This thesis has used bioinformatics and RNAi methods to investigate diverse aspects of liver fluke biology. In particular, new data on the diversity and expression of liver fluke ABC transporters has revealed previously unrecognised diversity/complexity of this protein superfamily. Some family members were found to be responsive to flukicide pressure suggesting that they may play a role in the normal worm response to drug treatment. Further, RNAi interrogation of their role in resistant fluke isolates supported this link by showing that the silencing of selective ABC transporters increased drug sensitivity - consistent with reduced drug efflux associated with silencing. Further work used RNAi to probe the potential of liver fluke calmodulins as new control targets. These efforts identified target-specific RNAi phenotypes that matched the phenotypes induced by exposure to calmodulin inhibitors, validating the specificity of both the RNAi and the inhibitory drugs. These data support calmodulin candidature as drug targets and further validate the use of RNAi as a tool for functional genomics in fluke. Finally, the work reported here reveals that the serum-induced growth of juvenile fluke speeds up silencing processes and may help researchers refine their approaches to post-RNAi phenotype analyses in the face of the unusually slow/long RNAi-dynamics displayed by liver fluke.

Using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and histology, the effects of artemisinin compounds on the tegumental surface and ultrastructure of the liver fluke, Fasciola hepatica, were assessed. The histology and SEM findings revealed that treatment of flukes with the artemisinin-type compounds, artemether, artesunate and OZ78, resulted in relatively little disruption to the external tegumental surface when compared with other fasciolicides. In contrast, changes to the ultrastructure, as examined by TEM, were rapid, severe and progressive, particularly in the gut and reproductive tissues. That changes in the gut occurred rapidly following administration, and were more extensive than those observed in the tegument, suggests that an oral route of uptake may predominate for these compounds. Experiments were undertaken using three different fluke isolates, two of which have been previously confirmed as being resistant to the current drug of choice, triclabendazole (TCBZ). The nature and extent of the changes in fluke tissues appeared to be similar across the isolates, confirming that artemisinin-type compounds have equal activity against both TCBZ-susceptible and TCBZ-resistant fluke. With each of the three compounds, the changes observed in fluke tissues were distinct from those induced by other known fasciolicides, being typified by widespread disruption to the mitochondria and the cisternae of the ger, particularly in the gut. As time progressed post-treatment, inhibition of spermatogenesis and oogenesis became evident, with widespread apoptosis observed in both tissues. The observed changes, particularly to the cisternae of the ger, link well to the suggested mechanism of action for these compounds, via inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), but suggest that a less-specific dual mechanism of action may be more likely. Finally, the current study established a time-scale for the action of artemisinin-type compounds against fluke, and substantiated previous concerns over the potential toxicity of artesunate when administered at high doses.

The malaria parasite is a plague of humanity. Understanding its biology and blocking essential parasite biochemical pathways are ways of controlling and possibly defeating malaria. During its complex life cycle, the parasite undergoes cycles of asexual replication in erythrocytes, then egresses from them to invade fresh erythrocytes. An essential cascade involved in egress, which includes at least two enzymes, has recently been discovered in P. Jalciparum . The parasite cGMP-dependent kinase PKG triggers the discharge of a serine protease called SUBl from secretory organelles called exonemes and its subsequent activation. SUBl cleaves several parasite substrates, enabling egress and preparing the merozoites for invasion. Blocking either enzyme inhibits egress, making them potential antimalarial targets. Whilst the most dangerous cause of human malaria is Plasmodium Jalciparum, the zoonotic species P. knowlesi can also cause severe disease and death. P. knowlesi has recently been adapted to in vitro culture in human erythrocytes, providing an excellent tool to study this human pathogen in depth. Shedding light on the specificity and regulation of P. knowlesi PKG and SUBl will provide information about an important biochemical pathwaywhich may be conserved across human Pla'smod~um species. In this study, P. knowlesi PKG was characterized using selective inhibitors of the enzyme that block egress of P. knowlesi parasites in vitro.PkSUB 1 was expressed in a recombinant enzymatically active form and new selective inhibitors of PfSUBl and PkSUBl were designed using a combination of in silico,in vitro and substrate based approaches. Some of these compounds were shown to inhibit both egress and SUB1-mediated processing of endogenous parasite substrates. The results show that the essential role of PKG and SUBl in egress is conserved in P .

