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The Gram-negative Vibrio cholerae is native to aquatic environments and an important human pathogen causing cholera disease. The induction of virulence in this bacterium is subjected to a wide variety of stimuli including environmental cues and quorum sensing. In this study, non-bactericidal cationic polymers were designed to capture Vibrio cholerae into clusters resulting in physiological changes. Poly(N-(3-aminopropyl) methacrylamide) (P1), poly(N-[3-dimethylamino)propyl] methacrylamide) (P2) or poly(acryloyl hydrazide) imidazole (P3) were synthesised via free radical polymerisation displaying amine groups that cluster cells mediated by electrostatic interactions. This binding resulted in a forced transition from planktonic to a sessile lifestyle. The clustering is accompanied by reduced motility, increased biofilm synthesis and repression of virulence since the expression cholera toxin was down-regulated. This avirulent phenotype was defective to colonise intestinal epithelial cells and the zebrafish digestive tract. Since the cell density increases locally as a result of the clustering, a quorum-sensing-controlled phenotype was observed as the lux operon was actively expressed. Overall, the bacterial physiology was modulated without genetic modification preventing virulence as the pathogen adapt its lifestyle during clustered lifestyle. This thesis highlights the use of polymeric materials as a mean to control pathogens beyond the use antibiotics.

Solar evaporite systems are man-managed ecosystems which are highly vulnerable to biological,physical and chemical disturbances. The problems encountered in such systems are in many cases found to be associated with the microbial ecology and the design of the system. This project focussed on investigating the nature of organic compounds contaminating soda ash produced at a solar evaporite production system located at Sua Pan in Botswana. Several years after the plant was commissioned, problems, including accumulation of total organic carbon (TOC) and discolouration of the soda ash product were encountered. The salt produced also retained high moisture content and was coloured pink. These phenomena impacted severely on the economic performance of the enterprise. This study was aimed at determining the origin and fate of these organic compounds within the system in order to elucidate the nature of the problem and also to conceptualise a remediation strategy suitable to reducing its impact. This was achieved by analysis of both dialysed and solvent extracts of the influent brine (well-brine), brine in the ponds (T-brine) and the bicarbonate filter cake. Although complete identification of the organic compounds isolated was not undertaken in this study, spectroscopic analysis of compounds isolated, by UV, IR, NMR and MS, strongly indicated that fulvic acids, a component of the influent well-brine organics, contribute to the organic contamination of the final product. Part of this component, however, is degraded during the ponding process. It was shown that an extracellular polysaccharide (EPS) produced by Dunaliella. spp., which proliferates in the evaporation ponds, contributes in a major way to the accumulation of TOC in the system. This was demonstrated by relating the sugar profile of carbohydrates isolated from the pond brine and final product, being arabinose, xylose, 2-o-methyl hexose, mannose, glucose and galactose. Studies reported show that EPS production was enhanced when algal cultures were exposed to stress conditions of high illumination, increasing salinity and temperature, and nitrogen limitation. Studies undertaken for the development of a remediation process for this system have shown that nutrient stripping and bacterial systems could be applied to deal with the dissolved TOC fraction, whereas adsorption systems could deal with the particulate fractions. Algal systems showed most potential for the removal of nutrients in the influent well-brine compared to chemical processes.Complete removal of ammonium and phosphorus removal efficiencies of pproximately 50% were achieved in an unoptimised pilot-scale Dunaliella-based HRAP. While similar effects were demonstrated for chemical processes, some economic constraints were noted. The potential of halophilic bacterial systems for the degradation of organic compounds in brine was also demonstrated. The limitations on the performance of such systems, associated with the low metabolic diversity, and poor immobilisation of physico-chemical processes were found to have a very low impact on the dissolved TOC fraction of the brine, the removal of the particulate material was found to result in a 35% TOC reduction in the final soda ash product and the production of a white final product.halobacteria, however, were noted. Although physico-chemical processes were found to have a very low impact on the dissolved TOC fraction of the brine, the removal of the particulate material was found to result in a 35% TOC reduction in the final soda ash product and the production of a white final product. Apart from a description of the microbial ecology of the ponds and the identification of major contributions to the TOC of the final product, a number of remediation strategies were evaluated and are described. These include chemical and biological stripping of nutrients sustaining microbial TOC production in the ponds, and also biological and physico-chemical processes for their removal once formed. Future studies to undertake the further development of these proposals has been described.

