This thesis presents quantitative studies of T cell and dendritic cell (DC) behaviour in mouse lymph nodes (LNs) in the naive state and following immunisation. These processes are of importance and interest in basic immunology, and better understanding could improve both diagnostic capacity and therapeutic manipulations, potentially helping in producing more effective vaccines or developing treatments for autoimmune diseases. The problem is also interesting conceptually as it is relevant to other fields where 3D movement of objects is tracked with a discrete scanning interval. A general immunology introduction is presented in chapter 1. In chapter 2, I apply quantitative methods to multi-photon imaging data to measure how T cells and DCs are spatially arranged in LNs. This has been previously studied to describe differences between the naive and immunised state and as an indicator of the magnitude of the immune response in LNs, but previous analyses have been generally descriptive. The quantitative analysis shows that some of the previous conclusions may have been premature. In chapter 3, I use Bayesian state-space models to test some hypotheses about the mode of T cell search for DCs. A two-state mode of movement where T cells can be classified as either interacting to a DC or freely migrating is supported over a model where T cells would home in on DCs at distance through for example the action of chemokines. In chapter 4, I study whether T cell migration is linked to the geometric structure of the fibroblast reticular network (FRC). I find support for the hypothesis that the movement is constrained to the fibroblast reticular cell (FRC) network over an alternative 'random walk with persistence time' model where cells would move randomly, with a short-term persistence driven by a hypothetical T cell intrinsic 'clock'. I also present unexpected results on the FRC network geometry. Finally, a quantitative method is presented for addressing some measurement biases inherent to multi-photon imaging. In all three chapters, novel findings are made, and the methods developed have the potential for further use to address important problems in the field. In chapter 5, I present a summary and synthesis of results from chapters 3-4 and a more speculative discussion of these results and potential future directions.

Trypanosoma brucei is the causative agent of both sleeping sickness in humans and the related veterinary disease, Nagana. Both diseases have a wide distribution across sub-Saharan Africa and affect some of the poorest areas of the world. T. brucei can be segregated into three morphologically identical sub-species based on host, geography and pathology. T. b. brucei is limited to domestic and wild animals throughout sub-Saharan Africa and is non-infective to humans due to trypanosome lytic factors found in human serum. T. b. gambiense and T. b. rhodesiense are human infective sub-species, named due to their relative geographic locations. T. b. gambiense is the dominant form of the disease, causing over 90% of reported cases. Study of T. b. gambiense is complicated in that there are two distinct groups. Group 1 is invariably resistant to lysis and by far the more prevalent group. Group 2 T. b. gambiense exhibit a variable resistance phenotype and are only found at a small number of Côte d’Ivoire disease foci. There are two trypanosome lytic factors in human serum (TLF-1 & 2), both containing the proteins Apolipoprotein L1 (ApoL1) and Haptogoblin-related protein (Hpr). It has been conclusively demonstrated that the lytic component of TLF is ApoL1, although Hpr is required for maximal lysis by facilitating uptake of TLF particles via the HpHbR cell surface receptor. This thesis has exposed several features of the human infectivity phenotype in both groups of T. b. gambiense, an area of research for which data has been lacking due to the difficulty of working with the organism. Fluorescence microscopy indicated that group 1 T. b. gambiense exhibit avoidance of TLF-1 particles by down-regulating HpHbR receptor expression and function. However, they are also able to resist the effects of recombinant ApoL1, suggesting an additional neutralisation or compensatory mechanism. Due to group 1 T. b. gambiense avoidance of TLF-1, TLF-2 is the more important lytic particle for this sub-species group and future research must take this into consideration. Unlike group 1, group 2 T. b. gambiense displays a variable human serum resistance phenotype that involves a neutralisation or compensatory mechanism for ApoL1, with no significant avoidance of lytic particles. Despite the high variability of the phenotype of group 2 T. b. gambiense, Quantitative Trait Analysis (QTL) using twenty-five F1 progeny from a T. b. brucei / group 2 T. b. gambiense cross indicated a strong heritable component to human serum resistance largely determined by a 30 gene locus on chromosome 8. Finally, a six multi-locus genotype population analysis of a Côte d’Ivoire T. b. gambiense focus was conducted, revealing little relationship between the two groups of T. b. gambiense in the field. The differences in the human serum resistance phenotypes and population genetics of both groups of T. b. gambiense revealed both prior and during this study make it appear likely that the two groups have evolved distinct human serum resistance strategies.

