This thesis addresses the challenges of measuring the acoustic (sound speed, attenuation, absorption and nonlinearity coefficients), thermal (thermal conductivity) and histological properties of soft tissues that lie within the HIFU beam during treatment. Differences in these tissue properties may affect the delivery of thermal therapies, but may also provide a basis for their monitoring with ultrasound. Novel measurement techniques have been developed and validated for each parameter. Measurements have been made in rat, bovine and human tissues. The finite amplitude insertion substitution (FAIS) method was used to obtain the attenuation coefficient and speed of sound. The absorption coefficients were determined using a thermometric method involving a Fabry-Perot fibre optic sensor. Nonlinearity coefficients were measured using the FAIS method with a large area hydrophone detector. Using these methodologies, a system that allows the measurement of all these parameters in the same region of a sample has been designed and built. Tissue thermal conductivity was determined using two methods: the first used a system designed to provide unidirectional heat flow between a source and heat sink. 35 MHz ultrasound (US) images were used to determine the volume of air trapped in the tissue sample, thus allowing corrections to the thermal conductivity to be made using the standard bio-heat equation model. The second method was a non-invasive method in which HIFU heating was combined with ultrasound thermometry. Finally, a Matlab code which allows quantification of important aspects of Haematoxylin and Eosin (H&E) stained histological images has been developed. This has been used to correlate the histological appearance of samples with their thermal and acoustic properties.

Acid-base balance is a dynamic process and the transition from one classification to another does not occur abruptly and, although the general principles for classifying such data are standard, the details of each algorithm can be subtly different. This research aimed to explore the use of self-organising maps as applied to the medical diagnostic problem of acid-base disorders. The relevance of using self-organising networks for the classification of this data particularly focused on the mapping from the multi-dimensional data to the network nodes. This maintained the spatial organisation of the data allowing a natural graphical representation which directly related to the more familiar diagrams used as aids in this subject. Furthermore, the network was not bound by rigid classificatory criteria and thus could give some meaningful category even to data samples excluded by the standard algorithms. It was, thus, an improvement on such rule-based diagnostic criteria. Other methods were investigated including supervised networks and clustering algorithms but were not as successful when presented with mixed samples and relied more strongly on the initial data classifications further strengthening the argument for the self-organising methods. The self-organising map has proved to be of potential use in the classification of acid-base disorders. It has several advantages over standard simple classificatory algorithms and such features can be used to provide a meaningful graphical representation of the data. Various methods can be used to overcome problems involved with the labelling of these maps and comparisons have been made between them. The use of these maps also shows potential in dealing with temporal data series.

Successful pregnancy requires tight regulation of two important processes: spiral artery remodelling and invasion of uterine tissue by placental extravillous trophoblast (EVT); disruption of these processes occurs in pregnancy complications such as sporadic miscarriage (SM). Maternal factors, including cytokines and growth factors, may play a role in regulating these fundamental events. The biological function of interleukins (IL)-6 and -8 in other physiological processes suggest that these cytokines maybe important mediators in uteroplacental tissues in early pregnancy. The aim of this study was to investigate intrauterine IL-6, IL-8 and their receptors at the fetal-maternal interface in early pregnancy; specifically, to investigate their role in regulation of EVT invasion and spiral artery remodelling events and determine whether levels of cytokines are altered in SM. IL-6, IL-8 and their receptors were localised in placental bed in the first half of pregnancy by immunohistochemistry. CD56+, CD14+, CD10+ and CD8+ cells were isolated from 8-10 and 12-14 weeks gestational age decidua using positive immunomagnetic separation and secretion of IL-6 and IL-8 was measured and compared by ELISA. All decidual cell types investigated secreted both IL-6 and IL-8. Decidual CD14+ cells were the most abundant source of both cytokines. Secretion of IL-6 by CD10+ cells (P=0.005) and IL-8 by CD56+ cells (P=0.02) was higher at 12-14 weeks compared with 8-10 weeks GA. Compared with healthy pregnant decidua, secretion of IL-6 and IL-8 protein by both CD14+ (IL-6 P=0.05; IL-8 P<0.0001) and CD56+ cells ( IL-6 P=0.02; IL-8 P=0.003) was reduced in SM (≤12+6 weeks gestation). The functional role of these cytokines in early pregnancy was assessed with invasion assays and a chorionic plate artery (CpA) model of artery remodelling. IL-6 did not stimulate EVT invasion in vitro but did cause phosphorylation of kinase proteins involved in trophoblast cell signalling and reduced EVT secretion of RANTES. IL-6 and IL-8 both contributed to misalignment of VSMC in CpA (P=0.04; P=0.001 respectively) and IL-6 increased the separation of CpA VSMC layers alone (P=0.02) and combined to its receptor sIL-6Rα (P=0.001). CD56+ and CD14+ leucocytes were observed in the walls of spiral arteries in the first half of pregnancy with CD14+ cells producing increased Ang-2 (P=0.002) and decreased Ang-1 at 12-14 weeks compared with 8-10 weeks GA (P=0.03). Production of IL-6 and IL-8 within the placental bed in early pregnancy and reduced levels in SM, together with their effects on EVT and VSMCs suggests a possible involvement in controlling trophoblast function and contribution in regulating spiral artery remodelling events.

