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The skin is one of the most common sites of exposure to chemicals. Some chemical entities are known to act specifically as skin allergens that can ultimately elicit the condition allergic contact dermatitis. This is defined as the presence of skin erythema and edema, resulting from a delayed-type antigen-specific T-cell-mediated reaction. One such chemical that has demonstrated immuno-stimulating capacity and is the focus of this thesis is p-phenylenediamine (PPD). PPD is widely used in the formulation of permanent hair dyes and is recQgnized as one of the most common chemical al1ergens. The sensitizing effects of PPD are believed to derive from the chemical's instability. Auto-oxidation in solution results in the formation of an electrophilic quinonediimine intermediate which is susceptible to sequential selfconjugation; the end product of these reactions is the trimer, Bandrowski's base (8B). Predictive animal models and patch testing have clearly identified PPD as a strong sensitizer; however, the role BB plays in the manifestation of allergic dermatitis remains uncertain. Thus, the aim of these studies was to investigate in detail, the chemical and cellular basis of PPD-mediated contact dermatitis. The purpose of initial experiments was to explore the relationship between PPD oxidation and functional maturation of human monocyte-derived dendritic cells in vitro. Exposure of dendritic cells to PPD (5-50 J,JM), but not BB, was associated with an increase in CD40 and MHC class II expression. These effects were not related to the direct toxic effects of PPD (neither apoptosis nor necrosis) or depletion of glutathione. PPD and 88 treatment did not alter basal cell surface expression of CD80, CD83, or CD86. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed PPD was rapidly oxidized in cell culture media to 88. Addition of glutathione inhibited 88 formation but did not prevent covalent binding of PPD to dendritic cells protein or dendritic cell activation. To investigate in greater detail the chemical basis of PPD-mediated increased CD40 expression, using the same DC methodology, a range of structurally related disperse dyes were studied. Dendritic cell exposure to all disperse dyes was associated with an increase in CD40 expression; the rationale for this up regulation is attributed to their complex structures that are prone to auto-oxidation. PPD was also shown to directly stimulate proliferation of lymphocytes isolated from allergic patients, but not volunteers. In contrast, BB-specific T-cells were characterized from blood of allergic patients and volunteers, but not cord blood. Their presence seems to reflect an acquired immune response which is not translated into an allergic reaction. A comparison of the functional characteristics of T-cells from patients and volunteers using Luminex and micro-array technology identified an important difference in cytokine secretion and cytokine gene expression when patient and volunteer samples were compared. Specifically, stimulated cells from allergic patients secreted high levels and expressed high levels of cytokine genes associated with an inflammatory Th2 response (IL-4, IL-5, and IL-13). In contrast, cells from healthy individuals exemplified a more regulatory phenotype (IL-2ra/CD25, TGF-131 and IL-10). In conclusion, these experiments have characterized the degradation of PPD in solution and binding of PPD-derived material to cellular and extracellular protein, and how this relates to the activation of both dendritic cells and T-cells. Data showing the PPD-specific stimulation of Th2 secreting T-cells from allergic patients, but not volunteers, represents an important discrimination between allergic and non-allergic groups.

The chemokine CXCL16 is selectively expressed by antigen presenting cells and unli ke most chemokines, is attached to the cell surface by means of a mucin stalk. This allows CXCL16 to function as an adhesion molecule, binding leukocytes such as T-cells which display the cognate receptor, CXCR6. Cleavage of CXCL16 by metalloproteinases can also release a soluble form of CXCLI6 which can induce the migration of CXCR6 expressing cells. In addition, membrane-bound CXCL16 can also ftinction as a scavenger receptor for a variety of ligands, including oxidised low density lipoprotein. Collectively, these functions support a role for both CXCL16 and CXCR6 in the process of atherosclerosis. The CXCL16:CXCR6 relationship appears to be monogamous and the nature of this interaction was investigated by a programme of mutagenesis used to examine CXCR6 function in vitro, assessed by adhesion, chemotaxis and ligand binding assays. CXCL16 function was also examined, in particular, the effects of a single nucleotide polymorphism associated with disease. In a third arm of the project, recombinant CXCL16 was produced in E. Coli using two different systems and subjected to assays of biological function. In contrast to other chemokine receptors, mutation of the CXCR6 N-terminus and inhibition of post-translational modifications were without effect on function. Likewise, N-terminal extension of CXCL16 by 24 amino-acids resulted in a protein with decent potency and efficacy in chemotaxis and not as anticipated, a CXCR6 antagonist. Cells expressing the CXCR6 mutants Asp-176-Asn and Glu274- Gln were unable to interact with soluble CXCLI6, suggesting that these residues are critical for ligand binding. Intriguingly, although unable to interact with soluble CXCL16, the Glu-274-Gln mutant was able ·to promote robust adhesion to membrane-anchored CXCL16, suggesting that the soluble and membrane-bound forms of CXCL16 possess distinct conformations. Glycosylation of CXCL16 was found to be critical for cell-cell adhesion function, and a Ala-181-Val polymorphic variant of CXCL16 was found to have no capacity for mediating cell-cell adhesion, suggesting a potential relevance of this mutation in disease. Collectively, the data suggests that the CXCL16:CXCR6 interaction is distinct from other chemokine-chemokine interactions described in the literature, and that current models for chemokine receptor activation, in which the chemokine N-terminus ligand protrudes into an intrahelical pocket, do not apply to the CXCR6:CXCLI6 interaction. This may have implications for successful antagonism of the receptor by small molecules.

