Introduction Alongside an ageing population with an increasing prevalence of chronic liver disease (CLD) is an improved survival rate for younger people with CLD who are surviving into older age. Older people often have different, more complex health service needs for which geriatric services exist. However, the rapidly expanding older CLD population may have specific needs or outgrow specialty specific services. Falls are more common with increasing age in the general population and represent a substantial burden for individuals, families, society and the economy. People with CLD have multiple theoretical risk factors for falling, generating the hypothesis that falls and fall related injury will be common in older people with CLD. However, in order for services to adapt to the changing demographic evidence is required; firstly for an evidence-based management approach for falls intervention and prevention studies and secondly to support setting up of services. Methods In Phase 1, self-complete data collection tools were mailed to existing, comprehensive databases of extant cases with primary biliary cirrhosis (PBC), primary sclerosing cholangitis and post-liver transplant (post-LT). The same tools were completed by patients with non-alcoholic fatty liver disease and alcoholic liver disease in the clinic. The tools provided data to define falls and fall related injury prevalence in an older (>65 years) and a younger CLD cohort. An existing database of older, healthy community-dwelling adults was used as a control group. In Phase 2, multidisciplinary falls assessments were performed in a group of people with CLD to explore possible modifiable fall associations in order to inform future intervention/prevention studies. Finally, potential barriers to multifactorial intervention were explored. Results Falls are common in older people with CLD with almost 50% having had a fall in the previous year, significantly more so than age- and sex- matched community controls. Falling was unrelated to liver disease but was independently associated with orthostatic dizziness, lower limb strength and fear of falling. Falls were most common in older people with PBC (58%) and least common in ALD (18%). Fall prevalence post-LT is similar to other CLDs and significantly greater than in community controls; it is associated with orthostatic dizziness and the nadir blood pressure on standing up. Transplantation appears to have no effect on falling. Several barriers to intervention exist, confidence to exercise, understanding the benefits of physical activity, fatigue and FOF which was independently associated with levels of physical activity. Conclusion As falls are very common in older people with CLD and represent a substantial problem on an individual and societal level, current services will need to adapt. Several fall associations have been identified, each of which is modifiable and provides evidence for future intervention studies and services. In the general population orthostatic dizziness is amenable to conservative measures and lower limb strength and fear of falling can be improved through physiotherapy. If future studies revealed these to be effective intervention methods a multidisciplinary team consisting of a physician, nurse and physiotherapist would be required.

Ageing as a biological process is ubiquitous in life. In humans, ageing and its related conditions are revealed with improvements in health care conditions. Evidence for ageing is also apparent in most other organisms including unicellular species. Many of the pathways and mechanisms involved in ageing are evolutionarily conserved across the tree of life which provides an exceptional opportunity to study simpler organisms and extend the results to more complex forms of life. There is a rapidly growing body of data from organisms of varying levels of complexity, but there is a shortage of attempts in coherently making use of these data. A systems-level approach is necessary to bridge the gap between different biological levels, integrate the available information, and enable the synthesis of unifying hypotheses. Also, given the evolutionary nature of the question at hand (i.e. ageing), a successful hypothesis needs to be able to account for evolutionary considerations. In this thesis, I take a theoretical approach and try to explain a number of aspects of ageing from a systems-level perspective in an evolutionary context. Among the topics that will be covered are the following: (i) intra-islet pancreatic beta-cell dynamics, (ii) antioxidant defence system in pancreatic beta-cells, (iii) metabolic evolution of the glucose homeostatic system, and (iv) asymmetric damage segregation in unicellular organisms. In (i), I investigate the dynamics of beta-cell number within pancreatic islets and link the results to pathophysiology of diabetes and its various stages. In (ii) and (iii), I provide a unifying hypothesis for the paradoxical and unequivocal observation that metabolically active beta-cells have a weak antioxidant defence system and interestingly, that they are particularly weak in females. In (iv), I show how asymmetric segregation of damage at the time of mitosis is a fundamental step toward ageing and then evaluate whether and by how much asymmetry is optimal in a given organism under certain environmental conditions. I use a variety of techniques including deterministic and stochastic modelling in this thesis. The shared essence of these projects is an attempt to put data of various sources together in a unifying, systems-level evolutionary framework in order to better understand some aspects of the ageing process and its consequences.