Pasteurella multocida is a Gram-negative bacterium that resides in the upper respiratory tract of domesticated animals. It is responsible for pneumonia in cattle, sheep and pigs, atrophic rhinitis in pigs, fowl cholera in poultry and haemorrhagic septicaemia of cattle and buffaloes. Different strains of P. multocida are associated with disease in different host species but little is known about the molecular basis of virulence and host-specificity in this pathogen. The outer membrane of P. multocida is at the interface between pathogen and host and it is highly likely that outer membrane proteins (OMPs) and lipopolysaccharide (LPS) are involved in disease pathogenesis and host-specificity. Outer membrane vesicles (OMVs) are derived from the outer membrane and there is increasing evidence of their involvement in disease pathogenesis. Hence, the present study aimed to compare the composition of OMPs and OMVs derived from different strains of P. multocida using proteomic approaches. Proteomic characterisation was carried out on the outer membranes of eight P. multocida isolates using gel-based and gel-free methods. A combination of gel-based and gel-free proteomic approaches identified a total of 67 OMPs associated with outer membrane biogenesis and integrity (11 proteins), transport and receptor activities (19 proteins), adherence (9 proteins), enzymatic activity (6 proteins) and unknown functions (22 proteins). Of these, 44 proteins were identified by both methods, 21 proteins were identified only by the gel-based method and two proteins were identified only by the gel-free approach. The study also characterised OMV production from the same P. multocida isolates and assessed the optimal OMV protein concentrations at different stage of growth. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that OMVs were enriched with OMPs and LPS. The OmpA protein was used as an outer membrane marker protein and its presence in OMVs was demonstrated by Western-blotting and immunogold-labelling. Proteomic analysis of the OMVs derived from the same eight P. multocida isolates was carried out by the gel-free approach in three biological replicates. A total of 200 proteins were identified and these included 58 OMPs, 43 periplasmic proteins, eight inner membrane proteins, 85 cytoplasmic proteins and six extracellular proteins. Interestingly, the number of proteins identified in the OMVs varied in different P. multocida isolates. Subsequently, OMPs identified in the outer membrane and OMV fractions were compared and the findings showed that OMPs associated with OM biogenesis and integrity were more likely to be present in the outer membrane, whereas OMPs associated with transport and receptor activities were highly enriched in the OMVs. These results highlight the potentially important role that OMVs of P. multocida play in disease pathogenesis and identified proteins for further evaluation as putative virulence factors and candidate vaccine antigens.

In this thesis, I have applied a complementary and alternative understanding of melanoma chemotaxis by incorporating the role of LPA and through a pseudopod-centred approach. I have demonstrated that pseudopods are self organising actin entities that undergo mainly bifurcation (splitting) from preexisting protrusions rather than through synthesis of de novo pseudopods for melanoma cell chemotaxis. This observation was also extended to mouse melanoblast migration in vivo. These superior split pseudopods influence cell steering by alignment, size and lifetime regulation, and biases retraction. In non-metastatic cells, they are no longer able to form stable split pseudopods in response to external stimuli. LPA signaling is also established as vital for the stability of split pseudopods. Hence, in the event of LPAR1 perturbation, metastatic cells formed more actin protrusions but they adopted pseudopod morphologies similar to the non-metastatic lines. The role of Rac, Ras, Paxillin and Ezrin in split pseudopod regulation was also explored in this thesis. Finally, the increased stability of split pseudopods acting through LPA signalling could emerge as a signature of metastatic cells.