Under oxidative stress condition, telomerase catalytic subunit can shuttle from the nucleus and localises within mitochondria. hTERT can improve mitochondrial functions and contribute to a decreased oxidative stress suggesting an entirely new function of telomerase in protecting mitochondria and cells under stress. However, there are still many questions about the mechanism and what factors influence the protective function of telomerase. In this study we investigated the kinetic exclusion of hTERT, the catalytic subunit of telomerase, in various cell lines under different oxidative stress conditions. We also used organelle specific hTERT localisation vectors to model hTERT localisation and investigated a correlation between hTERT location, nuclear DNA damage and ROS production. We found that cells excluded endogenous hTERT from the nucleus in a heterogeneous fashion independently of the cell types. Importantly, nuclear DNA damage showed a significant correlation with the localisation of hTERT. Cells where hTERT remained in the nucleus displayed high DNA damage while cells which excluded hTERT from the nucleus displayed no or very low DNA damage. Our results from specific hTERT localisation vectors specified that mitochondrial localisation of hTERT protects the nucleus from DNA damage and did not showed any sign of apoptosis induction while nuclear localisation of hTERT correlated with higher amounts of DNA damage and apoptosis. Moreover, mitochondrial localisation of hTERT decreased mitochondrial ROS generation levels directly after both endogenous and exogenous stress which we interpret as the reason for the prevention of nuclear DNA damage. Additionally, we analysed whether p53 status might influence the protective function of telomerase. Our results in an isogenic cell pair of glioblastoma cells showed that p53 status does not prominently influence the protective function of mitochondrial hTERT under low stress condition. However, nuclear hTERT of cells which contained inactive p53 displayed a significantly higher nuclear DNA damage than cells which contained an active p53 and this became more pronounced when stress levels were increased. We hypothesise that telomerase localisation might possibly interact with p53 when a cancer cell is under stress condition. However, the molecular mechanism for that is unknown. Our results demonstrate a novel link between mitochondrial localisation of hTERT, decrease of mitochondrial ROS generation and the protective capacity of hTERT to nuclear DNA from damage after stress treatments.

Electrical impedance tomography (EIT) is a medical imaging technology that provides a tomographic representation of the distribution of electrical impedance within the body. As the electrical impedance varies for different body tissues, it is possible to characterize tissues from the images and to detect physiological events. EIT systems have been developed from applying a single signal frequency to a range of frequencies. Imaging at multiple frequencies significantly improves the ability to characterize and differentiate heterogeneity within the region of interest. Applications of EIT are limited by its poor resolution as a consequence of limited number of electrodes and lack of independently published measurements. In a practical EIT system design the parallel structure is normally adopted as it provides a real time monitoring structure. However, there is a difficulty in expanding to a 2-dimensitional or 3-dimensitional high resolution imaging system, as the number of electrodes increase. In this thesis, a serial structure spectrum EIT system has been investigated and developed. Modelling of the electrical circuit has shown that the system bandwidth is degraded primarily by the signal transmission in the coaxial cable and multiplexer. To remove the capacitive effect of these components, a distribute system concept has been developed. The concept uses active electrodes in which a current source and a front end amplifier are embedded in the electrode which makes direct contact with the tissue being measured. The active electrode is based on the Howland current source. The required high output impedance of Howland current source can be realised by matching the two resistor arms. However, from the electrical equivalent circuit analysis the actual output impedance of this circuit was found to be degraded by the op-amp' s limited open loop gain, especially at higher frequencies. To solve the problem, the author describes in detail a novel method of compensating for the above effects. Subsequent circuit tests showed significant improvement after the compensation. Further, to improve the small signal noise ratio a programmable gain amplifier to adapt the frame data measurement was developed. These developments have led to the feasibility of active electrodes. The thesis describes in detail the development, of the MK2 EIT system which is presented as the output of this research.

