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This thesis evaluates the ability of ¹H nuclear magnetic resonance (NMR) spectroscopic and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) strategies to extract latent biochemical information from complex biofluid matrices from animal models and humans. ¹H NMR spectroscopy was applied to urine samples to investigate time-dependent biochemical changes in acid-base balance studies of metabolic alkalosis and metabolic acidosis and in a study of caloric restriction in order to establish normal physiological variation in organic acids including tricarboxylic acid (TCA) cycle intermediates. A method for profiling organic acids in human urine was developed, optimised and validated using UPLC-MS and then applied to targeted and untargeted metabolic profiling. A combined approach using both techniques was then applied in order to assess the relative efficiencies and merits of ¹H NMR spectroscopy and UPLC-MS for diagnosis of unknown inborn errors of metabolism (IEM) diseases using human urine samples in a blind fashion. Both NMR and UPLC-MS analytical platforms detected and identified all of the IEM correctly and revealed complementary biochemical information from IEM and controls. This evaluation was further extended and scaled up to assess the capability of the technologies for extracting latent biological information from urine samples collected in a large human population study. Chemometric methods such as Principal Component Analysis (PCA) and Orthogonal Projection to Latent Structure Discriminant Analysis (O-PLS-DA) were used to interpret and compare the two spectroscopic data types. These statistical methodologies allowed the detection of population specific biomarkers and the establishment of key metabolic phenotype differences in selected populations. In particular, both analytical approaches were able to discriminate Icelandic participants from other populations. The methods developed as part of this project should prove useful in widespread metabonomic applications in pharmaceutical and disease related areas.

Dendritic Cells (DCs) are key regulators in autoimmunity. DCs function as antigen presenting cells (APCs) and are present in trace amounts in virtually all organs of the human body. DCs activate and regulate naive T cell function by inspecting T cell function to identify specific T cells for the antigen presented on the DC surface as peptide-MHC molecules. Currently DCs harvested from blood and bone marrow possess individual variability in function and phenotype expression. Thus the need for standardised DC model is essential to enable more efficient and controllable studies into DC function as key immunoregulatory cells. Previous studies have shown myeloid DCs generated from MUTZ-3 myeloid leukaemic cell line precursors to be a potentially suitable model for DC function studies. In this study we aimed to establish MUTZ-3 as a model for DCs by incubating with various cytokines (TPO, IL-3, FLT3L, MCSF and SCF). The phenotype of the MUTZ-3 was analysed by flow cytometry to ensure the monocyte surface marker, CD14+, was optimally expressed. Further differentiation of these monocytes to immature DC was investigated by stimulating the MUTZ-3 with GM-CSF and IL-4. The results obtained are preliminary and require repeating, however, most have been shown to be reproducible. It was observed that cytokines can differentiate MUTZ-3 to optimise CD14+ monocytic expression. In addition, GM-CSF and IL-4 were shown to differentiate these monocytes to immature-DC. Finally, retinoic acid was used to differentiate the MUTZ-3. However, it is vital to continue investigating to establish the most efficient method for CD14+ expression is optimised.

