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T cell activation is critical for the initiation of the adaptive immune response against both pathogens and malignant cells. This process depends on a complex set of finely tuned interactions, not only between the T cell receptor (TCR) and peptide-MHC complexes, but also between numerous accessory molecules. Chapter 2 of this thesis examines CD6, a transmembrane glycoprotein predominantly found on the surface of T cells, and its interaction with CD166, another transmembrane glycoprotein found on the surface of antigen presenting cells (APCs), amongst others. This interaction is required for optimal T cell activation. I have solved the X-ray crystal structures of the three extracellular SRCR domains of CD6 and the two membrane distal IgSF domains of CD166. In collaboration with colleagues, the ligand binding sites on both CD6 and CD166 have been further defined. We have also shown that a single nucleotide polymorphism (SNP) in CD6 caused glycosylation, hindering the CD6/CD166 interaction. Finally, native mass spectrometry revealed competition between the heterophilic CD6/CD166 interaction and the homophilic CD166 interaction.

The advent of optical flow cytometry in the 1980s created a growing demand for flow cytometers in biomedical research and inspired scientists to build or modify commercial instruments. Oceanographers use flow cytometers to perform phytoplankton analysis in remote locations on board ships. Our current knowledge of the role of algae and phytoplankton and their impact on global climate is limited by the frequency with which it can be analysed. Cytometric analysis of phytoplankton life cycles in their natural environment has the potential to revolutionise our understanding, but requires the use of miniaturised devices to operate at remote locations. To date, many research groups have developed unique flow cytometer systems to enable in-situ phytoplankton analysis. The development and miniaturisation process continues, and there is still a need to improve detection limits, modularity, and maintenance requirements. Furthermore, recent advancements in image processing and the availability of high resolution and high sensitivity cameras enable combining flow cytometry with microscopy. Image flow cytometry offers better morphological information, and unlike traditional cytometry, has the potential to screen multiple particles at a time, increasing the throughput. High throughput is also highly desired in an early detection and monitoring of hematological diseases such as leukemia, and other cancers. The aim of this thesis is to contribute to the development process of the traditional micro flow cytometry and imaging cytometry. Two kinds of devices were described and investigated in this thesis. The first device was a micro-flow cytometer chip based on optical fibre coupled architecture with an integrated micro lens that was fabricated to demonstrate an integrated fluorescence collection method by using a miniature cylindrical lens with a graded refractive index profile. The fluorescence collection efficiency was found comparable to a standard microscope objective. The use of the micro lens represents a significant progress in miniaturisation and assembly without sacrificing of performance. Practical use of the device was demonstrated by optical differentiation of phytoplankton cultures by detection of fluorescence, forward, and side scattered light. In addition, the device was tested with plain polystyrene, and fluorescent particles. The results were compared against two commercial instruments. The second device presented in this thesis is an imaging flow cytometer that uses acoustic levitation to assemble particles into a sheet structure. A high resolution, low noise CMOS camera is then used to capture images of thousands of cells with each frame. While ultrasonic focussing has previously been demonstrated for 1D cytometry systems, extending the technology to a planar, much higher throughput format and integrating imaging is non-trivial, and represents a significant jump forward in capability, leading to diagnostic possibilities not achievable with current systems. A galvo mirror was used to track the images of the moving cells permitting exposure times of 10ms at frame rates of 50Hz with near-single-pixel motion blur. At 80 Hz, a throughput of over 200k particles per second was achieved with particle velocities reaching 104 mm/s. The factors affecting motion blur and throughput were studied, and demonstrated the system with fluorescent beads, leukocytes, chondrocyte cell lines, and algae.