The control of meticillin-resistant Staphylococcus aureus (MRSA) is a global healthcare priority. Screening patients for MRSA carriage occupies a central position in this control. Conventional culture methods for MRSA screening take 2-3 days to produce a positive result. Polymerase chain reaction (PCR) based systems can detect MRSA within one day ('rapid screening') though these tests are more costly than culture tests. It is hypothesised that rapid detection of MRSA carriers will lead to faster implementation of control procedures, reducing the transmission of MRSA. The first study in this thesis validates a rapid method, IDI-MRSA ™ for use on pooled and non-nasal specimens. The second paper is a controlled trial of the IDI-MRSA™ test. It investigated whether rapid MRSA screening leads to a reduction in MRSA acquisition and was set on ten wards of a London teaching hospital. The main outcome was the MRSA acquisition rate (proportion of patients negative for MRSA who became MRSA positive). Secondary outcomes included test characteristics and measures of resource use. The intervention was PCR screening for MRSA compared with conventional culture. 6888 (82.3%) patients had full data. The overall MRSA carriage rate on admission was 6.7%. Rapid tests led to a reduction in median reporting time from admission (46 to 22 hours, P<0.001) and reduced the number of inappropriate pre-emptive isolation days between the two arms (399 v 277, P<0.001). 108 (3,2%) patients in the control arm and 99 (2.8%) in the intervention arm acquired MRSA. When confounding factors were taken into account the adjusted odds ration was 0.91 (95% confidence interval 0.61 to 1.234). Rates of MRSA transmission, wound infection, and bacteraemia were not statistically different between the two arms. On these data it is unlikely that the increased costs of rapid tests can be justified compared with alternative control measures against MRSA.

Assessment of the taxonomy of the haemoplasmas is described using two protein encoding genes, gapA and dnaK, from a number of Mycoplasma and haemoplasma species and phylogenetically analyzing the concatenated data set. The resulting trees indicated that the haemoplasmas reside within their own clade, separated from the rest of the Mycoplasma species, adding more support for the suggestion that the haemoplasmas should occupy their own genus within the family Mycoplasmatacae . Immune responses and M. haemofelis copy numbers were analyzed following primary M. haemofelis infection and following re-challenge of M. haemofelis recovered cats. The results demonstrated that M. haemofelis recovered cats were protected from re-challenge. Analysis of the cytokine profile following primary infection showed increased levels of IL-12p35 and IL-12p40 mRNA suggestive of a Thl response. Although the mechanisms of protection could not be definitively determined, it was observed that proinflammatory cytokines may play a role. In vitro cultivation of M. haemofelis was attempted in both mycoplasma media and cell culture, by mimicking as closely as possible the blood environment and supplementation with possible haemoplasma metabolites, as identified by genome sequencing and metabolic pathway analysis. Suspected growth of M. haemofelis, as determined by qPCR, was associated with hypoxanthine supplementation, though the result could not be reproduced. A number of culture attempts, including the use of insect and mammalian cell lines, resulted in an initial decline in copy number followed by an apparent stationary phase of growth . Other species (mice, rabbit and guinea pig) were assessed for their ability to act as an alternative laboratory host for M. haemofelis, as well as to determine M. haemofelis host specificity. None of the animals became M. haemofelis infected, supporting previous suggestions of host specificity in immunocompetent animals.

