Skeletal muscles control the joints of the skeletal system and they allow human movement and interaction with the environment. They are vital for stability in balance, walking and running, and many other skilled motor tasks. To understand how muscles operate in general and specific situations there are a variety of tools at the disposal of research scientists and clinicians for analysing muscle function. Strain gauges for example allow the quantification of forces exerted during joint rotation. However, skeletal muscles are multilayer systems and often different muscles are responsible for the overall force generated during joint rotation. Therefore, strain gauges do not reveal the extent of the contribution of individual muscles during muscle function. The most widely-used and accepted muscle analysis tool is electromyography (EMG), which can measure the activation level of individual muscles by measuring the electrical potential propagating through muscle resulting from local activations of motor units. However, EMG does not linearly relate to any real physical forces, meaning that without prior knowledge of the force exertion on the level of the muscle, force cannot be estimated. EMG can measure superficial layers of muscle non-invasively by attaching surface electrodes (surface EMG) to the skin over the belly of the muscle. To measure the activity of individual muscle beneath the superficial muscle, a needle or thin-wire electrode must be inserted through the skin and into the muscle volume (intramuscular EMG), which is invasive and not practical in many situations. Furthermore, intramuscular EMG can only provide measurement of a very small volume (< 1mm³) which can have varying amounts of active motor units. Ultrasonography is a powerful cost-effective non-invasive imaging technology which allows real-time observation of cross-sections of multiple layers of dynamic skeletal muscle. Recent advances in automated skeletal muscle ultrasound analysis techniques, and advances in image processing techniques make ultrasound a valuable line of investigation for analysis of dynamic skeletal muscle. This aim of this thesis is to study and develop advanced image analysis techniques applicable to the analysis of dynamic skeletal muscle. The broader aim is to understand the capacity/limits of ultrasound as a skeletal muscle analysis tool. The ideas presented within offer new approaches to modelling complex muscle architecture and function via ultrasound. Tools have also been developed here that will contribute to, and promote ultrasound skeletal muscle analysis as a new and emerging technology which may be used by clinicians and research scientists to develop our understanding of skeletal muscle function. The main findings of this thesis are that automated segmentation of architecturally simple and complex skeletal muscle groups is possible and accurate, and that information about joint angles and muscle activity/force can be automatically extracted directly from ultrasound images without the explicit knowledge of how to extract it. The techniques used offer new possibilities for non-invasive information extraction from complex muscle groups such as the muscles in the human posterior neck.

Migration inhibition factor (MIF) was isolated from the mouse, rat, Xenopus laevis and the newt Triturus cristatus. These MIFs were used to investigate the specificity of the molecule and it was found that MIF is not species specific and that animals with an evolutionarily sophisticated immune response are responsive to MIFs produced by animals which show much less complex immune responses. Macrophages are one of the few normal adult cell types which show motility as part of their functional phenotype and this process can by manipulated in vitro by the presence or absence of MIF. Using LM and EM the effects of MIF and substrate on locomotion and macrophage-substrate adhesion were investigated. The question of macrophage heterogeneity was addressed to determine whether macrophages can be activated for a wide range or only a limited number of functions. The results obtained suggest that macrophages become committed along particular pathways depending on external stimuli from the micro-environment surrounding the cell and that performance of particular functions precludes the cell from performing certain other functions. Macrophage activating factor (MAF) co-chromatographs with MIF and was isolated with MIF. The effect of MIF/MAF and effector : target cell ratio on macrophage anti-tumour activity was also investigated. Macrophages were found to have a cytostatic effect at a much lower effector : target cell ratio than that required for cytolysis of transformed cells. Also, macrophages lyse target cells at a lower effector:target cell ratio when cultured on a collagen rather than a glass substrate. Macrophages are also attracted through collagen towards transformed cells preferentially. Pretreatment with MAF enhances the cytolyic response of macrophages. This response is increased further by pretreating macrophages with LPS and MAF when both pretreatment time and sequence are important in determining which functions macrophages perform.

