This study investigated the cell types infected with HIV in peripheral blood, and the relation of virus load in different cell subsets with disease progression. Two magnetic cell separation techniques were used to purify cell subsets from 34 HIV seropositive individuals at different stages of disease progression. HIV-1 DNA was detected in CD4 and CD8 lymphocytes, monocytes, natural killer cells and dendritic cells. The contribution to total proviral load by different subsets of peripheral blood mononuclear cells (PBMCS) was estimated by quantitative PCR combined with measurements of their relative frequency in peripheral blood. This analysis revealed that CD8 T lymphocytes are a major reservoir of HIV within the peripheral blood of individuals with AIDS. Further independent evidence for <I>in vivo</I> infection of CD8 lymphocytes was obtained by sequence comparisons of the V3 region of the envelope gene. Numerous sequences were obtained from each purified cell subset and initial sequence comparisons indicate that different virus populations may be present in CD4 and CD8 lymphocytes. The functional significance of this will remain unclear until such variants can be characterised virologically. An PCR based method was developed to detect spliced HIV mRNA transcripts whose presence within cells indicates active viral replication, and differentiates these from cells containing an inactive provirus. High levels of mRNA expression were detected in CD4 lymphocytes, CD8 lymphocytes and in one case in monocytes. This evidence indicates that mature CD8 lymphocytes can be actively infected with HIV-1 <I>in vivo</I> even although they do not express CD4.

This study tests the hypothesis that the Lewis<SUP>a</SUP> antigen, expressed by 80-90% of infants between the ages of 2-4 months, is one of the host receptors for two toxigenic bacteria species suggested to be involved in cot death, <I>Staphylococcus aureus </I>and <I>Bordetella pertussis. </I>Although respiratory viruses are often isolated from SIDS infants, there is no direct evidence for their involvement in these deaths; however, this study examined the effect of virus infection on binding of <I>S. aureus </I>and <I>B. pertussis </I>to epithelial cells in culture. By flow cytometry, binding of three toxigenic strains of <I>S. aureus </I>to buccal epithelial cells (BEC) from non-secretors (which usually express large amounts of Lewis<SUP>a</SUP>) was significantly greater than to cells of secretors. Pre-treatment of epithelial cells with monoclonal anti-Lewis<SUP>a</SUP>, anti-type 1 precursor or anti-Lewis<SUP>x</SUP> significantly reduced bacterial binding; and binding of <I>S. aureus </I>was significantly correlated with the amount of Lewis<SUP>a</SUP> present on the epithelial cells. Binding of <I>B. pertussis </I>to epithelial cells was also significantly inhibited by pre-treatment of the cells with anti-Lewis<SUP>a</SUP> or anti-Lewis<SUP>x</SUP>. A 67 kDa protein was isolated from cell membrane preparations of <I>S. aureus </I>(NCTC 10655) by affinity adsorption with synthetic Lewis<SUP>a</SUP> antigen conjugated to Synsorb beads. Pre-treatment of BEC with the purified protein reduced binding of staphylococcal strains to a greater extent than with the material not bound to the Synsorb beads. Respiratory Syncytial Virus (RSV) infects about 50% of infants by the first year of life and it is often isolated from infants with SIDS. RSV-infected HEp-2 cells bound significantly more <I>S. aureus </I>or <I>B. pertussis </I>than uninfected cells.

No description available.

No description available.

