Escherichia coli contributes to the commensal microbiota of most mammals by producing vitamins and aiding digestion. However, several strains of E. coli have evolved as pathogens and have become highly adapted for specific niches through the acquisition of pathogenicity islands. E. coli O157:H7 is a commensal bacterium of cattle but if transferred to humans, usually by contaminated food products, it can act as a pathogen. In humans E. coli O157:H7 causes diarrhoea, haemorrhagic colitis and in some cases haemolytic uremic syndrome which can result in death. Clinical treatment of this pathogen is difficult since the antibiotics currently available have been shown to worsen the clinical outcome of infection. Therefore, the discovery of novel anti-bacterial therapies is of high importance. A novel approach to limit pathogenesis is to target the factors used by bacteria to cause disease - their key virulence factors. In theory, such approaches should only compromise the ability of the pathogen to cause disease, rather than its ability to survive, thereby reducing the selective pressure for the development of resistance mechanisms. The type three secretion system (LEE T3SS) is a key virulence factor for E. coli O157:H7 as it facilitates tight attachment to host cells and the secretion of effector proteins. The importance of this virulence factor for the disease process makes it an attractive target for anti-microbial therapies. The salicylidene acylhydrazides (SA) are a class of compounds that inhibit the expression of the T3SS of several Gram-negative pathogens. When this study was started, the mode of action of these compounds was completely unknown. An affinity pull-down assay to identify the binding proteins of the SA compounds was conducted. Identification of the target proteins of the compounds was the first step in determining their mechanism for decreasing the expression of the LEE T3SS in E. coli O157. The pull-down identified nineteen putative targets, none of which had previously been linked to the regulation of the LEE T3SS. The aim of this thesis was to systematically investigate the putative targets using a combination of approaches: phenotypic studies of deletion mutants and structural and functional characterisation of the target proteins. From the nineteen putative targets seven were selected for further investigation namely Tpx, WbrA, FolX, FkpA, FklB, SurA and AdhE. Several of these proteins were shown to bind to the SA compounds however deletion of only one of the target proteins resulted in a decrease in LEE T3S. This target, AdhE, offers an exciting new lead in the search for novel targets for antibacterial therapies.

IL-33 and IL-17 family cytokines (IL-17A – IL-17F) have been shown to play roles in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. However knowledge of their role in periodontal disease pathogenesis is limited. The aim of this study was therefore to determine clinical associations between IL-33 and IL-17 family cytokines and chronic periodontitis. In addition, to begin to investigate the biological significance of these associations using in vitro model systems. 97 patients with chronic periodontitis and 77 healthy volunteers were recruited in Glasgow and Newcastle. Serum, gingival crevicular fluid (GCF) and saliva were analysed for levels of IL-33 and IL-17 family cytokines by ELISA. Periodontal tissues from 17 chronic periodontitis patients and 10 healthy subjects from Glasgow were also investigated for IL-33 and IL-17 family cytokines mRNA expression by real time PCR. Immunohistochemical analysis was also performed on tissue to investigate expression of IL-33 and IL-17E at the protein level. In vitro experiments were performed using the OKF6/TERT-2 oral keratinocyte cell line and primary human gingival epithelial (PHGE) cells. The cells were stimulated with either a live Porphyromonas gingivalis monospecies biofilm or recombinant cytokines and changes in expression of cytokines, chemokines and their receptors evaluated by real-time PCR, immunocytochemical analysis or ELISA. In addition, transcriptional activity was monitored by analysis of changes in the phosphorylation (activation) of the NF-κB p65 subunit transcription factor using serum, GCF and saliva. IL-17A and IL-17A/F levels were higher in chronic periodontitis patients, but serum IL-17E was lower. IL-17A, IL-17A/F and the serum IL-17A:IL-17E ratio correlated positively with clinical parameters. IL-33, and IL-17 family cytokine (except IL-17B) gene transcripts were higher in tissue of chronic periodontitis patients. In addition, IL-33, ST2, IL-17E and IL-17RB proteins are expressed in periodontal tissues. Furthermore, IL-33 protein expression is upregulated in tissue of chronic periodontitis patients. In vitro models showed that IL-33 and its receptors (ST2 and ST2L) are expressed by oral keratinocytes (OKF6/TERT-2 cells and PHGE cells) and IL-33 expression up-regulated in response to P. gingivalis. However, IL-33 failed to induce expression of a range of inflammatory mediators and receptors in OKF6/TERT-2 cells. In