Epstein-Barr Virus (EBV) is a human gamma herpesvirus that infects about 95% of world population. The life cycle of EBV can be divided into two stages: latent and lytic. During latency, the virus DNA is maintained as a nuclear episome with minimal protein expression to avoid immune recognition. Although primary EBV infection in childhood is usually asymptomatic, infection with EBV is associated with a wide range of proliferative diseases such as post-transplant lymphoproliferative disease (PTLD), Burkitt’s lymphoma (BL) and primary effusion lymphoma (PEL). Despite EBV infection being common, less is understood about the host control of the lytic virus life cycle or the sequence diversity of the viral genome. This thesis investigates the reactivation of lytic EBV replication in both PEL and BL cell lines and whole genome EBV sequence diversity. X-box binding protein 1 (XBP-1), a transcriptional activator that is essential for plasma cell differentiation, reactivates Kaposi sarcoma herpesvirus (KSHV), a closely related gamma herpesvirus from latency. With KSHV and EBV co-infection in PEL, the possibility of XBP-1 also reactivating EBV is investigated here. We show XBP-1 does not induce EBV into the lytic cycle in tumour B-cell backgrounds, either with or without protein kinase D. This contrasts previous observations. The lytic replication of EBV generates sequence diversity. To further understand EBV genomes and their association with the malignancies, a pipeline for sequencing EBV whole genomes using Next Generation Sequencing (NGS) has been developed. EBV whole genomes from mature Bcell tumour lines, such as PEL and BL have been sequenced assembled along with EBV from blood samples of PTLD patients. Examining the EBV whole genome sequences from PTLD blood samples through time suggests dynamic EBV sequence evolution can be observed in vivo. We conclude that EBV genomes contain higher variation than previously expected, and further study is needed to understand the relationship between EBV and the diseases.

The human cytomegalovirus (CMV) can cause significant morbidity and mortality in haematopoietic stem cell transplant (HSCT) patients. The immunosuppressed state of the hosts facilitates dissemination of the virus and disease. In contrast, CD8+ T cells in healthy individuals control viral dissemination and maintain a balance between antiviral host defence and replication of the virus that leads to viral latency with sporadic, harmless reactivations. Current pharmacological intervention in the HSCT setting is associated with significant toxicity and is counteracted by the occurrence of resistant viral strains. Alternative approaches, such as adoptive therapy of CMV specific CD8+ T cells are of great interest in the field. So far, levels of such cells correlating with protection against CMV disease in patients were only shown for cells targeting two different viral epitopes. The project described in this thesis investigates CD8+ T cell responses to several common CMV targets presented by different human leukocyte antigens in the HSCT setting. Results demonstrate significant differences between the numbers of different CMV specific CD8+ T cells that, in the presence of CD4+ T cell help, inversely correlate with the ability to detect CMV reactivation. Findings also demonstrate significant differences in the diversity of T cell receptors (TCRs) used by the different CMV specific CD8+ T cells isolated from HSCT patients. These findings are clinically relevant in that the quantity of cells shown to correlate with protection against CMV could be used as a marker for monitoring patients' immune status towards CMV. This may aid clinical decision making to limit pharmacological intervention to those patients at highest risk for the development of CMV disease. It may also aid the monitoring of the effectiveness of adoptive therapy trials. Therapeutic use of cells with high TCR diversity may be advantageous over other cells in that they may impede the development of CMV immune escape in patients.

We have found that a primary infection with the gastrointestinal nematode <i>Heligmosomoides polygyrus</i> elicits a potent CD4⁺CD25⁺ regulatory T cell (Treg) population within a generalised TH2 environment. This indicates that long-lived nematode parasites may exploit the host regulatory network to suppress protective immunity. We followed the expansion of CD4⁺CD25⁺ Treg cells within the mesenteric lymph nodes (MLN) and spleen over 70 days of infection. Over the time course, increased levels of IL-4, IL-5, IL-9, IL-13 and IL-10 were detected in both these sites by <i>in vitro</i> recall response to parasite antigen. In infected animals, Treg markers, such as surface bound TGF-β1 and CD103 were upregulated on CD4⁺CD25⁺ MLN cells. Surprisingly, however, Foxp3, a transcription factor necessary for Treg development and function, was not upregulated on CD4⁺CD25⁺ in infected animals. Additionally, we have demonstrated that this regulatory population has potent <i>in vitro</i> and <i>in vivo</i> suppressive activity. CD4⁺CD25⁺MLN cells from infected animals (day 28) can suppress mitogen-induced proliferation by both naïve and infected CD4⁺CD25^- cells, whilst naïve CD4⁺CD25⁺MLN cells can only suppress naïve CD4⁺CD25^- cells. Moreover, in a model of allergic airway inflammation, we observed that allergic inflammation was decreased in both infected animals and those cured of infection before airway challenge. Thus, infection is required to induce but not maintain this regulatory function. Finally, we found that concomitant <i>H. polygyrus</i> infection led to reduced expulsion of the related strongylid nematode <i>Nippostrongylus brasiliensis</i> in co-infected hosts. Regulatory markers, CD103 and TGF-β1, as well as TH2 cytokines were elevated in co-infected animals as compared to those with a single <i>N. brasiliensis </i>infection. However, re-infection with <i>H. polygyrus</i> of both cured and infected animals led to significant decreases in worm burden, so although the regulatory network generated by this parasite can increase survival of other nematodes, it does not suppress immune responses to itself upon re-infection.