Flow cytometry is an important tool for medicine and biology with applications from clinical diagnosis to investigations of fundamental cell biology. However, traditional flow cytometers are expensive, bulky and complex to operate. Miniaturised flow cytometers or microflow cytometers offer advantages over traditional devices, being compact, cheap and mass producible and would offer the user ease of operation and low sample consumption. A key challenge for developing a microflow cytometer is the integration of the optical components with the fluidics. Integrated optical waveguides offer an ideal method of light control in microflow cytometers as the waveguides are intrinsically aligned to the analysis region during fabrication. In this thesis the design, fabrication and demonstration of a silica-based microflow cytometer for bead-based immunoassays is presented. The device consists of a rugged monolithic glass chip with integrated waveguides which deliver excitation light to an etched microfluidic channel and collect light transmitted across the channel. The fluidics are designed to employ inertial focusing to reduce signal variation by bringing the flowing beads onto the same plane as the excitation beam. A fabrication process was developed using techniques common to the microelectronics industry which are scalable for mass production. This process allowed the realisation of devices with waveguides of a range of widths from 2.0 um and upwards allowing both single mode and multimode operation at the immunoassay analysis wavelengths of 532 nm and 637 nm. Microfluidic channels with rectangular cross sections suitable for inertial focussing with depths of 30 um were also realised. Inertial focussing was demonstrated to confine the flowing beads in two dimensions in the microfluidic channel which effectively reduced the fluorescence signal variation in the device to a CV of 29%. The application of the device was demonstrated by the detection of fluorescence from immunoassay beads incubated with the cytokine tumour necrosis factor alpha at 154 pg/ml. Additional functionality of the device was demonstrated with transmission based detection of flowing beads.

This study explores the expectations and experiences of newly qualified diagnostic radiographers during their transition into practice. This is a short, but important period in a professional’s career as he or she adjusts from being supervised to becoming an autonomous practitioner. It is during this period that they enhance their competence and confidence. This was a longitudinal study using interpretative phenomenological analysis methodology. Data was gathered from four students who participated in a focus group. This informed semi structured interviews with a further eight students who were interviewed prior to starting work and three times over the following twelve months. All the participants had undertaken a BSc (Hons) Diagnostic Radiography at the same higher education institution. Four main themes were generated from the data; experience, fitting in, identity and supporting the transition. A high proportion of clinical education, balanced with theoretical input had developed the participants to be autonomous, reflective practitioners. However, they did find it difficult when required to take responsibility for, and assess students. During the transition process their awareness of departmental culture increased as did their professional identity. The participants wanted tailored support and found that they could ask any colleagues for advice and support and found peer support useful. An excellent practice of organised scaffolding support was identified which can be adapted for use in different areas. This helped the participants build experience and confidence. This study brings to light the experiences of newly qualified diagnostic radiographers. The findings are open to theoretical generalizability and raise issues that may be used by academic staff in the preparation of students and managers who support newly qualified staff members. These include considering how to train and educate student radiographers in supervisory skills, how to build confidence in areas where it is difficult to gain clinical experience, and facilitating peer support in imaging departments.

Aim: The aim of this thesis was to develop a framework of quality in radiographic service delivery in Ghana. This aim was to fit the medico-socio-cultural context of Ghana so as to promote the radiographers with additional knowledge about quality issues in the imaging services and patient care. Methods: Mixed methods were used with a sequential explanatory design under two phases. Phase 1 was a quantitative design which employed participants’ and service perspectives. A patient satisfaction survey (questionnaire) was used to gather data from 90 adult participants who were radiographically examined and who gave voluntary consent. The service perspectives involved the basic quality assurance and quality control procedures: reject film analysis and diagnostic reference levels. Phase 2 was a qualitative design which used semi-structured interviews of six radiographers, six patients and three managers to further explore and explain the quality of service issues identified in Phase 1. Results: A reject film analysis rate of 14.55% was achieved. Diagnostic reference level values of 70 kVp and 18 mAs, and 60 kVp and 13 mAs were used to obtain images of diagnostic quality of postero-anterior (PA) chest for patients above and below average sizes. Diagnostic reference values of 75 kVp and 32 mAs, and 62 kVp and 21 mAs achieved images of diagnostic value of antero-posterior (AP) lumbar spine for patients above and below average sizes. Diagnostic values of 56 kVp and 5 mAs, and 52 kVp and 3 mAs also achieved quality images of AP knee for patients above and below average sizes. Four quality constructs were identified with diagnostic radiographic service provision both locally and nationally. The quality framework was built around the four main constructs: departmental management role, radiography workforce/staff role, quality and safety committee roles, and quality and safety outputs that would help improve efficiency and quality of radiographic service delivery in Ghana and to ensure patient satisfaction with the services delivered to them. Conclusions: The constructs contributed to service delivery theory by developing a unique quality framework that would provide policy-makers and managers a practical understanding of factors that affect quality of radiographic service delivery. Key words: Framework, quality service, reject rates, radiographic service, safety outputs.