Mitochondria are strictly maternally inherited, with all paternal mitochondria being destroyed following fertilisation. Women known to be carriers of pathogenic mtDNA mutations are therefore at increased risk of conceiving affected children. These women are currently offered the following options to aid in genetic counselling: oocyte donation, prenatal genetic diagnosis (PND) or preimplantation genetic diagnosis (PGD). One of the aims of this thesis, was to examine the feasibility of PGD for mtDNA inherited disorders, with specific emphasis on answering the following questions: how accurately does the mutation load observed in the biopsied blastomeres reflect the mutation load in the remaining embryo, are those mutation loads initially observed in the biopsied blastomeres maintained throughout preimplantation embryonic development and do mutation loads observed in the inner cell mass reflect those mutation loads observed in the extra-embryonic trophectoderm cells? In my thesis, I have now been able to provide data towards answering each of these questions through the examination of mutation loads in oocytes, embryos and blastocysts obtained from mitochondrial patients undergoing fertility treatment. Techniques, which have been developed in my current laboratory, have facilitated the characterisation of a nuclear transfer technique known as pronuclear transfer (PNT). This is a method to prevent the transmission of mitochondrial DNA disease from mother to child (Craven et al, 2010). As part of the work for my thesis, I have examined the reproducibility of the PNT technique by assessing whether the procedure could be performed by different operators, whilst maintaining levels of efficiency, survival and developmental outcome. Experiments are now being performed to examine the feasibility of PNT in normally fertilised human zygotes, created from donated oocytes. As it is unlikely that egg collection will be possible from two independent donors on the same day, the final purpose of this study was to examine the potential and feasibility of vitrification of eggs or fertilised embryos at both the pronuclear (PN) and Metaphase II (MII) stage for the purpose of the PNT technique. In summary, my studies has examined the reliability of current methods to reduce the likelihood of having a child affected by a mitochondrial DNA disorder and new techniques currently being developed to prevent the transmission of defective mitochondrial DNA, altogether. I hope this will provide fresh hope for patients with mitochondrial DNA disease.

This work considers novel image-processing and computer-vision techniques to advance the automated analysis of low-resolution, complex 3D volumetric Computed Tomography (CT) imagery obtained in the aviation-security-screening domain. Novel research is conducted in three key areas: image quality improvement, segmentation and classification. A sinogram-completion Metal Artefact Reduction (MAR) technique is presented. The presence of multiple metal objects in the scanning Field of View (FoV) is accounted for via a distance-driven weighting scheme. The technique is shown to perform comparably to the state-of-the-art medical MAR techniques in a quantitative and qualitative comparative evaluation. A materials-based technique is proposed for the segmentation of unknown objects from low-resolution, cluttered volumetric baggage-CT data. Initial coarse segmentations, generated using dual-energy techniques, are refined by partitioning at automatically-detected regions. Partitioning is guided by a novel random-forestbased quality metric (trained to recognise high-quality, single-object segments). A second segmentation-quality measure is presented for quantifying the quality of full segmentations. In a comparative evaluation, the proposed method is shown to produce similar-quality segmentations to the state-of-the-art at reduced processing times. A codebook model constructed using an Extremely Randomised Clustering (ERC) forest for feature encoding, a dense-feature-sampling strategy and a Support Vector Machine (SVM) classifier is presented. The model is shown to offer improvements in accuracy over the state-of-the-art 3D visual-cortex model at reduced processing times, particularly in the presence of noise and artefacts. The overall contribution of this work is a novel, fully-automated and effcient framework for the classification of objects in cluttered 3D baggage-CT imagery. It extends the current state-of-the-art by improving classification performance in the presence of noise and artefacts; by automating the previously-manual isolation of objects and by decreasing processing times by several orders of magnitude.