To investigate the frequency of transmission of ZDV resistant strains of HIV-1, samples obtained from antiretroviral-naive HIV-1 positive individuals were chosen from the MRC Molecular Epidemiology Project, Edinburgh. From this collection of over 600 individuals, 35 patients that had never received any drug therapy were analysed genotypically for the presence of any variation at codon 215 of reverse transcriptase, a position strongly associated with ZDV resistance. Automated sequence analysis of proviral DNA obtained from peripheral blood mononuclear cells revealed variation at this position in two individuals. In these individuals the amino acid encoded at this position was aspartic acid, which has never been observed during ZDV therapy. Further analysis showed this to be linked with a known ZDV resistance-associated mutation methionine41→leucine. Estimated seroconversion dates were August 1992 and August 1993 for these two individuals. As samples obtained in both 1994 and 1996 contained variation at resistance-associated sites, they appeared to have survived within the drug free environment for a period of up to 4 years. To determine the phenotype of strains with variation at codon 215, recombinant virus was produced in a common genetic background (HXB2-D), with the RT sequence replaced by patient derived RT fragments. In addition to phenotypic assays these recombinants were used in competition experiments to determine the fitness of the virus in comparison to wild type variants in the absence of drug. The presence of the GAC codon at 215 conferred at most a 1.6 fold increase in resistance to ZDV, when linked to the 41L mutation. This variant was rapidly outgrown by the wild type RT in assays where the two variants were grown together in one continuous culture, indicating some loss of fitness conferred by these changes. The initiation of an HIV-1 infection with already resistant viral strains has serious clinical implications in patient management, and treatment.

The use of core antigen (HBcAg) of hepatitis B virus (HBV) to present peptide epitopes to the immune system has been shown to enhance immunogenicity of the peptide epitopes. HBcAg fused to the first 8 amino acid residues of β-galactosidase was exploited to serve as a carrier protein to present the epitopes from the S, preS_1 and preS_2 regions of HBV as its truncated C-terminus. The emergence of an HBV escape mutant carrying an amino acid substitution from glycine to arginine at amino acid residue 145 of the S domain suggests that it may be necessary to modify future HBV vaccines. The immunodominant region of HBAsAg carrying mutant sequence at amino acid residue 145 was also fused to HBcAg. These HBcAg fusion proteins were expressed in <i>E.coli</i> and produced in high yields, and assembled into core-like particles morphologically indistinguishable from HBcAg itself. The largest multiple fusion protein, containing a dimer of the HBs_(111-156) sequence as well as sequences from preS_1 and preS_2 regions carried a total of 165 amino acid residues attached to the C-terminus of truncated HBcAg, and could still be accommodated in core-like particles. The HBcAg fusion proteins displaced similar HBc antigenicity and immunogenicity to the full-length HBcAg. Immunisation of rabbits with the HBcAg fusion proteins elicited T-cell-proliferative responses to HBcAg, HBsAg and preS_1 peptides. The T-cell responses to HBcAg were much higher and more consistent than those to HBsAg or preS_1 peptide. The HBcAg fusion proteins induced antibodies against the corresponding peptides. The fusions carrying the immunodominant region of HBsAg, either wild-type or gly_145 mutant with arginine, glutamic acid or lysine substitution, showed HBs antigenicity in the immunoblot analysis and the antigen-capture sandwich radioimmunoassay, albeit at a lesser extent, using two antibodies with different specificity.