vitro, IL-17E inhibited P. gingivalis monospecies biofilm and IL-17A induced expression of chemokines (IL-8 and/or CXCL5) by OKF6/TERT-2 cells at the transcriptional level by blocking the phosphorylation (activation) of the NF-κB p65 subunit. This study demonstrates clinical associations between IL-33 and IL-17 family cytokines and chronic periodontitis. The expression of IL-33 by oral keratinocytes and its up regulation upon exposure to P. gingivalis suggest it plays a role in the innate immune response to pathogens within the periodontium. However, the role of IL-33 in the periodontal inflammatory response remains to be elucidated. The negative correlations between serum levels of IL-17A and IL-17E and correlations with disease parameters, combined with their differing effects on the induction of expression of key neutrophil chemoattractants (CXCL5 and CXCL8), suggest opposing roles in periodontal immunity. Indeed, it can be hypothesised that the differential regulation of chemokine expression is due to IL-17A having pro- and IL-17E having anti-inflammatory properties. Indeed, as neutrophils play a key role in the early events associated with periodontal disease progression, the data suggests IL-17E is a rational target for therapeutic intervention.

Chemokines are the principle regulators of leukocyte migration in vivo and function during both normal (homeostatic) and inflammatory conditions to direct leukocytes to appropriate tissue locales. Chemokines mediate their affects by binding to their cognate G-protein coupled receptors (GPCRs) which are expressed on the surface of cells, and generate a signal upon ligand binding resulting in the initiation of a response such as chemotaxis. As well as the classical chemokine receptors which generate a conventional GPCR signal upon ligand binding, there exists a small family of atypical chemokine receptors that are characterised by an inability to mount classical receptor signalling. One of the most prominent members of this family is the atypical chemokine receptor, D6, which can bind at least 14 inflammatory CC chemokines with high affinity, but instead of the generation of a classical G-protein signalling response, D6 internalises ligands and targets them for lysosomal-mediated degradation. This functional attribute makes D6 a highly efficient binder, internaliser and scavenger of inflammatory CC chemokines that has been shown to be important for the resolution of inflammatory responses in vivo. Despite its well-studied biological role, very little is known about the structure/function relationships within and around D6 which regulate ligand binding and scavenging. Glycosaminoglycans have been demonstrated to be important for chemokine sequestration and presentation to many of the conventional chemokine receptors. Consequently, the role of glycosaminoglycans (GAGs) in chemokine presentation to D6 was studied using a cell line which is deficient in the synthesis of proteoglycans (CHO 745). Transfection of these cells with D6 and comparison to transfected WT CHO cells revealed that D6-mediated uptake and internalisation of chemokine is significantly reduced in the absence of GAGs. The N-terminus of D6 is thought to be the principle site for ligand binding, and the ability of D6 to bind all inflammatory CC chemokines makes this region an attractive target for therapeutic manipulation. Therefore a sulphated peptide representing the first 35 amino acids of D6 (D6-N (s)) was synthesised and investigated for its ability to bind D6 ligands. D6-N (s) was shown to neutralise the activity of the inflammatory CC chemokine CCL2 and prevent its interaction with its cognate receptor CCR2 in vitro. Importantly D6-N (s) was active, only in a specifically sulphated form, highlighting the importance of sulphated tyrosines for ligand binding. Considering the functional significance of the synthetic D6 peptide, attempts were made to identify a naturally ‘shed’ D6 N-terminal peptide which had been reported previously. Further study demonstrated the ability of the bacterial protease staphopain A, released from Staphylococcus aureus, to cleave the N-terminus of D6 and suppress its ligand internalisation activity. Finally, the conserved tyrosine motif present on the N-terminus of D6 was investigated more closely. Site-directed mutagenesis and sulphation inhibition of this region revealed the importance of post-translational tyrosine sulphation for ligand binding, internalisation and scavenging of inflammatory chemokines and alluded to the existence of an optimal sulphation pattern for ligand binding. Overall the results presented in this thesis shed new light on the nature of the molecules around, and the structural features within D6 that contribute to ligand binding and function of this extraordinary receptor. Furthermore, it was shown that a sulphated peptide derived from the N-terminus of D6 has the potential to be used therapeutically as a broad-based chemokine scavenger, which may be useful for dampening the effects of excessive chemokine production in chronic inflammatory conditions.