Leukocyte immunoglobulin-like receptors are a family of inhibitory and activating receptors found on a wide variety of immune cells, and are thought to play a significant role in determining immune responses. Their known ligands are HLA Class I molecules; HLA-G is the prototypic ligand which has high affinity for two inhibitory members of the family and mediates its immunosuppressive functions through them. The hypothesis of this study is that polymorphisms in the non-coding regions are likely to influence both the expression and response of the LILRs to various stimuli. Published data supports this view in that polymorphisms in the LILRs are associated with various immunologically mediated diseases. In addition, these molecules represent a potential target for therapeutic manipulation and attempts to develop a therapy using this route were undertaken. Expression levels of various LILR molecules on APCs at baseline and after stimulation have shown significant variation between subjects. Genomic variation in the promoter regions of LILRB2 was established by sequencing. The relationship between LILRB2 SNPs and cell expression levels was tested both directly and in a luciferase assay, but no significant associations were found. Engagement of LILRB2 by monoclonal antibodies was shown to affect TLR-induced cytokine secretion, but no relationship was found between LILRB2 expression levels and the effect on cytokine secretion. A synthetic construct (the “G-body”) utilising the LILR-HLA-G interaction has been developed for testing as a potential therapeutic molecule. This construct comprises two functional domains – an anti-HLA class I domain and an HLA-G domain. This construct would be expected to modulate allogeneic responses by localisation of an immunosuppressive signal (HLA-G) to the allogeneic HLA molecule; and (ii) potentially masking the allogeneic HLA class I molecule. Testing has shown that the construct suppresses lymphocyte proliferation with enhancement of this effect dependent on the presence of allogeneic HLA class I.

This thesis presents SAIF, (Spectral Analysis with Iterative Filter), a SA-based method for the quantification of PET data investigated with irreversible-uptake tracers. SAIF has been designed in order to maintain the main advantages of SA but providing a superior robustness to measurement noise. The final aim was to create a reliable and flexible PET quantification tool, offering a valid alternative to standard methodologies for functional quantitative imaging with PET and irreversible tracers.

Mice lacking the transcription factor T-bet in the innate immune system (Tbx21-/- x Rag2-/- Ulcerative Colitis, or TRUC) develop microbiota-dependent inflammatory bowel disease (IBD). The innate immune mechanisms responsible for causing disease are still to be resolved, yet potentially offer novel insights into the pathogenesis of IBD, and how T-bet might influence inflammation in the gut. In this thesis it was shown that chronic IBD in TRUC mice was dependent on interleukin-17A (IL-17A) producing IL-7R+ CD90+ innate lymphoid cells (ILCs). Depletion of ILCs or IL-17A neutralization cured disease. Cytokines responsible for driving innate IL-17A included IL-23, IL-6 and TL1A. IL-23 and IL-6 played functionally important roles in TRUC disease, since neutralisation reduced innate IL-17A production and attenuated disease. TNFα, predominantly produced by CD11chigh class II+ CD11b+ CD103- dendritic cells (DCs), synergised with IL-23 to drive IL-17A production by ILCs. Helicobacter typhlonius (HT) was identified as the key colitigenic bacterium driving TRUC disease. Four-five-four ribosomal RNA gene sequencing demonstrated that HT was consistently present in the faeces of all TRUC mice, but absent from a newly-derived colony of disease-free Tbx21-/- x Rag2-/- mice. Inoculation of pure HT cultures was sufficient to reinstate disease into this new colony of healthy mice. T-bet impacted on colitis in several ways. In the absence of T-bet, ILCs selectively produced IL-17A and were poor producers of interferon-γ. Second, T-bet deficient DCs produced excess TNFα in comparison with T-bet sufficient DCs. Finally, T-bet was a transcriptional repressor of the Il7ra locus. IL-7R was expressed by ILCs and signalling through this receptor was critical for TRUC disease. Selective IL-7R blockade attenuated disease. Together these data provide new insights into TRUC disease and demonstrate at least some of the mechanisms by which T-bet regulates the interplay between mucosal innate immune cells and the intestinal microbiota.