Local vascular cell activation occurs at sites of injury/inflammation. This results in the elaboration of cytokines, chemokines and, ultimately, the infiltration of interstitial tissues by peripheral blood leukocytes. Such vascular cell activation, while essential in host defence, has pathological consequences in a variety of human diseases. The immunopathology of these processes in the development of atherosclerosis, vasculitis, thrombosis, vasoconstriction and allograft rejection and their phenotypic expression is discussed in detail. The growing evidence linking systemic hypertension with the development of atherosclerosis, and glomerular hypertension/glomerulosclerosis is also reviewed. Evidence has been provided that mechanical strain acting on the vessel/glomerulus may be a trigger for antigen-independent vascular cell activation in these diseases. In the current study <i>in vitro</i> experiments were designed to focus specifically on the putative modulation of immune mediator expression by mechanical strain. Results demonstrate that mechanical strain can be either a proinflammatory or prosclerotic stimulus depending on the cell type used and provide further insights into the molecular mechanisms underlying the pathogenesis of these conditions. Finally, observations in both acute and chronic vascular disease suggest the existence of endogenous regulatory systems that act to restore homeostatic vascular tone, thrombogenicity and adhesiveness for leukocytes to normal. In this dynamic process a number of anti-inflammatory molecules are elaborated. Experiments focused on the actions of lipoxins, a group of rapidly-acting anti-inflammatory eicosanoids - specifically the signal transduction events triggered by the activation of lipoxin receptors in endothelial and mesangial cells. The suppressors of cytokine signalling (SOCS) proteins are a recently identified family of proteins which inhibit cytokine-activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) cascade. Experiments were undertaken to elucidate the role of SOCS in renal inflammation. In addition, the profile of SOCS expression, along with that of related immune mediators, was examined in a model of alloantigen-driven inflammation - experimental cardiac transplant rejection. SOCS expression was confirmed in both these models, implicating these endogenous inhibitors in the modulation of cytokine bioactivity in inflammation and host defence.

The major historical developments in clinical measurement and also in statistics and computing are surveyed, and an outline of the problems to be considered in the thesis is given. Before dealing with clinical measuring processes in general, some methods for handling measurements are examined. In particular, procedures for dealing with replicate measurements are investigated. The evaluation of a single clinical measuring process is discussed with special reference to continuous auto-analysers such as the Coulter Cell Counter Model S. The main facets of evaluation of a single clinical measuring device are presented. Designs and the corresponding analyses are given for the simultaneous estimation of the accuracy, precision and carryover in the presence of drift. In some situations, linearity is also investigated. The results of some practical experiments are presented. To complete the discussion of the evaluation of measuring processes, the comparison of different clinical measuring processes for the same parameter(s) is investigated. A number of different methods of analysis are examined. Using clinical data, the results obtained from these analyses are presented. In a survey of quality control the basic requirements for control in the clinical field relevant to haematology, and in the haematological laboratory, are discussed. Quality control is seen to have two components - Intra and Inter laboratory control. An examination of both types of control is presented. These incorporate discussions of methods and procedures of control and related topics. An example of an intra-laboratory scheme, currently in use, is presented. In the discussion of inter-laboratory control, the results of a survey of the participants of the Department of Health and Social Security and British Committee for Standardisation in Haematology. Haematology Quality Control-Trials are presented. Finally conclusions, further areas for investigation and areas of development are presented and outlined.

Aim Over the last two decades, ultrasound imaging and practice has undergone a significant transformation shaped by political, cultural, sociological, technological and educational influences. Furthermore, the continual rise in the demand for ultrasound examinations is outstripping supply. This has had several reverberations and ramifications for the dynamics, constitution and wellbeing of the sonography workforce. Moreover, the adoption of ultrasound technology by various professionals has resulted in professional fission and fusion with both vertical and horizontal substitution within this heterogeneous workforce. It is against this backdrop that newly qualified sonographers enter the workforce. One under-researched area is an understanding of the experiences of these newest members of this multi-disciplinary workforce. Methodology A case study methodology was utilised which explored the experiences of newly qualified sonographers in their first year of ultrasound practice. The newly qualified sonographers were from a multi-disciplinary background. A conceptual framework was initially developed to guide the discovery phase of this research. Data was collected from eleven participants using face to face interviews and work diaries. The data sets were analysed using thematic analysis. The findings of the research were then embedded into the conceptual framework. Findings Three main overarching themes from eight subthemes were identified, namely; transition, role development and maintaining competency and credibility. The knowledge and understanding generated has broader benefits not only for the individual professional but also for ultrasound educational curricula, individual organisations and professional bodies. Experiences of newly qualified sonographers can be improved by developing specific preceptorship programmes in the initial period of transition whilst drawing upon theories such as cognitive apprenticeship and situated learning to facilitate role development after qualification. For organisations with a multidisciplinary ultrasound workforce, the fostering of communities of practice should be encouraged as a social learning environment. These strategies will promote the future role and development of the sonographer and create a greater sense of professional pride, belonging and security whilst supporting both uni-professional and cross-boundary ultrasound working.