The principal aims of the present study were to determine, in an unselected general population: (a) whether chronic oro-facial pain co-occurs with other frequently unexplained symptoms (b) whether factors associated with chronic oro-facial pain are common across symptoms. A population-based cross-sectional study was conducted using 4200 randomly selected adults who were recruited from the age-sex register of a General Medical Practice in North West, England. The study examined the prevalence and co-occurrence of chronic oro-facial pain with three other chronic symptoms that are frequently unexplained: chronic widespread pain, Irritable bowel syndrome and chronic fatigue. Validated instruments were used to measure the occurrence of symptoms and to collect information on a variety of associated factors: demographic (age and gender), psychosocial (anxiety, depression, illness behaviour, life stressors and reporting of somatic symptoms), mechanical (teeth grinding, facial trauma, missing teeth and reporting that the teeth did not fit together properly). 2505 subjects returned completed questionnaires (adjusted response rate 72%). The prevalence of Chronic Widespread Pain was 15%. Chronic Oro-facial Pain 7%. Irritable Bowel Syndrome 9% and Chronic Fatigue 8%. The study found that 587 subjects (27%) reported one or more symptoms: 404 (18%) reported one symptom, 134 (6%) reported two, 34 (2%) reported three, whilst 15 (1%) reported all four symptoms. This study has shown that chronic symptoms (including Chronic Oro-facial pain) that are frequently unexplained co-occur in the general population and share common associated factors. These findings are consistent with the hypothesis that chronic oro-facial pain may share a common aetiology with other frequently unexplained symptoms although this needs to be confirmed in a prospective study.

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In this thesis I have investigated the profile and significance of nitric oxide metabolites in two human models: 1) Across healthy human coronary and pulmonary vascular beds 2) In the peripheral venous blood from patients with type 1 diabetes mellitus. Chapters 1-3 offer a comprehensive background detailing issues of relevance to the rest of this work. Chapter 4 outlines precise methodological protocols and materials used. The results of the above clinical and laboratory studies are presented and discussed in Chapters 5 and 6. Chapter 7 attempts to summarise the results obtained, drawing together conclusions and highlighting perspectives for future research. I found that nitric oxide was dynamically metabolised across the heart and that the compartmentalisation of its metabolites between plasma and erythrocytes was driven primarily by the oxygen saturation of the blood. Study of the changes in coronary arterial diameter and flow in response to exercise and inhibition of nitric oxide generation suggested the presence of an endothelium derived hyperpolarising factor-like activity in the epicardial coronary arteries. Among patients with type 1 diabetes, blood levels of nitric oxide metabolites were generally lower compared to controls and lower in those with microvascular complications comparing to those without. Vessel relaxation experiments suggested the existence of a red blood cell-related vasodilating factor (RRVF) which was present in both diabetics and controls but exerted stronger vasodilator activity when erythrocytes from the former group were added to aortic ring preparations in a hypoxic tissue bath system ex vivo. Another novel finding was a positive correlation between this RBC-related vasodilator activity and HbAic which was stronger in that group of patients who were generally younger with shorter duration of disease.

The aim of this project was to demonstrate and develop a confocal readout method for fluorescent immunosorbent assays and investigate its potential advantages in comparison to traditional immunoassays. The key point of a confocal immunosorbent assay is the ability to detect the thin layer of immunoassay in the presence of unbound fluorescent reagents without washing the overlayer. Heterogeneous and homogeneous sandwich immunoassays of human IgG model were demonstrated successfully followed by the use of an empirical decomposition method for quantitative separation of the signals of the thin fluorescent assay layer from the overlayer. The detection limits for the homogeneous and heterogeneous formats of the model were 2.2 and 5.5 ng/ml, respectively. The application of confocal microscopy in kinetic analysis of the antigen-antibody reaction of the human IgG model was studied for homogeneous and heterogeneous formats and two fluorescent labels antibodies (FITC and QDs). The association rates of binding of FITC and QD605 conjugated antibodies to human IgG on prepared surfaces were 5.7×104 and 2.6×104 (M-1s-1) respectively. Confocal detection immunosorbent assay enables the detection of more than one assay along the z-axis. By replacing standard substrates with multiple 30 :m layers of substrates, a high density array of immunosorbent assays was created within a stratified medium. Stacks of up to five modified thin mica substrates of model immunoassays were detected by confocal microscopy. When applied to model assays consisting of human and mouse IgGs on different layers, the z-axis multiplexing of immunosorbant assays was demonstrated. The arrays of multiplexed immunosorbent assays were extended to 3D format by using microcontact printing and the assay density was increased twice by detecting the stack of two substrates which each contained two IgGs assays.

