The research explores the spatial correlation between selected trace element abundance in soil and epidemiological data with regard to spatial disease (particularly cancer) patterns. Interpolation techniques within Geographical Information System (GIS) and geostatistics were used to map the Tellus geochemistry data and radon concentrations provided by Geological Survey Northern Ireland (OSNI). This enabled the spatial distribution of the identified potentially toxic elements (PTEs) including arsenic (As), cadmium (Cd), chromium (Cr), lead (Pd), nickel (Ni), selenium (Se), gamma dose rate and radon to be examined and rel ated to geological parent material and pedological sources. The unified BARGE bioaccessabi lity method was used to investigate the bioaccessible fraction of the PTEs. This provided a greater understanding of possible sources and exposure pathways. The incidence of twelve different cancer types (lung, stomach, leukaemia, oesophagus, colorectal, bladder, kidney, breast, mesothelioma, melanoma and non melanoma both basal and squamous skin cancer, were examined in the fonn of twenty-five coded datasets comprising aggregates over the 12 year period from 1993 to 2006. These data were provided by the Northern Ireland Cancer Registry (NICR). Age Stantardised Incidence Rates (ASIR)s were mapped and a local Moran's I analysis identified clustering of wards with high and low incidences of the different cancers and outlier wards of high or low incidences surrounded by wards exhibiting very different disease statistics. The use of a geographical weighted regression enabled the relationship between different cancers and PTEs in the soil and radon to be investigated. The results show comparisons of the geographical incidence of certain cancers (lung cancer and non melanoma skin cancer) in relation to concentrations of certain PTEs (arsenic and uranium levels in soils and radon). Northern Ireland fonns an important test case, as the complex geology is a microcosm for that encountered across the UK and Ireland.

Multidrug resistance (MDR) is the simultaneous acquisition of resistance by bacteria to a broad spectrum of structurally dissimilar compounds to which they have not been previously exposed. The principle cause of MDR has been identified as the efflux of antimicrobials from the cell by proteins which are integral membrane transporters. Escherichia coli MDR transporter, MdtM, is a previously uncharacterised, 410 amino acid residues protein from the ubiquitous major facilitator superfamily (MFS). This thesis focuses on the purification and characterisation of MdtM. Functional characterisation of MdtM using growth inhibition assays and whole cell transport assays revealed its role in intrinsic resistance of E. coli cells to a range of antimicrobial compounds. Functional assays also demonstrated that MdtM functions in alkaline pH homeostasis in E. coli. Inverted vesicle transport assays illustrate the antiport activity of MdtM. Site-directed mutagenesis studies of MdtM suggest that the residue D22 and the highly conserved residue R1 08 each play a role in proton recognition. MdtM was homologously overexpressed from a multicopy plasmid and purified to homogeneity in dodecyl-β-maltopyranoside (DDM) detergent solution. The oligomeric state and homogeneity of MdtM in a panel of detergent solutions was analysed using SEHPLC; MdtM is stable and monomeric in DDM solution. Fluorescence based assays determined the apparent binding affinity of purified MdtM in DDM solution for its substrates. This thesis indicates MdtM is an exceptionally versatile transporter, with a broad substrate range. This work provides a firm foundation for further biochemical and structural studies of this class of multidrug transporter protein.

Antimicrobial resistance is a critical issue in the continued treatment of infectious diseases. AraC-type transcriptional regulators, such as SoxS, RamA, MarA, and Rob, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria, in addition to playing diverse roles including metabolism and virulence. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes that extend beyond antimicrobial resistance. This thesis characterizes a novel homologue of these proteins, RarA, which is encoded in some members of the Enterobacteriaceae. In characterising this regulator, our data shows that the overexpression of, rarA results in a low level multidrug resistance phenotype, which is largely conferred through the upregulation of AcrAB and the recently-characterised OqxAB efflux pumps. Transcriptome analyses in K pneumoniae support the wider role of RarA in gene regulation, as RarA regulates genes involved in cell envelope biogenesis and posttranslational modification, previously uncharacterized transport proteins, energy production/conversion and amino acid transport/metabolism. In support of the transcriptome analyses, Biolog phenotype microarray profiling shows that RarA overexpression confers enhanced growth in the presence of several antibiotic classes, in addition to resistance to previously unreported disinfectant agents and antifungal compounds (c1ioquinol, B-hydroxyquinoline). The central role of RarA in resistance to myriad compounds is also relevant in the development of tigecycline resistance in K pneumoniae and ,Enterobacter spp. Intrinsic mechanisms offer microbial survival strategies in the face of continued antibiotic pressure and such mechanisms are not well characterised in K. pneumoniae. As such this study has characterised and described the role of a transcriptional regulator, RarA, which plays a role in the intrinsic K Pneumoniae resistome.

No description available.

