The use of multi-detector computed tomography and magnetic resonance imaging in autopsy practice is increasing, and there is an international push towards minimally invasive autopsy. A significant obstacle to replacing conventional autopsy with standard imaging is failure to yield detailed information concerning the coronary arteries. A previously established technique to outline arteries is the Swiss-developed ‘whole body angiography’ technique using a heart lung machine and up to 3 litres of contrast agent. This project concerns the novel development of a quicker and cheaper targeted cardiac post-mortem computed tomography angiography (PMCTA) method to allow higher throughput. This thesis introduces the current knowledge of PMCTA, presents the development of the novel method, including a patented task-specific device, and then studies the accuracy of PMCTA ‘cause of death’ against autopsy in 24 cases referred from HM coroners. Cause of death was recorded by three groups of reviewers using a ‘view scan and grant protocol’ of clinical history, external examination and postmortem angiography findings. A further study was then performed related to obtaining informed consent by telephone from the next-of-kin for post mortem imaging in 200 cases. The data showed that using this protocol 82% of cases had a comparably worded cause of death to the autopsy. The ability of PMCTA to detect ischemic heart disease was assessed with a specificity of 88.9% and sensitivity of 100 % (PPV = 92.31 %, NPV =100%). Recruiting for a 200 case study of PMCTA we achieved a 96% consent rate with considerable support from the next-of-kin. These data suggest that a “View, scan and grant” protocol could be used to replace the routine autopsy in certain Coronial cases, leading to a larger 200 case study. The high consent rate helps dispel the notion that there is public objection to post-mortem research.

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This thesis describes the development and characterisation of fluorescent optical nanosensors for pH, molecular oxygen and temperature for measurement in biological systems. Ratiometric pH-sensitive nanosensors were fabricated by entrapping a combination of two different pH-sensitive fluorophores and a pH-insensitive reference fluorophore into a polyacrylamide matrix. The combination of the two pH-sensitive fluorophores overcame the current limitations in pH sensing with fluorescent nanosensors by demonstrating a tuneable pKa and an extended dynamic range of pH measurement. Molecular oxygen nanosensors were synthesised by surveying a library of novel oxygen-sensitive metalloporphyrins. Through careful selection of fluorophores and a platinum cationic metalloporphyrin, hybrid quadruple fluorophore labelled nanosensors capable of simultaneously making ratiometric pH and oxygen measurements were fabricated. In addition, a custom designed calibration tool was engineered so that oxygen-sensitive nanoparticles can be continually imaged, whilst the oxygen concentration is being changed and monitored at the same time. Temperature-sensitive nanosensors were synthesised by conjugating a temperature-sensitive fluorophore to a photo and thermo-sta ble silica sol-gel matrix. These nanosensors were found to operate over a wide temperature range and can be calibrated through the use of custom designed microelectromechanical systems (MEMS) micro-hotplate. The MEMS micro-hotplate can be mounted onto virtually all fluorescence microscopes, due to its: (1) relatively small size, (2) high temperature operation and (3) uniformity in temperature distribution. Extended dynamic range pH-sensitive nanosensors were successfully delivered to the model organism Caenorhabditis e/egans and used to map the pH of the pharynx and intestine in real-time using an automated image analysis system. The automated image analysis system and the pH-sensitive nanosensors were validated against the gold standard of pH measurements, the pH electrode, to demonstrate high concordance and measurement resolution.

The introduction of hybrid imaging technology has begun to redefine working practice in nuclear medicine. Emerging hybrid imaging techniques and evolving roles have created opportunities and challenges for nuclear medicine practitioners, and this is coupled with changes to traditional professional relationships with other health care disciplines. Organisational ethnography was utilised as a research methodology to observe working practice at three clinical hybrid imaging environments and undertake interviews with nuclear medicine practitioners. The emerging culture and professional identity of the hybrid imaging workforce was explored using symbolic interactionism which highlighted opportunities and challenges associated with introducing new technology. Practitioner diaries (weblogs) were also used to supplement the data collection and understand the current position of the workforce. Observational and thematic analysis was undertaken in order to identify a number of key themes which included ‘cultural lag’ in terms of the delayed adoption of new technology, ‘occupational shift’ around traditional tasks now being undertaken by other health disciplines, a lack of clarity around ‘technological domain ownership’ and variable levels of ‘flat collaboration’. Incidences of technological determinism and reduced patient contact were also reported, along with a technocentric approach to the delivery of clinical services. Positive aspects of the study included evidence of role development, autonomous working practice and decision making capabilities. Innovative approaches to service delivery were documented, creating an occasion for reshaping the professional identity of the nuclear medicine practitioner. The emerging hybrid imaging environment has created an opportunity to provide a patient centric approach to service delivery and aligns to a number of government strategies focusing on innovation and improvement within the health care service. Professional reorder and an organisational shift of roles and responsibilities has begun to occur within hybrid imaging, which will undoubtedly continue to evolve over time. Appropriate training and education should to be considered for the emerging workforce, along with discussions around flat collaboration opportunities and mutual technological domain ownership.