Leishmania resides inside mammalian macrophages, from where it is thought to manipulate the host immune system by releasing virulence factors. The cysteine peptidase CPB has been shown to be secreted by the parasite and act as such a virulence factor. CPB is released through the flagellar pocket while being trafficked to the lysosome. Thus, in this project, the intracellular localisations of eight other L. major peptidases were analysed by fluorescence microscopy, after tagging the enzymes with green fluorescent protein (GFP). The candidate peptidases were chosen by bioinformatics analyses and predictions of N-terminal secretory signal peptides and potential transmembrane domains. The aim was to find a peptidase accumulating in the flagellar pocket of the cell, from where it could be secreted. Five candidate peptidases (a ubiquitin hydrolase, a CaaX prenyl protease, a zinc carboxypeptidase and two rhomboid peptidases) localised to the mitochondrion, which was unexpected. Another, a calpain-like peptidase, localised to the flagellum but not to the flagellar pocket. A serine carboxypeptidase was found very close to the flagellar pocket, possibly in small vesicles budding off or fusing with the pocket membrane, but did not co-localise with a flagellar pocket marker. The bioinformatics predictions differed from the experimental results here and, additionally, using different algorithms to predict protein properties resulted in contradictory predictions in several cases. This suggests that generic protein prediction programmes for mammalian or higher eukaryotic proteins can be unreliable and of limited usefulness for Leishmania proteins. This corroborates the notion that Leishmania may use novel, non-classical secretory pathways rather than or in addition to those characterised for higher eukaryotes. The L. major Bem46-like serine peptidase (LmjF35.4020) of the Clan SC (Family S9) was the only candidate peptidase that localised to the flagellar pocket when labelled with GFP. This was an indication that this enzyme may be released from the cell and could act as a virulence factor. Alternatively, it may be a resident protein of the flagellar pocket. Deleting the Bem46 gene in L. major did not have a measurable effect on promastigote growth or on footpad lesion development in mice inoculated with Bem46-deficient cells, so it does not appear to play a role as a major virulence factor. Apart from the secretion of virulence factors, rapid protein turnover, e.g. in the lysosome, is important for the infectivity of Leishmania. To investigate lysosome structure and function in L. major, a potential LMP (lysosomal membrane protein) was identified by bioinformatics. Thus far, no resident membrane proteins of the Leishmania lysosome are known and identifying such a protein would provide a useful marker for the closer investigation of this important organelle. In this project, the location and role of the LMP protein LmjF30.2670 was investigated using GFP-tagging and fluorescence microscopy. The experiments showed that LMP is not lysosomal in L. major, rather, it could be observed localising to a distinct, elongated and sometimes doughnut-shaped structure in close proximity to the kinetoplast. This structure was not directly associated with the flagellar pocket or the cell membrane, its position in the cell was variable within a certain area alongside the kinetoplast, it appeared to duplicate during cell division and it did not co-localise with the endocytic / lysosomal marker FM4-64. Deletion of the LMP gene did not have any effect on promastigote growth in cell culture and only a small and transient slowing effect on the development of mouse footpad lesions after inoculation with LMP-deficient L. major. Lysosomal membrane proteins can be targeted to the lysosome by the protein carrier complex AP3, which binds to tyrosine or dileucine motifs in cargo proteins. LMP contains two such tyrosine motifs at its C-terminus, but disruption of these by site-directed mutagenesis did not affect LMP localisation, suggesting that its trafficking is AP3-independent, which is in accordance with the non-lysosomal localisation of LMP. Finally, the lysosome-like acidocalcisome organelles have previously been shown to rely on the protein carrier complex AP3 for normal structure and function. In AP3-deficient Leishmania, the acidocalcisomes are defective and, at the same time, parasite virulence is markedly reduced (Besteiro et al., 2008o). To analyse how AP3 is important for acidocalcisome morphology and function, a proton pump of the acidocalcisomal membrane, the V-H+-PPase, was investigated by GFP-labelling and fluorescence microscopy. In wild type L. major the V-H+-PPase could be shown to localise to the acidocalcisomes, whereas in AP3-deficient cells it was not detectable, suggesting that the protein is mislocalised and likely degraded. The V-H+-PPase also contains several tyrosine motifs that may interact with AP3. The two most prominent of these were disrupted by site-directed mutagenesis, but this did not affect the localisation of the V-H+-PPase, suggesting that these two sites are not, or not solely, important for AP3 binding or that the V-H+-PPase is not bound by AP3 directly.