T-helper (Th) 17 cells are a subset of CD4+ T cells defined through the release of the cytokine interleukin-17a (IL-17a). Activation of these cells is critical for protection against some extracellular bacterial and fungal pathogens. However, a dysregulated Th17 response targeted against self is thought to play an important role in the immunopathology of a number of autoimmune conditions including Inflammatory Bowel Disease (IBD), Multiple Sclerosis (MS) or inflammatory arthritides. Further understanding of the mechanisms that influence the development of Th17 cells may aid future therapeutic targeting of these cells. Whilst the role of the cytokine milieu in Th cell polarisation is relatively well characterised, the degree of signalling through the TCR can also shape the form of the Th cell response. Both the density of antigenic peptide available and the affinity of the antigenic peptide for a particular TCR can contribute to the degree of TCR signalling. The hypothesis of this project was that TCR signal intensity could alter the development of Th17 cells from a naive precursor population. In particular, it was of interest to determine how citrullination of a putative TCR contact amino acid in an antigenic peptide could alter the Th cell response observed. The 5/4E8 T-cell receptor transgenic (TCR Tg) mouse provides a model in which >80% of T-cells specifically recognise an immunodominant epitope derived from the G1 domain of aggrecan – peptide-84-103 (p84-103). This model allowed for the examination of these factors and the underlying mechanism ex vivo using a purified naive CD4+ T cell population in coculture with LPS-matured dendritic cells (mDCs). The data presented in this thesis show the activation, proliferation and effector responses of naive 5/4E8 TCR Tg T cells to alterations in both cognate peptide (p89- 103) density and affinity through citrullination of a putative TCR contact residue (R93Cit). Interestingly, by reducing TCR signal strength the observed response shifts from one dominated by the Th2 phenotype to Th17 cells. Linking the degree of TCR activation to Th cell phenotype was the intensity of IL-2 signalling that in turn shaped the balance between phosphorylated STAT3 and STAT5. Compared to p89-103-primed T cells, T cells responding to R93Cit produced less IL-2, expressed lower levels of the ILiii 2 receptor subunit CD25, and showed reduced levels of STAT5 phosphorylation, whilst STAT3 activation was unaltered. IL-2 blockade in p89-103-primed T-cells selectively reduced STAT5 but not STAT3 phosphorylation, and concomitantly enhanced Th17 development. In summary, this work indicates the impact that changes to the intensity of TCR signalling can have on the murine Th17 response. Indeed, these data illustrate how a disease-relevant post-translational modification such as citrullination can promote Th17 development by altering the balance between STAT5 and STAT3 activation in responding T cells.

The increasing application of nanoparticles in medicine, particularly in imaging and therapy, is heavily reliant on fundamental understanding of their unique physicochemical properties. This thesis investigates the interactions that occur between nanoparticles and human cells, specifically their toxicity and the mechanism of their internalisation. Alkyl-capped silicon quantum dots (SiQDs) were chosen for this investigation because their physical properties have been well-characterised and they have very bright luminescence. These properties facilitate their detection inside cells by fluorescence microscopy and flow cytometry. Toxicity and uptake mechanisms for SiQDs in human cells were investigated. The intestinal cell line CACO-2 was used as a model for ingestion of nanoparticles. Uptake and accumulation of SiQS in CACO-2 cells was demonstrated by epi-fluorescence microscopy and confocal fluorescence microscopy. Cytotoxicity in CACO-2 cells was studied by oxidative stress measurement using a intracellular dye method (H2DCFDA) (ROS assay), cell viability determination (MTT and ATP assays) and DNA damage measurement (Comet assay). Exposure of CACO-2 to SiQDs resulted in low cytotoxicity with regard to cell viability and effects on ATP production. SiQDs did not induce intracellular ROS production or DNA strand breaks. Over time periods up to 14 days, SiQDs showed no evidence of acute or chronic cytotoxicity. Accumulation of SiQDs inside a selection of human cell lines (CACO-2, HeLa, HepG2, and Huh7) was studied in detail using flow cytometry. SiQDs were internalised by all four cell lines. The highest levels of accumulation were seen with HepG2 and HuH7 cells. Some evidence for a role for caveolin 2 in this process in HuH7 was obtained using inhibitors and by gene expression analysis. The reason why HepG2 cells showed the highest accumulation of SiQDs remains unclear as caveolin expression measured by quantitative reverse transcriptase-PCR in these cells is very low. Further work on a possible role for the protein clathrin in the endocytosis process in these cells is needed as the inhibitor used to investigate this was toxic to the cells and firm conclusions could not be obtained. iii In summary, SiQDs are a highly promising alternative to heavy-metal based quantum dots and as a nanoparticle model to study mechanisms of uptake. SiQDs appear non-toxic in CACO-2 cells. However, the internalisation of SiQDs is cell-line dependent and further studies on their toxicity in cells such as HuH7 which show high levels of internalisation and cells relevant to immune responses such as macrophages are needed.

