Objective: To assess the severity and predictors of fatigue in ANCA-associated vasculitis (AAV), and the contribution of peripheral and central mechanisms. Methods: Fatigue, anxiety/depression, sleep quality and pain were measured in 152 patients with AAV, 68 patients with CKD, andTl healthy conffols. Muscle mass, strength and endurance, cardio-respiratory fitness, perception of exertion, high-sensitivity C-reactive protein (hsCRP), and dehydroepiandrosterone (DHEA) were measured in 48 patients with AAV and 4l healthy controls. Results: Fatigue in AAV was more severe than in CKD 1p:g.gl3) or controls (p<0.001), and correlated with anxiety/depression, sleep quality and pain (all p<0.001). There was no difference in muscle mass (p:0.979) or strength (p=0.315) between AAV and conhols, but muscle endurance time was shorter in AAV (p=0.006), with greater muscle reserve (p=0.038) indicating central activation failure. Perception of exertion (p=0.006) and cardio-respiratory fitness (p:0.029) were worse in AAV than controls. Only perception of exertion independently predicted AAV fatigue (p:0.01). Sleep disturbance predicted altered perception of exertion (p:0.017). hsCRP was higher (p0.01 1) and DHEAS levels were lower (p<0.001) in AAV than controls, but neither predicted fatigue. Conclusion: Fatigue in AAV is more severe than in CKD or health, is due to central mechanisms, and may be amenable to intervention.

The costimulatory receptor CD28 and its inhibitory counterpart CTLA-4 share the same ligands and comprise a crucial checkpoint in T cell activation. CTLA-4 removes its ligands from antigen presenting cells by trans-endocytosis, which reduces the availability of costimulatory ligands for CD28 engagement to regulate T cell activation. This project examined the functional implications of reducing the availability of costimulatory molecules for CD4 T cell responses, and investigated the use of trans-endocytosis by other T cell receptors. Surprisingly, it was revealed that PD-1 and OX40 can also internalise their ligands, although perhaps not via the same mechanism as CTLA-4 trans-endocytosis. It was also shown that altering the availability of CD28 ligands affects the extent of T cell proliferation, suggesting that CTLA-4 trans-endocytosis can finely tune the T cell response. Furthermore, it was observed that CD28 costimulation is not always required for T cell activation and proliferation, but CD28 engagement is required for the optimal upregulation of a number of effector proteins and for TH2 cytokine production. Interestingly, T cells activated in the absence of CD28 signalling were not classically anergic. Strikingly, it was also found that memory T cells are dependent on CD28 costimulation.

The development, characterisation and testing of a novel all-optical, multi-modal preclinical biomedical imaging system is presented. The system aims to provide a new way of accurately visualising the spatial distribution and activity of molecular structures and processes in small animals by combining 3D bioluminescence tomography (BLT; reconstruction-based 3D imaging of internal bioluminescent reporter distributions), diffuse optical tomography (DOT; reconstruction-based imaging of optical parameter distributions) and optical surface capture techniques. The key principle of the imaging system is to use surface capture results to enhance the accuracy of DOT image reconstruction, and to use the results of both surface capture and DOT to enhance the accuracy of BLT. Presented experiments show that the developed system can reconstruct luminescent source distributions and optical parameters accurately and that small animal imaging is feasible with the system.

This thesis describes the impact of service delivery initiatives (SDIs) on patients’ waiting times within radiology departments. A systematic review of the literature (71 studies included) found the following broad type of SIDs: extended scope practice, quality management, productivity-enhancing technologies, outsourcing, pay-for-performance and multiple interventions. Ninety-six percent of the studies used either the pre- and post-intervention without control or the post-intervention only designs; but these designs are fundamentally weak and prone to bias. Furthermore, this thesis also described a case-study for the evaluation of the impact on patients’ waiting times of a 320-slice computed tomography (CT) scanner, speech recognition reporting and extended-working-hours within the Birmingham Heartlands Hospital (Heart of England NHS Foundation Trust), Birmingham. The evaluation combined the interrupted time series (ITS) design and qualitative interviews with healthcare professionals in a mixed methods approach. The mixed methods approach leverages the strengths of the quantitative and qualitative methods, so that the triangulation of the findings of one research method might be strengthened when supported by the findings of the other research method. The thesis used a distinctive implementation of ITS segmented regression which accounts for the changing trends of patients waiting times – an approach referred to as ITS ‘segmented spline’ regression.

