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Bdellovibrio bacteriovorus is an endobiotic bacterial predator that encodes multiple enzymes related to known peptidoglycan (PG) modifiers, which are necessary for predation to occur. Additionally, a plethora of cryptic proteins are expressed and upregulated during predation. It has been shown that L,D-transpeptidases (LDTs) are required for bdelloplast formation and stabilisation. These LDTs cluster in six groups in B. bacteriovorus HD100, nevertheless, no further LDT information has been previously described. Here we have determined and analysed the structures of LDTs Bd0566, Bd1075, Bd3334 and Bd3741, showing that even though they share a structurally conserved catalytic domain, they have acquired accessory domains and motifs characteristic to each LDT group. Moreover, we have integrated these novel structures with the previously solved structures of Bd0553, Bd0886 and Bd1402, generating a panel of seven LDTs representing the six groups of B. bacteriovorus HD100, allowing us to propose possible roles and the structural determinants defining the transferase/crosslinking or hydrolytic activities of each group. Further, we report the structure of two predatory NlpC/P60 amidohydrolases/peptidases, Bd0601 and Bd1177. We have determined that Bd0601 is a DUF1460 amidohydrolase, wherein the PG stem peptide binding groove is blocked by a tyrosine residue, suggesting the need of a conformational rearrangement for substrate binding. On the other hand, we have determined that Bd1177 is a bilobular NlpC/P60, wherein the active site is located in the interface between the catalytic N-terminal domain and a novel C-terminal regulatory domain, suggesting that Bd1177 fluctuates to an open conformation upon substrate binding. Finally, we have determined the crystallographic structure of the cryptic secreted protein Bd0675, depicting a novel fold. The solved structure depicts a prominent central acidic and aromatic groove delimited by a conserved disulphide-stabilised loop, suggesting that this protein has ligand binding functions. Based on the solved structure we have tested different candidate ligands; however further experiments are required to propose a function for Bd0675. In addition, due to its novelty, Bd0675 was used as target for multiple structure predicting algorithms in CASP14.

Biofilms are considered to be one of the most prevalent and successful modes of life on Earth, and the prevailing lifestyle for microorganisms. Enabling bacteria to adapt to an incredibly diverse array of environments and extreme conditions, biofilms are a major contaminant of both medical and industrial settings. Indeed, approximately 80% of microbial infections are associated with biofilm formation, whilst the damage caused by biofilms in industry is estimated at between 2 - 3% of global GDP per annum. In this body of work, the effect of two industrially-relevant shear conditions on biofilm formation by the reference Pseudomonas aeruginosa (Ps. a.) strains PA01 and PA14 was investigated, as well as the effect of growth conditions and growth medium components on curli gene expression in E. coli K12 PHL644. The CBC biofilm reactor was used to model low and high shear conditions at 75 RPM and 350 RPM respectively, and biofilms grown over a time period of 96 hours. High levels of the intracellular second messenger c-di-GMP are regarded as the determining factor for Ps. a. sessility and progression of the biofilm phenotype, thus the c-di-GMP-responsive cdrA::gfp reporter was used to measure intracellular c-di-GMP levels of PA01 and PA14 under low and high shear conditions. Biofilms were analysed via confocal laser scanning microscopy and staining of extracellular DNA (eDNA) and the exopolysaccharides Psl and Pel, which are all form crucial components of a self-produced and protective extracellular matrix that surrounds and enmeshes Ps. a. within a biofilm. Under high shear at 350 RPM, intracellular c-di-GMP levels of initially adhered (at 24 hours) bacteria were increased, resulting in increased production of exopolysaccharides and formation of early aggregative structures. Shear conditions were shown to impact upon biofilm development and maturation of three-dimensional structures: crucially, mushroom-shaped macrocolonies, which are archetypal of Ps. a. biofilm formation, did not form under high shear. Under low shear at 75 RPM, Psl and Pel were organised into networks of fibre-like structures that penetrated throughout well-established basal biofilms (> 72 hours), which is in agreement with the work of others (with respect to Psl), but is a novel observation of Pel morphology as produced by PA14. The work presented in this thesis therefore provides further insight on the variety of Psl and Pel morphologies that exhibit different structures, spatial and temporal organisation, and function across PA01 and PA14 biofilms in response to either low or high shear conditions. Whilst similarities were observed between the two strains, PA01 and PA14 exhibited distinct responses to the imposed shear regime, in terms of initial surface colonisation, time taken for mature structures to emerge, and exopolysaccharide production. Biofilms produced by E. coli PHL644 were grown using the Duran bottle model, via a method previously developed by the Overton laboratory. High levels of the transcriptional regulator CsgD are regarded as 'master switch' that determines biofilm formation in E. coli, thus the CsgD-responsive csgB::gfp reporter was used to measure curli gene expression in response to growth in LB broth (a rich medium) versus M63+ minimal medium, different concentrations of glucose (at 0 mM, 1 mM, 10 mM and 100 mM), and incubation at different temperatures (at 25 oC, 28 oC, 30 oC and 37 oC), and identify parameters which resulted in maximal expression of curli. Planktonic cell samples were taken from the tops of the Duran bottles, and sedimented cell samples taken from the bottom of the Duran bottles for comparative analysis of growth via spectrophotometry at OD600 and csgB::gfp fluorescence by flow cytometry. Curli gene expression was found to be highest in cultures grown in M63+ minimal medium, with a glucose concentration of 10 mM and at an incubation temperature of 30 oC, which is in agreement with comparable studies in the literature. Of interest was the fact that an inverse relationship between biomass concentration (as defined by OD600 values) and csgB::gfp fluorescence was observed. Curli gene expression in sedimented cell samples was consistently lower than that of planktonic cell cultures across all experimental subsets, suggesting that planktonic cells are physically more capable of surface attachment, and curli expression may be downregulated when in a sediment; exemplifying the importance and function of curli as the initial adhesin of E. coli K12. Overall, this body of work concludes that different shear conditions can impact upon Ps. a. biofilm development and induce distinct organisation of the ECM, and that the CBC biofilm reactor is a suitable experimental model for assessing the impact of turbulent flow regimes, akin to those experienced in industrial manufacturing plants, on biofilm formation and composition. Additionally, this body of work also demonstrated that the Duran bottle model is also a suitable method for biofilm formation, and for investigating the effect of a wide array of growth conditions on E. coli K12 biofilm formation and curli gene expression. Parameters that resulted in maximal curli gene expression in E. coli K12 PHL644 cultures grown via the Duran bottle method under the conditions tested were identified, and could be further used to optimise production of a physically robust E. coli biofilm for use in biocatalysis or certain industrial settings.

The golden age of antibiotic discovery has granted us a respite from incurable infections. However, bacteria can become tolerant to antibiotics by forming biofilms, and they can evolve resistance to antibiotics, rendering them useless. Finding new antibiotics is difficult, but looking into historical medicine may provide some answers. This PhD project has conducted an interdisciplinary investigation into a 10th-century remedy, Bald's eyesalve. This remedy calls for crushed onion and garlic to be combined with wine and ox gall, and left to stand for 9 days. This remedy was previously shown to have potent activity against S. aureus. Here, I have identified that many steps in the recipe create a more active remedy, highlighting the potential scholar behind these texts. I demonstrate this activity extends to a broad range of planktonic cultures (S. aureus, including MRSA, A. baumannii (including multi-drug resistant strains), E. coli, P. aeruginosa, B. cenocepa- cia). Importantly, a subset of these strains grown as established biofilms in a synthetic wound model were also eradicated (S. au- reus, all A. baumannii and B. cenocepacia). A. baumannii was unable to develop resistance to the remedy, but became resistant to antibiotics (meropenem and ciprooxacin) within 35 days. Chemical analysis (HPLC) has identified that although allicin is responsible for the activity against planktonic cultures, it was significantly less active against biofilms than Bald's eyesalve. Further analysis (LC-MS/MS) identified a combination of allicin with an aqueous wine fraction work synergistically to eradicate these S. aureus biofilms. To conclude, a potent antimicrobial semi-synthetic cocktail containing allicin and aqueous wine components has been developed from the historical remedy Bald's eyesalve. Additionally, these results stress the importance of 1) including biofilm models when exploring natural products for the anti-biofilm pipeline; and 2) researching mixtures of natural products, rather than isolated compounds, may generate potent therapies.

M. tuberculosis cell wall components are essential for host-pathogen interactions and modulating the immune response. M. tuberculosis may persist as pellicle-like biofilms within the peripheries of human cavities, and expectorated bacteria from these biofilms may establish infection in a new host. The complement system is an early cascade of the innate immune response, which plays an important role in M. tuberculosis access to alveolar macrophages. The impact of mycobacterial biofilms on complement activation has not been studied in-depth. This thesis aims to evaluate the role of mycolic acid maturation in pellicle biofilm formation and interactions of the mycobacterial cell envelope with the human complement system. Mycolic acids are a major cell wall component, and their accumulation is a hallmark of pellicle biofilm formation. M. smegmatis, a surrogate organism for M. tuberculosis, was mutated in gene MSMEG4722 (ortholog of M. tuberculosis Rv2509) (ΔMSMEG4722), causing the loss of mature mycolic acids for mycolic acid intermediates (Bhatt et al., 2008). As a result, ΔMSMEG4722 pellicle biofilms could form on the air-liquid interface but could not mature and produced significantly less extracellular material. Biochemical changes in the cell envelope of ΔMSMEG4722 were examined using thin layer chromatography. Changes included alteration to the distribution of glycolipids (TDM, TMM and GMM) within the apolar lipid fractions, occurring in bacteria cultured in Middlebrook 7H9 and as planktonic cells and pellicle biofilms. Within pellicle biofilms, mature free mycolic acids and free mycolic acid intermediates/ pre-mycolates decarboxylated to unsaturated ketones accumulated in the apolar lipid fractions for M. smegmatis mc2 155 and ΔMSMEG4722, respectively. Flow cytometry and ELISA demonstrated increased complement system activation, both on whole bacterial cells and the apolar lipid fractions of ΔMSMEG4722 compared to M. smegmatis mc2 155. Increased complement activator binding was also observed. When examining M. smegmatis mc2 155 pellicle biofilms, comparable complement activation was observed on planktonic cells and pellicle biofilm-derived cells. However, overall activator binding was lower, and there was decreased complement activation by the apolar lipid fractions. ΔMSMEG4722, in contrast, showed higher complement activation by both phenotypes compared to M. smegmatis mc2 155, but activator binding was only increased on biofilm-derived cells and both phenotypes demonstrated decreased complement activation by the outer apolar lipid fraction. The implications of these findings in understanding mycobacterial pellicle biofilm formation and deciphering mycobacterial cell wall component activation of the complement system are discussed.

The "race to the surface" describes the competition between bacteria and eukaryotic cells for colonisation of an implant device. This thesis aimed to address gaps in modelling the oral microenvironment surrounding a dental implant material, specifically focusing on three key elements: identification of a suitable hydrogel scaffold, development of a method for calculating biofilm viability on the surface of implant materials, and exploration of a computational model of surface adhesion. For strain values up to 30%, the mechanical stiffness of porcine oral mucosa under compression was determined, with porcine as a model tissue. Acellular alginate and alginate-collagen scaffolds were then characterised to determine the concentrations that were most similar to the stiffness of the porcine mucosa. Further investigations included scanning electron microscopy, rheology, and Fourier-transform infrared spectroscopy to fully characterise the surface topography, gelling behaviour, and chemical bonds within the polymers, respectively. Blended collagen-alginate hydrogels of 2.5 mg/mL collagen with 10 mg/mL alginate and 2.5 mg/mL collagen with 5 mg/mL alginate were found to closely match the mechanical stiffness of the native tissue, as well as demonstrating desirable gelling behaviour through the formation of collagen fibrils and low concentrations of cross-linking agent required. These hydrogels were then shown to support human dermal fibroblast growth upon encapsulation within the scaffold, with high cell viability. Matrix stiffness was found to influence fibroblast morphology, with a higher stiffness resulting in rounded fibroblast morphology and a lower stiffness resulting in a spindle shape. The presence of a stratified epithelium was not confirmed and therefore further optimisation of this model is required. Alongside the refinement of this 3D model, a novel analysis tool was developed for quantifying confocal micrographs of live/dead stained bioflms. The protocol was validated on different bacterial species, and the tool demonstrated a reliable measurement of bioflm growth and cell viability. Advantages of this tool include the low computational time to analyse images, ease of use, and transparency of the image processing methods employed. Finally, a computational model of initial bacterial adhesion to a surface was developed, based on a cellular automaton. The parameters that affected this initial stage of biofilm formation were investigated. The balance between the rate of migration to the surface, division rate, and death rate of a bacterial species had a significant input on the cells adhering to the surface. If the balance of these parameters can be controlled in vitro and in vivo, then this could inform the development of strategies for preventing surface colonisation. Ultimately, the work presented in this thesis will support the development of antimicrobial strategies and novel implant devices to prevent the occurrence of dental implant infection by providing improved methods of analysing the effect of such strategies in vitro.