Japanese encephalitis virus (JEV) is one of the primary global causes of viral encephalitis, with approximately 68,000 clinical cases and 20,000 deaths attributed to the virus annually. Between 30% and 50% of survivors suffer from debilitating neurological sequelae. Despite being a vaccine-preventable disease, no antiviral treatments are licensed and commercially available to counteract JEV infection. In order to quantify the neutralising antibody response raised against antigenic epitopes on flavivirus prME glycoproteins, conventional serological assays such as the plaque reduction neutralisation test (PRNT) can be employed. However, these assays often necessitate the handling of pathogenic wild-type virus in expensive high-biosafety laboratories, restricting the scope of their application, particularly in resource-deprived areas. Chimeric, replication-deficient pseudotype viruses can offer a solution to this problem, as they mimic wild-type virus entry mechanisms, enabling their use in pseudotype virus neutralisation assays (PVNAs). PVNAs bypass high biosafety requirements and permit vaccine evaluation and serosurveillance studies with no risk of inadvertent infection. This project focuses on the production of functional pseudotype viruses displaying the prM and E surface glycoproteins of the JEV flavivirus, for utilisation in serological neutralisation assays. Subcloning of the prME gene into an appropriate eukaryotic expression vector and insertion mutagenesis to produce prME with 15- and 24-residue upstream signal peptides are shown, before production of JEVpp with either HIV or MLV cores is attempted, via the conventional multi-plasmid co-transfection approach or the utilisation of constitutive gag-pol expressing cell lines. The impact of additional plasmid-derived furin protease expression and low glucose culture medium, as well as the construction of JEV/VSV chimeric prME glycoproteins and the introduction of Kozak consensus sequences upstream of the prME gene, to enhance the efficiency of JEVpp generation is also explored. Finally, the infectivity of lentiviral pseudotype viruses following lyophilisation, storage and reconstitution is confirmed, thus enabling their affordable global distribution.

The aim of this thesis was to expand upon the knowledge of the mechanism of action and resistance of antifolate drugs in African trypanosomes with a specific focus on transport mechanisms. A media deficient in folate and thymidine was established which enabled the assessment of their modulation on antifolate in vitro potencies and also screen a small set of antifolate compounds. The phenomenon of ‘thyminelessdeath’ was found to account for methotrexate toxicity, as well as the primary mechanism of raltitrexed toxicity. This was confirmed by cell cycle studies demonstrating cell cycle arrest in S phase which could be rescued with thymidine. Transport kinetics of folate and methotrexate were characterised and found to be competitive substrates for uptake in T. brucei. Transport of these substrates was inhibited by classical antifolates, but not by non-classical antifolates. Genome-wide RNAi library screens with methotrexate and raltitrexed identified the putative folate transporter genes to be involved in drug resistance. RNAi knockdown of the folate transport genes resulted in a substantial reduction in folate transport was seen. RNAi knockdown also led to cross-resistance to classical antifolates, whereas these parasites became hypersensitive to non-classical antifolates. Methotrexate-resistant trypanosomes were generated in which transport of methotrexate and folate was substantially reduced. Amino acid changes were evident in the putative folate transporter genes but no change in transcription or copy number was evident. Cross resistance to classical antifolates was demonstrated in these resistant parasites and cells become hypersensitivity to non-classical antifolates (a similar phenotype to folate transporter RNAi knockdown). Proteomic studies were performed in drugresistant trypanosomes; however, no conclusive findings were evident due to limitations of these experiments. In conclusion, these studies demonstrate good evidence of both transport-mediated drug action and drug resistance.

No description available.

No description available.

No description available.

The parasite Trypanosoma brucei is the causative agent of Human African trypanosomiasis and Nagana in cattle and millions of people in sub-Saharan Africa are at risk for both health and economic issues since current drugs are inadequate. N-linked glycosylation in Trypanosoma bruceidiffers from other eukaryotes and the parasite has several essential glycoproteins.Thus, the study of oligosaccharyltransferase activities is interesting for potential future drug discoveries. To assess whether TbSTT3A is essential in vitro(i.e. in culture), it was replaced by a drug resistance gene in the presence of a tetracycline inducible TbSTT3A ectopic copy. Although both endogenous TbSTT3A genes were replaced in the TbSTT3 locus, the TbSTT3A function was shown to be retained. These results might suggest that the cells have rearranged their genome during TbSTT3A replacement, which would imply that the tetracycline-inducible ectopic copy could not complement the loss of the endogenous TbSTT3A gene, suggesting that it is essential for cell viability in vitro. Additionally, in situ tagging of TbSTT3A enabled us to search for potential binding partners by performing blue native gel electrophoresis and immunoprecipitation using a Stable Isotope Labelling of Amino acids in Cell culture (SILAC) methodology. The results, further strengthened by co-immunoprecipitation, suggested that TbSTT3A forms a large multimeric complex with TbSTT3B in Trypanosoma brucei. To our knowledge, this is the first time two different STT3 enzymes have been reported to associate in an OST complex.