Uterine spiral arteries are remodelled in early pregnancy to supply sufficient maternal blood to the developing fetus. Inadequate remodelling is associated with the pregnancy pathologies pre-eclampsia and fetal growth restriction. Maternal natural killer cells within the decidua (dNK cells) are located near spiral arteries while remodelling events, such as disruption and loss of vascular cells and the extracellular matrix, occur. The effect of soluble factors produced by dNK cells on vascular smooth muscle cell (VSMC) apoptosis, differentiation status and matrix disruption was investigated to elucidate how dNK cells may contribute to the remodelling process. Microarray analysis was carried out to determine VSMC gene activity in response to dNK cell secreted factors. First trimester dNK cells were isolated from decidual tissue collected from surgical terminations of pregnancy and cultured to generate conditioned medium (CM) . Prior to termination, uterine artery Doppler ultrasound was carried out as a proxy measure for the extent of spiral artery remodelling. Pregnancies with a high resistance index (RI) are at increased risk of pre-eclampisa; therefore, comparisons were made between dNK cells isolated from women with a High RI and those with a Normal RI. The present study demonstrated that dNK CM does not induce VSMC apoptosis, nor does it alter susceptibility to apoptotic stimuli or VSMC differentiation status. Using a 3- dimensional vascular co-culture assay, it was established that dNK CM does not influence VSMC-mediated disruption of collagen. Decidual NK cells from High RI and Normal RI pregnancies behaved in a similar manner. Microarray analysis and verification identified an increase in interleukin-8 gene expression in VSMC stimulated with dNK CM, consistent with established roles for dNK cell secreted factors in trophoblast recruitment. In conclusion, soluble factors produced by dNK cells do not directly mediate the VSMC changes investigated here, but may alter VSMC production of chemoattractants.

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Ageing results in deterioration of the immune system which is associated with increased susceptibility to infection and impaired tissue regeneration. Phagocytosis by macrophages plays an essential role in both immune defence and regenerative processes such as repair of myelin sheaths in the CNS (remyelination). As such, age-related impairments in phagocytosis are likely to impact on the health of the elderly population. We hypothesised that ageing impaired phagocytosis by macrophages. Phagocytosis of fluorescent particles by peritoneal macrophages, bone marrow-derived macrophages and bone marrow monocytes was assessed. Ageing impaired phagocytosis by peritoneal macrophages. Bone marrow-derived macrophages and bone marrow monocytes did not exhibit age-related impairments in phagocytosis, indicating that there was no intrinsic defect in the phagocytic capacity of myeloid progenitors. We hypothesised that environmental factors in the peritoneum of old mice suppressed macrophage phagocytosis. Indeed, macrophages from young mice injected into the peritoneum of old mice exhibited impaired phagocytosis. This provides insight into the underlying cause of age-related reduction in phagocytosis by peritoneal macrophages. We observed a significant increase in B cells and B cell-derived IL-I 0 was found. These factors could potentially play a role in the impairment of phagocytosis by peritoneal macrophages in old mice. Myelin debris clearance is essential for efficient remyelination to occur. Ageing impaired phagocytosis of myelin debris by both peritoneal macrophages and CNS-derived microglia. As such, age-related decline in remyelination efficiency could be in part due to a lack of myelin debris clearance by macrophages and microglia. Age-related impairments in phagocytosis by tissue-resident macrophages such as peritoneal macrophages and microglia have implications for defence against infection, regenerative processes and also maintenance of homeostasis in the older population. As such, boosting macrophage phagocytosis may represent an important therapeutic target in order to maintain health in the older population.

Digital pathology is now underpinning biomarker and drug discovery, clinical diagnosis via companion diagnostics and provides valuable tools for the current move towards providing personal stratified medicine. The potential digital pathology has for giving better insight into many disease mechanisms is due to firstly it's digital (and therefore linkable/indexable) nature, and secondly the sheer amount of data that can now be generated by imaging hardware and image analysis methodologies. The work carried out in this thesis was an examination of the current barriers to verifying and exploiting the large amounts of information that is available via the use of digital pathology. The large size of whole slide images generated in digital pathology means that lossy image compression becomes a necessity due to storage limitations. Lossy image compression however discards data in a non-linear transformation, e.g. JPEG image compression, and therefore changes the values of pixels in the images. As image analysis algorithms use these values to perform quantification and segmentation of regions within the image, the first piece of this thesis examines the affect image compression has on fundamental image calculations common in image analysis algorithms. Tissue imagery data is complex but the' visual pathological information provided is the basis of clinical diagnosis. These patterns are currently used to show the symptoms of disease. This thesis details a system that can make this information easily searchable using these patterns as the input criterion for the search. A system that may highlight tissue with specific malignant morphological properties could clearly provide a valuable resource in large clinical trials where manual analysis of specimens requires many work hours. The imaging information provided by digital pathology is not independent from other experimental and clinical data and the final piece of this thesis provides a framework for connecting clinico-pathological, genetic and imaging information in an attempt to provide a much more detailed and connected view of related diseases. This work highlights the considerations necessary in designing studies employing image analysis and underpins the future importance of digital pathology applications in modern tissue-based translational research.