The use of biomacromolecules, e.g. peptides and antibodies, as therapeutics, so-called ‘biologics’, is experiencing an increase in interest as more and more small molecule therapeutics fail to meet the regulatory requirements for human use. To expedite the examination of such biologics as useful therapeutics, positron-emission tomography (PET) is being used as an early stage non-invasive in vivo imaging modality to rule in, or to rule out, candidates from the drug discovery pipeline. To realise this goal, small, reactive radiolabelled compounds, termed prosthetic groups, are often used to label the biologic of interest. Furthermore, prosthetic groups may also be incorporated into small molecule therapeutics, thus enabling rapid elucidation of candidates in a pre-clinical environment from a single appropriate prosthetic group precursor/apparatus set up. Diaryliodonium salts have been considered as useful precursors to prosthetic groups, as this class of compound may facilitate the one-step introduction of a nucleophilic radiolabel, e.g. [18F]fluoride ion, onto any position of a given arene. However, direction of the radiolabel onto the target arene is generally dictated by stereoelectronics, where the nucleophile is substituted upon the least electron-rich arene. To achieve this effect, electron-rich arenes, such as 2-thienyl, are employed. However, a potentiated product where the second arene is also electron-rich is the poorly characterised 2-[18F]fluorothiophene. To ensure that the products of radiofluorination processes reported herein are correctly assigned, an authentic sample of 2-[19F]fluorothiophene has been prepared on a 750 mmol scale (Scheme A). Scheme A: Synthesis of 2-[19F]fluorothiophene. Unambiguous verification of the detection of 2-[18F]fluorothiophene has herein been achieved for the first time by radiofluorination of the highly electrophilic electron-rich dithienyliodonium trifluoroacetate, using a microfluidic apparatus. iii Microfluidic apparatus is a technology which is experiencing an emergence as a useful means of probing the conditions advantageous to the production of radiolabeled material. We investigated the use of such apparatus in the optimised preparation of regioisomers of a key prosthetic group, [18F]fluorobenzaldehyde ([18F]FBA). The successful optimisation of the radiolabeling process has delivered 4-[18F]FBA in excellent radiochemical yields (>95%), as well delivering the associated regioisomer, 3-[18F]FBA, which is unavailable by convention means, by use of diaryliodonium salt precursors (Scheme B). Scheme B: Radiosynthesis of 4-[18F]FBA and 3-[18F]FBA from diaryliodonium salt precursors. Another prosthetic group considered bears the thiol reactive maleimide. An appropriate synthesis of a suitable diaryliodonium salt precursor (Scheme C) is reported herein. Scheme C: Synthesis of the precursor to the prosthetic group N-(4-[18F]fluorophenyl)maleimide.

Experimental and GEANT4 simulated Compton camera images and results are presented. This thesis aims to assess the contrasting performance of two seperate detector configurations which include a two germanium Compton camera (GeGe) and a germanium caesium iodide (GeCsI) Compton camera. GEANT4 software has previously been validated. All images were reconstructed using an existsing image reconstruction algorithm which utilises a simple back projection method.