Laboratory-based evolution has become a tool that is widely used to understand an organism's response to stressful environments through linking the genotype to the phenotype. Within laboratory evolution, the role that loss of function mutations play in adaptation is a topic of debate, with recent observations suggesting that adaptive loss of function mutations are a common adaptive strategy. One limiting factor of this technique is that the time taken to conduct a single experiment can be extensive. With these points in mind, we proposed to see if a short term selection experiment on a high density transposon library, using Transposon Directed Insertion-site Sequencing (TraDIS) to analyse the data, would produce results which correlate with those from long-term evolution experiments. Since TraDIS provides a measure of relative contributions to fitness of each gene, in principle it should be possible to use TraDIS to identify genes whose loss of function provides a fitness benefit on a significantly shorter timescale. Previously in our laboratory, five populations of E. coli K-12 MG1655 were evolved in a dynamic pH environment by daily passaging over five months in unbuffered LB, starting at pH 4.5. Whole genome resequencing of the final populations and clones revealed many striking similarities in the evolutionary trajectories of these populations. Therefore, to explore the hypothesis that short term selection of a high density transposon library could identify genes that were also found in the five month evolution experiment, an E. coli K-12 MG1655 transposon library was constructed and passaged for 10 days under similar conditions as the evolution experiment at both pH 4.5 and pH 7. TraDIS analysis showed that, within these populations, insertions in a few genes had accumulated, suggesting there was a fitness advantage for a strain carrying these insertions. These genes showed a significant overlap with the ones identified in the evolution experiment. These results highlight a possible alternative approach to laboratory evolution when attempting to understand an organism's response to stress, providing a foundation for future work to explore different conditions. Research data supporting this thesis can be found on the University of Birmingham eData repository at: https://doi.org/10.25500/edata.bham.00000750.

The emergence of life on Earth developed a system of DNA containing the information inherited in the reproduction of organisms, directing also the variability of life as this changes. The language using for deciphering the DNA is the genetic code, which is degenerated as 20 amino acids are codified by 64 codons, or triplets of nitrogenous bases (adenine, guanine, cytosine and thymine) in the nucleotides that form the DNA. 59 codons have other/s synonymous codons, and those are not used randomly but are a product of different phenomena. The drivers of codon usage bias can be mutations, genetic drift and natural selection processes. The study of these codons can provide evolutionary information, like gene family evolution and organisation, can offer an insight in population size, and also can be used to create software to predict ORFs and coding regions. Codon study also can be important in biotechnology for heterologous expression, as the codon used have a control over the efficiency and accuracy during translation, resulting in determinant for protein folding, growth rate and avoidance of defective proteins. Opisthokonta is a group of organisms, close to Apusomonadida, and gathering Holozoa (Ichthyosporea, Filasterea, Choanoflagellatea and Metazoa) and Holomycota. The present study uses the transcriptome of 20 choanoflagellates species, Sphaeroforma arctica (Class Ichthyosporea), Thecamonas trahens (class Apusomonadea) and five early branching metazoans (Amphimedon queenslandica, Mnemiopsis leidyi, Nematostella vectensis, Thelohanellus kitauei and Trichoplax adhaerens) to study the codon usage bias and anticodon related phenomena in the mentioned species. Analysis revealed optimal codons for each organism, with a GC ended codons enrichment in T. trahens, unicellular Opisthokonta, and retained after the transition to pluricellularity (with divergences in M. leidyi and N. vectensis). All the unicellular species showed evidence for natural selection upon translational accuracy and efficiency, even the less biased ones. In metazoan only translational efficiency was evidenced, with lower strength of selection, and poor support for translational accuracy in ctenophoran and cnidarian species. For other strategies giving advantages on protein translation, indirect evidence of deamination in high degenerated amino acids was found in all the species studied, while direct evidence was found in S. arctica, A. queenslandica, M. leidyi and N. vectensis. Additionally, tRNA presenting isotype-anticodon disagreements were found in apusomonadean, ichthyosporean, metazoan species and in S. rosetta. Deamination, and possibly chimeric tRNA appeared early in evolution, before the divergence of Apusomonadea and Opisthokonta, benefiting the use of optimal codons through evolution until the apparition of the latest metazoan organisms.

Lyme disease is the most common vector-borne infection across temperate zones of the Northern Hemisphere and is caused by the spirochete, Borrelia burgdorferi sensu lato (s.l). The outer membrane (OM) of Borrelia possesses many unique features, including an abundance of lipoproteins and few integral β-barrel membrane proteins. The predicted small β-barrel OMPs from Borrelia burgdorferi s.s; BB_0027, BB_0405, BB_0406 and BB_0562, are all predicted to form an 8-stranded transmembrane β-barrel. Three truncations were made to each OMP to increase the chance of crystal formation and these, along with potentially soluble targets, were cloned and recombinantly expressed in E. coli. Following purification, a BB_0406 truncation was soluble in DDM and showed promise for crystal formation. Factor-H binding was investigated using a far-Western blot. No binding was detected, in agreement with the most recent literature. The Borrelia genome consists of a linear chromosome and numerous linear and circular plasmids. While the chromosome of Borrelia has been subject to various searches for β-barrel OM proteins, the plasmids have so far remained unexplored. This research utilised a computational-framework approach to identify potential OM β-barrel proteins in the plasmid proteome of B. burgdorferi s.l. This approach identified two plasmid-encoded proteins: the annotated porin OMS28, and a previously uncharacterized protein BBJ25. Orthologs of BBJ25 are detected in two other major spirochaete families, Brachyspira and Treponema. Within the Borrelia species complex, BBJ25 is found within a predicted 7-gene operon at either the 3rd or 7th position, resulting in two allelic variants. Phylogenetic analysis of the allelic variants is inconsistent with vertical descent alongside the chromosome suggesting that horizontal transfer and/or recombination has occurred after the divergence of Relapsing Fever/Lyme Disease-type Borrelia. While the precise role of BBJ25 remains unknown, the identification of a novel conserved outer membrane protein is of interest as a diagnostic or therapeutic target.

Concussion occurs when a "biomechanical force, transiently disturbs normal brain function causing neurological, cognitive and behavioural signs and symptoms". However, the diagnosis of concussion is imprecise and challenging. Currently, there is a great deal of attention on concussion in contact sports due to the immediate and potential long-term effects on the brain. The transient and complex array of symptoms that occur, mean patient compliance is essential, to quantify the neurological deficit that enables an experienced assessor to diagnose accurately. The future in concussion diagnosis therefore lies in the development of a simple, relevant, rapid and objective assessment of the trauma to the brain. The most likely candidates are either brain imaging e.g. MRI, or measurement of a relevant biomarker or group of biomarkers. Rapid diagnosis is particularly needed in contact sports such as rugby and football to enable players to safely return to play. The development of an objective diagnostic test may also help to explain the longer-term impact of head injury, and be applicable to other sports and situations e.g. military or household accidents etc. In this thesis, I have hypothesised that changes in small non-coding RNAs (sncRNAs) and extracellular vesicles (sEVs) in saliva may be used for the diagnosis of concussion. Changes in either or both of these in saliva could reflect rapid changes in the brain and systemic circulation due to the concussion. Understanding of mechanism through which these changes could occur is beyond the scope of this thesis. The Study of Concussion in Rugby Union through MicroRNAs (SCRUM) (ISRCTN16974791) has used next Generation Sequencing (NGS) and qPCR validation steps to analyse 390 saliva samples from the top two tiers of professional rugby in England. Samples were collected during training, 3 hours and 48-72 hours post-injury. Controls were taken from groups with musculoskeletal injury, uninjured players, and players in whom concussion had been suspected but ruled out on. I have identified 12 sncRNAs that undergo up-regulation in saliva of players with concussion (ten in the immediate aftermath, two 48-72 hours post injury). Two sncRNA families had more than one member responding to injury, namely the RNU-6 snRNA and Let-7 miRNA families. Further work is required to confirm whether measurement of these, or a combination of sncRNAs, can be used to diagnose concussion. sEVs were measured in saliva using a new assay, the ExoView, but no clear pattern of change was observed. This is likely to reflect the complexity of sEVs which vary markedly in size and can be generated from multiple cell types. Studies on miRNAs, were extended to a deceased group of patients who had been diagnosed with Alzheimer's disease (AD), in view of a possible historical link to concussion. Brain samples were obtained from the South West Dementia Brain Bank (SWDBB, University of Bristol). Two progressive stages of AD and a control group were assessed using qPCR. The Let-7 family miRNAs, hsa-let-7i-5p and hsa-let-7a-5p, were found to be differentially expressed in the brains of AD patients. Further work is required to understand the function of these two mRNAs in the brain. In conclusion, this study has identified several sncRNAs that may change in saliva in response to concussion and which could form the basis of a future diagnostic test. This work has also provided preliminary evidence of changes in the Let-7 family of miRNAs in AD. Further work is required to understand the significance of changes in sncRNAs in concussion and AD, and a possible link between their role (if any) in these two disorders.

Cocoa flavanols (CF) are bioactive compounds that exert antioxidant and anti-inflammatory properties and can aid overall health as a result. This PhD looked to investigate the efficacy of CF as an ergogenic aid for muscle recovery following exercise-induced muscle damage (EIMD). As strenuous exercise can elicit oxidative stress and cause the muscle to enter an inflammatory state, CF may aid recovery by blunting the overproduction of reactive oxygen species and limit the pro-inflammatory response. A systematic review of the literature was carried out, resulting in 14 studies, identifying that acute and sub-chronic consumption of CF blunts exercise-induced oxidative stress and, likely through a similar mechanism, may delay fatigue during exercise. It was identified that the most pertinent area of research related to CF and EIMD as only three studies currently existed, with evidence of the benefits unclear due to methodological issues, such as ineffective muscle damaging protocols. Not only that, but within sporting settings, optimal recovery is crucial for maintaining high levels of performance whether in training or during competition. A failure of full recovery can reduce athletic performance, e.g., reductions in force output or sprint ability, and can increase injury risk. The first experimental study investigated the impact of an acute dose of CF (either 830mg or 1245mg) on muscle recovery following EIMD. Overall, 23 participants completed the study. Participants performed maximal voluntary isometric contractions (MVIC) of the knee flexors to assess muscle function and a visual analogue scale (VAS) and lower extremity functional scale (LEFS) to assess perceived soreness. To induce muscle damage five sets of 10 maximal concentric/eccentric hamstring curls were performed on each leg using an isokinetic dynamometer, with muscle function and soreness being measured immediately post, 24, 48 and 72 hr following EIMD. It was observed that the highest dose of CF (1245mg) may have a minimal effect on the recovery of MVIC and muscle soreness; although not statistically significant. The second experimental study was a sub-group inter and intra-sex analysis of the data gathered from the first study. Overall, no significant differences were observed between males and females for measures of muscle recovery. The third experimental study investigated the impact of daily consumption of 1245mg of CF on muscle recovery following repeated bouts of strenuous exercise, separated by 72 hours. In addition to MVIC, VAS, and LEFS, electromyography was included within the measures to assess muscle recovery. This study had 9 participants complete the seven-day protocol (one baseline and six consecutive days) ingesting 8 beverages throughout the period. No overall significant differences were observed between the groups, however at the final time point (48 hr post the second EIMD protocol) large effect sizes were observed and a statistically significant difference at that time point for MVIC data, VAS, and LEFS. The data contained within this thesis provides novel information on the potential of CF as an ergogenic aid for muscle recovery. It appears that CF does not offer a significant benefit for muscle recovery when compared to a recovery drink containing only carbohydrate and protein. However, the large effect sizes observed in all three studies imply there may be a small effect of CF on recovery, as such the data from this PhD needs to be corroborated by future research to further justify the use of CF as an ergogenic aid for recovery.

Bread quality depends on the aerated structure of the baked loaf, which arises from the unique rheology of wheat flour doughs as a result of the viscoelastic behaviour of wheat gluten proteins. Development of this dough structure is an essential element of dough preparation, generally achieved in modern processes via high speed mixing; however, sheeting of doughs is potentially a more effective and energy efficient approach that gives superior bread quality. Meanwhile, inclusion of bran in the dough formulation enhances the healthiness of bread, but hinders the development of the gluten structure and the resulting quality and palatability of the bread. Using sheeting to enhance dough development could help to offset the damaging effects of bran and allow production of more appealing high fibre breads. Implementing sheeting in a commercial breadmaking operation is more difficult than the use of high-speed mixing; however, recently there have been moves to implement this technology commercially. There is therefore a need to understand in greater detail the development of bread dough by sheeting, and its interactions with bran and with the development of the aerated structure of bread.