Cell Wall-Deficiency in Staphylococcus aureus and its Role in Antibiotic Resistance. Elizabeth R. Fuller. Cell wall-deficient bacteria (CWDB) induced from Staphylococcus aureus ATCC 9144 (Oxford strain) were generated on medium with elevated osmolality in the presence of sublethal levels of penicillin G. On removal of antibiotic pressure the cell wall-competent (CWC) revertants along with these CWDB exhibited high-level penicillin and methicillin resistance, which was stable in the revertants. The revertants looked visually different, had an altered Gram stain and growth rate. Their matrixassisted laser desorption/ionisation time-of-flight (MALDI-TOF) `fingerprint' was also altered and they were more resistant to lysis by lysostaphin in comparison to the wild-type. Reversed-phaseh igh-performance liquid chromatography( RP-HPLC) showed that the revertants' cell walls had shorter glycan chains and more pentaglycine cross-bridges. A rapid,r eproduciblem ethodu sing liquid mediaw ase stablishedu singt he same medium and sublethal levels of penicillin G. The revertants produced using this method had the same characteristics as those cells produced from the original method. The high-level resistance seen in the revertants was homogenous and confirmed to be due to the transient CWD state, along with not being strain-specific. Transmission electron microscopy showed that the CWD cells and the revertant cells, when grown in penicillin, had a very disordered cell wall with areas where the cell wall appeared absent and were indistinguishable. The revertant cells were mecA-negative,ß -lactamase-negativea nd did not contain any mutations in the coding regions of pbp genes. The CWD cells and revertant cells, when grown in penicillin, were resistant to lysis by lysostaphin but were very sensitive to lysis with Triton X- 100. These data indicate that the resistant cells are not dependent upon an intact cell wall for osmotic stability and they are able to switch readily to this mode of growth in the presence of penicillin G.

In bacteria, multidrug efflux systems have been identified as significant determinants of resistance recently. These resistance pumps are widely distributed in bacterial species and many pathogenic bacteria posses them, which play an important role in their intrinsic and acquired multidrug resistances. The RND and MATE family transporters have also been shown to be involved in the pathogenicity of bacteria. Knowledge of the structure and mechanism of these transporter proteins would be exceedingly useful in the design of inhibitors. In Gram-negative bacteria, multidrug resistance is conferred in part by the tripartite multidrug efflux pumps that are composed of an inner membrane transport protein, a membrane fusion protein and an outer membrane protein. One such tripartite pump, VceCAB of Vibrio cholerae, is composed of an inner membrane H(^+)-antiporter VceB, a membrane fusion protein VceA and an outer membrane channel VceC. To investigate the role of this pump in the multidrug resistance of Vibrio cholerae, we have characterized functionally and structurally the three components of the VceCAB pump and the regulator VceR. The crystal structure of VceC was determined at 1.8 Â resolutions. Despite the very low degree of sequence identity between them, VceC shares the same overall architecture as TolC, consisting of three domains: the ß-domain, the α-domain and the equatorial domain. The trimeric VceC packs in laminar sheets in the crystal that resemble membranes. Like TolC, the α-barrel of the VceC channel at the periplasmic end is closed through the packing interactions of coiled-coil helices, but the residues that maintain the closed state of the channels of VceC and TolC are different. The ß-barrel region of VceC is also closed, whereas the ß-barrel region of TolC is open to the extracellular medium. The channel interior of VceC is generally electronegative and contains two rings of clusters negative charge. The ring made by residues Glu(^397) and Glu(^303) is conserved in OprM, but is not in TolC. Mutagenesis assay of this negative charged ring indicated its functional role during transport. The optimal desolvation area (ODA) on the surface of VceC is different from that of TolC, suggesting distinct architectures of VceC-based and TolC-based tripartite pumps. Sub-cellular fractionation of cells expressing full length and truncated VceA suggested that VceA is anchored to the IM via a transmembrane helix. Analytical gel filtration chromatography experiments revealed that the periplasmic domain of VceA that was expressed in the periplasm of E.coil forais a trimer, which could represent its oligomeric state in the VceCAB pump. The three components of tripartite pumps are easy to dissociate in vitro, making it difficult to co-crystallize them. We overproduced the protein complex in which VceA (12-406) is in complex with the VceB-VceA fusion protein. This complex was stable during purification, which could provide an invaluable way for co-crystallization of these two components of the VceCAB pump. An analytical gel filtration and DLS experiments indicated that the basic functional unit of VceR is a dimer; the binding of substrate СССР to VceR has been determined to occur with a Hill coefficient of about four, and thus each VceR dimer binds four СССР molecules. This stoichiometry of drug/VceR-subunit is different from that of other transcriptional regulators in the TetR/CamR family. There are differences between MtrD and other RND family multidrug efflux pumps. The knowledge of difference will be important for understanding the mechanism of these family transporters. In this study, we successfully overexpressed, purified and crystallized MtrD. The resolution of MtrD crystals was optimised to 7-10 Å at present.

No description available.

