Dysregulated IL-1 activity has been implicated in the pathogenesis of several acute and chronic inflammatory as well as autoimmune diseases. Genomic analysis has identified additional IL-1 family members and also expanded the IL-1 receptor family. To date, there are eleven IL-1 family members and nine IL-1 receptor family members. Information regarding the biological activity and immunological role of the newer IL-1 family members is still very limited. The novel IL-1 cytokines, IL-1F6, IL-1F8 and IL-1F9 (recently renamed IL-36α, IL-36β and IL-36γ respectively) have been shown to signal via IL-1 receptor related protein 2 (IL-1Rrp2, recently renamed IL-36R). The main aims of this study were: (i) to investigate the expression of IL-1Rrp2 by human myelomonocytic and non-myelomonocytic immune cells as well as other human cells, (ii) to determine the possible function of IL-1Rrp2 and (iii) to determine the effect of IL-1Rrp2 expression on T lymphocytes. Results reported in this thesis indicate that among human myelomonocytic cells, constitutive IL-1Rrp2 expression is unique to dendritic cells (DCs). IL-1Rrp2 was expressed by monocyte- derived DCs (MDDCs) and plasmacytoid DCs (pDCs) but not by peripheral blood type 1 or type 2 myeloid DCs (mDC1 or mDC2). IL-1Rrp2 expression was regulated in response to both classical (IL-1β) and novel IL-1 cytokines (IL-1F8 and IL-1F9). Similarly to IL-1β and bacterial LPS, novel IL-1 cytokines mediated DC maturation as shown by DC phenotypic changes (e.g. upregulation of HLA-DR expression and decreased CD1a expression following culture of DCs with IL-1F8) and cytokine production. Among non-myelomonocytic cells, constitutive IL-1Rrp2 expression was observed in lamina propria tissue, T lymphocytes, NCI/ADR-RES cells and HT 29 cells. IL-1F8-matured human DCs were fully functional as they induced proliferation of IFN-γ-producing TH1 subsets. Results suggest that novel IL-1 cytokines play a role in inflammatory responses involving human DCs, with possible implications for inflammatory disease.

The synthesis of ligand systems and complexes thereof suitable for use as positron emission tomography (PET) agents is studied herein. The ligands 6,6’-(1,4-diazepane- 1,4-diyl)bis(3-aminobenzoate) (HPR), 6,6’-(piperazine-1,4-diyl)bis(3-aminobenzoate) (PipR.2HCl), 6,6’,6”-(1,4,7-triazonane-1,4,7-triyl)tris(3-aminobenzoate) (TACNR), 1,4-bis(2-amino-4-tolyl)- 1,4-diazepane (HPTol), 1,4-bis(2-amino-4-trifluoromethylphenyl)-1,4-diazepane (HPCF3 ), di-tert-butyl 4,4’-(1,4-diazepane-1,4-diyl)bis(3-aminobenzoate) (HPtButyl), 1,3-bis(2-amino- 4-tolyl)-1,3-diazacyclohexane (PipTol), 1,4,7-tris(2-amino-4-trifluoromethylphenyl)-1,4,7- triazacyclononane (TACNCF3), have been studied. Ni(II), Cu(II), and Zn(II) complexes of these ligands have been synthesised, DFT calculations have elucidated structural configuration, whilst x-ray crystal structures of NiHPtButyl, and CuTACNCF3 have been determined. EPR measurements of CuHPR have been taken. A series of triazine core compounds have been prepared for use as model compounds for targeting molecules. The compound Tz(EtGly)(BocGuan)Cl has been synthesised with the required arms to selectively bind to the integrin αvβ3. DO3A moieties have been added to these ligands, and mass spectra analysis of the coordination products with a series of metals has been determined. NO2A derived compounds 2,2’-(7-tosyl-1,4,7-triazonane-1,4-diyl)diaceticacid (TsTACNA2) and 2,2’-(7-(4-nitrophenyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NPhTACNA2) have been studied,with the x-ray crystallography of the copper complexes completed. Positron emmision tomography (PET) studies of the complexes have also been undertaken and show high levels of complexation at ng scales, with moderate stability in human serum. Glutaric acid functionalised compounds (2S,2’S)-2,2’-(1,4-diazepane-1,4-diyl)dipentanedioic acid (HPGlut), 2,2’,2”-(1,4,7-triazonane-1,4,7-triyl)tripentanedioic acid (TACNGlut), and 2,2’-(4,11-dimethyl-1,4,8,11-tetraazacyclotetradecane-1,8-diyl)dipentanedioic acid (DMiii iv CGlut) have been synthesised, with the copper complex of DMCGlut affording structural determination. The Mn(II) and Gd(III) complexes of the DMCGlut ligand system have been studied by xylenol orange UV titration for their metal-ligand binding ratio, with the Mn(II) system showing a 1:1 binding ratio, whilst the Gd(III) complex showed no binding at all. Synthesis of the trispyrazylborate analogue, tris(4-methyl-2-(2-pyridine)pyrazyl)borate (MeTpPy), was unsuccessful, however, the bis- (MeDpPy) and tetra- (MeQpPy) substituted analogues were successfully synthesised.

