Engineered nanoparticles (ENPs) are increasingly being exploited in a whole range of applications and products due to their novel physicochemistry. Hence it is inevitable that ENPs will enter the environment at an increasing rate over the coming years. The consequential impact following interaction between ENPs with plants and soil microbial communities is of great concern given that they play fundamental roles in the environment and food production. In this study, the impact of capped silver nanoparticles (cAg NPs) on terrestrial (Arabidopsis thaliana and Vicia faba) and aquatic (Lemna minor) plants was investigated. In addition, due to the important role of bacteria in plant survival and growth, this study also assessed the effect of cAg NPs on plant-associated soil microbial community structure. cAg NPs demonstrated varied toxicity towards plants and the associated soil microbes. Whilst the aquatic plants and soil microbial communities investigated in this study were not affected by cAg NPs up to 100 mg/L, for the terrestrial plants evaluated here, cAg NPs above 12 mg/L (specifically 50 and 100 mg/L) demonstrated differential toxic responses. Based on the results of this study, it is clear that concentration, exposure method, released ions, plant species, light intensity and growth mediums are key factors that influence the toxicity of cAg NPs. Although the cAg NP concentrations applied in this study are not yet environmentally relevant, with continued and uncontrolled commercial production of Ag NPs and/or in the event of spillage, such concentrations could occur in the environment in the future. Chlorophyll fluorescence and gas exchange are valuable techniques for analysing the toxicity of ENPs on plants, due to their rapid and reliable results. Further studies in the interactions between plants and Ag NPs are urgently needed and would benefit from the use of different application methods such as aerosolization.

Cultured motile cells show a highly enriched belt of actin at the tip of their lamellipodial protrusions, termed the leading edge. It is at this leading edge that researchers have uncovered pools of highly localized mRNAs encoding actin and actin-regulatory proteins, including β-actin, Profilin, Cofilin and all seven subunits of the Arp2/3 complex, whose local translation is then required for proper cell migration. The localization and local translation of these mRNAs are regulated by RNA-binding proteins, including Imp, which localizes β-actin mRNA at the leading edge. However, published studies have so far failed to determine if Imp is required to localize β-actin mRNA and/or other mRNAs at the leading edge of migratory cells in vivo. Our examination of Drosophila embryonic macrophage migration in vivo revealed that actin is not enriched at the leading edge, compared with cultured macrophages, demonstrating that a single cell population employs different mechanisms of cytoskeletal arrangement when migrating in vivo, compared with an ex vivo migration along a 2D substrate. It is therefore not surprising that we did not observe Imp at the leading edge of macrophages in vivo. However, overexpression of Imp reduced both the velocity and directionality of macrophages and inhibited cell-to-cell contact inhibition, suggesting a defect in microtubule dynamics, although we have yet to establish a mechanism for this. We show that Imp binds the 3’UTR of β-actin, profilin, and β-integrin mRNAs and reveal three sites of primary sequence that are required for Imp binding to β-actin mRNA. Our results suggest that, in contrast to cultured migratory cells, β-actin mRNA is unlikely to be localized to, and locally translated, at the leading edge of macrophages in vivo. However, cytoplasmic mRNA regulation is likely to play some kind of role in cell migration, as revealed by overexpression of Imp, which impairs macrophage motility. This thesis highlights a crucial requirement for studies to determine the mechanisms of cytoplasmic mRNA regulation in motile cells in vivo, which appear to be distinct from those employed in some cultured cells.