The aim of this study was to examine how caregivers manage their day-to-day living and health care needs, care for themselves and their sick children with HIV and AIDS in rural Malawi. The study used a longitudinal descriptive qualitative research design, using the “lens” of a narrative analysis theoretical framework to explore the experiences of caregivers looking after a child living with HIV and AIDS in rural Malawi. In-depth Interviews (IDIs) with women caregivers (n=20) recruited from Mponela Rural Hospital catchment area were conducted and all twenty women caregivers participated in in-depth interviews. Direct Observations (DOs) of the environment where interviews were conducted and at a local rural hospital were used to explore the primary care and support available for these women caregivers and their children. Data were analysed manually using the thematic analysis of the narrative accounts , combined with a detailed narrative analysis of one carers experiences to better understand how women constructed their stories in their own particular cultural context. A summary of the narrative analysis accounts of the rest of the 19 participants has been done to exemplify the main key issues each one of them had told in her story of caring for a child living with HIV and AIDS.

Kinetoplastid protozoans have unusual cell cycles. Three unitary organelles need to be replicated and segregated to the daughter cells at each cell division; the nucleus, kinetoplast and basal body, associated with the flagellum The replication and segregation of these organelles requires to be co-ordinated. Inthis study the timing of nuclear and kinetoplast cell cycle events was examined in two species of kinetoplastids, Trypanosoma brucei and Leishmania mexicana, which are major pathogens of humans, to ascertain the degree of co-ordination between the two organelles in the cell cycle. Particular attention was paid to three main features; the duration of S-phase for each organelle, the relative timing of mitosis and kinetoplast division and the lengths of the post-mitotic/division cytokinesis periods. Two life cycle stages were examined for each species i.e. procyclic and bloodstream forms of T. brucei and promastigote and amastigote forms of L. mexicana. The objectives of this study were to establish firstly whether a similar pattern of events occurs for both stages within each species examined and secondly, whether or not T. brucei and L. mexicana share common features to their cell cycles. Studies of cell cycle events were conducted using immunofluorescent labelling to detect S-phase in both organelles, enabled by use of an anti-bromo-deoxyuridine (BrdU) antibody as an S-phase marker. Division of organelles was identified by staining the cells with DAPI, a DNA intercalating dye. Initial studies confirmed that the cell cycles of both species were consistent with the general pattern of events of the eukaryotic nuclear cycle. Also, as has been described previously in kinetoplastids, the kinetoplast DNA (containing mitochondrial DNA) has a pattern of discretely separated phases of replication and division analagous to the nuclear cycle. Analysis was made of the relative timing of S-phase, mitosis/division and cytokinesis for each life cycle stage, producing data which were statistically testable. For both life cycle stages of T. brucei a similar pattern of events was observed. Three populations of BrdU labelled cells were identified; cells which were BrdU labelled in the nucleus only, labelled in the kinetoplast only and labelled for both organelles. These observations indicate that there was a non-co-ordinate start and finish to S-phase, with an overlap in the timing of the S-phase periods. Kinetoplast division was initiated and completed before the start of mitosis in the nucleus and an extended cytokinesis period for each organelle was identified, although of longer duration in the kinetoplast. For both life cycle stages of L. mexicana, three populations of cells labelled with BrdU were observed as in T. brucei, again indicating that there was a non-co-ordinate start and finish to S-phase, with an overlap in the timing of both periods. The sequence of division for the organelles for both life cycle stages differed to that observed in T brucei. The nucleus divided before the kinetoplast and there was a much shorter cytokinesis period for both organelles in comparison to T brucei. These differences in the pattern of events may reflect either a difference in the control of cell cycle timing in each species or in