The oxygenated volatile organic compound, acetaldehyde, plays a fundamental role in atmospheric chemistry by altering the atmosphere's oxidative status or "self-cleaning capacity". Acetaldehyde flux from freshwater, estuarine, and marine environments is thought to be under significant microbial control, yet the identity and diversity of microbial acetaldehyde degraders, and the mechanisms of acetaldehyde degradation, are largely unknown. In this thesis, the key acetaldehyde-degrading microorganisms in the Colne Estuary, the River Colne and the River Gipping were identified using metagenetic sequencing and quantitative PCR. Bacteria were primarily responsible for acetaldehyde degradation, with members of the genera Pseudomonas and Arcobacter identified as key acetaldehyde degraders. Seventeen bacterial isolates were cultivated from the Colne Estuary and degraded acetaldehyde at a concentration of 2.27 mM, with Halobacillus strains A4 and A5, Rhodococcus strain A14, and Bacillus strain A17 identified as important acetaldehyde degraders. Fourteen previously identified bacteria, belonging to the Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia, also degraded acetaldehyde, suggesting that the microbial acetaldehyde sink is highly diverse. Using genomics and proteomics, the pathway of acetaldehyde metabolism used by Rhodococcus strain A14 was proposed. Acetaldehyde is oxidised to acetate by aldehyde dehydrogenase enzymes, with acetate converted to acetyl-CoA via acetyl-CoA synthetase. These reactions are thought to be conserved amongst acetaldehyde-degrading bacteria. Under favourable growth conditions, acetaldehyde is dissimilated to CO2 for energy production via the oxidative decarboxylation steps of the tricarboxylic acid cycle. Acetaldehyde-derived carbon is assimilated into biomass by upregulating the glyoxylate shunt and gluconeogenesis pathway, ensuring carbon is conserved for growth. Using 14C-radiolabelling, rates of microbial dissimilation were shown to be significantly higher than assimilation rates in the freshwater rivers, estuaries, and coastal seas of south-west England, with summer rates significantly higher than winter. These findings suggest that microorganisms exert considerable control over acetaldehyde emissions and represent an important component of the global acetaldehyde cycle.

The evaluation of how Bacteria respond to oil-contamination, and the application of dispersants and biosurfactants, in North Sea seawater microcosms is the focus of Chapter Two. Analysis revealed that dispersants and biosurfactants, which significantly reduced the interfacial tension between oil and water, significantly increased growth of obligate hydrocarbonoclastic bacteria (OHCB) in 24 hours, translating into significantly enhanced alkane-biodegradation. Early sampling of microcosms revealed how the OHCB Oleispira, hitherto considered a psychrophile, can dominate bacterial communities at the relatively high temperature of 16oC. Bacterial response to oil-pollution is examined further in Chapter Three, where an in situ oil-slick is compared to a chemically dispersed oil-slick in the North Sea. Results suggest a lack of hydrocarbon-degrading bacteria (HCB) growth, even in samples with measurable hydrocarbons, could potentially be attributed to phosphorous limitation. Whilst the Ecological Index of Hydrocarbon Exposure, which quantifies the proportion of a bacterial community with hydrocarbon-biodegradation potential, revealed an extremely low score, highlighting a limited capacity for the environment, at the time of sampling, to naturally attenuate oil. Analysis of sediments contaminated by the Agia Zoni II oil-spill (Greece, 2017), in Chapter Four, demonstrated significant growth of HCB five-days post-oil-spill. Whilst the relative abundance of HCB declined as oil was removed, a legacy effect was observed, with the OHCB Alcanivorax and Cycloclasticus persisting for several months after the oil-spill. Finally, analysis of sediments around a North Sea shipwreck (HMS Royal Oak), in Chapter Five, revealed low levels of pyrogenic polycyclic aromatic hydrocarbons and little evidence of HCB, indicating sediments showed no long-term impact by previous oil-pollution from the shipwreck. This thesis not only advances our understanding of microbial response to oil-spills, remediation strategies, and shipwrecks, in a range of marine environments, but also highlights the importance of harnessing such knowledge and data to advance post-incident monitoring guidelines and models.

Conventional environmental hazard assessment practices focus on determining intrinsic danger of chemicals via induction of crude apical endpoints. A novel chemical safety assessment paradigm is pioneering the understanding of molecular mechanisms fundamentally driving such endpoints to increase quality of future chemical safety assessment. To achieve stakeholder acceptance of new technologies such as omics to drive discovery of toxicological mechanisms, there is a need for evaluation of experimental approaches tailored towards this end, using environmentally relevant models such as microalgae. The research goal of this thesis was to propose the development, characterization and application of an experimental design and a suitable physical algal exposure system tailored towards hypothesis of acute chemical mode of action (MoA) and key events in adverse outcome pathways (AOPs) driven by time-resolved multi-omics profiling of algae. An experimental design aligned with the AOP framework was delineated, and a customized algal vial exposure system developed and rigorously validated. The developed system generated highly similar metabolomics profiles in a repeat-exposure study, and existing differences could be mitigated via an internal normalization strategy. Markers driving the adverse phenotype after exposure could be reproducibly identified from the study. Applying the experimental design via the developed exposure system, large-scale and highly time- resolved multi-omics (metabolomics, lipidomics and transcriptomics) exposure experiments of Chlamydomonas reinhardtii to the baseline-toxicant chlorobenzene and the herbicide norflurazon were conducted. Acute chemical MoA and draft AOPs were successfully constructed, using generated and retrieved literature data. Results emphasized discrete (primarily chloroplast-based oxidative stress for norflurazon, general metabolic inhibition and membrane-disruptive effects for chlorobenzene) however to a degree shared molecular stress signatures (inhibition of cellular motility, activation of autophagy and apoptosis). Finally, the utility and limitations of the presented discovery approach for application in hazard assessment were discussed, suggesting high utility to future prioritization of testing and to chemical read-across of hazard using omics-signatures, pending further validation of the method, including application to new model toxicants, putative biomarker identification from the omics datasets, and targeted manual validation of the latter.

Choanoflagellates are ubiquitous and one of the most important groups of heterotrophic pico- and nanoplankton. They are the closest relatives to metazoans and are considered a very valuable group for the study of the early evolution, in animals, of some key life traits as important as sexual reproduction or multicellularity. The phylogeny of freshwater choanoflagellates remains poorly understood, with only one major group containing most of the diversity of freshwater cultured species. Conversely, previous studies have estimated a high hidden biodiversity amongst freshwater choanoflagellates that have led to the emergence of clades composed solely of environmental sequences. Such disparities between the known and estimated diversity of freshwater choanoflagellates pose a major barrier towards the understanding of the evolutionary history of marine:freshwater transitions in choanoflagellates. By a combination of exhaustive phylogenetic analysis, ancestral sequence reconstruction, and phylogenetic placement of environmental sequences, the present study has sought to investigate these discrepancies, in order to unravel the evolutionary history of marine:freshwater transitions in choanoflagellates. Furthermore, the present study has focused on the assessment of the tolerance to salinity changes in several species of choanoflagellates, in order to identify candidates for future studies focused on the molecular mechanisms of adaptation to varying salinities. Additionally the present study has involved extensive efforts towards the isolation of new species of choanoflagellates species from rare freshwater habitats, more specifically acidic and hyperalkaline environments. As a result of the analysis carried out, the present study has revealed that three independent colonisations of freshwater environments have occurred throughout the evolutionary history of Clade 2 choanoflagellates. Similarly, the results have identified three major hotspots of hidden diversity within choanoflagellates. Such hotspots are located at basal positions of Acanthoecida, Craspedida, and within Clade 2 salpingoecids. The estimated hidden diversity within Clade 2 salpingoecids is of marine origin and indicates that, within this group, freshwater colonisations events might be much more frequent than previously thought. The estimated hidden diversity of the other two hotspots is of both marine and freshwater origin indicating that multiple lineages of both marine and freshwater choanoflagellates remain unknown and that marine:freshwater transitions have occurred independently several times throughout the evolutionary history of choanoflagellates. The results from the autoecology of Microstomoeca roanoka revealed that this species is a prime candidate for the study of the molecular mechanisms of adaptation to varying salinities in choanoflagellates. Lastly, the present study contains the description of four morphospecies of choanoflagellates, two from hyperalkaline and two from acidic environments.

Here a report shows a new class of biological three-dimensional (3D) metamaterials (MM) architected from M13 bacteriophage, using a facile and scalable evaporative front deposition methodology (EFD). The highly anisotropic structures achieved tuneable negative refraction from the visible to the mid-infrared (MIR) ranges of the electromagnetic spectrum. Optical MM are considered composite materials in which sub-wavelength features, engineered from constituent materials, can control the macroscopic electromagnetic properties of the material. EFD afforded architecture of one-dimensional (1D) arrays of 3D ridges on substrate surfaces, whereby transmission, reflection, optical imaging and modelling were utilised to further characterise topography, and also demonstrate their activity as optical MM, in particular as negative index materials (NIM). Therefore, a new class of biological MM with exciting potential for defence systems and wave guiding over a tuneable wavelength range has been proposed. The high surface area and loading capacity, provided by the hierarchical internal structure, allowed for pre-templating surface modifications of M13 to display functional nanomaterials. This extra functionality of the 3D architecture to act as a colorimetric detection system, highlights the flexibility of the dynamic system to be tailored towards a multitude of research areas, from cloaking to diagnostics and drug delivery.

Tissue engineering is proving a promising tool as an alternative to relying on donor availability in restorative tissue therapeutics. Tissue engineering concepts entail combining cells and materials in vitro to generate 3D parts that replicate in vivo tissue environments. Much focus has been spent on researching biopolymer hydrogels as scaffolds within tissue engineering constructs as there is vast potential in their tailorability to simulate the extracellular matrices within native human tissues. Although many features within hydrogels may be finely tuned when developing tissue engineering implants, such as water content, mechanical properties, and microarchitecture, current studies in this area are seldom able to recapitulate the physicochemical and mechanical gradients that are exhibited in human tissues, within a single construct. Moreover, the hydrogel precursor solutions used in 3D bioprinting tissue engineering constructs are usually manipulated towards enhancing either print fidelity (by increasing solution viscosity) or to preserve the viability of suspended cells (by decreasing the viscosity of precursor solutions). 3D bioprinted tissue engineering constructs therefore often fail to harbour both optimal print fidelity in conjunction with viable encapsulated cells thus leaving the intended tissue engineering ECM environment poorly reproduced. The work presented in this thesis addresses these problems and demonstrates that the success of a tissue engineering construct need not be compromised by the usual trade-off between print fidelity and cell viability and can in fact possess both of these features that are crucial to the function of the part. This technology uses a supporting gel bed within which multiple cell/hydrogel printing solutions are layered to keep the construct in shape prior to solidifying. The mechanisms behind the function of the supporting gel bed as a suspending agent were first analysed, showing that its shear-thinning behaviour allows material to be deposited within it followed by rapid restructuring to uphold the construct shape. A range of contrasting hydrogels and cell types were then 3D bioprinted into multiple layers and configurations, pushing the boundaries of what can usually be achieved when conventionally 3D printing low viscosity solutions onto a planar surface. As an example of the potential of this technique in becoming commonplace in the clinic, a chronic-depth skin equivalent was 3D printed, complete with hypodermis, dual compartment dermis and epidermis. The construct contained gradients in material chemistry, mechanical properties, microarchitecture, and cell phenotype, and served as an example of the capacity that this system holds in generating highly sophisticated human tissue mimics.

Coral reefs are the result of long-term net calcium carbonate (CaCO3) accretion and the balance between carbonate production and erosion, referred to as the carbonate budget. Coral calcification is influenced by environmental conditions (e.g. temperature, depth and ocean acidification) and depends on a symbiotic partnership between the coral and its dinoflagellate algae in the family Symbiodiniaceae. When a coral encounters stress, the symbiosis breaks down resulting in coral bleaching which impacts reef calcification. To effectively monitor reef health, it is essential to determine the current status of reef environments and whether reef frameworks are actively accreting CaCO3 or being eroded away. This study aims to determine reef accretional health in three bioregions: Bahrain (located in the hottest sea on the planet – the Arabian Gulf), the Seychelles (with an environment susceptible to regular El Nino disturbances - Western Indian Ocean) and Indonesia (located within the epicenter of marine biodiversity, the Coral Triangle - Central Indo-Pacific). In addition, genetic diversity of selected coral symbionts is investigated. In light of predicted warming trends, which present a threat to the structural integrity of reefs worldwide, questions arise whether reefs will be able to maintain a positive carbonate budgetary state to keep pace with future sea level rise and maintain the stability of island-nations such as Bahrain, the Seychelles and Indonesia. This thesis explores the past and present trajectories of reefs within these bioregions whilst providing important insights for consideration to model future trajectories of these reefs.