Protection against the highly contagious foot and mouth disease virus (FMDV) coincides with neutralising antibody titres. Infection in cattle is characterised by 100% morbidity of an acute vesicular disease whilst infection of their closest relative, the African buffalo, is sub-clinical despite having diverged only 5.7 – 9.3 million years ago (Glanzmann et al., 2016; 1). The germline and antibody repertoire in African buffalo has not previously been characterised and so the cause of their differential disease response may be the production of a more specific and / or avid antibody response to FMDV than cattle. The cattle and African buffalo antibody germline was sought to characterise the recombinatorial potential of the antibody loci and their subsequent primary antibody repertoire. Expression of the antibody heavy chain (IGH) and antibody lambda light chain (IGL) was investigated with qPCR and RNA-seq. The antibody repertoire in response to FMDV infection was interrogated in African buffalo infected with SAT1 FMDV and compared to the cattle IGH repertoire inoculated with highly purified SAT1 FMDV antigen. The recombinatorial potential of the cattle and African buffalo IGH and IGL is severely limited compared to other species such as mice and human. The characterisation of the cattle IGH and IGL is the most accurate to date and reveals internal duplications of the IGH, disrupting the expected IGHV-IGHD-IGHJ-IGHC ordering seen in mammalian immune loci and resulting in four IGHD regions, containing long and ultra-long IGHD. These IGHD provide a novel diversification mechanism that can compensate for limited germline diversity by forming long and ultra-long CDR3H loops that are highly diverse in their length and amino acid composition. The African buffalo antibody repertoire also forms highly diverse long and ultra-long CDR3H, despite lack of evidence for the existence of the duplications in their IGH. Limited variability is seen in the length and amino acid composition of the IGL in both species, suggesting they are playing a structural role to support these unusual long and ultra-long CDR3H. In response to FMDV infection in African buffalo, a dramatic increase in specific long and ultra-long CDR3H sequence abundance occurs but this change in frequency of specific transcripts is absent in cattle. The differential antibody response may account for the protection of African buffalo against FMD.

ERp27 is a 27.7 kDa redox-inactive member of the protein disulphide isomerase (PDI) family. It was found to interact with another PDI member, the well-known thiol oxidoreductase ERp57 (58 kDa) in vitro. Although it is known that ERp57 interacts with ERp27 in vitro this interaction was not investigated in living cells. In this research project we applied in vitro and in cellulo approaches to investigate the same interaction of ERp57 and ERp27 then to compare it to the interaction of ERp57/calnexin (CNX)/calreticulin (CRT) complex to determine if the ERp57 interaction with ERp27 competes with the ERp57/CNX/CRT complex. Additionally, we investigated the physiological role of ERp27. Protein expressions and purifications were carried out by the Nickel agarose affinity chromatography to obtain sufficient amount of proteins for analysis. Additionally, proteins were purified by gel filtration-chromatography. The interaction between purified ERp27 and ERp57 was determined using isothermal titration calorimetry (ITC) and by chemical cross-linking. The ITC results confirmed the interaction between ERp57 and the lectin CRT. However, we could not detect an interaction between ERp57 and ERp27 possibly due to low protein concentrations. Moreover, the in vitro cross-linking results were in agreement with the previous research and verified the binding of ERp57 with ERp27. However, in cellulo chemical cross-linking suggested that the same interaction does not occur in living cells. Nevertheless, this investigation revealed that ERp27 binds to other proteins in cellulo. Mass spectrometry results have identified protein candidates that interact with ERp27 in living cells which are the PDI homologous P5 and the ER oxidoreductin Ero1. These results provide new insights of the role of ERp27 and provide suggestions for further research.

