No description available.

No description available.

No description available.

No description available.

No description available.

Low-dosage ultraviolet confocal fluorescence microscopy and filipin staining showed that cholesterol accumulated at <i>Salmonella</i> entry foci, and was retained by <i>Salmonella-</i>containing vacuoles (SCVs) following bacterial uptake. This was particularly evident in the previously uncharacterised membrane ruffles induced on cultured fibroblasts, when viewed by scanning electron microscopy and three-dimensional fluorescence rendering. Cellular cholesterol redistribution required bacterial effector delivery directed by the SPI1 encoded TTSS. These results showed that host cholesterol is essential for <i>Salmonella </i>uptake, and indicated the involvement of host cell rafts in the entry process. Purified SipB was identified as a cholesterol binding protein in novel gel filtration assays <i>in vitro</i>. Upon cholesterol depletion of cultured eukaryotic cells, addition of purified SipB was sufficient to restore correct localisation of the raft marker, caveolin-1. Fluorescence microscopy, immunogold electron microscopy and biochemical assays demonstrated that cellular binding of purified SipB was reduced in cholesterol depleted cells compared with untreated cells. Localisation of SipB to rafts was observed in <i>Salmonella </i>infected cells, or cells treated with purified SipB. Enhanced localisation for purified SipB was observed when added to cells in complex with SipC, or following treatment of cells with cytoskeleton-disrupting drugs. In combination, these data indicate that SipB specifically targets hosts rafts during entry. New insights into the uptake of <i>Salmonella</i> and their attached flagella were gained by immunoflourescence microscopy. Flagella are lengthy, rigid, helical structures and their fate, following cell entry, was previously unknown. The flagella filament was observed to adopt novel, uncharacterised conformations within the host cell and whilst further flagella assembly was not apparent, pre-existing flagella exhibited long term intracellular stability. This study addresses the issue of the fate <i>Salmonella</i> flagella after cell entry and the nature of the SCV microenvironment.

Antisera raised against the C-terminal decapeptide of G<SUB>s</SUB>α, (RMHLRQYELL or RM), immunodetected polypeptides of 52, 45 and 42 kDa species consistent with identified mammalian species. The specificity of the immunoreactivity was demonstrated by competition with preincubations utilising the decapeptide RM against which the antiserum was raised. Antibodies to G<SUB>i</SUB> and G<SUB>q </SUB>and their respective competing peptides were also utilised. These indicated the presence of a 41 kDa polypeptide cross-reactive with anti-G<SUB>i</SUB> and two polypeptides of 43 and 42 kDa cross-reactive with anti-Gq in trypanosome extracts. GTP binding proteins of <I>T. b. brucei</I> were additionally identified by [α<SUP>32</SUP>P]-GTP binding. This method elucidated polypeptides of 65, 52, 45 and 41 kDa as well as low molecular weight polypeptides (less than 30 kDa) typical in size and abundance of members of the ras superfamily. GTP-binding proteins were also labelled by a photoaffinity reaction by UV irradiation in the presence of [α<SUP>32</SUP>P]-GTP, identifying polypeptides of 160, 140, 65, 52 and 45 kDa in trypanosomes which co-localised with CS1-immunoreactive species. Cholera toxin catalysed ADP-ribosylations of 52 and 45 kDa polypeptides while pertussis toxin mediated the ADP-ribosylation of 41-43 kDa polypeptides; consistent with observations for mammalian species and the immoreactivity data presented. The role of tyrosine kinase signalling pathways was also studied. By stimulating conditions required for transformation of the bloodstream form to the procyclic, increases in autophosphorylating kinase activity and tyrosine phosphorylation were observed. Phosphorylations on polypeptides of 138, 87, 66, 60 and 46 kDa were found to be alkaline stable, identifying them as possible tyrosine kinases. Phosphoamino acid analysis confirmed phosphorylation of tyrosine of polypeptides of 138, 66 and 46 kDa. The autophosphorylating kinase activity of a 138 kDa polypeptide was found to be 1) stimulated by EGF; 2) shown to be alkaline resistance and; 3) to be phosphorylated on tyrosine which indicate it might be the putative EGF receptor tyrosine kinase of <I>T. brucei</I> while a 98 kDa polypeptide was identified by immunoblotting as a possible insulin receptor kinase.

The first steps in the life cycle of HSV-1 are attachment of the virus to the cell, followed by fusion of the virion envelope with the plasma membrane. Both of these events are mediated by virally encoded glycoproteins present in the virion envelope which interact with receptors on the cell surface. Evidence for the involvement of certain glycoproteins in each step has come from the use of viruses which are unable to correctly express the glycoprotein in question. To date four glycoproteins (gB, gD, gH and gL) have been shown to be essential for viral infectivity of fibroblast cells, and gC has been implicated in their efficient infection. However, during a natural infection the primary targets of the virus are polarized epithelial cells, and therefore such cells represent a more realistic system with which to study viral infectivity. Polarized epithelial cells are characterised by distinct apical and basolateral domains, each with a unique protein and lipid composition, and can be cultured <I>in vitro. </I>After demonstrating the polarity of the epithelial cells used in these studies, I was unable to demonstrate any phenotype for a range of viruses which did not express individual glycoproteins. Notably viruses which were unable to express gC were able to infect the apical surface of the polarized cells, conflicting with published data. Detailed characterisation of these viruses was undertaken in non-polarized cells, and no evidence for the involvement of gC in infectivity was observed, again contrary to current dogma. However, a role for gC in the efficient secretion of virus from cells was observed.

The mouse model has been extensively used in studies of HSV pathogenesis and has led to a greater understanding of many aspects of the virus life cycle and viral replication <i>in vivo</i>. The current study makes use of the mouse model to investigate two aspects of viral pathogenesis. The initial project concerns viral resistance to the antiviral drug Aciclovir (ACV) and the pathogenesis of a resistant clinical isolate. The aim of the second project was to investigate the effects of disrupting antigen presentation by MHC class I on the replication and pathogenesis of HSV-1. Aciclovir is a highly effective drug in the treatment of herpetic infections. Its effectiveness, however, is undermined by the emergence of resistance virus isolates that can cause serious disease in immunocompromised patients. In the majority of cases resistance occurs through a mutation that disrupts the TK gene of HSV and is normally associated with attenuation of neurovirulence. In this study a clinical isolate resistant to ACV (4B) was found to display a high level of neurovirulence despite a double G insertion mutation that would theoretically disrupt all TK activity. Following inoculation of the mice the virus was able to replicate efficiently in dorsal root ganglia during acute infection and was able to reactivate from latency. Furthermore, plaque purified isolates of 4B uniformly expressed a very low level of TK activity. Disruption of the TK gene of 4B led to a loss of the low level of TK activity and a loss in the ability to replicate in the ganglia of mice and to reactivate from latency. Further investigation showed that the ability of isolate 4B to replicate in the nervous system of mice was due to the virus gaining an additional G residue, rescuing the original frameshift mutation and allowing the virus to express wild type levels of TK. The neurovirulence associated with 4B is therefore due to phenotypic reversion. The immune response of the host plays an important role in shaping the pathogenesis of HSV. To counteract the antiviral effects of the immune system HSV encodes a number of genes involved in immune evasion.

No description available.