The Trypanosomatidae are a family of parasitic protozoa that infect various animals and plants. Several species within the Trypanosoma and Leishmania genera also pose a major threat to human health. Among these are Trypanosoma cruzi and Leishmania infantum, aetiological agents of the highly debilitating and often deadly vector-borne zoonoses Chagas disease and American visceral leishmaniasis. Current treatment options are far from safe, only partially effective and rarely available in the impoverished regions of Latin America where these ‘neglected tropical diseases’ prevail. Wider-reaching, sustainable protection against T. cruzi and L. infantum might best be achieved by intercepting key routes of zoonotic transmission, but this prophylactic approach requires a better understanding of how these parasites disperse and evolve at various spatiotemporal scales. This dissertation addresses key questions around trypanosomatid parasite biology and spatial epidemiology based on high-resolution, geo-referenced DNA sequence datasets constructed from disease foci throughout Latin America: Which forms of genetic exchange occur in T. cruzi, and are exchange events frequent enough to significantly alter the distribution of important epidemiological traits? How do demographic histories, for example, the recent invasive expansion of L. infantum into the Americas, impact parasite population structure, and do structural changes pose a threat to public health? Can environmental variables predict parasite dispersal patterns at the landscape scale? Following the first chapter’s review of population genetic and genomic approaches in the study of trypanosomatid diseases in Latin America, Chapter 2 describes how reproductive polymorphism segregates T. cruzi populations in southern Ecuador. The study is the first to clearly demonstrate meiotic sex in this species, for decades thought to exchange genetic material only very rarely, and only by non-Mendelian means. T. cruzi subpopulations from the Ecuadorian study site exhibit all major hallmarks of sexual reproduction, including genome-wide Hardy-Weinberg allele frequencies, rapid decay of linkage disequilibrium with map distance and genealogies that fluctuate among chromosomes. The presence of sex promotes the transfer and transformation of genotypes underlying important epidemiological traits, posing great challenges to disease surveillance and the development of diagnostics and drugs. Chapter 3 demonstrates that mating events are also pivotal to L. infantum population structure in Brazil, where introduction bottlenecks have led to striking genetic discontinuities between sympatric strains. Genetic hybridization occurs genome-wide, including at a recently identified ‘miltefosine sensitivity locus’ that appears to be deleted from the majority of Brazilian L. infantum genomes. The study combines an array of genomic and phenotypic analyses to determine whether rapid population expansion or strong purifying selection has driven this prominent > 12 kb deletion to high abundance across Brazil. Results expose deletion size differences that covary with phylogenetic structure and suggest that deletion-carrying strains do not form a private monophyletic clade. These observations are inconsistent with the hypothesis that the deletion genotype rose to high prevalence simply as the result of a founder effect. Enzymatic assays show that loss of ecto-3’-nucleotidase gene function within the deleted locus is coupled to increased ecto-ATPase activity, raising the possibility that alternative metabolic strategies enhance L. infantum fitness in its introduced range. The study also uses demographic simulation modelling to determine whether L. infantum populations in the Americas have expanded from just one or multiple introduction events. Comparison of observed vs. simulated summary statistics using random forests suggests a single introduction from the Old World, but better spatial sampling coverage is required to rule out other demographic scenarios in a pattern-process modelling approach. Further sampling is also necessary to substantiate signs of convergent selection introduced above. Chapter 4 therefore develops a ‘genome-wide locus sequence typing’ (GLST) tool to summarize parasite genetic polymorphism at a fraction of genomic sequencing cost. Applied directly to the infection source (e.g., vector or host tissue), the method also avoids bias from cell purification and culturing steps typically involved prior to sequencing of trypanosomatid and other obligate parasite genomes. GLST scans genomic pilot data for hundreds of polymorphic sequence fragments whose thermodynamic properties permit simultaneous PCR amplification in a single reaction tube. For proof of principle, GLST is applied to metagenomic DNA extracts from various Chagas disease vector species collected in Colombia, Venezuela, and Ecuador. Epimastigote DNA from several T. cruzi reference clones is also analyzed. The method distinguishes 387 single-nucleotide polymorphisms (SNPs) in T. cruzi sub-lineage TcI and an additional 393 SNPs in non-TcI clones. Genetic distances calculated from these SNPs correlate with geographic distances among samples but also distinguish parasites from triatomines collected at common collection sites. The method thereby appears suitable for agent-based spatio-genetic (simulation) analyses left wanted by Chapter 3 – and further formulated in Chapter 5. The potential to survey parasite genetic diversity abundantly across landscapes compels deeper, more systematic exploration of how environmental variables influence the spread of disease. As environmental context is only marginally considered in the population genetic analyses of Chapters 2 – 4, Chapter 5 proposes a new, spatially explicit modelling framework to predict vector-borne parasite gene flow through heterogeneous environment. In this framework, remotely sensed environmental raster values are re-coded and merged into a composite ‘resistance surface’ that summarizes hypothesized effects of landscape features on parasite transmission among vectors and hosts. Parasite population genetic differentiation is then simulated on this surface and fitted to observed diversity patterns in order to evaluate original hypotheses on how environmental variables modulate parasite gene flow. The chapter thereby makes a maiden step from standard population genetic to ‘landscape genomic’ approaches in understanding the ecology and evolution of vector-borne disease. In summary, this dissertation first demonstrates the power of population genetics and genomics to understand fundamental biological properties of important protist parasites, then identifies areas where analytical tools are missing and creates new technical and conceptual frameworks to help fill these gaps. The general discussion (Chapter 6) also outlines several follow-up projects on the key finding of meiotic genetic signatures in T. cruzi. Exploiting recently developed T. cruzi genome-editing systems for the detection of meiotic gene expression and heterozygosis will help understand why and in which life cycle stage some parasite populations use sex and others do not. Long-read sequencing of parental and recombinant genomes will help understand the extent to which sex is diversifying T. cruzi phenotypes, especially virulence and drug resistance properties conferred by surface molecules with repetitive genetic bases intractable to short-read analysis. Chapter 6 also provides follow-up plans for all other research chapters. Emphasis is placed on advancing the complementarity, transferability and public health benefit of the many different methods and concepts employed in this work.

Presently, Tuberculosis (Tb), caused by Mycobacterium tuberculosis (Mtb), remains a global health threat, particularly with the emergence of drug-resistant strains and HIV and Tb co-infection that challenges the current Tb treatment. Preventative measures and several anti-Tb drugs target the lipids of the mycobacterial outer membrane consisting of mycolic acids amongst an array of glycolipids. In Mtb, there are thirteen outer membrane lipid transporters designated mycobacterial membrane protein large (MmpL) proteins. These proteins are also involved in heme acquisition and possibly drug resistance and nitrosative stress adaptation, contributing to mycobacterial survival and pathogenesis. MmpL proteins are structurally and phylogenetically classed as resistance-nodulation-division (RND) transporters that use protonmotive force (PMF) to mediate substrate translocation. Unlike their RND counterparts in Gram-negative bacteria the structure and mechanisms of these transporters in Mycobacteria are yet to be determined. This work aimed to investigate the mechanism of three transport systems: MmpL3, MmpL7 and DrrABC (encoded by mmpL3, mmpL7 and drrABC genes) using molecular modelling, genetics and lipid and protein biochemistry. mmpL3 is essential for trehalose monomycolate (TMM) transport. While mmpL7 is required for the export of two structurally related lipids, phthiocerol dimycocerosates (PDIM) and phenolic glycolipids (PGL). With the use of different membrane extraction agents, we were able to identify and propose improvements for the structural characterisation of MmpL3 and MmpL7. Molecular analysis and the MmpL7 homology model facilitated the identification of residues in the transmembrane and periplasmic domain that were verified by genetics and lipid analysis as critical for PDIM synthesis and transport. Even though MmpL7 is an RND transporter, there was no evidence of conserved PMF sites or a network forming proton translocation channel. Conveniently within the same genetic context is drrC, part of the drrABC cluster, that has a co-dependent relationship with mmpL7 in PDIM transport. Interestingly the overexpression of only drrAB genes function as a multi-drug ATP-binding cassette (ABC) pump. Structural modelling and molecular analysis revealed that drrABC encodes a heterodimeric ABC transporter where drrB and drrC encode the transmembrane spanning domains that create a pore or channel for substrate entry/exit, while drrA encodes the nucleotide binding domain for ATP binding and hydrolysis mediated transport suggesting a potential mechanism for PDIM/PGL transport. Genetics and biochemical analyses verified that all drrABC genes were required for PDIM transport across the membrane. Indeed, this work provides evidence that MmpL7 does not operate as an independent transporter but instead serves as a scaffold linking lipid synthesis and transport. The knowledge gained from such investigations related to MmpL proteins endeavours to aid in better understanding of anti-Tb drug development.

Respiratory tract infections of domesticated livestock is a global issue with a huge economic burden. Bovine respiratory disease presents one of the largest economic issues facing livestock industries and is responsible for the vast majority of illnesses and deaths in cattle. Many bacterial and viral agents are involved in the development of bovine respiratory disease, of which, Mannheimia haemolytica is one of the most prevalent bacterial agents. The interactions between M. haemolytica, respiratory epithelial cells and immune cells, and the factors governing lower respiratory tract colonisation of the pathogen are not well understood. This lack of understanding surrounding the specific events resulting in pathogenesis is hampering the development of successful new therapeutics and preventative measures. Hence, there is currently an urgent requirement for the development of in vitro models of the bovine respiratory tract which can mimic the in vivo environment to gain a more in depth understanding of the pathogenesis of bovine respiratory disease to develop improved strategies for managing this complex disease. In the present study, a co-culture model was developed, comprising differentiated BBECs grown at ALI and bovine neutrophils. The parameters required for successful attachment and migration of neutrophils were optimised including cell density of neutrophils, concentration of stimulants, appropriate pore density of transwell inserts, application of neutrophils into the culture and attachment time of neutrophils to cultures. Following optimisation, the effects of different stimulants on neutrophil migration was assessed. The pathogenic serotype A1 strain of M. haemolytica elicited greater migration of neutrophils across the bronchial epithelium compared to the non-pathogenic serotype A2 strain of M. haemolytica. A leukotoxin mutant strain of M. haemolytica failed to elicit substantial migration of neutrophils compared to the wild type strain. Stimulation of neutrophils with LPS resulted in lower neutrophil migration numbers compared with the pathogenic strain of M. haemolytica. However, migration was greater for LPS compared to the non-pathogenic strain and the leukotoxin mutant. Stimulation of neutrophil migration with BRSV resulted in lower neutrophil migration numbers compared to both pathogenic and non-pathogenic strains of M. haemolytica. The ability of the pathogenic strain of M. haemolytica to adhere to and colonise the bronchial epithelium in the presence of neutrophils and antiserum was also assessed. Colonisation of M. haemolytica to the epithelium was substantially reduced in the presence of either neutrophils or antiserum compared to M. haemolytica alone. Furthermore, substantially lower colonisation was observed in the presence of neutrophils and antiserum together within co-cultures. Experimental in vivo infection was conducted to assess the response of the entire respiratory tract to infection with M. haemolytica, BRSV and to co-infection with these two pathogens. Calves were infected via aerosol with either a pathogenic strain of M. haemolytica, BRSV or co-infected with both M. haemolytica and BRSV. Samples of tissue were removed from the nasal turbinate, pharyngeal tonsil, trachea, bronchi and lung post mortem for histological assessment. Infection with M. haemolytica and BRSV alone led to development of macroscopic lesions and symptoms of pneumonia. However, co-infection resulted in greater severity of symptoms with development of more extensive macroscopic lung lesions. Co-infected animals also exhibited extravasation of cells resulting in large airway obstructions, vascular congestion, epithelial necrosis and alveolitis. Assessment of the relationship between the infection status of the URT and the LRT highlighted inconsistencies, questioning the reliability of utilisation of the URT as a predictor of respiratory disease. Substantial invasion of both the URT and LRT was observed for the majority of infected animals. Conversely, invasion of the upper respiratory tract was observed for a number of mock infected control animals, although the lower respiratory tract exhibited no signs of invasion. Additionally, the applicability of the in vitro BBEC co-culture model was validated through comparison to in vivo assessment of the response to infection. The transport properties and antimicrobial activity of antibiotics frequently used in the prevention and treatment of bovine respiratory disease were assessed within cultures of differentiated BBECs grown at ALI. Five different classes of antibiotics were assessed, fluoroquinolones (marbofloxacin), macrolides (tilmicosin), β-lactams (amoxicillin), tetracyclines (oxytetracyline) and amphenicols (florfenicol). All five antibiotics exhibited differing rates of transport across the bronchial epithelium determined via bioassay analysis and MALDI-TOF MS. The lipophilic drugs, florfenicol, marbofloxacin and tilmicosin exhibited the fastest transport rates. Conversely, amoxicillin and oxytetracyline which have lower lipophilicities, were transported at relatively slower rates. Metaphylactic (preventative) and therapeutic (treatment) application of the antibiotics florfenicol and marbofloxacin to BBECs infected with a pathogenic strain of M. haemolytica was conducted to assess their antimicrobial efficacy. Marbofloxacin provided a more immediate antimicrobial effect with lower numbers of bacteria present within the wash samples than for florfenicol for metaphylactic application and lower colonisation efficiency as well as lower numbers of bacteria present within the wash samples than for florfenicol for therapeutic application. Metaphylactic treatment of infected cell cultures with either florfenicol or marbofloxacin resulted in an immediate reduction in bacterial colonisation of the epithelium. Colonisation of the epithelium was successfully prevented by 24 h, with complete elimination of bacteria from the cultures by 48 h. Therapeutic treatment of infected cell cultures with either florfenicol or marbofloxacin did not result in an immediate reduction in bacterial adherence where an initial increase in colonisation was observed. However by 48 h post treatment, prevention of bacterial colonisation and complete elimination of bacteria from the cultures was observed. The results herein demonstrate the development of an in vitro co-culture model of the bovine respiratory tract for studying the pathogenesis of infectious disease. Infections assessed in vivo validated this co-culture as a model which can successfully mimic the in vivo environment and response to infection which may provide new insight into the interactions between pathogens, epithelial cells and immune cells. In addition, this study demonstrated that cell cultures of the bovine bronchial epithelium can be utilised to study antibiotic transport and activity, presenting a tool for potentially screening new antibiotics.