Staphylococcus aureus pathogenicity islands (SaPIs) form part of the wider family of phage inducible chromosomal islands (PICIs), which are extremely mobile phage satellites. SaPIs are clinically relevant, encoding and disseminating virulence and fitness factors to promote bacterial evolution and pathogenesis. These islands are innately linked to their helper phage’s life cycle, requiring phage-mediated induction and packaging for transfer between bacterial hosts. The inherent association between SaPIs and helper phages is one of the best characterised examples of virus satellite interactions in prokaryotic cells. The SaPIs sit passively in the host chromosome, controlled by the SaPI-encoded master repressor protein; the Stl. Interaction between the SaPIs and their helper phages begins when a phage-encoded protein binds to the Stl to de-repress the island. Different SaPIs encode different Stl repressors, which require diverse phage-encoded proteins for derepression. The best characterised phage-encoded inducers are the trimeric dUTPases (Duts), which traditionally prevent the misincorporation of uracil into DNA, but have been proposed as regulatory proteins that induce SaPIbov1. Recently, the structurally dissimilar phage-encoded dimeric Dut of NM1 has also been shown to mobilize SaPIbov1, but how this is accomplished remains unsolved. Here, this work expands the family of SaPIbov1 phage-encoded inducers to include the subset of phage-encoded dimeric Duts. This develops upon the theory that specific phage-proteins are required for SaPI induction, broadening this vision to show that SaPIs instead target conserved phage mechanisms. Using SaPIbov1 as an example, this study indicates the StlSaPIbov1 can interact with the structurally distinct dimeric and trimeric Duts, whose only shared value is their role for the phage. Furthermore, this study highlights that, although these two proteins are completely unrelated, they both encode one highly variable region (named motif VI). Indeed, the mechanism for the dimeric Dut interaction with the StlSaPIbov1 is investigated here and a number of parallels to the trimeric Dut mode of action are apparent. These results indicate that the structurally divergent dimeric and trimeric Duts interact with the SaPIbov1 Stl using analogous, but distinct mechanisms that represent a fascinating example of convergent evolution. Furthermore, investigations into the SaPIbov1 Stl indicate that Stl accomplishes these interactions with the dimeric and trimeric Duts by using separate domains for each interaction. This intriguing Stl evolution is an elegant strategy utilised by the SaPIs to overcome phage-encoded inducer mutation or exchange. This highlights the continual arms race between the phages, which aim to escape SaPI induction, and the SaPIs, which must overcome these changes for island induction to occur. Furthermore, these results highlight the potential for Stl targeting of structurally diverse, functionally related phage proteins to facilitate intra- and inter-specific transfer. Overall, these results highlight the SaPIs as a fascinating subcellular parasite of the phages, which have evolved a novel strategy to target their phage inducer proteins and achieve transfer. Likewise, this study shows the biological significance of the dimeric Duts as regulatory molecules.

Given the recent discovery of RUNX1 somatic mutations in biopsies of breast cancer patients, the overall purpose of the present thesis consists of using different in vivo and ex vivo experimental systems in the attempt to answer two main questions: firstly, if the Runx1 gene plays any causative role in the context of breast cancer; and secondly, if its putative function is symptomatic of a tumour suppressor gene and/or of a pro-oncogene. By characterizing the effects of Runx1 deletion in two different breast cancer mouse models (i.e. the MMTV-PyMT and the Wnt/β-catenin-driven models of mammary tumourigenesis), this thesis provides the first in vivo evidence of a dualistic role played by the gene in the context of breast cancer. Runx1 would in fact appear to act as a tumour suppressor at early stages of the disease, whilst as a pro-oncogene at later stages of mammary tumourigenesis. To fully comprehend the significance of these major findings, the introduction will first provide a brief description on the RUNX family of genes, as well as on the state-of-the-art knowledge of RUNX1’s role in both mammary gland and breast cancer biology. As such, particular attention will then be given not only to the ontogeny, endocrine regulation and composition of the murine mammary gland, yet also to the high degree of heterogeneity, the putative “cell-of-origin(s)” and the different experimental models commonly used to study breast cancer. Through the aforementioned rationale, it is hoped that the introduction will serve as a platform which may hold the key for unveiling the controversial role played by RUNX1 in the context of breast cancer.