Cytokines and chemokines control the fine balance of immunity and inflammation within the body. Some tumours are capable of utilising these, to promote their own growth and survival. This is particularly apparent in Hodgkin's lymphoma (HL) where the malignant Hodgkin Reed-Sternberg (HRS) cells comprise 1% of the total cellular infiltrate. The rest of the infiltrating mass resembles chronic inflammation, probably under the influence of cytokines and chemokines produced by the HRS cell. A proportion of the HL cases are associated with the Epstein-Barr virus (EBV). EBV can de-regulate cytokine and chemokine expression and might therefore contribute to the immune infiltrate. In this study a modified antigen retrieval method was developed to enable the investigation of expression of cytokines and chemokines by immunohistochemistry. This method was used to show that interleukin-6 (lL-6) expression by HRS cells is associated with the presence of 'B' symptoms and with an increased likelihood of failure to achieve complete remission in patients with advanced HL. The same approach was used to demonstrate that Fractalkine (FKN) is expressed by the majority of HRS cells. An association between FKN expression and increased frequency ofGranzyme B+ (GrB) cytotoxic T-cells (CTLs) was also seen. Affymetrix gene profiling was used to identify cytokines and chemokines whose expression was influenced by EBV infection of HL cell lines. This analysis showed that CXCR4 and CCL20 were up-regulated by EBV infection; immunohistochemistry on HL cases showed a similar association. EBV+/CXCR4+ cell lines were shown to be capable of migrating towards a CXCLl2 gradient. Transwell assays using conditioned media from EBV+ICCL20+ cell lines, showed a chemotaxic effect on a population of peripheral blood mononuclear cells (PBMC) which was three fold greater than media from EBV-ICCL20-. In conclusion, this study has gathered more important information concerning the role of cytokines and chemokines in HL, this may also assist in identifying targets for future therapeutic treatments of the disease.

Many solid tumours and other pathological sites such as infected wounds are characterised by hypoxic regions (defined by an O2 tension of <1%), which are often heavily infiltrated by macrophages. Macrophages respond to hypoxia by up-regulating a number of genes likely to promote tumour growth and spread, including MMP-7. MMP-7 has been shown to be up-regulated in various tumours and it also has important roles in protection against microbial infections including release of the pro-inflammatory cytokine TNF and activation of pro-defensins. The aim of this project was therefore to determine the mechanisms of hypoxic up-regulation of matrix metalloproteinase-7 (MMP-7) in primary human macrophages. An important aspect of this project was the analysis of the MMP-7 promoter in an attempt to identify the DNA elements required for hypoxic up-regulation, using wild-type and mutated luciferase reporter constructs transfected into primary human macrophages. A - 296 bp construct was shown to be up-regulated 3-fold in primary human macrophages exposed to 5 days of hypoxia. The luciferase expression from the constructs containing mutations in the Ets and AP-1 transcription factor binding sites was not detectable, suggesting that these sites were essential for basal MMP-7 gene expression. In this project, it was shown that MMP-7 mRNA was indeed up-regulated in severe hypoxia (0.2% O2 for 18 hrs) in primary human macrophages. However, further experiments produced the surprising finding that hypoxia alone was not able to up-regulate MMP-7 mRNA; rather, the gene was induced by co-stimulation with hypoxia and TLR ligands such as LPS. The use of Polymyxin B, which neutralises LPS, blocked MMP-7 hypoxic up-regulation. Therefore, my data indicate that the observed and previously published “hypoxic” up-regulation of MMP-7 mRNA is actually most likely to be due to the synergistic interaction of hypoxia with LPS or other TLR ligands. MMP-7 has previously been shown to be induced by TLR ligands, but my finding of synergy between these and hypoxia in up-regulation of MMP-7 mRNA and protein is novel, and challenges current opinion on MMP-7 regulation by hypoxia. Since the PI3K/Akt pathways is involved in TLR signaling and has been reported to be involved in hypoxic up-regulation of MMP-7, this pathway was investigated using two inhibitors, LY294002 and wortmannin. LY294002, and to a lesser extent wortmannin, inhibited LPS-induced MMP-7 up-regulation, linking MMP-7 LPS-regulation with the PI3K pathway. Another TLR signaling pathway, NF-κB, was investigated as a possible MMP-7 regulating pathway. NF-κB seems to be involved in MMP-7 up-regulation. Therefore, both PI3K and NF-κB pathways can be essential in MMP-7 up-regulation. These findings regarding the regulation MMP-7 expression will expand knowledge of its important role, especially in innate immunity in the context of hypoxia and infection.