The repair of damaged articular cartilage remains a clinical problem despite the development of numerous surgical approaches for cartilage regeneration. As result new options for therapeutic approaches are being sought. One of the candidate cell types for cartilage repair are mesenchymal stem cells (MSCs). These cells can be isolated from a number of different tissues and have the ability to differentiate down several different mesenchymal lineages. This thesis focused on the use of MSCs for repairing damaged articular cartilage. Specifically I investigated the effect of producing regenerative medicine type constructs containing different populations of MSCs on the induction of chondrogenesis in response to mechanical load, compared the induction of chondrogenesis in MSCs through the application of exogenous TGF-β1 and multiaxial mechanical load and identified potentially novel markers of MSC chondrogenesis. The results presented in this thesis show that the induction of chondrogenesis in MSCs can be manipulated by producing constructs that contain separate populations of MSCs. The work demonstrated that seeding a layer of MSCs on the loaded surface of a fibrin-poly(ester-urethane) scaffold could increase the deposition of histologically detectable matrix. However, it was not possible to determine the mechanism responsible for this. Comparison of the secretomes of MSCs stimulated with TGF-β1 and mechanical load showed that these two forms of chondrogenic stimulation are not analogous and that a number of markers, including GRO and MMP13 may be useful for monitoring the progression of MSCs through chondrogenesis and hypertrophy. These data provide further insights into the effect of joint-like load on MSCs within tissue engineering/regenerative medicine style constructs, and the chondrogenic response of MSCs to this stimulation, which may prove to be useful for the development of constructs for cartilage repair.

The work in this thesis examines genome-wide and local changes in the patterns of nucleosome positioning throughout the human genome. Nucleosomes are the fundamental repeating unit of chromatin. Their properties and positioning in the genome dictate whether and how proteins involved in gene regulation can access DNA. Nucleosomes are dynamic; their positions can vary considerably at some loci from one cell type to another. Chromatin remodelling complexes can change the structure and the positions of nucleosomes. Their mis-regulation leads to congenital defects affecting pre-natal and early childhood development and is associated with neuro-psychiatric disorders. As mutations in genes that encode chromatin remodelling proteins are associated with human mental health disorders, the work in this thesis focusses on changes that occur in chromatin structure during early neural development. I have used MNase-seq data to construct genome-wide, high-resolution chromatin particle positioning maps from undifferentiated human induced pluripotent stem cells (hiPSC) and following differentiation to the neuro-progenitor cell (NPC) stage. These maps reveal that a small proportion of the pluripotent genome possesses well-positioned nucleosomes, the number of which increases approximately 8-fold during neural cell development. This is accompanied by changes in the distribution and localisation of nucleosomes between iPS and NPC cells. Differences in nucleosome positioning during neural cell differentiation were investigated at regulatory regions. Loss and gain of positioned nucleosomes at TSS of pluripotent and neural-specific genes was detected and correlated with gene expression. In addition I investigated the chromatin structure at the binding motifs of two important genome regulators REST and CTCF in detail. Nucleosome positioning is maintained at REST binding motifs during neural cell development. In contrast, at CTCF sites nucleosome repositioning occurs during neural cell development. This work provides insight into the role of chromatin structure in the regulation of human neural cell differentiation.

Agricultural land use is a leading cause of habitat degradation and biodiversity loss in streams. Understanding the mechanisms by which land use change affects stream ecosystems is essential for their effective management. Despite this, the consequences of agricultural intensification for community composition and ecosystem functioning in streams remain poorly resolved. Using national-scale monitoring data and new field data from upland streams in South Wales, this study investigated the effects of pastoral intensification on the community composition, functional diversity and feeding interactions of stream macroinvertebrates. A combination of analytical tools were used, including propensity modelling, ecological traits, stable isotopes and Next Generation DNA sequencing to quantify diet. Taxonomic and functional diversity had non-linear relationships with pastoral intensity, declining beyond a threshold of 4 mg L-1 nitrate and 8% fine sediment cover. This decline occurred as a non-random loss of species possessing specific traits, including large body size and lack of resistance forms. Although monitoring data showed that at a UK-wide scale pastoral agriculture (cf. other land uses) had a positive effect on richness and sensitive species representation, the threshold intensity at which effects become negative is exceeded in many locations within the U.K. and globally. Invertebrates that feed by grazing algae were particularly vulnerable to agricultural stressors. Combined with changes in the availability and quality of basal resources with pastoral intensification, this decline in grazer representation resulted in invertebrate communities becoming increasingly reliant on detrital resources. Further, there was indication that methane-derived carbon contributed to the food web in high intensity sites, which has not previously been observed in upland streams. Although only relatively minor changes were observed in predator-prey interactions across the intensity gradient, there was a suggestion of simplification of the food web in high intensity sites. Together these changes could radically alter ecosystem properties such as secondary production, nutrient processing and resilience. Overall, the results highlight the management priorities of reducing fine sediment and nutrient inputs to agricultural streams. The identification of a threshold at which agricultural effects become deleterious will assist in guiding mitigation efforts. Further work is required to determine the generality of this threshold across stream ecosystems.

