The interaction between antigen presenting cells (APCs) and T cells is one of the pivotal events in initiation and regulation of an immune response. Central to this is a tripartite molecular interaction between antigen (Ag), molecules of the major histocompatibility complex (MHC) and the Ag specific T cell receptor (TcR). In this study, factors that influence Ag presentation, other than TcR/Ag-MHC interaction, have been studied using a combination of in vitro and in vivo model systems. The results were evaluated in terms of T cell immune responsiveness. An in vitro mixed leucocyte reaction was used to demonstrate that freshly isolated potentially alloreactive murine splenic T cells can be induced, in primary culture, to develop a state of long-lived hypo-responsiveness, both at the level of proliferation and interleukin two secretion. First, this can be induced, in an allo-specific manner, by exposure to allogeneic APCs modified by a chemical cross-linker. This hypo-responsiveness is associated with markedly reduced T cell/APC adhesive clustering interactions despite the lack of a detectable change in lymphocyte function associated antigen one (LFA-1) and intercellular adhesion molecule one (ICAM-1) on the surface of modified APCs. Second, L cells were used which express TcR ligand (transfected MHC class II molecules), but do not express two of the crucial receptor/counter receptor pairs for T cell-APC binding, viz. LFA-1 and ICAM-1, and do not express functional co-stimulatory molecules. These cells could also induce T cell hypo-responsiveness. The results indicate that APCs which do not express "co-stimulatory" signals induce T cell inactivation. The modified allogeneic APCs, which induce T cell hypo-responsiveness in vitro, primed T cells successfully in vivo when they were injected into hind footpads of mice. This apparent paradox was clarified by examining the migratory behaviour of modified and unmodified labelled APCs in vivo. Only a small proportion of label was detected in draining lymph nodes; the kinetics of label recovery were unaffected by fixation of APCs, suggesting that there is no active migration of APCs. Thus, during in vivo experimental allo-sensitization via the subcutaneous route, indirect priming of allogeneic T cells may be the dominant pathway. During the course of these studies, it has become increasingly evident that the interaction of APCs with T cells is a dynamic multimolecular signalling mechanism. Most other studies have focused upon TcR engagement by Ag-MHC molecules which endows Ag specificity to presentation interactions. However, other components of Ag presentation, which are collectively termed co-stimulatory signals (and may include some cell adhesion molecules as well as unidentified factors) may be equally important since they are essential for the full activation and clonal expansion of T cells. Furthermore, these latter pathways may provide regulatory switches for the T cell to differentiate into functionally divergent states, and hence are potentially important for the rational design of immunomodulatory therapies.

Medical image processing has experienced dramatic expansion, and has been an interesting research field that attracted expertise from applied mathematics, computer sciences, engineering, biology and even medicine. This work is concerned with developing image processing techniques to automate the detection and classification of cells in digital images of day 2 embryos for suitability for In Vitro Fertilization (IVF) treatment. In IVF treatment eggs are removed from the ovaries of the woman and injected with sperms of the man in a dish in the laboratory so that fertilization can take place and yield embryos. The embryos are then graded and examined to decide which embryos are the best to be re-implanted into the woman's womb again. The grading system used in this work involved day 2 embryos, and a dataset of 40 images was provided by Al Agyal clinic in Alexandria. At this stage of development the embryos should have 4 approximately circular cells with similar sizes in order to be considered as suitable for re-implantation. The work develops an automated image processing system which firstly locates the embryo in a microscope image, and then detects the cells in the embryo and matches their properties against the criteria for re-implantation. Although the main problem was the overlapping of the cells in the images, it was also found that the size (magnification) and the brightness also varied from one image to another and these factors had to be taken into consideration during the development of the detection algorithms. Once the perimeter of the embryo had been located, several edge detection techniques including the Sobel, Prewitt and Canny operators were examined as pre-processing for the circular Hough Transform. From 94 cells, only 62 cells (65%) were detected, but at the same time 226 of false cells were also detected. As an alternative approach, template matching was investigated, using templates with a range of sizes which were selected to match the acceptable size criteria for re-implantation and at the same time take into consideration the different magnification scales of the images used. The Sum of Added Differences (SAD) and the Normalized Cross Correlation (NCC) were used as a measure of the match. The NCC technique gave better results than SAD, which failed to detect any true cells. NCC technique only detected 50% of true cells, and further refinement to this approach was made. This involved binarisation of the images and templates, and the creation of two new edge-detection algorithms, one of which was based on the convolution technique while the other was based on the difference of the grey level between the border of the cell and its background. These changes have increased the cell detection accuracy to 80%, and reduced the detection of false cells from 118 to 39. Of the 40 images available, 30 images were used to develop the automated system while 10 images were left to test the performance of the system. In the case of the 10 images, 5 had larger embryos and 5 smaller ones than the 30 images, where the embryos had similar sizes. It was found that 85% of the cells in the 10 images were properly detected with only 6 false cells found. As the missed cells and false cells were distributed among the 40 images, only 8 were analysed correctly (all true cells detected and no false cells found) but these were all correctly identified as suitable or not suitable for re-implantation. Further work is required to improve the cell detection algorithm, and to decrease further the number of false cells detected and hence improve the classification of the embryo.