Autoimmune neuropathies are a group of conditions resulting from inflammatory attack of the peripheral nervous system (PNS). Guillain-Barré syndrome (GBS), an acute autoimmune neuropathy, presents in both axonal and demyelinating forms. Axonal forms of GBS are caused by autoantibodies which target gangliosides on peripheral nerves. Here, they cause axonal damage via activation of the complement pathway. In ex vivo preparations, anti-ganglioside antibodies have been shown to cause complement-mediated injury to the node of Ranvier and the motor nerve terminal. Of these two vulnerable sites, the motor nerve terminal has recently been shown to be able to internalise anti-ganglioside antibody while the node of Ranvier does not. Rabbit models have demonstrated that immunisation with ganglioside results in a motor axonal neuropathy but no such model currently exists in mice. This is partially due to the fact that wildtype mice respond poorly when immunised with ganglioside and partially due to the requirement of an exogenous complement source to cause injury. This laboratory has recently developed GalNAcT-/--Tg(neuronal) and GalNAcT-/--Tg(glial) mice with complex ganglioside expression restricted to neurons and glia, respectively. This thesis aimed to develop a mouse model of GBS by actively immunising these mice with ganglioside liposomes. Subsequently, the aims were expanded to look at the clearance of the antibodies by membranes which express their ganglioside target. To complete these aims, wildtype, GalNAcT-/-, GalNAcT-/--Tg(neuronal) and GalNAcT-/--Tg(glial) mice were compared. Following immunisation with liposomes containing GD1b, wildtype mice responded poorly as demonstrated by low presence of immunoglobulin in their sera. Conversely, GalNAcT-/- mice, which lack complex gangliosides, showed high presence of immunoglobulin in their sera. GalNAcT-/--Tg(neuronal) and GalNAcT-/--Tg(glial) mice showed intermediate levels. This was assumed to be due to varying levels of tolerance to “self” lipids among the mice. However, when normal human serum was introduced, GalNAcT-/--Tg(neuronal) and GalNAcT-/--Tg(glial) mice did not show any complement-mediated injury. Based upon evidence from ELISpots from the splenocytes of these mice, there did not appear to be any major differences in anti-GD1b antibody-producing cells among genotypes. The possibility that the antibodies produced by these mice were being removed by internalisation was investigated at the NMJ ex vivo and in vivo. Ex vivo, antibodies against complex gangliosides were demonstrated to be cleared in a receptor-dependent and activity-dependent manner. Following passive immunisation with pathogenic monoclonal antibody in vivo, serum levels of the antibody were cleared rapidly in wildtype mice but remain elevated at 7 days in GalNAcT-/- mice. GalNAcT-/--Tg(neuronal) mice cleared antibody at an intermediate rate. Non-pathogenic antibodies were not cleared from the circulation over the 7 days from any of the three genotypes. These results have demonstrated that levels of anti-ganglioside antibody can be regulated by receptor-dependent internalisation, especially at the motor nerve terminal. These studies have highlighted this structure as a novel regulator of anti-ganglioside antibody in vivo. The clearance of antibody is also dependent on the ability of the antibody to bind to living tissue; therefore antibodies detected in patient serum may not represent pathogenic, disease-causing antibodies. These factors may profoundly influence host vulnerability to antibody-mediated disease by affecting circulating levels of pathological antibodies.