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P. aeruginosa is a versatile microorganism with high levels of antimicrobial resistance and a common contaminant of home and personal care (HPC) products. Relatively little is known about P. aeruginosa in industrial settings and to further investigate this, four areas were considered: (i) culture-dependent and –independent detection of P. aeruginosa; (ii) P. aeruginosa and bacterial diversity in HPC products; (iii) preservative susceptibility and phenotypic characterisation of industrial P. aeruginosa strains, and; (iv) P. aeruginosa adaptation to a preservative combination. A large collection of P. aeruginosa and non-P. aeruginosa industrial isolates was utilised to evaluate five selective agars for the detection of P. aeruginosa. Media using negative selection performed best overall, but media using positive selection had potential as enrichment media. Culture-independent detection of P. aeruginosa and overall bacterial diversity was achieved via direct DNA extraction from contaminated HPC products, species specific PCRs and 16S rRNA gene sequencing analysis. The bacterial diversity in HPC products was low, with less than three contaminating genera in each product. The diversity of P. aeruginosa strains from industrial, clinical and environmental sources was investigated using five genotyping techniques, ranging from PCR-fingerprinting methods to whole genome sequencing, and phenotypic assays examining preservative susceptibility, growth dynamics and motility. P. aeruginosa strain diversity was high and there was no association between genotype, phenotype and isolation source. The development of adaptive resistance of P. aeruginosa to a preservative combination used in HPC products was modelled using planktonic growth and biofilm assays. P. aeruginosa PA14 grew in elevated levels of the preservatives chloromethylisothiazolinone, methylisothiazolinone and dimethylol dimethyl hydantoin but it was unknown whether the increased tolerance was stable. Whilst eradication of P. aeruginosa from the industrial environment is unlikely, improved detection methods and understanding of the species will inform industrial practices and preservative formulations to minimise HPC product contamination.

Staphylococcus aureus is a major cause of infections within hospitals. Its spread among patients in hospitals is one of the major problems that require urgent attention. Staphylococcus aureus has the ability to interact with other microorganisms such as the free-living amoebae Acanthamoeba which acts as a host for various pathogens. This interaction is highly complex and may be beneficial to one of them or both. The main aims of this project were to study the interactions of Acanthamoeba castellanii with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA), and to understand the molecular mechanisms involved. A large variety of molecules play a role in growth, function, migration and apoptosis. The role of Phosphoinositide 3-kinase (PI3K) in Acanthamoeba/bacteria interaction was studied through the use of inhibitors such as LY294002 and wortmannin, while the involvement of protein tyrosine kinase was studied through genistein and protein tyrosine phosphatase by sodium orthovandate (Na3VO4). The role of serine proteases of Acanthamoeba was investigated by using its inhibitor Phenylmethylsulfonyl fluoride (PMSF). The results showed that MRSA exhibited more invasive abilities towards Acanthamoeba, when compared with MSSA. In addition, inhibition of PI3K and tyrosine phosphatase subsequently resulted in the reduction of MRSA and MSSA invasion. However, PMSF increased bacterial uptake by Acanthamoeba. Two dimensional gel electrophoresis was performed to separate and fractionate the Acanthamoeba conditioned medium and the Universal Protein Resource Uniprot database was used for the analysis and identification of protein for the forty three spots identified. This study further stresses the need for antimicrobial drugs that are effective without inflicting damage upon the host, as a result of the resistant nature of MRSA towards a variety of antibiotics. One possible method that can be used is Photodynaic Therapy, due to its effectiveness against microorganisms in a localised infected area, through the use of local combined photosensitizers and light. In addition, to simplify the interaction process, heat-killed fluorescein isothiocyanate-labelled (FITC)-labelled MRSA and MSSA were used. The findings suggest that trophozoites Acanthamoeba trophozoites uptake of MRSA was significantly higher than MSSA which may act as "vehicle" that facilitates the spread of MRSA to susceptible hosts. This study showed that photodynamic therapy had a significant impact on bacterial survival. Photosensitizers m-Tetra (hydroxyphenyl) chlorin (m-THPC) and psoralen were very effective in eradicating bacteria. In antimicrobial assays, natural product eugenol derivatives demonstrated significant inhibitory activity on the growth of MRSA and exhibited dose-dependent cytotoxic effects on Acanthamoeba trophozoites. Future work should continue to identify more molecular mechanisms of Acanthamoeba interaction with MRSA and finding new strategies for effective antimicrobial therapies.