No description available.

Training of future endoscopists within the UK has shown to be of variable quality. Those learning endoscopy have the opportunity to attend short courses but much of their training occurs within base hospitals around the UK; this training tends to occur via an apprenticeship style model on a one-to-one basis with an experienced endoscopist, the ‘trainer’. These trainers have the opportunity, and are encouraged, to attend ‘Training the trainer’ courses but then receive no ongoing validated feedback about their training. Evidence suggests that gaining feedback on teaching performance can improve subsequent performance; therefore this project aimed to create a validated feedback tool which could be completed by trainees, peers or trainers as a self-reflection exercise. This tool can then be used to give formative feedback to endoscopy trainers.

Dendritic cells (DC) are potent antigen presenting cells which have been implicated in a number of autoimmune diseases. Tumour necrosis factor (TNF) is a key mediator of inflammatory diseases such as rheumatoid arthritis (RA) and plays a central role in DC biology. My aim was to identify the individual contributions of the two TNF receptors (TNFR1 and TNFR2) in regulating the maturation and survival of human inflammatory monocyte-derived (moDC) and steady-state myeloid DC. To address this, I have made use of TNFR-selective ligands in order to dissect the individual contributions of the two receptors. In moDC, TNFR1-selective, but not TNFR2-selective stimulation resulted in increased expression of DC maturation markers CD83 and CD86, and enhanced T cell stimulatory capacity. A DNA binding assay was used to demonstrate that in moDC TNFR1, but not TNFR2, activates the classical p65 NFB pathway whereas both TNFR1 and TNFR2 activate the alternative p100/p52 NFB pathway, highlighting differences in signalling downstream of the receptors. Furthermore, moDC survival was prolonged by selective stimulation of either TNFR1 or TNFR2 as shown by reduced intracellular levels of active caspase-3, indicating that innate signals can promote DC survival in the absence of DC maturation. Accordingly, the p65 NFB pathway was involved in the pro-survival effect of TNFR1 whereas the Bcl-2/Bcl-xL pathway (identified by the use of small molecule inhibitors) was essential to survival mediated by both TNFR. In contrast, in myeloid DC, maturation was mainly mediated through TNFR1, whereas TNFR2 was superior in protecting DC from cell death. Antagonistic TNFR1-specific antibodies were used to confirm that cell death protection via TNFR2 was independent of TNFR1-mediated signalling and vice versa confirming that the two receptors can act independently of one another. Understanding the immunoregulatory properties of signalling through these two TNF receptors is important for the design of more targeted anti-TNF therapy.

Oxidative stress is a key feature of inflammatory diseases. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated by many cell types during inflammation. ROS are known to induce chemokines, however it is increasingly apparent that RNS also impact on inflammation. This study was designed to investigate the effects of tissue stress on both chemokine production and function. How stress alters chemokine production in epithelium was established by qPCR. A distinct tissue, stress and chemokine specific response was elicited; of those studied, CXCL8 showed the greatest induction. The chemokines produced by epithelial cells were functional but post-translational modification occurred and so these chemokines may not have their predicted function. The effects of stress in vivo were also assessed. Immunohistochemistry showed association between RNS activity and ischaemic time in a model of kidney ischaemia-reperfusion injury. These observations were extended to human inflammatory liver disease, with increased RNS activity at sites of inflammation, a situation in which chemokines such as CCL2 are also present. RNS also modulates inflammation by post-translational modification. CCL2 was nitrated by RNS creating a chemokine, nCCL2, with decreased chemotactic activity in a diffusion gradient. Similar results were seen for nCCL5 and nCXCL8. Recruitment of HEK-CCR2b cells was decreased following CCL2 nitration and radio-ligand binding experiments confirmed there was some loss of receptor binding. However, the biological significance of this was uncertain. Glycosaminoglycan interactions were prevented by CCL2 nitration as was proportion of transendothelial migration. The ability of nitration to decrease the chemotactic potential of CCL2 was confirmed by in vivo assays. These data show the complexities of the chemokine system. Increased chemokine production by oxidative stress and concurrent modification of those chemokines by RNS represents a powerful paradigm for the regulation of inflammation.