Reduced sulphydryl thiols (RSH): cysteine, homocysteine and glutathione are fundamental cellular components having important biological functions, including roles within the pathogenesis of a variety of clinical conditions. Independent analysis of these species is problematic and analytical difficulties relating to instrumental selectivity and sensitivity need to be overcome. This thesis describes the work carried out on the development and characterisation of a range of systems that could be used to facilitate thiol detection, ideally at the point-of-care, focussing largely on electrochemical techniques. Silver-thiol interactions were studied as a route to assist the sample processing. Here a novel controlled silver release mechanism was assessed. Silver release was found to be dependent upon the thiol structure. This has possible future applications to the development of methods to prevent biofilm formation, although the full mechanism of silver-thiol release requires further understanding. The development of unique molecular imprinted polymers was attempted. These would facilitate the detection of amino acids and the relevant thiol species via the amine functionality. The polymers proved unstable in the presence of hydroxylamine. However, this property makes the polymers suitable for use as protective or sacrificial polymers which can potentially be exploited in the manufacture of patterned electrodes. The nucleophilic substitution reaction between thiols and quinones, or quinone type materials, was explored as a possible route to assist selective thiol detection via electrochemical or colorimetric methods. Development of such reagentless sensing platforms would be beneficial in clinical analysis. Selectivity of thiol determination was achieved, although sensitivity issues will restrict real-world applications. A pH sensor utilising uric acid redox sensitivity was developed and was integrated within a disposable electrode assembly to enable wound pH monitoring. This platform was adapted as a prototype generic sensor for thiol analysis.

One of the hallmarks of successful immunotherapy is the generation of high avidity cytotoxic T cells which can recognise and respond to very low concentration of antigens. This sensitivity of T cells is usually determined by peptide titration ELISpot assays. Even though these assays are generally useful, they are laborious and sample demanding. The assays become even more difficult if the peptide(s) accountable for the generation of vaccine specific responses are unknown such as whole protein or cell vaccines. Therefore, there is a need to identify markers which can quickly and reliably identify a high avidity T cell response in cancer vaccination settings. To achieve this goal, this study utilised a C57Bl/6J mouse model which could efficiently generate high and low avidity T cell responses, when immunisation was undertaken with two form of vaccines to deliver the target antigens. The antigenic epitopes used for this study were derived from TRP-2 ‘self’ and ovalbumin (OVA) ‘foreign’ antigens. Immunisation of animals with these antigens in a DNA vaccine format induces a high avidity T cell response, in contrast to the response when these are administered in the peptide vaccine format. However, both the immunisations produced same number of peptide specific CD8+ T cells, which was assessed my multimer staining. When these cells were subjected to in vitro stimulations with the target peptides, the functionality of the low avidity T cells was restored whereas the high avidity T cells failed to respond to lower peptide concentrations. This showed the plasticity of antigen specific T cells and their ability to modulate their functionality according to the stimulation they have received. In order to identify markers that are associated with the high avidity T cell responses in vivo, antigen specific CD8+ T cells were isolated from high and low avidity groups using MHC multimer sorting. A global transcriptional profiling was conducted on the mRNA isolated from these cells. Analysis of expression data identified several differentially expressed genes between the groups. Six differentially expressed genes (Granzyme A, Granzyme B, FAS Apoptotic Inhibitory Molecule, Telomerase RNA Component, CD5 Antigen-Like, Spi-C Transcription Factor) were further selected and confirmed using qRT PCR. Expression of three genes were correlated with the microarray gene expression data. Among these, two genes (Granzyme A & B) were further confirmed at the protein level using flow cytometry. Further to this, studies of gene expression activation kinetics of TCR signalling using pentamer sorted cells with anti-CD3/CD28 monoclonal antibody coated microbeads, revealed that ImmunoBody® derived high avidity T cells are more signal competent with rapid up-regulation of genes involving in T cell receptor signalling pathway. Proteomic characterisation of MHC multimer sorted cells using LC-MS profiling identified several proteins uniquely associated with Peptide or ImmunoBody® pentamer positive T cells. Many of these proteins were associated with important T cell functional properties. Further confirmation of these markers and their role in T cell avidity is required, however these studies were limited by the lack of availability of proteins from the low number of peptide-specific cells and antibodies.