The human fetus is donated passive immunity, via the placenta, in the form of maternal IgG class antibodies. This provides protection against pathogens while the fetus is immunologically immature. Immunocytochemistry has been performed on placental tissues to localise endogenous IgG and three subtypes of IgG receptor designated FcgammaRI (CD64), FcgammaRII (CDw32) and FcgammaRIII (CD16). Ultrastructural immunocytochemistry has been used to investigate the subcellular distribution of IgG. FcgammaRI is expressed by mesenchymal cells of term and first trimester placentae. FcgammaRII is expressed by endothelial cells and FcgammaRIII is expressed by syncytiotrophoblast. Endogenous IgG is restricted to the syncytiotrophoblast in first trimester villi indicating that transport is inhibited in early stages of gestation. At term IgG is associated with extracellular matrix, serum, endothelial cells, mesenchymal cells and syncytiotrophoblast suggesting that it is efficiently transported across the trophoblast. Ultrastructural observations of IgG distribution indicate that it is taken up by receptor-mediated endocytosis into the syncytiotrophoblast and reaches an endosome-like compartment. In term amniochorion FcgammaRI, FcgammaRII and FcgammaRIII are expressed by leucocytes in the maternal decidua and heterogeneous populations of mesenchymal cells in the fetal connective tissues suggesting that they possess an immunoregulatory role. Endogenous IgG is restricted to the extracellular matrix and cells possessing Fcgamma receptors. Cytotrophoblast and amniotic epithelial cells, which do not possess Fcgamma receptors, contain no endogenous antibodies. The hypothesis is proposed that cytotrophoblast cells are a barrier to IgG transmission across the placenta. This accounts for the paradox that there are low levels of transport in the first trimester when the syncytiotrophoblast expresses Fcgamma receptors. Cytotrophoblast cells form an almost continuous layer underlying the syncytiotrophoblast in the first trimester which becomes attenuated as the placenta matures. Furthermore, IgG may diffuse non-specifically between cytotrophoblast cells of the amniochorion and thus contribute to the acquisition of passive immunity. These data suggest that IgG is transmitted across the vascular system of the placenta and is not acquired via amniotic fluid.

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Competent sonography includes a unique combination of visuospatial perception and psychomotor skills not yet identified. Little is known about the relationship between these two abilities and scanning ability and this feasibility study explores possible relationships. A sample of thirty experienced sonographers and thirty trainees before and after training were administered five visuospatial perception tests, two psychomotor tests and an Obstetric Structured Assessment Test (OSAT) to asses scanning ability. The two psychomotor tests employed were the Perdue Pegboard Test (PPT) and Zig Zag Tracking (ZZT) test. The PPT measured dexterity for gross movement of arms, hands, and fingers and fingertip dexterity. The ZZT measured hand to eye coordination or speed and accuracy of hand movement. No significant relationship between trainees' visuospatial ability or psychomotor abilities and scanning abilities were found. The results for visuospatial perception tests suggested that the Snow Pictures Test, Surface Development Test and Gestalt Completion Test were influenced by sonography training so they were not measuring innate skills. However, the Mental Rotation and Abstract Reasoning Tests scores were not influenced by training therefore they measured the innate skills of sonographers and therefore may be useful in selection processes for admission to training programs or, to identify those trainees who may benefit from more intense training and support. However abilities require further investigation. The ranges of scores for each of the eight tests for the profession of sonography were established. The range of scores for sonographers for the PPT and ZZT gave a measure of dexterity for gross movement of arms, hands, and fingers and fingertip dexterity for this particular group adding to the current body of knowledge in this area. Performance on the OSA T pre-training may give an indication of post training scarming performance so this may be a useful tool for initial assessment of potential trainees but, further studies with a larger sample would be required before this could be endorsed, validated or implemented.

Electrical Impedance Tomography (EIT) is a medical imaging technique which reconstructs the internal conductivity of an object from boundary measurements. EIT has the potential to provide a novel means of imaging in acute stroke, epilepsy or traumatic brain injury. Previous studies, whilst demonstrating the potential of the technique, have not been successful clinically. The work in this thesis aims to address fundamental limitations including measurement drift in electronic hardware, lack of an anatomically realistic tank phantom for rigorous testing, poor electrode-skin contact and mis-location of scalp electrodes. Chapter 1 provides an introduction of the principles of bioimpedance and EIT, as well as a review of previous clinical studies. Chapter 2 details the development of a novel anatomically realistic head phantom, simulating the human adult head with scalp electrodes, using a 3D printer and cylindrical holes to provide simulated conductivity. This replicated the varying spatial conductivity of the skull within 5 % of the true value. Two multifrequency EIT systems with parallel voltage recording were optimised for recording in the adult head with scalp electrodes, in chapter 3. Measurement drift was reduced by better case design and temperature control and data quality was improved with an updated interface to the current source and signal processing. The UCL ScouseTom system, performed best, with lower noise in all resistor and tank measurements, but the differences were masked during scalp recordings. Further, both systems produced similar results in the realistic adult head tank from chapter 2. Recent advances in EIT imaging coupled with the developments in chapters 2 and 3 provided opportunity to reassess the feasibility of monitoring epilepsy with EIT. Biologically representative perturbations was localised to within 8 mm in the head tank, with less than half the image error of previous studies. However, the key limitations of application time and measurement drift with scalp electrodes had yet to be addressed. Therefore the focus of the work in chapter 5 and chapter 6 was the design and testing of a novel self-adjusting electrode helmet. Skin-electrode impedance was continuously optimised by constant pressure, rotation and feedback control, and position sensors returned the co-ordinates of electrode tips. Finally, experiments with this helmet were undertaken to assess the feasibility of future clinical recordings.