B-cell autoreactivity is a characteristic abnormality in several autoimmune diseases. In systemic lupus erythematosus (SLE), inappropriate activation of TLR7 and TLR9, accompanied by high serum levels of BAFF, are implicated in disease pathogenesis. In murine B-cells, TLR9 activation resulted in up-regulating BAFF expression, while such a direct effect has not yet been established in normal human B-cells (nhB-cells). Therefore, the effect of CpG-2006, a synthetic TLR9 ligand, on the expression and function of BAFF and its receptors (BAFF-R, TACI and BCMA) in nhB-cells was studied. BAFF expression, in response to CpG-2006, was significantly upregulated at the level of mRNA, intracellular and membrane-bound protein in nhB-cell. In contrast, enhancement of BAFF secretion in culture supernatants of CpG-2006-stimulated nhB-cells was not detected. CpG-2006 treatment of nhB-cells significantly enhanced the expression of TACI and BCMA, but did not affect BAFF-R expression. CpG-2006-stimulated B-cells, in co-cultures with freshly isolated nhB-cells, co-stimulated B-cell receptor-induced cellular proliferation of the freshly isolated nhB-cells. This effect was completely blocked by a BAFF-specific monoclonal antibody. CpG-2006 treatment of nhB-cells sensitised them to proliferate in response to exogenous BAFF, whereas exogenous BAFF per se had no effect on the proliferation of untreated nhB-cells. This effect was inhibited by an anti-BAFF-R blocking antibody, but was not inhibited by anti-TACI or anti-BCMA antibodies. CpG-2006 mediated BAFF expression through NF-κB and ERK1/2 pathways, and partially through p38MAPK. Simultaneous treatment of nhB-cells with CpG-2006 and either of the inhibitory ODNs (ODN-TTAGGG or ODN-2088), at 1:5 ratio (CpG-2006: INH-ODN), inhibited CpG-2006-induced BAFF expression in nhB-cells. Finally, BJAB, RPMI and RAMOS B-cell tumour lines failed to represent nhB-cell’s responses to CpG-2006 treatment. Taken together, these novel findings demonstrate a functional cross-talk between TLR9 and BAFF in nhB-cells, and have possible implications for the roles of TLR9 and BAFF in the pathogenesis of SLE.