The enteric microflora represents one of the densest microbial populations in the biological world; as a consequence, the intestinal immune system is constantly exposed to high concentrations of antigenic materials. One of the major frontline defences in the innate immune system is the intestinal epithelial layer, which presents both a physical barrier and an immune sensor to the antigens of the lumen. The latter function is performed by the expression of pattern recognition receptors, which recognise a wide variety of bacterial antigens, and the production of inflammatory cytokines, which stimulate, or inhibit, inflammation. The overall aim of the present study was to investigate the immunomodulatory potential of extracellular products, from non-pathogenic bacteria, with intestinal epithelial cells. Two in vitro human intestinal epithelial cell lines HT29-19A and Caco-2 were shown to exhibit different expression levels of Toll-like receptors (TLRs) and the inflammatory cytokines, interleukin (IL)-8 and IL-10. These differences were reflected in their sensitivity (monitored by IL-8 release) to known TLR agonists, isolated from pathogenic bacteria. Caco-2 cells were also shown to form physiologically active tight junctions, with the formation and maintenance of domes. Both cell lines exhibited sensitivity to the cytotoxic extracellular products of the enteropathogen Clostridium difficile. Extracellular products, in crude cell-free supernatants and bacterial sonicates, from the commensal Gram-negative bacterium Escherichia coli C25, significantly increased IL-8 release in both cell lines. Lipopolysaccharides and membrane vesicles were shown to contribute to the proinflammatory effects of C25-derived extracellular products. These extracellular products were also shown to regulate bacterial internalisation in both cell lines. Crude cell free supernatants and bacterial sonicates from two lactobacilli strains Lactobacillus acidophilus 5e2 and Lactobacillus helveticus sp. Rosyjski were also found to be biologically active, stimulating IL-8 release and TLR expression modification in both intestinal epithelial cell lines. In addition, ultrapure EPSs, isolated from these lactobacilli strains, were also found to possess immunomodulatory potential. HT29-19A cells, pre-treated with EPSs, were found to be ‘primed’ to bacterial agonists, peptidoglycan and flagellin, with a significantly potentiated release in IL-8 observed. Finally, EPSs were also found to modify bacterial adherence and internalisation in both cell lines. In conclusion, data presented in this investigation has shown that the use of the intestinal epithelial cell lines, HT29-19A and Caco-2, presents a reasonable model for investigating the interaction of bacterial extracellular products with the intestinal epithelium. Additionally, it has demonstrated that extracellular products, isolated from non pathogenic, enteric-associated bacteria, possess immunomodulatory potential in vitro. If these effects were also to occur in vivo, then they could potentially contribute to intestinal homeostasis and the innate ‘priming’ of the epithelial layer to pathogens and their products.