β-lactam hypersensitivity reactions can be severe and are extremely difficult to predict. Drug-specific T-cells have been identified in blood of patients presenting with cutaneous and hepatic hypersensitivity reactions, indicating that they play a role in the disease pathogenesis. Animal models are highly effective tools that have been used extensively to dissect mechanisms of disease and pathways of disease progression; however, animal models of drug hypersensitivity reactions have proven difficult to develop. The aims of this thesis were to utilize 3 β-lactam antibiotics amoxicillin, piperacillin and flucloxacillin to explore antigen-specific T-cell responses in the mouse and to attempt to develop a model of T-cell-mediated drug-induced liver damage. The project utilized the C57/Bl6 CD4+ T-cell deficient mouse with a mutation in the αβ gene encoding for MHC class II molecules, which has previously been used to investigate skin sensitization to drugs. In initial experiments, amoxicillin-specific CD8+ T-cell responses were detected both in vivo and ex vivo. Sensitization was obtained through painting of the drug onto the skin of mice that had been depleted of CD4+ T-cells, which are thought to exert regulatory/suppressor functions. On completion of the sensitization protocol, draining lymph node cells were removed and the drug-specific T-cell response was detected through analysis of proliferation and IFN-γ release. In contrast, proliferative responses and cytokine release were not detected with cells from vehicle control mice. The study was expanded to include 3 β-lactam antibiotics. Activation of CD8+ T-cells was readily detectable following sensitization with flucloxacillin. In contrast, only weak ex-vivo proliferative responses were detected following sensitization with piperacillin, which may relate to the fact that piperacillin preferentially activates CD4+ T-cells in hypertensive human patients. Drug-specific T-cell clones from human patients were generated and tested alongside murine counterparts to provide a detailed assessment of cross-reactivity and variability in the drug-specific T-cell response between species. Amoxicillin and flucloxacillin demonstrated cross-reactivity with both human and murine drug-specific T-cells. Piperacillin cross-reactivity was difficult to assess in mouse. However, human piperacillin-specific T-cells displayed no evident cross-reactivity with amoxicillin or flucloxacillin. The ex vivo activation of flucloxacillin-specific CD8+ T-cells from sensitised mice was discovered to be dependent on the presence of APCs. The concentration of APCs added to cultures of drug-specific draining lymph node cells was directly correlated with the amount of CD8+ T-cell activation. In fact, the removal of APCs ablated the proliferative response and IFNγ secretion when APCs were added to flucloxacillin re-challenged ex vivo cultures of flucloxacillin-specific CD8+ T-cells from the draining lymph nodes of sensitised mice. There are currently no animal models of drug-induced liver injury where the adaptive immune system has been shown to damage hepatocytes. It is therefore difficult to explore the mechanistic basis of the tissue injury. Thus, an aim of the project was to characterize the immunogenicity of flucloxacillin and explore whether flucloxacillin-responsive CD8+ T-cells damage hepatocytes. In initial experiments sensitization was achieved through epicutaneous application. CD8+ T-cells from draining lymph nodes of the flucloxacillin-treated mice proliferated in a concentration-dependent manner following ex vivo secondary stimulation. The proliferative response was associated with IFN-γ and granzyme B release. Flucloxacillin-specific hepatocyte toxicity and apoptosis was observed when CD8+ T-cells were cultured with dendritic cells and flucloxacillin for 24h, washed and transferred to the hepatocyte cultures. In contrast, hepatocyte killing was not detected in with T-cells from vehicle control mice. In separate experiments, flucloxacillin-specific T-cells were forced to migrate to the mesenteric lymph nodes using retinoic acid, prior to administration of oral flucloxacillin for 10 days, followed by analysis of liver histology and plasma biomarkers of liver injury. Oral exposure resulted in gall bladder swelling, hepatic mononuclear cell infiltration (especially around the bile ducts) and mild elevations in plasma ALT. This work has highlighted the usefulness of animal models in studying disease whilst also acting as evidence to the difficulty in developing such models. The experiments show successful sensitization of mice against different β-lactam antibiotics and a promising model to study the role of the adaptive immune system in flucloxacillin-induced cholestatic liver injury.