Actin is a highly abundant structural protein in eukaryotes that is critical for several cellular processes. In the apicomplexan parasite Toxoplasma gondii, actin is critical for the completion of the lytic cycle and, thus, parasite survival. Only recently, actin structures were visualised in Toxoplasma by exploiting actin-chromobodies, revealing an extensive actin network within the parasitophorous vacuole (PV) (Periz et al. 2017). This network consists of intravacuolar filamentous structures that connect individual parasites within the PV. In addition, parasites possess a cytosolic actin centre (cAC) anterior to the nucleus. The study presented here aimed at exploiting actin visualisation to investigate actin dynamics in unprecedented detail in vivo. For this purpose, I established a conditional CRISPR/Cas9 that allows for rapid and efficient gene disruption in Toxoplasma. Combining this system with the actin-chromobody technology granted detailed insights into the actin dynamics in intracellular parasites. I identified the actin depolymerisation factor (TgADF) as an important factor in the disassembly of the intravacuolar F-actin filaments prior to parasite egress from the host cell. Furthermore, this study revealed TgFormin2 to be critical for maintaining the cAC. Since cAC loss severely impaired actin distribution and peripheral actin flow in intracellular parasites, I concluded that TgFormin2 represents a major key player in mediating proper actin dynamics. TgFormin2 also appeared to be important for apicoplast inheritance and positioning. In summary, data presented in this thesis significantly contribute to the understanding of actin dynamics in Toxoplasma. Further insights into apicomplexan actin dynamics will be gained by exploiting the conditional CRISPR/Cas9 technology for phenotypic screening approaches.

Obligate hydrocarbanoclastic bacteria (OHCB) typically dominate microbial communities after marine oil spills due to their distinct ability to utilize hydrocarbons as sole carbon and energy sources. They perform key roles in the natural attenuation of oil pollution, but also have the potential to be used in bioremediation strategies. To understand the enzymes involved in alkane degradation in three key OHCB (Thalassolituus oleivorans MIL-1, Alcanivorax borkumensis SK2, and Oleispira antarctica RB-8), LC-MS/MS shotgun proteomics was performed on cells growing on different hydrocarbons. T. oleivorans grew equally well on all substrates tested (n-C10-n-C32) as it utilised a subterminal alkane oxidation pathway whilst growing on long chain-alkanes, evidenced by significant upregulation of Baeyer-Villiger monooxygenase and an esterase, proteins catalysing ketone and ester metabolism, respectively. Whilst growing on the branched alkane pristane A. borkumensis not only biosynthesised a cytochrome P450, an alcohol oxidase and an alcohol dehydrogenase but also expressed a set of enzymes for β-oxidation of the resultant fatty acids generated during hydrocarbon degradation, overcoming potential steric hindrance. The mechanisms O. antarctica utilises to provide it with ecological competitiveness in cold environments were identified by an increase in spectral counts for proteins involved in flagella structure/output to overcome higher viscosity, flagella rotation to accumulate cells, and proline metabolism to counteract oxidative stress, during growth at 4°C compared to 16°C. Overall, this thesis gives insight into understanding the physiology and ecology of hydrocarbonoclastic bacteria, the process of natural remediation of oil, underpins future ‘omics’ studies, and identifies enzymes with potential industrial applications and may aid rational decisions in the design of future bioremediation strategies for oil pollution.

HNSCC is the sixth most common cancer in the world and over the last decade its genetic landscape has been extensively investigated. However, due to the heterogenic nature of HNSCC tumours, which is complicated by HPV infection, the effectiveness of current therapies and the development of new therapies have been hindered. In this thesis, by using a combination of genome-wide methods, the cis-regulatory network was investigated in HNSCC cell lines and compared to non-tumourigenic primary oral cells. The analyses revealed a common core regulatory network shared by both the HNSCC and non-tumourigenic cells. The network included pathways, which have previously been found to promote HNSCC such as the EGFR, MAPK, TGF-b and WNT pathways. A novel finding was the involvement of the Hippo pathway and its transcription factor effector, TEAD4. The network appears to be driven primarily by the p63-AP1-TEAD4 transcription factors and in conjunction with the mentioned pathways, appears to form the underlying network required for epithelial function but also holds the potential to become deregulated, leading to HNSCC. Disruption of Hippo-driven gene transcription can reduce HNSCC cell viability therefore pointing to potential therapeutic targets and mechanisms to elucidate HNSCC function.

Epstein-Barr virus (EBV) has been associated with several lymphoid and epithelial malignancies, such as Burkitt's Lymphoma and Nasopharyngeal Carcinoma, due to latent infection properties. The only EBV protein produced in all latency profiles of EBV is Epstein-Barr virus nuclear antigen 1 (EBNA1), a protein that forms a homodimer that is responsible for ensuring the replication and persistence of the EBV genome in cells to ensure a lifelong infection. EBNA1 has been explored as a driver for oncogenesis due to its ability to interact with host proteins and cellular promoters, whilst evading the immune system. The EBNA1 domain called the Glycine-Alanine repeat (GAr) causes translation suppression, resulting in the prevention of MHC class I EBNA1 antigen presentation. The GAr mediated self-regulatory properties of EBNA1 may be oncogenic; the full mechanism of which requires elucidation but appears to utilise a host stress response mechanism. This study focuses on revealing possible therapeutic agents for controlling EBV disease through the inhibition of EBNA1 expression or function. In order to inhibit EBNA1 dimerisation function, eight peptides, attached to cell penetrating peptides, were synthesised and designed to mimic the region of EBNA1 responsible for stabilising homodimers. Peptides were applied to human B cell lines to determine toxicity, specificity to EBNA1 cell survival, and the effect on dimerisation through an optimised dimerisation assay. The peptides did not affect EBNA1 specifically and no effect on cell viability or homodimer level was observed. Confocal microscopy revealed that the peptides were unable to enter the nucleus and instead aggregated, indicating they were unsuitable for use as a therapeutic agent. To investigate the GAr mediated self-inhibition mechanism, epithelial cell lines were transfected with GAr containing or lacking EBNA1 constructs, and cellular protein expression level evaluated. Pathway analysis and the evaluation of protein expression changes in latency I and III cell lines and Eμ-EBNA1 transgenic tumour cells revealed PI3K signalling components, C-MYC, E2F1, and MDM2 as candidate host proteins involved in this mechanism. Drug inhibition of proteins involved in these pathways and the effect on EBNA1 was analysed in human BL cell lines and Eμ-EBNA1 tumour cells in culture. C-MYC, PI3K, and MDM2 inhibition caused a reduction in EBNA1 expression, with PI3Kδ inhibition appearing to cause cell death in EBNA1 dependent cell lines. CAL 101, a PI3K p110δ inhibitor that is FDA approved, was then analysed for potential therapeutic properties in vivo using transplanted Eμ-EBNA1 tumour cells that were tagged with RFP to facilitate analysis. Whilst PI3K inhibition was successful in causing Eμ-EBNA1 tumour cell death in culture, this was not translated in vivo. Two in vivo studies, one that targeted dissemination of tumour cells and the other for treating established tumours, both resulted in progressed but variable tumour infiltration that did not differ significantly from the vehicle treated controls. The link between the proteins MDM2, E2F1, C-MYC and PI3K reveals new therapeutic routes to target EBNA1 function and suggests that MDM2 may be involved in the EBNA1 self-inhibition mechanism. In summary, these findings contribute to understanding the mRNA translational stress response caused by the GAr of EBNA1, which acts to increase proliferation signals in EBNA1 containing tumour cells.

In 2017, an estimated 219 million people became infected with a Plasmodium parasite, resulting in malaria. Of the 435,000 deaths, 61% were in children aged 5 years and under. With increasing incidences of resistance to standard anti-malarial therapies, and the unprecedented failure of rapid diagnostic tests in Sub-Saharan Africa, there is an onus on the scientific community to identify new mechanisms by which parasites of the Plasmodium genus can be detected and targeted for eradication. In the present study, the epigenetic mechanisms by which the rodent malaria parasite, Plasmodium berghei, regulates its transmission were examined. In malaria parasites, transmission from a mosquito vector to a mammalian host is mediated by a stage of sexual development known as gametocytogenesis. Using reverse genetics approaches, in combination with fluorescence-assisted cell sorting, a previously undetermined role for histone acetyltransferase 1 (HAT1) in Plasmodium intraerythrocytic growth and gametocytogenesis was determined. In a similar manner, histone deacetylase 1 (HDA1), a protein previously shown to be transcribed alongside proteins AP2-G and LSD2 prior to schizont egress, was determined to be essential to optimal asexual growth of P. berghei. In addition to these functional studies, acid extraction of histones and tandem mass spectrometry were used to determine the array of histone post-translational modifications that define the mature asexual schizont from sexual-stage gametocytes. To examine some of these sex-specific histone modifications further, a novel chromatin-immunoprecipitation/high-throughput sequencing technique, called 'ChIPmentation', was used to identify the genomic targets of four epigenetic modifications in mature asexual parasites, with a view to future comparison of these histone marks to those found at gametocyte stage. This study also represents the first time that H3K122 acetylation has been examined at any stage in any Plasmodium species. The insights gleaned from this study add to our current knowledge of the landscape of epigenetic modifications and their regulatory enzymes at asexual and sexual stages of Plasmodium parasite development.

The annexin superfamily is comprised of a range of Ca2+ dependent phospholipid binding proteins. Each of these proteins are metamorphic, which means that they possesses the capacity to dynamically and reversibly refold themselves into two or more stable conformations. Annexin A5 (ANXA5) has been linked with a variety of cellular functions including: endocytosis, Ca2+ transfer, anticoagulation, membrane repair and exocytosis. Although this protein has been characterised as having pH-dependent and Ca2+-dependent phosphatidylserine (PS) binding the full extent of these mechanisms are not understood. In this study the changes in ANXA5 conformation as it transitions through its binding states was investigated. Recombinant human ANXA5 was produced in E. coli and subjected to analysis by circular dichroism (CD) and Förster resonance energy transfer (FRET). This revealed that the Ca2+ driven metamorphic refolding of ANXA5 involves minor adjustments of the protein to facilitate trimerisation upon PS binding. However, these experiments also revealed that ANXA5 at acidic conditions undergoes a significant change in conformation upon exposure to liposomes. When under these conditions ANXA5 was demonstrated to bind both PS-containing and PS-free liposomes independent of Ca2+. This indicates that ANXA5 membrane association at acidic conditions relies upon hydrophobic interactions. This variance between the Ca2+- and pH-dependent binding mechanisms is suggestive of alternate cellular localisation and function. Furthermore, this study also examined the mechanisms behind the Ca2+ channel activity of ANXA5. In particular, the necessity for ANXA5 trimerisation. This investigation demonstrated that Ca2+ flux could be readily induced, with a delayed onset, across PS-containing liposomes in the presence of nanomolar concentrations of ANXA5, but was lost when trimer formation was disrupted. Furthermore, this investigation also observed the formation of a transmembrane ANXA5 complex by changes in the rate of FRET across a membrane. Similar to the ANXA5-mediated Ca2+ flux this structure was lost upon the prevention of ANXA5 trimerisation, which suggests that the ANXA5 trimer is able to sufficiently alter the structure of the membrane to allow for the formation of a Ca2+ channel.