Patients living with many cancers, including ovarian cancer (OC), often suffer from a lack of adequate treatment options. In the case of OC, primary debulking surgery followed by platinum and paclitaxel chemotherapy has led to a vast improvement in patient survival over the past few decades, however, rates of drug-resistant recurrence remain high. Research into new, experimental treatment options is therefore warranted for OC and other cancers. Oncolytic viruses (OVs) are replication-competent viruses that can selectively infect and destroy cancerous cell types, while leaving healthy cells unharmed. OVs do this by exploiting differences between cancer and normal cell phenotypes. Herpes simplex virus (HSV)-1, strain 1716 is one example of this type of virus that has shown selectivity for cancer cells in previous preclinical studies, as well as high levels of safety in humans. One prominent area of current OV study seeks to investigate the ability of OVs to induce immunogenic cell death (ICD) – this term describes multiple modes of programmed death pathways that culminate in release of proimmunogenic factors, which facilitate a modification of the host immune system. Two of the most prominent of these pathways are necroptosis and immunogenic apoptosis (IA). Here, I show that while many OV cell lines express the necessary components for necroptosis, they are unable to undergo classical necroptotic death (induced by TSZ). Despite this, HSV-1716 can infect and kill a range of OC lines successfully. I showed that HSV-1716-induced cell death displays two markers of IA yet does not seem to rely solely on apoptosis to kill cells. In addition, it appears not to rely on any components of the necrosome in order to kill cells, even in cells that are competent to typical necroptosis. However, when RIPK3 is overexpressed in HeLa cells, virus-induced cell death increases, as do markers of both necroptosis and IA. To investigate the role of ICP6 in HSV-1716-induced ICD, viral and cell mutants were made possessing various forms of the protein. Full-length ICP6 protein expressed in cell lines had the effect of blocking cellular response to TSZ, but constructs lacking a region known as the RHIM did not. A functionally similar mutation was produced within the RHIM of live HSV-1716 using CRISPR/Cas9 technology, which was shown to have the effect of disrupting ICP6/RIPK3 binding – thought to be the determinant of necroptotic cell death. Despite this, no changes in cell death signalling could be determined between the viruses at all. Interestingly, when cells were infected in combination with TNF-α, or TNF-α in addition to SMAC mimetic, the RHIM-modified virus produced significantly more death than HSV-1716. This suggests that while loss of RIPK3 inhibition is not sufficient to lead to increased necrosis alone, cells infected with this virus are more sensitive to further necrosis induction. This finding may prove to have great utility for producing the next generation of oncolytic viral therapeutics which can induce greater levels of proimmunogenic cell death. From this we can conclude that HSV-1716 is capable of inducing IA in OC cells. Death is not dependent on necroptosis, however additional RIPK3 seems to sensitise cells to death by other means. Cellular binding of viral ICP6 and RIPK3 can be disrupted by modification of the RHIM, although this change has no bearing on ICD signalling alone but can sensitise cells to TNF-α-induced death.

The Drosophila Toll receptor is crucial for dorsoventral patterning in embryos, and for innate immunity. Toll also functions during central nervous system development, promoting neuronal survival and targeting. There are nine Toll paralogues in Drosophila, and it is unknown whether any of these also function in the CNS. Toll’s ligand, Spz, has an NGF domain. NGF is a vertebrate neurotrophin - a growth factor that regulates the development and function of the nervous system. Drosophila Neurotrophin 1 (DNT1), identified by homology to the vertebrate neurotrophin BDNF, and DNT2 are paralogues of spz. The three DNTs – DNT1, DNT2 and spz – are structural and functional homologues of vertebrate neurotrophins, and they promote neuronal survival, targeting and synaptogenesis in Drosophila. However, the receptors for DNT1 and DNT2 are unknown. Here, using a combination of in situ hybridisations and reporters that drive GFP expression, I investigate the expression of Toll paralogues in the Drosophila nervous system. By generating null mutant flies and gain-of-function transgenic flies, I examine genetic interactions between Tolls and DNTs. I also investigate the rolls of these receptors in adult locomotion, axon targeting and cell survival. Finally, in cell culture, I test whether DNTs can signal through Tolls to activate NFκB.