Germinal Centres (GCs) play a central role in adaptive immunity; involving processes of cell migration, clonal expansion, hypermutation, and selection. To elucidate the role of affinity in regulating these processes, a technique for measuring B cell receptor affinity maturation in GCs in situ was developed. To facilitate interrogation of individual antibody-antigen interactions, atomic force microscopy (AFM) was chosen, offering nanometre positional resolution, and pico-Newton force sensitivity. Specificity of gold-coated AFM cantilevers towards the targeted receptors was achieved via a bespoke modification scheme, using self-assembled amine-terminated alkanethiol to facilitate attachment of the receptor specific antigen NP. Influences on molecule deposition and subsequent NP addition were investigated, as were control measures facilitating identification of successful modifications (Chapter 4). Effects of sample preparation techniques on AFM adhesion measurements were investigated (Chapter 5). Subsequently, the developed AFM technique was applied in interrogation of B cells and hybridomas – expressing receptors of varying affinity towards NP – and two GCs in tissue sections (Chapter 6). For the automated and unbiased evaluation of large amounts of varying AFM data, bespoke data analysis methods were developed. The project finds that AFM is capable of quantifying specific antibody-antigen interactions, but was unable to measure these in tissue sections. Possible reasons preventing such measurements are discussed.

Neutrophils constitute the main immune defense against microbial invasion. When activated, they migrate towards the site of infection where they eliminate any foreign material in an effort to prevent wide-spread tissue damage and ultimately resolve infection. Previous work on neutrophil function in the elderly has highlighted a number of neutrophil effector functions, including phagocytosis, superoxide production and migration that exhibit decreased efficiency suggesting the potential for reduced pathogen clearance in older adults. This thesis reveals a migratory phenotype distinctive of neutrophils isolated from healthy elderly donors (> 60 years) and characterized by a maintained speed of migration (chemokinesis) but with significantly reduced directional migration (chemotaxis) and overall migratory accuracy in response to a range of chemoattractants. This migratory phenotype was shown to be associated with a constitutive basal activation of PI3Kinase in neutrophils isolated from older donors and appears to be a causative factor as treatment of neutrophils with inhibitors selective for PI3Kinase-γ and –δ, was able to restore migratory dynamics. The ‘old-migratory’ phenotype was amenable to correction by pre-incubation with 1nM Simvastatin in vitro and a two-week prescription of 80mg/day Simvastatin in vivo in healthy older adults. The ability of simvastatin to modulate migratory dynamics potentially provides a safe, cost effective intervention to reduce morbidity and mortality from infections in the elderly population.

The research in this thesis is concerned with the effect of chronic stress, caregiving and bereavement, and ageing on immune and endocrine parameters. First, there was no difference in serum anti-CMV antibody titre between younger caregivers and matched controls, but CMV seropositive caregivers with more negative health behaviours had higher CMV antibody titre. Second, there was no difference in neutrophil function between caregivers and controls in both younger and older group, while only younger caregivers showed a higher serum cortisol:cortisone ratio than controls. Further, those caregivers that reported higher anxiety and burden symptoms had lower neutrophil phagocytosis. Third, caregivers had more senescent KLRG1\(^+\) T cells than controls, but comparable number of “exhausted” PD-1\(^+\) T cells and thymic output. Finally, young bereaved adults showed similar neutrophil function and serum cortisol and DHEAS levels as non-bereaved controls, whereas older bereaved adults had impaired neutrophil function and a higher cortisol:DHEAS ratio. These findings suggest that chronic stress can have differential effects on immune and endocrine parameters, but in some cases, presence of immunosenescence is required for immune decrements to be observed. Further, they emphasise the importance of focusing on the individual's response to chronic stress rather than their chronic stress status, per se.