the morphology of each cell type. Trypanosomes have to re-arrange their organelles, such that they occupy sites on either side of the division furrow, hence the extended cytokinesis phase. In L. mexicana, however, this requirement for organelle re-positioning is reduced or absent and therefore the cytokinesis periods are much shorter. Antigenic variation is the key strategy which allows African trypanosomes to evade the effects of the host's immune response. The switching from expression of one variant surface glycoprotein (VSG) to that of another has been indirectly linked to the cell cycle, as bloodstream slender forms divide and undergo antigenic switching, whereas bloodstream stumpy forms are non-dividing and do not appear to switch. An attempt to examine directly the proposed link between antigenic switching and the cell cycle was made using cell cycle markers (anti-BrdU antibody as an S-phase marker and DAPI staining to determine the configurations of the organelles), as well as VAT specific antibodies against VSGs expressed in the cloned lines studied. Unfortunately, trypanosomes expressing two VSG coats simultaneously and therefore undergoing antigenic switch, could not be detected in any of the cloned lines examined. This unresolved difficulty was attributed to a deficiency in the detection system employed. Differentiation in kinetoplastids is thought to be linked to the cell cycle, as progression through the life cycle involves transitions from proliferative to nonproliferative forms. The differentiation of Leishmania major promastigote forms, from dividing non-infective stages to non-dividing infective meta cyclic forms, involves major molecular and morphological changes. Using three metacyclic-specific markers (non-agglutination by peanut agglutinin, a monoclonal antibody (3F12) against metacyclic-specific epitopes of the major surface molecule lipophosphoglycan (LPG) and a rabbit anti-serum (ab336) against a metacyclic-specific surface protein, the Gene B protein) metacyclic production in in vitro culture was examined. Observation of population growth CUIVes suggests that differentiation is in part intrinsically programmed within the parasite but may also be inducible by environmental changes. Comparison of these data with mathematical models indicated that the promastigote population was likely to be heterogeneous, containing sub-populations that replicate and differentiate at different rates. Two major assumptions of the heterogeneous model are that meta cyclic forms are both non-dividing and incapable of dedifferentiation. These two possibilities were tested experimentally and indicated that meta cyclic forms are indeed non-dividing, but are also capable of de-differentiation, albeit at a very low rate. Examination of meta cyclic production at the cellular level also indicated that the event which causes commitment to differentiation occurs at least one cell division before symmetrical production of two daughter meta cyclic forms. It has been found that in the presence of a chronic infection the growth of a secondary infection is significantly inhibited (Turner et al., 1996). The available data suggests that there appears to be an overall down-regulation in the growth of the entire mixed population. The aim of this component of the project was to select for 'growth' mutant trypanosomes i.e. mutants which overcome growth inhibition. Bloodstream trypanosomes which had been mutagenised in vitro with ethyl methane sulphonate (EMS) were inoculated into mice in the presence of a pre-existing chronic infection. Populations of mutagenised trypanosomes which grew significantly in comparison to controls where only low rates of growth occurred, were selected by optical cloning. This rationale led to the generation of growth mutant clones which stably expressed the altered growth phenotype. These studies may permit the development of methods for the investigation of the regulation of growth and virulence in these parasites. In conclusion, the analysis of the relative timing of cell cycle events in T. brucei and L. mexicana has highlighted both common and novel features to the cell cycles of these kinetoplastids. The study of meta cyclic production in L. major has also revealed the intrinsic and extrinsic nature of regulation of differentiation in these parasites, as well as the commitment to differentiate at the cellular level. Furthermore, the generation of 'growth' mutant trypanosomes provides an additional tool for the future study of growth regulation in trypanosome infections.

