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The <I>Saccharomyces cerevisiae</I> <I>STE6</I> gene product mediates the export of the peptide mating pheromone a-factor. The Ste6 polypeptide (Ste6p) belongs to the ABC transporter superfamily whose members include many proteins of medical significance. Ste6p is a short-lived transmembrane protein which is produced in low levels in wild-type yeast. This thesis describes the construction, expression and partial purification of a recombinant form of Ste6p from <I>S. cerevisiae</I>. In the absence of a functional assay for Ste6p its presence was detected by Western blot analysis using polyclonal antibodies raised in this study. The antibodies were produced in rabbits immunised with a recombinant Ste6p-ProteinA fusion protein. Purification of wild-type Ste6p was hindered by the very low levels at which the protein was being produced. As an alternative to conventional purification techniques, Ste6p was affinity-tagged at its extreme N-terminus with a six histidine residue (to produce N(His)<SUB>6</SUB>Ste6p) so that it could be absorbed from a dilute solution by its high affinity to Ni-NTA (nickel-Nitrilo-Tri-Acetic-acid) resin. The chimaeric protein was expressed under the control of the <I>GAL</I> promoter in a <I>MAT</I>a, protease deficient strain of <I>S. cerevisiae</I> against a background of wild-type protein. Purification of N(His)<SUB>6</SUB>Ste6p failed due to an apparent inability of the chimaeric protein to bind to the resin. Extracts of cells expressing N(His)<SUB>6</SUB>Ste6p were Western blotted and probed with an anti-histidine-tag monoclonal antibody. The antibody failed to detect any protein of the correct size for the Ste6p chimaera. These results suggested that the N-terminus of N(His)<SUB>6</SUB>Ste6p had been removed during post-translational modification of the protein. The third approach to the purification of Ste6p involved tagging the C-terminus of the protein with eight histidine residues to produce C(His)<SUB>8</SUB>Ste6p. As with the N-terminally tagged Ste6p, this chimaera was expressed under the control of the <I>GAL</I> promoter, however in this case the protein was expressed in a <I>MATαpep4</I> strain of <I>S. cerevisae</I>. This protein could be detected by the anti-histidine tag monoclonal antibody and was able to bind to the Ni-NTA resin.

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This project set out to isolate and characterise anaerobic cellulolytic bacteria from the estuarine sediments of the River Don in Aberdeenshire. The project developed one aspect of a previous, more general study of the microbiology of the Don estuary. Initial studies showed the media used to be unsuitable for the purpose of isolating pure cultures, as did the methodology for the preparation of the anaerobic media. The methodology for the preparation of the media, and the composition of the media were therefore modified. Using the improved media prepared by the modified methodology successful techniques for the isolation of pure cultures of anaerobic, cellulolytic bacteria were developed. Nine pure cultures of anaerobic, cellulolytic bacteria were isolated: eight from the estuarine sediments of the River Don and one from a sample of marine, sediment taken from the North Sea All were characterised as members of'the genus Clostridium. One isolate was characterised as a new species and was provisionally named Clostridium papyrosolvens in view of its "paper-dissolving" properties. No other isolates were sufficiently characterised to enable their identification as species. A field test lasting for one year showed that the improved solid medium was significantly superior to that used in the previous study for the enumeration of anaerobic cellulolytic bacteria from the estuarine sediments of the River Don. Based on the results obtained during a yearlong study of the estuarine sediments of the River Don a hypothesis for the regulation of microbial activity in the sediment was proposed. This hypothesis was that the movement of solutes in the sediment was principally by diffusion and hence the microbial activity of the sediments was also regulated by diffusion.

The prokaryotic domain Archaea represents one of the three major evolutionary lineages of cellular life. Cultivated Archaea are represented by organisms limited to environments of extreme temperature, salinity or anoxia. However, molecular surveys have recently revealed uncultivated Archaea to be globally distributed and active in a variety of marine, freshwater and terrestrial habitats, with a lineage associated with the hyperthermophilic Crenarchaeota kingdom being the most ecologically diverse. The diversity of rhizosphere soil Archaea from three grassland types, associated with different management practices, was examined at a site in the Border region of Scotland, using 16S rRNA and rDNA methodologies. DGGE and sequence analysis revealed the archaeal community to be dominated by two distinct lineages of nonthermophilic Crenarchaeota, with sequences associated with methanogenic Euryarchaeota being retrieved only after anaerobic enrichment. Analysis of the distribution of archaeal communities in the rhizosphere demonstrated a large amount of spatial variability within and between replicate plots. However, grassland management was demonstrated to affect both community structure and activity and, the same dominating band or ribotype was present in all three grassland soils examined. Analysis of 16S rRNA-derived amplicons from managed and natural grasslands at sites in the north of England and the north of Wales also revealed crenarchaeotes to be the predominantly active Archaea. Again, management specific differences were observed, with one dominating ribotype the same as that at the Scottish site. Soil microcosm experiments examined the effects of three environmental parameters associated with the transition of natural to managed grassland; an increase in pH, increased urine addition due to intensive sheep grazing, and fertiliser application. In direct contrast to the bacterial community, profiles associated with the active members of the archaeal community were highly stable exhibiting little change to perturbation.

Commensal bacterial were screened for their ability to modulate inflammatory gene expression in epithelial cells. The influence of intestinal pathogens on cytokine responses was also investigated using co-culture models. It was demonstrated that Bacteroids thetaiotaomicron and Bacteroides vulgatus, which are both dominant members of the human colonic microflora, had the ability differentially to modulate inflammatory responses in intestinal epithelial cells. These observations were confirmed in vivo using a minimal flora rat model. The human pathogen Salmonella enteritidis, when incubated with the Caco-2 human intestinal epithelial cell line, upregulated mRNA expression of the pro-inflammatory cytokines TNF-, MIP-2 and IL-8, but down regulated mRNA expression of the anti-inflammatory cytokine TGF-. In contrast, co-culture of Salmonella enteritidis with Bacteroides thetaiotaomicron, resulted in attenuation of TNF- , MIP-2 and IL-8 mRNA expression and restoration of TGF- to control levels. The viability, growth, attachment and invasion characteristics of the bacteria were not allowed during the time course of the experiments. The attenuating effect of Bacteroides thetaiotaomicron on the expression of pro-inflammatory genes, in particular IL-8, was shown significantly to reduce polymorphonuclear neutrophil (PMN) recruitment in vitro, using an epithelial cell/neutrophil transwell co-culture system. These results were also confirmed in vivo using minimal flora (isolator-reared) rats. PMN recruitment both in vitro and in vivo was assessed using myeloperoxidase activity. The immunosuppressive activity of B. thetaiotaomicron was found to involve down regulation of the nuclear translocation of the transcription factors NF-B/p65 and phosphorylation of AP-1 sub-unit proteins, both of which regulate the expression of TNF- and IL-8 mRNA in intestinal epithelial cells. This effect required viable Bacteroides thetaiotaomicron. The influence of Bacteroides vulgatus, which augmented expression of pro-inflammatory cytokines TNF- and IL-8, when compared to Salmonella alone was functionally distinct from that of Bacteroides thetaiotaomicron. Furthermore, the presence of Bacteroides vulgatus increased the nuclear translocation of the NF-B/p65 subunit, when compared to Salmonella alone.

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No description available.