Rapidly proliferating cells, such as cancerous cells, show increased reliance on deoxyribonucleic acid (DNA) salvage pathways for producing nucleotides required for DNA synthesis. As such targeting these salvage pathways using radiolabelled nucleosides can provide a means of imaging the extent of proliferation within a tissue using positron emission tomography (PET). This permits the detection of malignant growths. Thiothymidine and 2'-deoxy-2',2'-difluoro nucleoside analogues are currently being evaluated for theoretically superior properties in comparison to 3'-deoxy-3'-[18F]-fluorothymidine (FLT), the current standard PET proliferation marker. Investigations towards the design and synthesis of radiolabelled analogues of these nucleosides for evaluation as PET tracers were carried out. Synthesis and radiosynthesis of the 2',2'-difluoro nucleoside analogue i was completed using a Stille coupling to introduce the carbon-11 radiolabel. The synthesis of ii, a precursor to a carbon-11 methylated gemcitabine analogue and iii, an intermediate in the synthesis of thiothymidine nucleosides are also reported. [Molecular diagram appears here. To view, please open pdf attachment] Based on the synthetic route developed for accessing iv an analogue of ii suitable for radiolabelling with fluorine-18, a series of difluoro nucleoside analogues with potential uses in PET, HIV therapy and fluorescent imaging were also synthesised v-viii. [Molecular diagram appears here. To view, please open pdf attachment].

Positron Emission Tomography (PET) is emerging as a powerful method for imaging cancer through the design and development of new radiotracers. Antibodies have promising properties as ligands for targeting cancer, as they have the advantage of displaying high affinity for their respective receptors. However, the use of antibodies as radiotracers is limited to the use of long-lived isotopes, as these large biomolecules additionally display slow blood circulation and clearance. The use of short-lived isotopes such as 18F or 68Ga, in combination with antibodies, would provide the ideal balance between targetability and clearance. This may be achieved by use of a two-step pretargeting strategy, whereby a reactive tag is conjugated to the antibody and allowed to localise in the tissue to be imaged, before systemic administration of a chemical reporter (e.g. a labelled reactive partner) which allows the 'pretargeted' tissue to be imaged. The Strain-Promoted Azide/Alkyne Cycloaddition (SPAAC) reaction between cyclooctynes and azides was evaluated as an appropriate bioorthogonal reaction for application to a pretargeting strategy using short-lived isotopes. The synthesis of a library of cyclooctyne precursors was carried out, which were evaluated in terms of their reactivity with azides, and their suitability for in vivo applications. An 18F-labelled version of the SPAAC reaction was developed, demonstrating the ability of the reaction to be carried out under different conditions. This model reaction was translated to in vivo pretargeting using a cyclooctyne modified Herceptin monoclonal antibody and an 18F-labelled azide. These initial experiments indicated that the SPAAC reaction may not be fast enough to occur at the low concentrations which are found in vivo. The reaction was thoroughly examined in terms of kinetics at different concentrations, and a high concentration-dependence upon rate of reaction was confirmed. This was supported by a 68Ga-labelled SPAAC reaction, which was carried out using reportedly more reactive cyclooctynes than those used in the initial experiments. In general, the reaction showed a greater preference to be carried out in organic solvents such as acetonitrile, and under closer to physiological conditions the reactions were less likely to proceed. The Inverse-electron-Demand Diels-Alder (IeDDA) reaction between tetrazines and strained alkenes was evaluated as an alternative bioorthogonal reaction for demonstrating in vivo pretargeting. A series of 68Ga-labelled IeDDA reactions between a 68Ga-labelled tetrazine and a series of norbornene analogues demonstrated the superior reaction kinetics and biocompatibility of the IeDDA reaction. The initial translation of the IeDDA reaction to a proof-of-concept for pretargeting using cyclic RGD pentapeptides was initially unsuccessful, attributed to the surprisingly poor reactivity of norbornene-modified cyclic RGD pentapeptides towards a 68Ga-labelled tetrazine. The reaction between a 68Ga-labelled tetrazine and a Cetuximab antibody, which had been modified with the more reactive trans-cyclooctene (TCO) moeity, was successfully demonstrated. The hypothesised pretargeting strategy using this model reaction was achieved on high EGFR expressing cells, validating the IeDDA reaction in this context. These results suggested tantalising opportunities for application of the IeDDA reaction to in vivo pretargeting for PET imaging using short-lived isotopes such as 18F and 68Ga.