Plasmodium chabaudi has been shown to undergo antigenic variation during the course of infection in mice. The importance of this model is the similarity and applicability of its features to infection of humans in P. falciparum. This thesis presents work performed using P. chabaudi to study various aspects of antigenic variation in asexual erythrocytic malaria parasites. The course of infection of P. chabaudi in N1H mice shows an initial acute parasitaemia which clears to subpatency. This is usually followed, after a period of days, by a second, and occasionally a third, recrudescent parasitaemia of lesser magnitude and duration. A cloned parent parasite population and cloned parasite populations derived from a recrudescence of the parent were tested in an indirect fluorescent antibody test on live, schizont-infected RBC (live IFAT) using a panel of hyperimmune sera raised against these populations and against one of the recrudescent clones after mosquito transmission. This test can detect antigens on the surface of parasitised RBC. The results of this analysis indicated that all the recrudescent clones were antigenically different from the parent and some were different from each other. In total, including the parent, six variant antigen types (VATs) were identified. Some of these also appeared to vary in immunogenicity. The effects of mosquito transmission on expression of variant antibodies was also examined using the panel of hyperimmune sera in the live IFAT. Mosquito transmission of two antigenically distinct recrudescent clone populations resulted in a change in antigenicity of both types to an apparently similar VAT, which had the same apparent identity as that of the original, post mosquito transmission but pre-cloning, parent population.

CD8+ T-cells are essential for the immune control of pathogens and the natural eradication of cancer. CD8+ T-cells also play a major role in the pathogenesis of autoimmunity and alloreactivity. CD8+ T-cells recognize short peptide fragments (8-13 amino acids) presented at the target cell surface bound to Major Histocompatability Class I (MHCI) molecules. Tcell antigen recognition is unique in nature because it involves the binding of a single ligand (peptide–MHC [pMHC]) by two receptors (TCR and CD8). The CD8 glycoprotein, which serves as the coreceptor on MHCI-restricted T-cells, acts to enhance the antigen sensitivity of T-cells by binding to a largely invariant region of MHCI at a site distinct from the TCR docking platform. CD8 has been shown to have multiple roles including enhancing effects on early T-cell activation events and also in controlling the level of T-cell cross-reactivity. The pMHCI/CD8 interaction is classified as having a very weak binding affinity and very fast kinetics. I discovered that this low solution binding affinity is essential in maintaining homeostasis as dramatically increasing the strength of this interaction resulted in total loss of T-cell specificity and activation independent of TCR engagement. This led me to examine the possibility that anti-CD8 antibodies could also bypass the normal requirements for T-cell activation. I identified one specific clonotype of antibody capable of this phenomenon but simultaneously discovered multiple effector phenotypes of other anti-CD8 antibodies. These included both enhancing and inhibitory effects on pMHCI tetramer binding and CD8+ T-cell activation. Subsequently, I explored the possibility of using these inhibitory anti-CD8 antibodies to block T-cell function in systems which are highly dependent on CD8 such as autoreactive CD8+ T-cells. I demonstrated that targeting CD8 can be used as a strategy to block autoreactive CD8+ T-cell activation in the absence of any effect on pathogen specific immunity. This highlights a novel therapeutic strategy that warrants further investigation. Finally, I demonstrated that CD8 can alter the functional avidity of a CD8+ T-cell for its agonists and act to re-arrange the relative potencies of each of its potential agonists, a novel “focussing mechanism” for CD8 in T cell activation. These results provide new insight to the biological role of CD8 in T-cells and even predict a novel mechanism for CD8 in controlling T-cell function. My results also highlight the potential of targeting CD8 for immunotherapeutic design in autoimmune disorders.