Many studies of visual perception of diagnostic medical images have traditionally used modalities that are static and two-dimensional. These studies display single images, for example chest x-rays or related images grouped in a particular layout, such as mammograms. An eye tracker is used to capture eye gaze information from the observer as they view the image. Continuing advances in digital medical imaging and workstation design now permit more complex modalities to be used in diagnostic practice. In addition to standard 2D images, sectional imaging using CT or MRI can be used to compose a full 3D rendering of the subject. Image sets can also be stacked in sequence, with the observer controlling the navigation up and down the stack. Such a modality, with a discrete z-axis, can be thought of as having 2.5 dimensions. These new modalities have an interactive characteristic; searching the complete image set can no longer be performed by eye movement alone. The observers search strategy now has to include changing the data that is visible on the display at anyone time, and recalling the location of details that might not be currently visible, in addition to visual searching. This thesis addresses the issue of capturing an observer's perception of a scan as they navigated using the stack-mode visualisation. The work uses CT Colonography (CTC) as an example modality, which is very dependent on the stack mode interpretation to detect polyps in the colon. CTe is a relatively young modality, with no film-based equivalent, and relies heavily on the properties of modern eT scanner technology to generate hundreds of images for a patient volume. A key aspect of eTe interpretation is inspecting the colon wall. By tracking observer gaze, and mapping it into the scanned patient volume, this work, for the first time, calculates how much of the colon wall has been seen by an observer preforming an axial interpretation. Eye tracking in medical imaging is by no means new, but it has tended to be based around static project x-rays, or the interpretation of non-moving images. This work shows how to eye track an observer as they navigate though an axial representation of a patient volume, and how to calculate how the eye gaze path would appear in the 3D scan. This work represents the intersection of three research areas. The first is the developing field CT colonography, an imaging technique that has developed rapidly in the first decade of the 21st Century. Secondly the field of medical image perception, and the use of eye tracking to assess how a medical professional interprets a radiograph. This field has focused on film or film-equivalent imaging in its 30 year history but has not transitioned assessing multi-dimensional imaging techniques. The act of linking these two fields requires the glue -of computing technology and Human Computer Interaction (HC!), which make the third field in this multidisciplinary work. In this thesis the reader is introduced to two human body parts: the colon and the eye. An understanding of the colon and colon cancer is necessary to fully appreciate the subtleties of a particular type of imaging and interpretation. Measuring this interpretation, and indeed interpreting the interpretation, requires some familiarity with the human eye and its movements.

This thesis has medical image perception as its central theme and it contains selected publications in that discipline from 1998 to 2010. Other aspects of medical imaging and related subjects have played their part in my research and teaching over a longer period but through my PhD and in the postdoctoral years the subject area dominating my academic thinking has been the. sources of error in the interpretation of medical images. Medical image perception is a mature science and several authors have published historical accounts of its origins and development. Probably the most comprehensive of these appeared in the Journal of the American College of Radiologists (Kundel 2006) based on material used in a keynote lecture to the Medical Image Perception Society Conference in 2003. Although it is a personal account it captures the dynamic of empirical research in medical image perception from the first recorded formal experiment in radiological error (Birkelo et al194 7) to the present. The covering work to the thesis emphasises the importance of multidisciplinary approaches to medical image perception and radiological error. I This has been the guiding principle behind the published work contained here, drawing on ideas and methods from physics, radiology, psychology, computer science and ergonomics. Through this broad-fronted approach the body of work has made original contributions to the field which have been extended and continue to be developed by others.

The field of metabonomics is beginning to grow rapidly due to the ability to analyse biofluids, providing a 'snapshot' of biological processes that have happened (cf: proteomic/transcriptomic studies, which predict what may happen), making it possible to profile responses over time. The work described in this thesis was motivated by the aim of profiling clinical urine samples obtained from fracture patients, with a view to identifying potential biomarkers related to failed fracture healing. This led to the need to develop and evaluate metabonomic approaches, specifically a orthogonal separation approach complementary to the commonly-used reversed phase (RP) separation methods, namely hydrophilic interaction liquid chromatography (HILI C). Urine samples from healthy volunteers were collected and used to develop an LCMS 'metabonomic toolbox'. This development evaluated various aspects of a· metabonomic study that are commonly poorly reported within the literature: 'study design, sample collection storage and handling considerations, data extraction, normalisation and scaling methods, and multivariate data analysis tools. From the literature, the commonly-used method of normalising to creatinine was ' found to be unsuitable due to perturbations in the urinary excretion of creatinine due to factors such as illness. Methods used to evaluate system ~tability were also developed and added to the 'toolbox'. HILIC was successfully used as a separation technique orthogonal to RP, producing comparable results but using different metabolites; this highlights the fact that much potential information is P?ssibly being lost when only RP-LC-MS methods are used for analysis. The need to use both modes of ionisation polarity were also addressed for an increased coverage in biofluid metabolite profiles. , The knowledge gained in the development of the 'metabonomic toolbox' was used for the analysis of clinical urine samples. Despite the lack of properly time-setted samples and none of the recruited patients suffering delayed fracture healing, potential metabolites related to fracture healing were found. However, the samples were very different to previously-analysed samples from healthy volunteers; they , showed very large amounts of protein, which had a large range of molecular weights. These were' identified- proteomically. Finally, ESI-Q-o-ToF MS/MS, MALDI-ToFlToF MS/MS and racemic amino acid analysis were used for the structural determination of a pseudomonad biosurfactant, which was identified, unexpectedly, as the cyclic Iipopeptide white line inducing principle, WLIP.