Inflammation is a central component of the immune response. In its acute form it aids the transition from disease to health via the activation of numerous immune cells, enabling them to reach the site of infection/injury and orchestrate themselves to combat pathogens, facilitating resolution and repair to restore the host to health. However, chronic inflammation is deleterious to the host and differs from the “classical” acute inflammatory process in that the inflammation is not necessarily so readily obvious and is not self-limiting; rather, the immune system is in a constant state of low-grade activation and when challenged by pathogenic or sterile injury the response is heightened, resulting in prolonged tissue damage and a failure of efficient resolution mechanisms. Neutrophils are important mediators of acquired innate immune responses but may also contribute to the pathogenesis of chronic inflammatory diseases. Neutrophils are heavily involved in antimicrobial defence; their primary role is the localisation and elimination of pathogenic microorganisms. This, combined with their relatively short lifespan, has resulted in a traditional view of them as limited “kamikaze” cells. However, as detailed here, neutrophils have been shown to act with complexity and sophistication, orchestrating the immune/inflammatory response but also inadvertently contributing to tissue damage in different disease states. This thesis includes the study of neutrophil function in acute inflammatory episodes such as gingivitis and more chronic long-term health conditions such as obesity, chronic periodontitis and Papillon-Lefèvre Syndrome. The findings outlined here support the role of neutrophils as important contributors to both acute and chronic disease, showing these cells to be far more sophisticated than previously regarded.

The humoral immune response is characterised by the production of antibody secreting B cells. Some of these cells have cycled through the germinal centre, diversifying and optimising their antigen receptors to produce affinity-matured, class switched antibody. As this is a relatively slow process, the first class switched antibody is produced by non-mutated B blasts that have differentiated independently of the germinal centre reaction outside B-cell follicles. Extrafollicular foci of plasmablasts provide the first line of defence within the adaptive antibody response. IRF4 has been shown to be essential for Ig class switching and plasma-cell differentiation. Two expression levels of IRF4 were reported, with intermediate levels proposed to regulate CSR in the GC, while high levels regulate plasma-cell differentiation. We have correlated the two phases of IRF4 induction with specific stages of B-cell differentiation. Following immunisation of quasi-monoclonal mice with NP-Ficoll, intermediate levels of IRF4 protein are expressed by all B blasts as they move to the outer T zone and before the formation of germinal centres or plasma cells. This is followed by expression of AID and CSR. In contrast, plasma-cell differentiation occurs with high level expression of IRF4, expression of Blimp1 and complete suppression of AID and CSR. The NFκB family signalling molecules C-REL and NFκB1 have been shown to be required for the induction of IRF4 protein in B cells following stimulation. We show that these signalling molecules are not necessary to induce the early rapid intermediate level expression of IRF4, and extrafollicular expression of AID and CSR occur normally when they are absent. IRF4-high expression and plasma-cell differentiation, however, are blocked in the absence of these molecules.

Paper partition chromatography has been used extensively to study the aminoacid excretion in the urine of healthy children, of newborn infants and of patients suffering from eight different metabolic disorders. The purpose of this investigation was to discover the aminoacid pattern in the urine and the extent to which it is characteristic of each condition, and to establish the differences between the various aminoacid patterns. In order to gain some insight into the mechanism of the various forms of aminoaciduria a comparison was made between the aminoacid pattern of the plasma and that of the urine. In some cases microbiological assay has been used to establish the plasma level of certain aminoacids more exactly than was possible by paper chromatography. [From p92, Summary and conclusions] The main diseases investigated were: Lignac-Fanconi disease Cystine-lysinuria Phenylpyruvic oligophrenia Liver cirrhosis and hepatitis Steatorrhoea Galactosaemia Wilson's disease [From p4, Introduction]