In order to achieve accreditation for their services, pathology departments must meet international recognised standards and comply with national guidelines, issued by accrediting bodies such as Clinical Pathology Accreditation (United Kingdom Accreditation Service), the Medicines and Healthcare products Regulatory Agency and the Human Tissue Authority in United Kingdom. Objectives: An evaluation of existing Quality Management System (QMS) and implementation of a quality standard and a centralised electronic document management software (Q-pulse) in a pathology department of a district hospital. Method: A service evaluation utilising an adapted version of the Context, Input, Process, and Product model, (with the addition of the implementation stage). This consisted of context evaluation (an evaluation of the department’s decentralised QMS), input evaluation (an assessment of the various types of electronic document management system (EDMS), implementation (the implementation of a centralised QMS and EDMS), progress evaluation (monitoring, documentation and assessment of the progress of the QMS) and finally impact evaluation (measuring the impact of the newly implemented system). Data collection included quality audits, non-participant observation and qualitative interviews. The implementation strategies involved technical support training and feedbacks. Results: The implementation of a continuous QMS and EDMS within the department, using the adapted CIPP model, increased the involvement of all staff in quality matters. It enabled the department to improve quality control processes, procedures and performance. Staff and quality teams now meet regularly and multidisciplinary workings groups have been put in place to continual improve the quality matters. The level of compliance and awareness to standards and guidelines has increased through education and training of staff in within the QMS, and there is an increase in top level management involvement in quality activities within the department. As a result of this evidence of compliance, the department’s Quality management system, was accorded, an unconditional accreditation, by the accrediting bodies. (Clinical Pathology Accreditation (United Kingdom Accreditation Service), the Medicines and Healthcare products Regulatory Agency). Conclusion: The quality management system is functioning in all the departments. All staff involved in the evaluation processes, judged it to be useful for them and the department. Further research is required to establish the longevity of the implemented products and other modules within Q-Pulse.

A polymorphism in the essential autophagy gene ATG16L1 is associated with susceptibility to inflammatory bowel disease. However, the role of autophagy in maintenance of intestinal immune homeostasis remains unclear. Using targeted deletion of Atg16l1 in T cells, we demonstrate critical requirement for autophagy in generation of appropriate adaptive immune cell responses within the mucosa. Selective deletion of Atg16l1 in T cells resulted in spontaneous intestinal inflammation, characterized by accumulation of Th2 cells and aberrant antibody responses towards dietary and microbiota antigens. Foxp3+ Treg cells were dependent on autophagy for their survival and function within the intestinal lamina propria, where they acted to limit Th2 cell accumulation. In addition, we demonstrate a novel role for autophagy in limiting Th2 cell survival in a cell-intrinsic manner. The distinct requirements for autophagy in the survival of different intestinal CD4+ T cell subsets reveals a novel role for autophagy in mucosal T cell homeostasis, with potential implications for understanding and treatment of chronic inflammatory disorders.

The CD8+ T (CTL) cell response is an important component of the immune response to HIV-1 infection. Presentation of peptides by the human leukocyte antigen (HLA) class I is key in directing that response. HLA-B*27:05 and HLA-B*57:01 have been consistently associated with immune control of HIV. To gain novel insights into the determinants of an effective CTL response mediated by these alleles, this work investigated cases of HIV disease control and progression in HLA-B*27:05 and B*57:01-expressing subjects. These subjects were either infected with clades of HIV infrequently found in combination with these alleles (C clade in HLA-B*27:05, and CRF01_AE clade in HLA-B*27:05/B*57:01) and/or represented the rare scenario of dual expression of both alleles. Transmission pair studies, viral replicative capacity assays and full-length ultra-deep sequencing were used to characterise CTL-mediated intra-host viral evolution, viral fitness and their effect on disease outcome. In the context of HLA-B*27:05 expression, this work demonstrated that the patterns of CTL immune escape and the associated viral fitness are considerably different in C clade compared to B clade HIV infection. This work also showed that rapid intra-host viral evolution and selection of escape may lead to disease progression despite expression of both favourable HLA class I alleles. The critical role of the Gag-specific CTL response in maintaining disease control, despite applying an unbiased genome-wide approach to detecting viral evolution, was demonstrated. This work has described the interactions between expression of HLA-B*27:05 and/or HLA-B*57:01, intra-host viral evolution and HIV disease progression, contributing to our understanding of CTL-mediated immune control. This is an important approach for elucidating correlates of protective immunity against HIV infection that may inform the design of intervention strategies.

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