Leishmaniasis is a neglected tropical parasitic disease that causes several debilitating manifestations. No commercially available vaccine exists against this disease, and treatment strategies are far from ideal with the emergence of resistance, coupled with toxic side effects of many of the drugs available. Rational drug design relies on knowledge of the cell biology of the parasite and the interplay between the parasite and its hosts. Production of secreted proteins, the secretome, has become a known strategy for parasite invasion and persistence in host cells, however, host-parasite interaction is still not well defined. Virulence factors secreted by the parasite mediate the host-parasite interaction and create a niche permissive for parasite proliferation. They therefore represent potential therapeutic targets and vaccine candidates. Here, the use of secretomics was implemented to investigate these virulence factors. Parasite conditioned culture supernatant, containing the secretome, was characterised by morphological, immunochemical and proteomic analyses. Here, we optimised and extended current methods and applied them to the medically relevant amastigote stage. Method development and validation was implemented to extract a reproducible secretome in vitro. Induction of cell stress was managed and cell viability maintained to minimise interference of intracellular proteins. A total of 256 proteins were reproducibly identified in the secretome of promastigotes and 36 proteins were reproducibly identified in the secretome of amastigotes. Analysis of their protein abundance index (emPAI) allowed comparison of the relative abundance of proteins and functions of the secretome throughout the parasite life cycle. Differences in the putative functions of nutrient salvage, protease production and antioxidant activity were observed. Analyses reveal that many proteins lack a signal peptide and as such are thought to be released by nonclassical secretion mechanisms. Several exosome-associated proteins and membrane proteins were also detected in the secretome, suggesting the occurrence of secretion by exosomes or microvesicles. Extended comparative analyses between the secretome of parasites with differing phenotypes allowed us to infer functionality of the secretome in the parasite's survival but also variations within the same species which result in differing disease outcomes. Dysregulation in the secretion of various proteins in attenuated parasites implicates these proteins in the virulence of the parasite. An increase in the secretion of pro-inflammatory mediators and destructive proteases by parasites isolated from patients with chronic cutaneous leishmaniasis compared to those from patients with self-healing lesions, indicates the role of the parasite in the chronicity of cutaneous leishmaniasis. Here, we demonstrate an applicable method for the study of the Leishmania mexicana promastigote and amastigote secretome. Results suggest that the secretome plays a role in disease progression and virulence. Proteomic analyses of the secretome, like this study presented here, provide crucial information on the host:parasite interaction for the identification of therapeutic targets and potential vaccine candidates for the provision of safer treatments and new vaccines for eradication of this disease.

Background: Cardiovascular disease (CVD) is currently the leading cause of mortality world-wide, responsible for approximately one-third of all global deaths. Atherosclerosis, the primary cause of CVD, is a chronic inflammatory disease characterised by lipid accumulation in the arterial wall. Despite the success of current therapies, the incidence of CVD continues to rise and the search continues for alternative therapeutic agents. The Lab4, Lab4b and CUL66 probiotic consortia have been shown to possess lipid-lowering and immunomodulatory capabilities, highlighting their anti-atherogenic potential. The aim of this project was to assess the anti-atherogenic effects of Lab4, Lab4b and CUL66 probiotic consortia using in vitro and in vivo model systems. Results: Experiments using in vitro model systems demonstrated probiotic-induced attenuation of macrophage foam cell formation at the cellular and molecular levels. In addition, probiotics reduced monocyte migration, increased phagocytosis, and reduced proliferation of key atherosclerosis-associated cell types. Mice supplemented with a live combination of Lab4 and CUL66 demonstrated reduced atherosclerotic plaque formation, and an improved plasma lipid profile compared to the control. In addition, numbers of macrophages and T-cells were reduced in both the plaque and the bone marrow of probiotic-treated mice. Liver gene expression analysis revealed probiotic-induced attenuation of several key pro-atherogenic genes. Conclusion: Findings from this study demonstrate many anti-atherogenic effects of Lab4, Lab4b and CUL66 probiotic consortia in both in vitro and in vivo model systems, and highlight their potential as candidates for inclusion in clinical trials and future atherosclerosis intervention strategies. Underlying mechanisms of action have also been investigated and proposed, thereby contributing to the understanding of probiotic action.

Copper (Cu) is essential to the growth and the morphological development of Streptomyces lividans. Understanding how Cu regulates the key development switches in this Gram-positive bacterium has been an area of extensive research. In particular, how Cu homeostasis and regulation are controlled and how metalation of enzymes important for morphological development is achieved have been previously investigated. To this end, this thesis reports the discovery of a cytosolic copper storage protein in S. lividans and offers new insight into intracellular Cu regulation, which is not under direct control of the Cu regulator protein CsoR (copper sensitive operon repressor). This copper storage protein belongs to a family of recently discovered cytosolic proteins known as Csp3. These members are exclusively found in the bacterial cytosol and comprise of a four-helix bundle that assemble into homotetramers and can bind between 70-80 Cu(I) ions through mainly Cys thiolate coordination. In Chapter 2 bioinformatic analyses reveals the phylogenetic distribution of Csp3 across Bacteria and Archaea and confirms the presence of Csp3 in S. lividans. Furthermore, the Csp3 in S. lividans is located in a gene environment that is sensitive to elevated Cu levels. Taxonomic distribution of these genes reveals a possible link to a novel transmembrane Cu export system that could facilitate removal of Cu from Csp3. X-ray structures of the apo and Cu(I) bound forms of the Csp3 from S. lividans have been determined and confirm a homotetramer assembly that can bind up to 80 Cu(I) ions (Chapter 3). The binding of Cu(I) ions in Csp3 is found to be cooperative with a Hill coefficient of 1.9 and Cu(I) can be transferred to Csp3 from a CopZ-like Cu(I) chaperone (Chapter 3). A Δcsp3 null-mutant in S. lividans reveals that Csp3 is operable at high Cu levels and this suggests it acts to provide an additional level of protection against Cu toxicity once the CsoR system becomes saturated (Chapter 3). The mechanism of Cu(I)-loading to Csp3 has also been investigated through X-ray crystallography, site-directed mutagenesis and stopped-flow reaction kinetics using aqueous Cu(I) and Cu(I) chelated by a donor. A clear role for a His residue (His107) leading to the formation of a tetranuclear [Cu4(μ2-S-Cys)4(Nδ1-His)] cluster is observed, followed by the loading of Cu(I) in a fluxional and dynamic manner (Chapters 4 and 5). Finally, over-expression studies of a putative transmembrane protein (SLI_RS17250) that is encoded by a neighbouring gene to the S. lividans Csp3 gene and could be part of a novel Cu export system, identified in Chapter 2, is described (Chapter 6).

Decomposing meat produces a characteristic profile of Volatile Organic Compounds (VOCs) that changes as a function of time. This has been considered by the forensic community as a possible means of estimating Post Mortem Interval (PMI) and thus could be of importance in criminal investigations. Previous work has focussed on obtaining VOC profiles for the decomposition stage and the key chemicals identified include alcohols, aldehydes, acids and sulphur compounds including Dimethyl disulphide (DMDS) and Dimethyl trisulphide (DMTS). The type and levels of these VOCs change throughout the decomposition of mammalian cadavers and are of considerable interest in the forensic community in a range of areas including 'time profiling' as a means of estimating PMI, locating clandestine graves and to assist in the training of Victim Recovery (VR) dogs. VOCs can accumulate in the air surrounding a cadaver (the 'headspace') and the current research focussed on methods to collect, detect and identify these. This research was carried out using Thermal Desorption Gas Chromatography Mass Spectrometry (TD-GC-MS) and Headspace Gas Chromatography Mass Spectrometry (HS-GC-MS).TD-GC-MS uses an adsorbent tube and pump system to concentrate a sample and HS-GC-MS uses a sealed vial system to introduce and analyse solid or liquid without the requirement for extensive sample preparation. GC-MS was used for this study because of its ability to combine separation and identification, which is particularly appropriate for complex matrices, such as those seen in the decomposition processes. A combination of pure chemicals and pork were used to develop methodology for TD-GC-MS and HS-GC-MS where the sampling parameters (such as mass of meat, size on container, presence of insects, type of substrate) were investigated in detail. No Certified Reference Material (CRM) was available so a standard mix of chemicals was devised based on a literature review and the results of preliminary experiments. For pork studies, it was found that optimum results were obtained using 500 g pork samples placed on sand as a substrate and contained within clip lock plastic boxes. The results confirmed those of other workers that the accessibility of insects to the meat plays an essential role for a realistic decomposition profile. A key aim of this project was the development of Standard Operating Procedures (SOPs) for the detection of VOCs produced in the headspace above a cadaver during its decomposition. SOPs are a way of reproducing methodology of an, often complex, but routine activity. The SOPs were developed as an iterative process throughout the project and then further tested and validated using repeatability studies and intra-laboratory evaluation. Validation studies involved repeatability studies by the author and two competent analysts. It was shown that the SOPs fulfilled their basic requirement in terms of ease of use and suitability for detecting VOCs associated with decomposition. The robustness of these SOPs was successfully demonstrated using chemically similar systems in the phyto-kingdom. The robustness was evaluated by applying them, after some adaptation to the sample collection procedure, to plants that mimic decomposition by producing chemicals that attract insects. The presence of DMDS and DMTS was detected in plants known to attract blowflies which are of key entomological importance in decomposition events. The research was extended to carnivorous plants particularly the lifecycle of Sarraceniaflava, which has not been previously studied in this way. In addition to the work on plants, a short study on a commercial odour reduction system was undertaken which further confirmed the scope of the SOPs. The SOPs were found to be applicable to these systems and the robustness was shown by being able to withstand minor modifications while still being fit for purpose. Overall, limitations were seen in terms of repeatability for the technique and due to the complexity of the decomposition matrix. However, the SOPs showed great promise to be used as a starting point for forensic practitioners to test their own methodology and instrumentation for decomposition studies.

For almost a century, antibiotics have been used as treatment agents in the combat of infections caused by pathogenic microbes. For a short while, antibiotics were heralded as miracle drugs; their discovery and introduction into the clinic has no doubt saved countless animal and human lives. The use of antibiotics in surgical procedures has ushered in the modern medical era where surgeons are afforded a whole host of cutting edge treatment and ground-breaking surgical techniques thanks to the benefits and treatment options antibiotics offer. The dark times of the pre-antibiotic era where many lives were lost through trivial illnesses have long been forgotten. However, what was once a trickle of reports of antibiotic resistance has since turned into a flood of pandemic proportions; the once lethal actions of antibiotics are threatened by the seemingly unstoppable march of resistance. Pathogenic microbes, once susceptible to the killing mechanisms of antibiotics, are now equipped with several mechanisms capable of shrugging off the previously deadly antibiotics. The main weapon in our antibiotic armamentarium is β-lactam antibiotics, owing to their cheapness, efficacy, and limited side effects. It is perhaps unsurprising then that the main driver of antibiotic resistance is by the production of β-lactamases, enzymes capable of efficiently catalysing the hydrolysis of the β-lactam ring present in β-lactam antibiotics, rendering them useless. Unfortunately, soil which was once a rich source of novel antibiotic producing bacteria has long been described as an over-mined resource and attempts to match the success of natural and semi synthetic antibiotics with truly synthetic antibiotics has largely been a failure. It is known that the majority of bacteria in situ are unculturable in laboratory conditions. The work in this thesis explores novel methods to increase the in vitro growth rate of bacteria in an attempt to revitalise traditional antimicrobial discovery methods, by use of a novel incubation tool known as the isolation chip or "iChip" for short. The iChip is designed in the hope to increase the in vitro growth rate of previously unculturable bacteria. The work in this thesis describes the characterisation and antimicrobial activity of a previously little described strain of Pseudomonas baetica, a bacterium of little interest, only noted for its pathogenicity towards certain species of fish. Given the dearth of novel antibiotics, β-lactamase inhibitors represent an alternative mechanism for overcoming antimicrobial resistance as compared to novel antimicrobials. This thesis also explores the potential of novel β-lactamase inhibitors, in the hope of extending the shelf life of existing "resistance vulnerable" β-lactam antibiotics. Several ring-opened carbamate ester analogues of avibactam, a novel β-lactamase inhibitor, were synthesised and their effectiveness as serine-β-lactamase inhibitors were assessed. One of the inhibitors was found to be even more effective than avibactam itself. Also, the effect of pH upon inhibition of serine-β-lactamases by these inhibitors and the catalytic activity of these enzymes was measured showing that the catalytic machinery is the same for both hydrolysis and inhibition. Finally, several other existing compounds were tested for their inhibitory effectiveness against β-lactamases. Ellagic acid was found to be a good inhibitor of serine-β-lactamases and showed an interesting inhibitory profile dependent upon pH demonstrating that both the mono-and di-anions are effective inhibitors.

In this work, the intramolecular cycloaddition of furan dienes was investigated. At equilibrium, amide-tethered substrates form acyclic precursor : cycloadduct mixtures. Higher portion of cycloadduct proved to be favoured by bulky nitrogen-groups, polar solvents, higher temperature and the presence of some Lewis acids. It was demonstrated that the equilibrium can be displaced by coupling a reversible Diels-Alder cycloaddition to an irreversible and chemoselective reaction, such as Pd-catalysed hydrogenation or mCPBA epoxidation, resulting in the complete transformation of the mixture starting material to the functionalised cycloadduct. A range of asymmetric transformations were also investigated to resolve the racemic mixture derived from the Diels-Alder reaction. The Sharpless dihydroxylation showed a high level of chemoselectivity but it was not able to discriminate between the cycloadduct enantiomers. The Jacobsen epoxidation was not chemoselective but, in absence of Diels-Alder starting material, produced cycloadduct epoxides in modest enantioselectivity via kinetic resolution and dynamic kinetic resolution. Non-symmetrical transformation of the olefin, by asymmetric hydroboration-oxidation, provided regioisomeric alcohols in good enantiomeric excess by regiodivergent resolution.