This thesis describes advances in the characterisation, calibration and data processing of optical coherence tomography (OCT) systems. Femtosecond (fs) laser inscription was used for producing OCT-phantoms. Transparent materials are generally inert to infra-red radiations, but with fs lasers material modification occurs via non-linear processes when the highly focused light source interacts with the materials. This modification is confined to the focal volume and is highly reproducible. In order to select the best inscription parameters, combination of different inscription parameters were tested, using three fs laser systems, with different operating properties, on a variety of materials. This facilitated the understanding of the key characteristics of the produced structures with the aim of producing viable OCT-phantoms. Finally, OCT-phantoms were successfully designed and fabricated in fused silica. The use of these phantoms to characterise many properties (resolution, distortion, sensitivity decay, scan linearity) of an OCT system was demonstrated. Quantitative methods were developed to support the characterisation of an OCT system collecting images from phantoms and also to improve the quality of the OCT images. Characterisation methods include the measurement of the spatially variant resolution (point spread function (PSF) and modulation transfer function (MTF)), sensitivity and distortion. Processing of OCT data is a computer intensive process. Standard central processing unit (CPU) based processing might take several minutes to a few hours to process acquired data, thus data processing is a significant bottleneck. An alternative choice is to use expensive hardware-based processing such as field programmable gate arrays (FPGAs). However, recently graphics processing unit (GPU) based data processing methods have been developed to minimize this data processing and rendering time. These processing techniques include standard-processing methods which includes a set of algorithms to process the raw data (interference) obtained by the detector and generate A-scans. The work presented here describes accelerated data processing and post processing techniques for OCT systems. The GPU based processing developed, during the PhD, was later implemented into a custom built Fourier domain optical coherence tomography (FD-OCT) system. This system currently processes and renders data in real time. Processing throughput of this system is currently limited by the camera capture rate. OCTphantoms have been heavily used for the qualitative characterization and adjustment/ fine tuning of the operating conditions of OCT system. Currently, investigations are under way to characterize OCT systems using our phantoms. The work presented in this thesis demonstrate several novel techniques of fabricating OCT-phantoms and accelerating OCT data processing using GPUs. In the process of developing phantoms and quantitative methods, a thorough understanding and practical knowledge of OCT and fs laser processing systems was developed. This understanding leads to several novel pieces of research that are not only relevant to OCT but have broader importance. For example, extensive understanding of the properties of fs inscribed structures will be useful in other photonic application such as making of phase mask, wave guides and microfluidic channels. Acceleration of data processing with GPUs is also useful in other fields.

Individuals within the aged population show an increased susceptibility to infection, implying a decline in immune function, a phenomenon known as immunosenescence. Paradoxically, an increase in autoimmune disease, such as rheumatoid arthritis, is also associated with ageing, therefore some aspects of the immune system appear to be inappropriately active in the elderly. The above evidence suggests inappropriate control of the immune system as we age. Macrophages, and their precursors monocytes, play a key role in control of the immune system. They play an important role in host defence in the form of phagocytosis, and also link the innate and adaptive immune system via antigen presentation. Macrophages also have a reparative role, as professional phagocytes of dead and dying cells. Clearance of apoptotic cells by macrophages has also been shown to directly influence immune responses in an anti-inflammatory manner. Inappropriate control of macrophage function with regards to dead cell clearance may contribute to pathology as we age. The aims of this study were to assess the impact of lipid treatment, as a model of the aged environment, on the ability of macrophages to interact with, and respond to, apoptotic cells. Using a series of in vitro cell models, responses of macrophages (normal and lipid-loaded) to apoptotic macrophages (normal and lipid-loaded) were investigated. Monocyte recruitment to apoptotic cells, a key process in resolving inflammation, was assessed in addition to cytokine responses. Data here shows, for the first time, that apoptotic macrophages (normal and lipid-loaded) induce inflammation in human monocyte-derived macrophages, a response that could drive inflammation in age-associated pathology e.g. atherosclerosis. Monoclonal antibody inhibition studies suggest the classical chemokine CX3CL1 may be involved in monocyte recruitment to apoptotic macrophages, but not apoptotic foam cells, therefore differential clearance strategies may be employed following lipid-loading. CD14, an important apoptotic cell tethering receptor, was not found to have a prominent role in this process, whilst the role for ICAM-3 remains unclear. Additionally, a small pilot study using macrophages from young (<25) and mid-life (>40) donors was undertaken. Preliminary data was gathered to assess the ability of primary human monocyte-derived macrophages, from young and mid-life donors, to interact with, and respond to, apoptotic cells. MØ from mid-life individuals showed no significant differences in their ability to respond to immune modulation by apoptotic cells compared to MØ from young donors. Larger cohorts would be required to investigate whether immune modulation of MØ by apoptotic cells contribute to inflammatory pathology throughout ageing.