In this thesis methods of accurately and reproducibly measuring intestinal content and transverse relaxation to study the fate of food in the gastrointestinal tract are reported. In addition to this a technique for measuring relaxation parameters in the brain at ultra-high field is also investigated. Several methods can be used when quantifying the fate of food through the gastrointestinal tract; there are many factors that can be measured using MRI and these are discussed along with other non-MRI techniques. In this work a method for quantifying small bowel water content (SBWC) is optimised and validated for use at 3.0 T and a technique for measuring T2 in the abdomen is developed and optimised called T2-prepared balanced turbo field echo (T2-prep bTFE). These two methods are then used, in conjunction with other established MRI techniques, to study the fate of food in the gastrointestinal tract from the stomach all the way through to the colon. A hybrid gradient echo-spin echo (GESSE) sequence is also investigated and optimised for measuring T2 and T2* simultaneously in the brain at 7.0 T. This sequence is also proved to have applications in the liver at lower field strengths. The GESSE sequence is used to measure the first T2 values in deep grey structures in the brain at 7.0 T. In this work cross-field (1.5, 3.0 and 7.0 T) variations in T2 are studied. Also differences in T2 and T2* are measured in the brain to determine variations between white matter tracts and to ascertain any effects of Parkinson’s disease on deep grey matter structures.

A review is presented of the major cancer surveys conducted in trans-Saharan Africa since the end of the First World War, starting with the more numerous relative ratio or frequency studies before proceeding to the four recent incidence rate surveys which have taken place in Johannesburg, Kivu and Ruanda Urundi, Kampala, and Lourenco Marques. An account is then given of the three years cancer incidence rate survey in Ibadan, Nigeria, which extended from April 1960 until the end of March 1963. In describing the organisation and running of the Ibadan survey particular attention is paid to a number of sociological studies designed to elucidate special local circumstances which might be affecting the validity of the eventual results. One of these subsidiary surveys was concerned with the attitude of the population towards modern medical facilities and the extent of their continued reliance upon traditional modes of treatment. Studies of prominent local tumours made in the course of the main survey also receive individual mention. Among them was an attempt to assess the prevalence of Kaposi's Sarcome throughout Nigeria, an exercise which served to demonstrate the complexities of epidemiology in such a setting. The incidence rate survey in the town of Ibadan showed low crude annual rates of approximately 45 per 100:000 of the population for all cancers in both sexes. However, during much of their life span Ibadan people seem to have age specific cancer incidence rates which are only slightly lower than those of the U.S.A., with the notable exceptions of the 5 - 9 age group and the over-fifties. Young male children have a very high incidence of the Burkitt Tumour, making the overall rates for this age much higher than those in the United States whilst in older people in Ibadan the cancer incidence rate, which had been rising with age, falls off steeply. Apart from the Burkitt Tumour and tumours of the reticulo-endothelial system generally, the striking feature of the Ibadan cancer scene is the high incidence of primary liver celled carcinoma among males. Finally, attention is drawn to the unsatisfactory nature of relative ratio surveys in a continent with rapidly developing medical services and suggestions are made regardint the interpretation of some of the Ibadan incidence survey results. It is emphasised that caution must still be exercised even when considering cancer incidence rates if these have been based entirely upon hospital diagnosed cases, drawn from a population whose ages are not accurately known and whose elderly members may be disinclined to come to hospital.