Pseudomonas aeruginosa is a Gram negative pathogenic bacterium that has the notable ability to inhabit a broad range of environments, including humans as an opportunistic pathogen. P aeruginosa secrets outer membrane vesicles (OMVs) which contain virulence factors such as: pro-elastase, hemolysin, phoshpolipase C, protease, alkaline phosphatase and B-lactamase that can damage the cells of host and also other bacteria. P aeruginosa commonly colonises wound beds and this can result in the development of a chronic wound which is an important cause of major pathology. Two strains of P aeruginosa PS3 (hospital strain), which was isolated from the dressing of an established chronic wound and a reference laboratory strain (L) that had no known pathogenic factors were cultured in different media including a simulated wound fluid. The secreted extracellular products were isolated and used to test their virulence on a cultured human keratinocyte cell line (HaCaT) and primary human keratinocytes (NHK). Vesicles derived from the outer membrane of the bacteria were isolated and the protein expression in these outer membrane vesicles (OMV) were compared between the two strains of P aeruginosa and with the outer membranes isolated from both strains of bacteria. The virulence of these vesicles and the outer membrane was tested on the keratinocytes. The initial part of the study demonstrated that HaCaT cells grown for either 4 or 10days expressed a range of TLRs required for the recognition and response to a wide range of bacterial antigens TLR1, TLR2, TLR4, and both aged cells. In contrast TLR5, TLR9 were found just in 10 days cells. The expression of a range of TLRs was investigated and the most expression at the mRNA level was found for TLR2 and TLR4 when the cells were grown for either 4 or 10 days. Both strains of P aeruginosa produced OMVs that appeared to contain a similar protein profile and this was similar to that of the isolated outer membrane. The yield OMVs from PS3 and the lab strain was generally similar but was higher when the bacteria were treated with gentamycin before isolation and also when the bacteria were grown in the presence of ethanol. Cultured keratinocyte cells secreted of IL-8 in response to exposure to cell free extracellular material from both strains P aeruginosa with little effect on the cell biomass. HaCaT cells produced a much higher concentration of IL-8 in response to OMVs than did NHK or an adapted more proliferative derivative of HaCaT (HaCaTa) with OMVs from the hospital strain stimulating slightly more IL-8 secretion compared to lab strain. NHK also produced significant amounts of IL-8 in response to challenge and with OM of PS3 or the lab strain. P. aeruginosa grown in SWF produced OMVs that stimulated a greater production of IL-8 in keratinocytes comparted to bacteria grown in normal bacterial broth (TSB). These data indicate that P aeruginosa which are a common bacterium isolated from the bed of chronic wounds secrete virulence factors that stimulate an inflammatory phenotype in cultured keratinocytes. This pathogenic response is largely driven by the secretion of vesicles derived from the outer membrane and the hospital strain has a slightly greater pathogenicity than the lab strain this has implications for the treatment of wounds.

The presence of hypoxia within tumours has significant biological and pharmacological implications leading to resistance to therapy and poor prognosis. The eradication of hypoxic cells has been a major goal for many years but despite decades of research, no hypoxia specific therapies have been approved for use in humans. The purpose of this thesis is to explore the possibility of targeting the glycolytic phenotype of cancers with the aim of specifically eradicating hypoxic cells. Initial studies using a series of glycolytic inhibitors (3-bromopyruvate, 2-deoxyglucose and sodium oxamate) did not show hypoxia selectivity in vitro and in several cell lines, resistance under hypoxia (0.1% oxygen) occurred. In the case of the pyruvate dehydrogenase kinase 1 (PDK1) inhibitor dichloroacetate (DCA) however, hypoxia selectivity was observed in vitro but the magnitude of the sensitisation was much less than that of the hypoxia activated prodrug tirapazamine. Nevertheless, DCA did inhibit PDK1 leading to reduced phosphorylation of the pyruvate dehydrogenase complex and hypoxia selective cell kill. These results questioned whether glucose was indeed important for the survival of hypoxic cells but studies where glucose was removed from culture media confirmed that glucose is required for the survival of hypoxic cells in vitro. Using this model, mannose was also shown to be an important monosaccharide that could support the growth of cells under hypoxia opening up a novel avenue for future research. Finally, a series of novel organometallic complexes based on the metal silver were explored as potential glycolytic inhibitors. The silver N-heterocyclic carbene Ag8 was shown to be a potent and selective inhibitor of lactate dehydrogenase A (LDH-A) leading to reduced export of lactate from cells. Ag8 has a complex mechanism of action with inhibition of LDH-A being a novel mechanism. In conclusion, this study has demonstrated that targeting key enzymes in the glycolytic pathway does not generate hypoxia selective cytotoxic effects. This conclusion is surprising and reflects the complexity of targeting cellular metabolism. The identification of mannose as an important fuel for hypoxic cells opens up new avenues for research and the inhibition of LDH-A by organometallic complexes such as Ag8 requires further studies.

In cereal-based biorefineries, Distillers Dried Grains with Solubles (DDGS) is the main by-product, which is used in relatively low value animal feed formulations. A major component of DDGS is arabinoxylan (AX), which is a potential food ingredient and source of novel prebiotics. The production of AX uses large amounts of ethanol, which gives scope for feasible integration of AX extraction within a biorefinery. Meanwhile, enzymatic production of prebiotic arabinoxylan oligosaccharides (AXOS) and xylo-oligosaccharides (XOS) is of interest to animal feed formulators, as these prebiotics enhance feed conversion significantly. The integration scenario would be implemented on the in-process streams, the Distillers Wet Grain (DWG) and the Solubles before they are combined. However, these are not readily available to study, as biorefineries are closed processes that do not allow in-process sampling of these streams. Therefore, this project used the GUNTCE-640 bioethanol unit to produce wet by-products representative of commercial DWG. Fermentation often batches of 6kg of wheat yielded on average 1275g (db) of DWG with 16% AX content and 800g of dry Solubles with 11.4% AX content. Enzymatic treatment of the DWG with commercial endoxylanase yielded less than 3%w/w of the prebiotic oligosaccharides. AX polysaccharides were extracted by alkaline oxidation, with and without further enzymatic purification, to give AX contents of 44% and 19%, respectively. Enzymatic treatment converted only 6% of the AX into AXOS/XOS, with significant production of undesired monosaccharides. Limitations in the analytical methods used prompted the development of a new HPAEC-PAD method for the simultaneous measurement of mono-and oligosaccharides and uronic acids. The new method was used to quantify the profiles of XOSin commercial materials and following enzyme treatment of biomass materials and oligosaccharide standards up to DP6, the latter to reveal the xylanase mode of action. The commercial xylanase investigated showed a preference for larger XOS molecules and was incapable of acting on branched (AXOS) molecules. Two types of AX with different branching patterns were produced; highly branched AX was extracted chemically from DWG with further purification to give 51% w/w purity, and less branched AX was produced from the Solubles fraction by ultrafiltration to give 74% purity. Enzyme treatment converted 46% of the Solubles AX into oligosaccharides, showing this to be a promising feedstock for XOS production.

The family Hantaviridae, of the Bunyavirales order, contains many important human pathogens of which Puumala virus (PUUV) is the most widely distributed member in Europe. It causes nephropathia epidemica, a milder form of haemorrhagic fever with renal syndrome and mortality rates of up to 1% have been reported. They are enveloped viruses, with a tripartite single-stranded negative sense RNA genome, that replicate solely in the cell cytoplasm. Several factors have been proposed to play a role in hantavirus pathogenicity, including regulation of innate immune responses, cell signalling and enhancement of endothelial cell permeability. The work presented in this thesis describes biological and molecular characterisation of the mechanisms behind a hantavirus infection. Transcriptome analysis was a valuable tool that allowed the investigation of the broader picture of the effect of PUUV on the host cell. 549 and as many as 7,283 genes were differentially expressed at 24 and 48 hours post infection, respectively, in PUUV-infected cells, revealing extensive transcriptional change. By 48 hours normal cellular function appeared severely disrupted. Most genes involved in mitochondrial functioning were down-regulated, suggesting a reduced cellular energy level. Dysregulation of an important signalling hub such as mitochondria might have a more global impact on cellular functions, consistent with findings in this study. Intrinsic apoptosis pathway, which is mediated by mitochondria, appeared inhibited. Whereas, death receptor signalling was activated. Pathways associated with actin formation, organisation and signalling also appeared inhibited. Members of Rho family of GTPases, which are key regulators of actin dynamics, were down-regulated overall. Furthermore, integrin signalling, which mediates Rho GTPase activity, was also inhibited. Immunofluorescence studies revealed marked morphological changes in mitochondria and substantial remodelling of the actin cytoskeleton. Further analysis revealed a direct interaction between PUUV N protein and anillin, a scaffolding protein that mediates formation and organisation of actin filaments, suggesting a potential novel mechanism behind actin cytoskeleton reorganisation. Biological interferon (IFN) assays enabled the identification of two IFN antagonists encoded by PUUV, the cytoplasmic tail of the Gn glycoprotein and the non-structural protein, NSs. The Gn tail inhibited type I IFN induction at the level of TRAF3-TBK, in agreement with previous studies on other hantaviruses. Whereas, NSs was found to block IFN induction downstream of IRF3, suggesting it was able to disrupt transcription or translation. Utilising immunofluorescence and chromatin immunoprecipitation methods, it was found that PUUV NSs possessed a potential mechanism to inhibit transcription by blocking serine 2 phosphorylation at the C-terminal domain of RNA polymerase II in a similar manner to the previously described Bunyamwera virus NSs. The data presented in this thesis illustrates the broad range of mechanisms employed by PUUV to alter cell function to aid virus replication and subvert innate immune responses.

Polar regions are subjected to rapid climate change, with increased air temperatures and precipitation being predicted during future decades. Rising temperatures and precipitation will have an effect on saprotrophic soil fungi, microbes key to nutrient cycling and decomposition processes that are dominant in polar soils owing to their abilities of remaining physiologically active at low temperatures and water availabilities. Here, a combination of field warming experiments and laboratory experiments are used to investigate the effects of warming, water and nutrient availability on the abundance, growth and enzyme activities (cellulase, chitinase, acid and alkaline phosphatase and leucine aminopeptidase) of a range of saprotrophic fungi in Arctic and Antarctic soils. In a five-year-long maritime Antarctic field experiment, the abundance of Pseudogymnoascus pannorum DNA was reduced in soil warmed with open top chambers (OTCs) that had been enriched with nutrients. Laboratory experiments confirmed the inhibitory effect of warming to > 21 °C on the growth and enzyme activities of P. pannorum, but only when water was not a limiting factor. In contrast, in an Arctic field experiment, OTCs and watering had no effects on the abundance of DNA of seven Arctic soil fungal taxa after three years of treatment. The growth of five Arctic taxa (Acremonium sp., Isaria sp., Leptosphaeria sp., Phialocephala sp. and Mortierella spp.) was increased by warming to > 21 °C, with only that of P. pannorum being inhibited by warming to 24 °C. Warming did not affect the enzyme activities of Arctic fungi, with activities mainly being influenced by changes in water potential. The research here suggests that the growth and enzyme activities of Antarctic P. pannorum may be inhibited by future warming arising from continued greenhouse gas emissions to the atmosphere, but that Arctic saprotrophic soil fungi appear to be more resilient to environmental changes.

The activities of competing wood decay fungi bring about the decomposition of deadwood. The rate of decomposition is determined by the community composition, which is shaped by competitive interactions. Fungi compete with one another for territory and resources within 3 dimensional space via the production of an arsenal of inhibitory chemical compounds, yet interspecific interactions and chemical warfare within communities have never before been studied in 3 dimensional resources. This thesis, therefore, aims to determine how interactions cause metabolic processes to change, and, by the use of novel 3 dimensional systems, how those processes are affected by increased species diversity and spatial heterogeneity. Overarching hypotheses which have driven investigations are: (1) secondary metabolism and interaction outcome are inherently linked, both of which are affected by environmental conditions, (2) changes to spatial distributions and increased species diversity cause interaction outcomes and community dynamics to alter, and (3) changes to metabolic strategies for antagonism and resource utilisation will reflect alterations to community dynamics. Transitive interactions in pair wise 2 dimensional systems often became intransitive in species richer 3 dimensional systems, and the extent of patch fragmentation determined the length of coexistence of individuals within communities. Production of secondary metabolites comprising the fungal chemical weaponry, namely extracellular lignocellulolytic enzymes and volatile organic compounds (VOCs), was significantly different between 2 and 3 dimensional systems, between systems of varied species diversity, and between different spatial distributions of fungi, reflecting the observed changes to community dynamics. Furthermore, the production of intracellular metabolites which function in the biosynthesis pathways of antimicrobial compounds and agents of decay, was affected by species diversity, spatial distributions and territory fragmentation. Additionally, secondary metabolism and interaction outcome were found to be inherently linked, and affected by abiotic conditions. Specifically, interaction outcomes changed under different temperatures, and when wood was pre colonised for different lengths of time. Changes to the production of both enzymes and VOCs reflected the different outcomes, as either the cause or effect of outcome changes. These conclusions not only confirm the thesis hypotheses, but also demonstrate the importance of environmental conditions, species diversity and spatial dynamics to fungal community dynamics and forest ecosystem functioning.