Metal and metal oxide nanoparticles including calcium oxide, gold, and superparamagnetic iron oxide nanoparticles (SPIOs) were synthesised using a range of techniques including reduction, co-precipitation and spinning disc technology. SPIOs were primarily synthesised via a co-precipitation method using iron (II) chloride, iron (III) chloride and ammonia; a spinning disc reactor and gaseous ammonia were trialled successfully for scale up, producing spherical particles of 10-40 nm in diameter as analysed by TEM. Nanoparticles were coated via a novel solvent-free grinding process which was successful for drug molecules, immunogenic peptides and amino acids; the mode of binding theorised to be taking place via an electrostatic interaction between the SPIO and the carboxyl, amine or hydroxyl groups on the coating materials. Recrystallisation of the coating materials to form HCl salts, was found to increase the binding efficiency with no detrimental effects to the particles. These coated SPIOs were found to be stable in a range of buffered solutions as well as blood and cell culture media. Separation of particles by size exclusion chromatography (SEC), dialysis and magnetic separation was only effective for a small range of coatings, with high speed centrifugation at a speed of 60 000 rpm being confirmed as the only universally successful method. Imaging of citrate capped gold nanoparticles using a CT phantom revealed that gold concentrations of 3700 mg.l-1 were required for in vivo use and so this was not continued. Coated SPIOs however produced relaxation times comparable with Endorem®, as well as being non-toxic. SPIOs coated in this way were more stable than Endorem® and stayed in solution retaining their superparamagnetic properties for periods in excess of 72 days with only a negligible degree of degradation. Various peptides were synthesised using an optimised microwave assisted solid-phase peptide synthesis (SPPS) method using double the suggested coupling times, all of which were analysed for purity and structure using MALDI-TOF MS and HPLC. A cell-penetrating peptide (CPP) synthesised via this method was coated onto SPIOs and mixed into a gel for transdermal delivery using porcine skin. An NMR profile of the skin using a 0.25T NMR MOUSE® before and after application of the gel showed that after an incubation period of 2 hrs the CPP-SPIOs had penetrated the skin leading to a reduction in signal. This has potential applications for subcutaneous drug delivery and hyperthermia. Cell studies using U937 and BMDCs indicated by both ICP and fluorescence microscopy that SPIOs coated with fluorescently labelled peptides were successfully taken up into cells. SPIOs were further investigated as vectors for delivery of immunogenic peptides, namely p53(105) using female C57BL/6 mice. Results indicated that mice immunised with SPIO as a vector showed similar levels of immune response to the p53(105) following immunisation as when incomplete Freund’s adjuvant (IFA) was used. However, SPIO immunisations produced a significantly increased specific response compared to the condition using IFA. Results indicate that SPIO could be successful as a vector for cancer vaccines.

Candida albicans is a human fungal pathogen responsible for superficial mucosal infections and life-threatening systemic disease. Despite the availability of potent antifungal drugs, mortality rates of systemic candidiasis remain high. Alternative therapies and vaccines are therefore desirable; however their generation depends on a comprehensive understanding of antifungal immunity. Innate recognition of fungi by the immune system is primarily mediated by a class of myeloid-expressed molecules termed the C-type lectin receptors (CLRs). CLRs mediate a variety of functions including phagocytosis, fungal killing, initiation of inflammation, and the generation of adaptive immunity. Adaptive responses are required for long-term memory and are shaped by the initial innate immune response controlled by CLR-expressing myeloid cells, yet the influence of CLRs on fungal-specific adaptive immunity is not well understood. In this thesis, the generation of C. albicans-specific T-cell immunity, particularly the CD4+ T-cell response, was investigated using OT.II transgenic T-cells and an ovalbumin-expressing strain of C. albicans. Using this model system, a novel role for the CLR, Dectin-1, was found in controlling CD4+ T-cell viability and recruitment to the GI tract. No roles for Dectin-1, or the related CLR Dectin-2, were found in controlling T-cell responses in the C. albicans infected kidney. However, it was found that, surprisingly, antigen-specific CD4+ T-cells did not migrate into the kidney during infection. Artificial restoration of this defect using immunoliposomes could significantly protect against infection, thus highlighting the importance of these lymphocytes to antifungal immunity. Furthermore, this thesis also explores the generation of better tools to study C. albicans-specific T-cell responses, and the roles of uncharacterised CLRs in the generation of adaptive immunity. Collectively, this research provides new insights into the generation and regulation of antigen-specific CD4+ T-cell immunity during C. albicans infections.