Streptococcus pneumoniae (pneumococcus) is a leading cause of childhood morbidity and mortality around the world. The available polysaccharide-based vaccines are limited by either low efficacy in young children or narrow serotype coverage. Recent research has focused on developing protein vaccines. Pneumococcal pilus-1 proteins play an important role in pneumococcal adherence to host respiratory epithelium and subsequent bacterial colonization or invasion; and may therefore be an effective vaccine candidate. This PhD project investigated natural immunity in humans to pneumococcal pilus-1 proteins and its association with nasopharyngeal carriage of pneumococcus. Nasopharyngeal carriage of pneumococcus was analysed by bacteriological culture of nasal swabs on blood agar. Pneumococci were identified based on their colony morphology and optochin sensitivity; and further confirmed by PCR detection of pneumolysin gene in the isolates. Pneumococcal carriage was found to be common in young children, which gradually decreased with advancing age. PCR detection of pilus islet 1 (PI-1) gene revealed that percentage of pilus-1 positivity was low among these carriage isolates. This low prevalence may be associated with the recent introduction of pneumococcal conjugate vaccination, which covers the common piliated serotypes. ELISA based measurement of serum and salivary antibodies to pilus-1 proteins detected significant antibody levels to both RrgA and RrgB, presumably developed as a part of natural immune response in children and adults. An age-dependent increase in serum antibody levels to both RrgA and RrgB was also observed, and anti-RrgA appeared to develop earlier in childhood than anti-RrgB. Moreover, higher levels of antibody, especially anti-RrgA were found in children who were culture-negative than in those who were culture-positive for pneumococcus. It suggests that these naturally developed antibodies may contribute to the protection against pneumococcal carriage in humans. Using an in vitro model of human NALT, the study revealed that adenotonsillar tissues are important induction sites for immune response to these antigens, by priming B cell memory. The induction of antibody secreting cells in NALT was enumerated by ELISpot assay; and the antibody production was measured by antigen-specific ELISA. In vitro stimulation with a wild type (TIGR4) pneumococcal culture supernatant (containing both RrgA and RrgB proteins) induced a significant memory B cell and antibody response in adenotonsillar cells. Current carriage in vivo enhanced the memory B cell response and antibody production. Flowcytometric analysis of CFSE labelled adenotonsillar MNC suggested that pneumococcal pilus-1 proteins RrgA and RrgB were capable of stimulating CD4+ T cell proliferative response in human NALT. Stimulation with pneumococcal CCS induced Th17 related cytokines production in both human adenotonsillar MNC and PBMC culture supernatant. Importantly, this in vitro production of Th17 cytokines after stimulation with TIGR4wt CCS was significantly higher than that of CCS derived from its isogenic RrgA-/- and RrgB-/- mutants, indicating a contribution from both of these proteins. The ability of these pilus-1 proteins to stimulate CD4+ T cell proliferation and Th17 response may contribute to the natural immunity to pneumococcus in humans. This study has revealed that both of these pilus-1 proteins, RrgA and RrgB are immunogenic and are capable of priming for memory B and T cell response in human NALT. These findings aid to our understanding on the naturally developed immune response to pilus-1 proteins, and may inform future vaccination strategy with intranasal immunisation containing protein antigens against pneumococcal infection.