Genetic mapping, positional cloning and reverse genetics provide an alternative to the biochemical and molecular approaches used to date, to determine the basis of arsenical resistance in Trypanosoma brucei. Genetic mapping of loci determining phenotypes of relevance to diseases has proved to be a powerful approach in a number of organisms including humans and Plasmodium falciparum, particularly when coupled with a full genome sequence. In this thesis, this approach has been established in T. brucei by determining the genetic basis of naturally occurring arsenical resistance and undertaking linkage analysis using our recently developed genetic map. I adapted a simple, verified screening assay for assessing drug sensitivity based on the use of AlamarBlue. Three stocks used as parents in genetic crosses differed in drug sensitivity; STIB 247-Sensitive, STIB 386-Resistant and TREU 927-Resistant. Genetic linkage analysis using 101 polymorphic markers Identified from the extensive sequence available from the genome project was then used to examine inheritance of the drug resistance phenotype in T. brucei. From this, the co-segregation (into F1 progeny) of markers and the resistance phenotype was determined using crosses, 247 x 386 and 247 x 927. Inheritance of resistance in both crosses was compatible with a simple single locus genetic model with one dominant allele determining resistance. Linkage analysis showed that the locus conferring resistance lay within an ~25 kilobase region on Chromosome II, which contains 6 open reading frames (ORFs). A reverse genetic approach was then used to disrupt alleles for each of the six ORFs in turn. An allele of one gene, Tb927.2.2380, was shown to determine resistance and this was confirmed by transfecting the resistance allele into a drug sensitive stock to generate an arsenical resistant line. This gene also determines cross-resistance to the major veterinary trypanocide, diminazene aceturate and has been named the arsenical and diamidine (ard) resistance gene.

The problem of increasing rates of antibiotic resistance has become a global concern, particularly among multi drug resistant Gram-negative nosocomial pathogens. These organisms display non- susceptibility to the majority of routinely used antibiotics, causing infections which are more difficult to treat and increase the duration of patient recovery. Due to the plethora of resistance determinants and the molecular machinery which facilitates their dissemination, new strategies are required to investigate the mechanisms that confer antibiotic resistance. Proteomic techniques allow the global analysis of the expressed proteome, providing a more holistic view of the current physiological state of the bacterial cell. The techniques used in this investigation cover the separation, quantification and identification of proteins present in cellular extracts from resistant organisms. These included the use of 2-D electrophoresis, DIGE and LC-MS/MS mass spectrometry applied to multidrug resistant Escherichia coli, Klebsiella pneumoniae, Enterobacter c/oacae, Serratia marcescens and Acinetobacter baumannii. In summary, these investigations revealed that the Tol-Pal membrane protein system and susceptibilities to polymyxin antibiotics and biocides are altered upon acquisition of a resistance plasmid in E. coli. Furthermore, it revealed that non-carbapenemase-mediated carbapenem resistance in K. pneumoniae involved the loss of fimbriae proteins, the increased expression of OmpK26 and the resistance proteins ErnrA and APH(3"), in addition to OmpK35/36 porin loss. The upregulation of a multi drug efflux pump in E. c/oacae, A. baumannii and S. marcescens involved the differential regulation of many proteins, spanning a broad range of functional classes, including the MinCDE cell division inhibitors, iron acquisition proteins such as FepA and FhuA and proteins involved in biofilm and LPS formation such as PapC, LptD and GmhA. Overall this project has highlighted the complex and dynamic changes in protein expression upon acquisition of a resistance phenotype and the importance of using genetically related isolates when undertaking proteomic analyses. This work also emphasised the advantages of using proteomics for profiling the expression of resistance proteins, including the detection of specific enzymes, such as CTX-M ESBLs.

Host-pathogen interactions have critical implications for the establishment of disease and for determining adaptive immune responses of the host. This study has conducted global Aspergillus fumigatus transcriptional analyses throughout the initiation of murine infection using a wild-type and an attenuated ΔlaeA isolate. A novel data analysis protocol was applied from which three time-series datasets were generated between 4, 8 and 14 hours post infection. This approach identified distinct temporal gene expression profiles during disease initiation whereby numerous secreted enzymes, including proteases and antigens, were upregulated between 4 and 8 hours, while a striking upregulation of genes in secondary metabolism clusters and subtelomeric loci was observed between 8 and 14 hours. In order to test the role of several upregulated secondary metabolite genes on host-pathogen interactions and virulence, two isolates mutated in non-ribosomal peptide synthetase encoding genes (ΔftmA, Δpes3), and mutants of a hybrid non-ribosomal-polyketide synthase (ΔpsoA) and a putative secondary metabolite transcription factor (ΔregA) were analysed in murine models of infection. These analyses suggest fumitremorgin C, pseurotin A and, putatively, a pigment augment A. fumigatus virulence. In contrast, the pes3 gene product favours pathogen clearance, possibly by facilitating recognition of host innate immunity. There was no evidence to support the view that defective secondary metabolism is causative of the attenuated virulence phenotype observed for the LaeA mutant. Alternative hypotheses regarding the attenuation of ΔlaeA include upregulation and downregulation of Th1 and Th2 associated antigens respectively, and deficiencies in plasma membrane transport relative to wild-type isolates. A hypothesised deficiency in maintenance of genome stability, due to LINE-1 mobilisation in the ΔlaeA isolate was tested, and subsequently discredited by a discovered lack of isogenicity among the tested strains. Work presented in this thesis also assesses the use of a mass-spectrometric method for detection of epitope tagged A. fumigatus protein in the host.