, one variant of Matrix metalloproteinase 9, one variant of Glutathione S-tronsferase mu-1, one variant of Glutathione S-transferase theta-1 and one variant of Glutathione S-transferase pi-1. A total of eight variants were investigated. Variants were analysed for effect on multiple proxy measures of asthma severity, including asthma exacerbations, asthma treatment steps, pulmonary function and quality of life. Variants were also analysed for their effect on allergy.