There are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O2 tension. In high O2 tension (normoxia) the PHDs hydroxylate HIFα subunits on 2 conserved proline residues inducing binding of the von-Hippel-Lindau (VHL) tumour suppressor, the recognition component of a multi-protein ubiquitin-ligase complex, initiating HIFα ubiquitylation and degradation by the 26S proteasome. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIFα or as a multi-protein complex. In this thesis, data are presented that shows that the tumour suppressor protein LIMD1 acts as a molecular scaffold simultaneously binding the PHDs and pVHL into a normoxic protein complex (normoxiplex), increasing their physical proximity in order to enable efficient and rapid sequential modifications and thus degradation of HIF1α. Data are presented which indicates that increased LIMD1 expression down regulates HIF transcriptional activity, by promoting HIF1α degradation via the oxygen dependent degradation domain in a manner dependent on hydroxylase and 26S proteasome activities. However, degradation of this domain is not wholly dependent on the well characterised proline residues subject to hydroxylation, suggesting that LIMD1 may alter proline hydroxylation specificity or modulate HIF via a different mechanism. Furthermore, endogenous depletion of LIMD1 results in the converse, leading to HIF1α stabilisation and accumulation, enhancing HIF transcriptional activity. Moreover, Limd1-/- MEFs show increased HIF transcriptional activity. One mechanism by which this is achieved involves the binding of PHD2 within the N-terminal portion of LIMD1 while allowing concurrent binding of VHL to the C-terminal zinc-finger LIM domains. However, the LIMD1 mediated mechanism regulating HIF1α independently of proline residues 402 and 564 is still unclear. Finally, data are presented that show that the LIMD1 family member proteins Ajuba and WTIP all bind specifically to VHL but differentially to PHDs 1, 2 and 3 and thus these three LIM domain containing proteins represent a new group of hypoxic regulators.

In plastic surgery, flap reconstruction has been utilised to repair defects in every part of the body, in an effort to restore form and function to patients. The basis of every flap is its blood supply, therefore this series of studies investigates the vascular territory of named arteries, veins and even perforators, utilizing computer tomography (CT) and TeraRecon software. The latter two is technology which allows appreciation of vascular flow in 3D and 4D (dynamic studies), whereas previous studies of vascularity has only been static and in 2D. Vascular anatomy studies were performed using fresh cadavers. Perforator flaps on the anterior trunk studied were the internal mammary artery perforator (IMAP) flap, the transverse rectus abdominis musculocutaneous (TRAM) flap, the deep inferior epigastric artery perforator (DIEP) flap and the superficial inferior epigastric artery (SIEA) flap. Posterior trunk flaps included the posterior intercostal artery perforator flap, the lumbar artery perforator flap and the superior gluteal artery perforator (SGAP) flap. In the upper extremity, we studied the supraclavicular artery perforator flap. In the lower extremity, we studied the gracilis musculocutaneous flap. Trends and characteristics are noted in the vascular analyses, and four major principles drawn are discussed in the last chapter.

Leukocyte recruitment to sites of inflammation is a crucial event in host defense against pathogens and tissue injury. Although there is at present much interest in deciphering the mechanisms of leukocyte transendothelial cell migration, little attention has been paid to the subsequent steps, i.e. leukocyte migration through the pericyte layer and the venular basement membrane. In this context, results from this group previously demonstrated that neutrophils preferentially transmigrate through gaps between adjacent pericytes, regions associated with sites of low matrix protein expression within the vascular basement membrane. The aim of this thesis was to extend these findings by investigating the impact of inflammatory mediators on pericyte morphology and vascular basement membrane deposition using both in vitro and in vivo models. Flow cytometry analysis of pericyte-like C3H/10T1/2 cells and primary lung pericytes revealed the expression of key pro-inflammatory molecules on their surface (including cytokine receptors and adhesion molecules) and the regulation of these molecules upon cytokine stimulation. Using the murine cremaster muscle model it was further demonstrated that key neutrophil chemoattractants (i.e. LTB4, KC, C5a and fMLP) induced neutrophil transmigration that was associated with a change in pericyte morphology (as quantified through enlargement of gaps between adjacent pericytes). These changes in pericyte gap size were neutrophil-dependent and mediated by endogenously generated TNF as demonstrated in neutrophil-depleted mice and TNFR-/- mice, respectively. In addition, TNF appeared to mediate post-inflammatory BM deposition in response to LTB4 and was required for chemoattractant-induced vascular permeability. Hence, the results of the present work have demonstrated the ability of pericytes to respond to both cytokines and chemoattractants, suggesting an active role for pericytes in the regulation of inflammatory responses. In addition, findings provide the first evidence for chemoattractant-induced changes in vascular morphology and barrier functions of venular walls in vivo via the release of endogenous TNF as a secondary mediator, effects that may contribute to the pro-inflammatory properties of these stimuli.