Chronic systemic immunosuppression in pancreatic islet transplantation restricts its clinical application. This study aims to explore the potential of cell-mediated immune-modulation as an alternative to conventional immunosuppressive regimens; specifically investigating the innate immunosuppressive properties of human amniotic epithelial cells (AEC). Cell constructs composed of human islets and AEC (islet:AEC) were bio-engineered in rotational culture. Insulin secretory capacity and immuno-modulatory potential were characterised using appropriate in vitro assays. Fluorescence immunocytochemistry and multiplex arrays was used to identify putative mediators of the immunosuppressive response in isolated AEC monocultures. Islets and islet:AEC constructs demonstrated sustained, physiologically-appropriate insulin secretion. Resting peripheral blood mononuclear cells (PBMC) were activated on exposure to human islets but this response was significantly (p<0.05) attenuated in islet:AEC constructs. Phytohaemagglutinin (5\( \mu \)g/ml)-induced PBMC proliferation was sustained on contact with unmodified islets but abrogated in AEC and islet:AEC constructs. CD4+ and CD8+ T-cell proliferation was responsive to AEC; their in vitro expansion both in response to CD3/CD28 activation and contact with human islets being suppressed by the presence of AEC. Transplanted islets may thus benefit from an immune-privilege status conferred on them as a consequence of their close proximity to human AEC. Such an approach may diminish the requirement for generalised systemic immunosuppression in islet transplantation.

Rheumatoid arthritis is an autoimmune disease that affects approximately 1% of the population, where chronic inflammation of the synovial joints can lead to active destruction of cartilage and bone. New therapeutic targets are discovered by investigating genes or processes that exacerbate or ameliorate disease progression. Mouse models of inflammatory arthritis are commonly employed for this purpose, in conjunction with biomedical imaging techniques and suitable measures of disease severity. This thesis investigated the hypothesis that a statistical model of non-pathological bone shape variation could be used to quantify bone destruction present in micro-CT images. A framework for constructing statistical shape models of the hind paw was developed, based on articulated registration of a manually segmented reference image. Successful registration of the reference towards ten healthy hind paw samples was followed by statistical shape analysis. Mouse models of inflammatory arthritis were then investigated and compared by identifying bone abnormalities as deviations from the model statistics. Validation of the model against digital phantoms and clinical scores indicates that the method is largely successful in this effort. Application of the method in a novel study of macrophage-mediated inflammation shows promising results that are supportive of previous findings.

Enterohaemorrhagic E. coli (EHEC) are important gastrointestinal pathogens of humans. E. coli serotype O157:H7 is the EHEC most commonly associated with human illness. E. coli O157:H7 is carried asymptomatically by cattle which form an important reservoir for the bacterium. E. coli O157:H7 has been found to colonise at the terminal rectum of cattle in preference to other sites in the bovine gastrointestinal tract. The first objective of this work was to characterise the roles of bacterial secreted components responsible for key functions in the modulation of host defences against EHEC. Data presented here reaffirms the role of flagellin in the elicitation of a proinflammatory response in a cultured human epithelial cell line; however, the response of a bovine epithelial cell line to bacterial secreted products was not affected by the presence or absence of flagellin. A role in the modulation of the host response for the StcE protease was also investigated: although its role in interaction with the bovine host was not established, bovine secretory antibodies to StcE were detected in rectal mucosal scrapings from an E. coli O157:H7-challenged calf, suggesting that StcE is expressed and recognised in vivo. The second key objective was to isolate E. coli from the bovine intestinal tract in order to define the colonisation patterns of E. coli within the bovine intestinal tract and relate this to bacterial genotype and to provide bovine E. coli isolates to test for inhibitory activity against E. coli O157:H7 which may yield bacteria with potential as probiotic agents with a view to reducing the prevalence of EHEC in cattle. Genotypic analysis of bovine resident E. coli confirmed that these strains carry a variety of virulence factor-encoding genes; however, certain dominant genotypes were identified and the genomic structure of representative isolates was predicted by genomic microarray. EHEC-related genotypes were found to be positively associated with colonisation at the rectum, whereas non-EHEC genotypes were found to colonise multiple intestinal sites without showing any apparent site-specificity. The third and final objective of this analysis was to carry out genotypic analysis of Scottish EHEC strains in order to predict whether increased incidence of EHEC infection in Scotland may be related to the presence of EHEC strains carrying altered complement of virulence factor-encoding genes. The analysis of EHEC isolated in Scotland revealed that these strains exhibit a genomic profile which is largely typical of EHEC isolated elsewhere, although there were certain differences in the carriage of a certain genomic elements. The results presented here support the proposal that bacteriophages are the key mediators of genetic variability among E. coli isolates.