A successful kidney transplant is the best treatment for established renal failure, yet around 300 patients per annum are denied transplants because they have antibodies, most notably directed against donor HLA or ABO in their blood, which have the potential to cause acute and chronic rejection of the transplant. Such antibodies are present in 25% (roughly 1750 of the 7000 on the kidney transplant waiting list) of the patients listed for a deceased donor transplant. Programmes to remove antibody and transplant patients across HLA antibody barriers have been developed, but are limited by a high rate of acute rejection. This thesis explores the factors which may impact upon the pathogenicity of HLA-specific antibodies and also aims to enhance the understanding of the techniques used in the laboratory to define these antibodies. A range of studies were carried out examining factors such as the IgG subclass composition of the anti-HLA response. Assay variations were designed to enable a higher definition of antibody specificity to be achieved, and for the first time in the literature the direct quantification of HLA-specific antibodies in patient sera was performed. In addition, proof of principle design and testing was carried out on a novel prototype therapeutic device for the selective depletion of HLA-specific antibodies directly from patient plasma and sera. The antibodies isolated from this approach were also used in studies to examine the factors which determine serum cytotoxicity.

Using an IBV reverse genetics system a series of recombinant viruses were generated to investigate the potential for utilizing IBV as a vaccine vector. Through the replacement of non-essential regions of the IBV genome, with eGFP or hRluc, factors influencing the stability of recombinant viruses expressing heterologous genes were determined. Expression of heterologous proteins was possible from a variety of virus constructs. The stability of recombinant viruses varied depending on the genome location of the heterologous gene, with replacement of Gene 5 proving to be most stable following passage in cell culture. Stability was strongly influenced by the MOI at which viruses were passaged, with low MOIs resulting in increased stability. The replacement of Gene 5 with a heterologous virus gene may be a suitable target for development of a bivalent vaccine capable of protecting against IBV and a second avian viral disease. Analysis of recombinant IBV mRNA expression profiles led to an investigation into an uncharacterized RNA species, and its link to the IBV intergenic region. A novel subgenomic mRNA was identified associated with the intergenic region that was shown to be transcribed via a non-canonical transcription regulatory sequence. In contradiction to the current model of coronavirus transcription this mRNA has a transcription regulatory sequence derived mainly from the leader, and not the body, transcription regulatory sequence. The non-canonical sequence was shown to be responsible for reduced transcription levels of the intergenic region mRNA. This project proposes the presence of an additional IBV subgenomic mRNA, transcribed via a non-canonical mechanism, and encoding a novel 5th accessory protein of IBV and closely related gammacoronaviruses.

The effects of Mirazid® (MZD), an extract of myrrh, on Schistosoma mansoni were investigated and compared to those of praziquantel (PZQ). The effects of both drugs on the soluble proteome and the immune responses to schistosome infections were also investigated. In vitro studies showed that MZD was more effective than PZQ against the schistosomula stage. However, its effects against adult worms were less than those of PZQ. Lengthy exposures to high concentrations of MZD were required for the drug to be effective. In vivo studies showed that MZD had no anti-schistosomal activity against S. mansoni in mice. In vitro exposure of adult worms to either PZQ or MZD caused significant changes in the expression of some proteins. Interestingly, some vaccine candidates including paramyosin and actin were among the proteins that showed differential expression. Treatment of human S. haematobium infection with PZQ favoured the development of protective immune responses that render the individuals resistant to reinfection. Sera from putatively resistant individuals showed distinct recognition patterns of soluble worm antigen, after exposure to reinfection, compared to sera from susceptible individuals. Interestingly, many putative vaccine candidate antigens were recognised by antibody isotypes that are found in individuals with some degree of resistance to infection. In conclusion, although the in vitro studies revealed some promising effects of MZD, the results of the in vivo studies do not support the use of this drug in the treatment of schistosome infections.