Previous work has shown that subcutaneous (s.c) specific immunotherapy (SIT) using the myelin protein peptide MBP Acl-9 [4Y] induces protection against EAE in the TCR transgenic Tg4 mice. Repeated administration of MBP Ac1-9 [4Y] was found to induce IL-1O expressing TH1 like cells which were termed IL-l0 Tregs. To further the understanding of how IL-1O transcription is regulated, the expression of IFN-y, IL-4, IL-1O, c-Maf, NFIL3 protein and mRNA, Gata3 mRNA and epigenetic modifications at the Il10 promoter were characterised for each dose of s.c MBP Ac 1-9 [4 Y] administration. The analysis revealed that c-Maf and NFIL3 positively correlated with Il-IO expression, whilst levels of H3K27me3 at the Il10 promoter, an epigenetic mark associated with transcriptional repression, correlated inversely with IL-IO expression. To investigate the therapeutic potential of non-antigen specific immunoregulatory small molecules, GSK3 inhibitors were co-cultured with murine TH1, TH2 and human memory T cells. The analysis shows that GSK3 inhibitors also induce IL-10 expression via transcriptional mechanisms in both murine Th cells and human memory T cells. Similar to IL-l0 Tregs, murine TH1 GSK3 inhibitor treated cells expressed higher levels of NFIL3 and c-Maf whilst murine TH2 GSK3 inhibitor treated cells expressed higher levels of NFIL3 only. In contrast to IL-10 Tregs both THI and TH2 cells expressed higher levels of Gata3 in response to GSK3 inhibitor treatment. In addition, epigenetic changes also take place in the Il10 promoter of GSK3 inhibitor treated murine TH1 and human memory T cells. Collectively these results show that it is possible to alter epigenetic modifications and induce IL-1O expression in murine TH1 and TH1-like cells with in vivo and in vitro methods. Furthermore, we propose combining the antigen specific s.c SIT with GSK3 inhibitors to improve the efficacy and safety of both treatments.

This thesis presents an investigation of depth‐resolved absorption imaging by scanning objects through an annular beam of X‐rays. The resultant ring shaped projections record depth dependent parallax. The study focuses on two different scanning methods. First, a single axis scan has been employed to record curvilinear stereoscopic images. Second, a two axis raster scanning approach enables focal plane images to be produced via tomosynthesis. These novel absorption contrast methods have been carefully designed to complement X-ray diffraction considerations. Prior work employing an annular beam concerned the collection of X­ray diffraction data for structural analysis/phase identification. This method, termed Focal Construct Geometry (FCG), provided orders of magnitude increase in the intensity of the diffracted X-rays within the beam’s dark interior to enable rapid processing. However, this work relied upon a priori sample position. The research presented in this thesis establishes a complementary absorption imaging modality, which is designed to support and inform the analysis of the diffraction data by providing quantitative spatial information. This approach is essential for the autonomous interrogation of unknown objects at unknown positions within an inspection volume e.g. aviation security baggage screening. Two novel approaches have been developed and investigated. The first, termed Annular Beam Scanning (ABS), treats each annular field as a pair of semi‐annular projections as defined by an axis perpendicular to the scan direction. The resultant curvilinear stereoscopic image pairs contain depth dependent parallax along this scan direction. Algorithms to extract three‐dimensional information from the curvilinear images have been derived and validated. It has been shown that range information is proportional to the opening half‐angle ϕ of the annular beam but inversely proportional to the angular position σ of each matched element pair on the semi‐annular sensors. The second approach reassembles pixel data from a matrix of annular projections to form Multidirectional Perspective Views (MPV). These views encode object range as circular parallax. The application of Digital Tomosynthesis (DTS) employing a Shift‐and‐Add process enables focal plane images to be recorded. Unlike conventional DTS the minimum resolvable axial increment is independent of focal plane position within the inspected volume. While both approaches provide three‐dimensional data, MPV/DTS is the optimal method for combination with FCG diffraction datasets. The final part of this thesis describes results combining DTS with simulated diffraction image spaces to autonomously determine 2θ diffraction angles. This research programme has established a new annular beam imaging modality, which contributes significantly to the body of knowledge in the field of analytical 3D X‐ray imaging.

This thesis describes some of the observations made in a study of various factors believed to be involved in the aetiology of postoperative wound infection. An attempt has been made to compare two different ward and operating theatre environments with a view to assessing their relative contributions to the development of wound sepsis. Many different factors have been investigated, the previous literature of interest and relevance being reviewed in the introduction to each chapter. It is necessary to emphasise that the thesis has been written by a general surgeon in training. There has been no attempt to present a learned bacteriological treatise, rather the intention being to bring out features of practical importance to the clsinical surgeon. In order to facilitate the reading and comprehension of this work, the material is presented in two volumes. The first volume contains the main text of the thesis, while, in the second volume are included tables, figures, bibliography, and appendix.