Background: Antigen presenting cells (APCs) such as macrophages play a crucial role in orchestrating immune responses against foreign materials. The activation status of macrophages can determine the outcome of an immune response following implantation of synthetic materials, towards either healing or inflammation. A large range of biomaterials are used in the fabrication of implantable devices and drug delivery systems. These materials will be in close contact with APCs and characteristics such as surface chemistry may have a critical role in polarising macrophages towards pro- or anti-inflammatory immune phenotype. Each phenotype can be characterised by their cytokine profile, transcription factors, surface markers or even morphology. Objectives: The overall objective of this study was identifying novel chemistries that are able to induce differentiation of human monocytes towards macrophages with distinct pro or anti-inflammatory phenotypes. To achieve this, a combination of different surface chemistries has been generated using oxygen plasma etching as well as acrylate and acrylamide polymer libraries. Methods: Fluorescent microscopy, real time-PCR, multiplex assay, ELISA, macrophage phagocytic activity were used for macrophage phenotype identification. Libraries of acrylates and acrylamide polymer microarrays (first generation microarray of 141 polymers and second generation of 442 polymers), and oxygen plasma etching of polystyrene used as two different techniques for making different surface chemistries. CellProfiller software was used for analysing images and was used for machine learning for phenotype identification. Results: polystyrene with highly hydrophobic surfaces are shown to suppress expression of M1-associated surface markers and cytokines while promoting M2-associated markers. However, highly hydrophilic surfaces seem to have the opposite effect as evidenced by promoting M1-associated marker expression and pro-inflammatory cytokine production while suppressing M2-associated marker expression and anti-inflammatory cytokine production. Also, the protein thickness was proportional with the hydrophilicity of the surface, which had impact on cell polarisation. Furthermore, co-polymers 157 from the second generation array was the most M2 biased polymer among the first and second generation of microarray polymers by induction of MR (M2 marker) cell expression, while co-polymers 217 and 123 from the second generation had impact to increase calprotectin (M1 marker). Also, cell adherence and morphology were affected by polymers surface chemistry. Conclusion: Surface chemistry without using polarising cytokine can polarise macrophage towards pro-inflammatory and anti-inflammatory phenotypes.

The identification and classification of tissue abnormalities for the purpose of disease diagnosis have been greatly served by the discipline of histopathology, and Immunohistochemistry (IHC) in particular. The advent of digital slide scanners and computerised slide viewing software have opened the door for introducing automated algorithms into what has traditionally been a predominantly manual discipline. Multi-IHC analysis is one potential area of interest for automation, which will be discussed in detail in this work. Analysis occurs on serial sections of tissue, which must be realigned before their IHC marker expressions can be compared directly. This requires a robust method of serial section registration. Two methods of automated serial section registration are present, which are each designed to align a particular tissue type: breast core biopsy sections or resected colorectal cancer sections. Automated multi-IHC analysis is presented from the perspective of two case studies: Scoring of Oestrogen Receptor and Progesterone Receptor (ER/PR) on breast core biopsies and IHC scoring and colocalisation of resected colorectal cancer (CRC) sections. For each case study the background of the problem is introduced, followed by a discussion of how each type of analysis is performed in clinical practice, and it is then explained how this is implemented as an automated algorithm. For the scoring of ER/PR, it is shown that the algorithm can achieve good agreement with a pathologist on a sample of 50 cases, which suggests that automated ER/PR scoring is suitable for clinical practice. For the analysis of CRC, the results of scoring and colocalisation are shown in the form of localised maps with a discussion into how they may be used for further analysis. As part of this framework a number of additional steps must be carried out before the goal of multi-IHC analysis can be realised. Two pre-processing steps, both of which are key to ensuring that the end results are of the highest quality, are presented: Tissue Segmentation and Out of Focus Area detection. A complete Out of Focus Area detection system is presented, which has led to the development of a Windows software that is currently being used in a local hospital. In addition, we present an automated method of Stain Separation, based around Independent Component Analysis, which allows us to extract and process the IHC marker expressions directly. This method includes a novel correction process to improve any faults in the primary analysis.