Chitosan(CS), a natural cationic polymer obtained by the partial N-deacetylation of chitin, has been investigated widely for its potential in the development of food and drug delivery systems and pharmaceutical applications, however it has not generally been considered in forensic applications for example fingerprints (fingermarks). The purpose of this study was to prepare chitosan micro/nanoparticles through cross-linking with tripolyphosphate (TPP) utilising the ionotropic interaction between positively charged amino groups (CS) and negatively charged counter ions (TPP). The investigation into the potential of these particles was divided into two parts: forensic and pharmaceutical applications. Firstly, these formulations were characterized (relative viscosity, zeta potential, particle size, FT-IR, XRD, SEM) and evaluated for forensic applications (fingermark visualisation). This can be controlled by the charge density of CS and TPP, which depends on the pH and ionic strength of the solution. Secondly, the combined effects of three independent variables (pH, ionic strength and CS: TPP ratio) on three important physico-chemical properties (viscosity, zeta potential andparticle size) during the preparation of microparticles were investigated. CS: TPP microparticles (CSMPs) were prepared using experimental design and equations were generated and used to predict relative viscosity, zeta potential and particle size under different conditions. This gives us the ability to design tuneable CS: TPP microparticles with desired size for specific pharmaceutical or forensic applications e.g. latent fingerprint visualisation. Fingerprints are a very common form of physical evidence. The most commonly used procedure for revealing the ridge pattern is powder dusting, which relies on the mechanical adherence of fingerprint formulation to the fatty components of the skin deposit that are secreted by sweat pores that exist on friction ridges. The development of latent fingermarks using IICSMPs was analysed by using a 23 factorial design, which considered simultaneously three main factors: pH, ionic strength and CS: TPP (v/v) ratio. CS: TPP ratio has the strongest effect on fingerprint quality. The best conditions for fingerprint visualisation were microparticles prepared using a buffer of pH 4.8, 0.2 M ionic strength at a CS: TPP of 2:1. Although we have demonstrated that CSMPs can be used to develop latent fingermarks there are limitationsin that they are only applicable as a powder and are only sensitive up to the third depletion level for a fingermark aged for one day. In the final sections of this thesis, chitosan nanoparticles were prepared and characterized for potential applications in drug delivery (using ibuprofen as a model drug) and in terms of their interactions with mucin (mucoadhesion). It has been demonstrated that chitosan nanoparticles can incorporate appreciable quantities of ibuprofen into nanoparticles (CS-IBU-TPP), although the order addition of the individual components is important. The carboxylate ions of the ibuprofen (negative charge) and could bind strongly to the ammonium group (positive charge) of chitosan, thereby allowing greater drug-loading capacity in the chitosan nanoparticles. In addition, the interaction between different ratios chitosan nanoparticles (CS: TPP) and mucin were evaluated based on relative viscosity, zeta potential and particle size. It has been suggested that chitosan nanoparticle-mucin interactions are driven by electrostatic forces. The results conclude that interactions between CS: TPP nanoparticles and mucin occur, with a CS: TPP ratio of 4:1 displaying the strongest interaction with mucin. This is observed through differences in relative viscosity, zeta potential and particle size.

Neuroinflammation and pro-inflammatory mediators play key roles in the pathogenesis of neurodegenerative diseases such as Alzheimer disease (AD). Studies have suggested that oestrogen has anti-inflammatory and neuroprotective activities. However, risks of tumour development, heart disease, stroke and blood clots associated with oestrogen replacement therapy limit its application in neurodegenerative disorders. This has switched interest to the use of alternatives such as the selective oestrogen receptor modulators (SERMs) and phytoestrogens which retain the neuroprotective effects of oestrogen with fewer side effects. Formononentin (FMN) is a phytoestrogen which showed significant anti-inflammatory activity through inhibition of nitric oxide (NO) released from lipopolysaccharide-stimulated mouse macrophage RAW cells. However, the ability of formononetin to produce anti-neuroinflammatory activityin LPS-activated microglia is unknown. In addition, the molecular mechanisms involved in the action of the compound in neuroinflammation is unknown. It is also not clear if the anti-inflammatory and neuroprotective effects of formononetin are mediated through estrogen receptors. This study has evaluated the effects of formononetin on neuroinflammation in BV2 microglia. Also the roles of ERβ activation in the anti-inflammatory activity of formononetin in the microglia was investigated. BV2 microglia were activated with lipopolysaccharide (LPS) with or without formononetin (2.5, 5 and 10μM), followed by measurement of TNFα, IL-6, nitrite and PGE2 levels in culture supernatants. Also, protein levels of iNOS, COX-2, total IkBα, phospho-IkBα, phospho-p65 subunit, phospho-IKKα, phospho-p38,phospho-JNK, phospho-ERK 1/2, phospho-MLK3, phospho-TAK1 and ERβ were detected with immunoblotting. A reporter gene assay was used to evaluate NF-kB and ERβ transcriptional activation in HEK293. Electrophoretic mobility shift assay (EMSA) and DNA binding assays were used to examine the effect of formononetin on DNA binding of NF-kB. HT22 neuronal cells were used to study the neuroprotective activity of formononetin. The role of estrogen receptor in the anti-inflammatory activity of formononetin was investigated with western blotting for ERαand ERβexpression in BV2 microglia The effect of formononetin on transcriptional activity of oestrogen-response element (ERE) was also investigated. Small interfering RNAs (siRNAs) targeted at ERβ were used to silence ERβ geneand levels of TNFα, nitrite and PGE2 were detected in ERβ-silenced BV2 microglia, while immunofluorescence was used to detect NF-κB p65 subunit. Results show that formononetin significantly suppressed TNFα, IL-6 and IL-1β without affecting viability of BV2 microglia cells. Furthermore, formononetin inhibited the production of nitrite and PGE2, and reduced protein levels of COX-2 and iNOS in LPS-activated microglia. In addition, formononetin prevented HTT22 neuronal toxicity produced by microglia-conditioned medium. Formononetin targeted NF-kB activity by inhibiting the phosphorylation of IKKα, IkBα and nuclear translocation of the p65 subunit. The compound also suppressed NF-kB/DNA binding activity in LPS stimulated BV2 microglia. Further results showed that formononetin inhibited p-38, JNK and ERK1/2 MAPKs activation by targeting upstream proteins TAK-1 and MLK3. The study revealed that BV2 microglia express ERβ, but not ERα. The study also demonstrated that formononetin increased transcriptional activity of ERE in HEK293 cells. Results further showed that this compound increased protein levels of ERβin BV2 microglia. RNA interference experiments revealed that NF-kB-mediated anti-inflammatory activity of formononetin was abolished following transfection of BV2 microglia with ERβ siRNA. Taken together, this study has demonstrated for the first time, that formononetin inhibits neuroinflammationin BV2 microglia, and neuroinflammation-mediated neurotoxicityin HT22 mouse hippocampal neurons by targeting upstream convergence proteins in both the NF-kB and MAPK signalling pathways. This study also established that the ability of the compound to interact with microglial ERβ possibly contributes to the anti-inflammatory action of formononetin.

Transposable elements (TEs) are almost ubiquitous components of eukaryotic genomes that have long been considered solely deleterious or ’junk DNA’. They are split into two main forms, retro-transposons and DNA transposons, depending on the method of replication employed. Hosts have developed strategies for combating TEs including RNAi, methylation and copy number con-trol. TEs have also evolved ways of persisting in the genome in order to survive, such as target site specificity. Two additional ways which may be utilised by TEs, positive selection and horizontal transfer, were investigated here primarily using the budding yeasts in the Saccharomyces sensu lato complex. These species typically contain up to five families of retrotransposons, designatedTy1-5, and multiple subfamilies, all of varying transpositional activity. Discoveries of insertions evolving under positive selection and providing benefits to their hosts have been sporadic and serendipitous findings in a number of organisms. Full genome screenings for such insertions are rarely published, despite the impact TE insertions have upon their hosts. A population genomics approach was performed to address this issue in the genomes of Saccha-romyces cerevisiae and sister species S. paradoxus. Signatures of positive selection acting upon Ty insertions were identified using Tajima’s statistical D test. Neighbouring genes were also analysed to ascertain the true target of selection where hitchhiking linked the two. A subset of LTR-gene pairings were explored using qPCR in order to identify any effects on host gene expression the occupied loci may cause. Two genes displayed significantly increased levels of expression, which may be due to the presence of positive selection candidate LTRs, which in turn may contribute to improving host fitness. This thesis further documents the systematic screening for Ty-like elements of all available genomes of budding yeast and related species. Extensive phylogenetic analyses estimated evolutionary relationships and possible horizontal transfer events of elements between the species. Evidence for in excess of 75 horizontal transfer events was uncovered here, around half of which were successful in propagating in new genomes. The occurrence of horizontal transfer of TEs in the genomes of budding yeast is therefore far more common than previously documented. During screening of genomes, a further potential method of avoiding host defences was uncovered. The divergence of the highly active Ty4 family, which coincided with population isolation of multiple Saccharomyces species into subfamilies, was surprising given previous reports of this family being of particularly low activity. Such events are rarely recorded in eukaryotic genomes, and may also illustrate the compulsive spread of a new subfamily via horizontal transfer. The investigations reported here represent the first genomic screening of Ty insertions in Saccharomyces for signatures of positive selection, and an updated, comprehensive search for evidence of HT between species of budding yeast. Both may act as methods for TE families to persist in the genomes of their hosts, and represent far more than simply ’junk DNA’.

Radioactive waste disposal in the UK is expected to be managed via the construction of a deep geological disposal facility (GDF) back filled with cementitious grouts. Post closure the repository is expected to have a hyper alkaline, anoxic environment. The UK's intermediate-level radioactive waste (ILW) inventory contains significant quantities of cellulosic materials that expected to undergo alkaline hydrolysis under repository conditions to form Iso-saccharinic acids (ISA). The isomers of ISA (α-and β-)are able to form water-soluble complexes with radioelements, increasing their solubility and enhancing their migration. The biological removal of ISA through microbial activity would therefore have a positive impact on repository performance by reducing complex formation. This research aimed to isolate pure cultures capable of degrade ISAs under anaerobic and alkaline conditions analogous to GDF. The microcosms of mineral media and CDPs-fed cycle as a sole carbon source inoculated by alkaline soil samples from the Buxton site. The degradation process monitored under a fermentation and an anaerobic respiration by adding terminal electron acceptors. All processes carried out under alkaline and strict anaerobic conditions. Different types of agar plate media used to obtain pure culture. The results of the current study indicate that alkaliphilic bacterial communities were capable of fermenting ISA to acetate up to pH 11.0. In addition, the ISA (α-and β-isomers) degradation through terminal electron acceptors at pHs (7.0,8.0,9.0and 10.0) were tested, resulted to a significant amount of ISA was degraded in Nitrate-reducing culture, and small amount in Iron (III)-reducing culture, whilst there was no sign of degradation in Sulphate-reducing culture. A 16SrRNA gene sequencing showed a significant reduction of the bacterial community in the microcosms compared with the crude soil sample. The phyla that detected in all microcosms at different pH values dominated by Firmicutes, followed by Proteobacteria, Bacteroidetes, Actinobacteria and Archaea Euryarchaeota. However, in pure culture many of the bacteria isolated were unable to degrade ISA even though they had been isolated and enriched under ISA degrading conditions. Only two bacterial strains purified which were capable of degrading ISA. These strains Macellibacteroides fermentans HH-ZS a Gram negative, strictly anaerobic bacterium and a strain of Aeromonas sp. The biochemical analysis, metal and NaCl tolerance, pH profile, biofilm and extracellular polymeric substances detection, fatty acid methyl ester profile, and whole genome sequencing analysis used for characterization of the some isolated Alkaliphiles. The phylogenetic and biomarker results led to identifying a novel strain of Macellibacteroides fermentans HH-ZS strain is the first Gram negative, strictly anaerobic bacteria able to degrade ISA. Future work; the genome of the M. fermentans HH-ZS strain harboured a number of carbohydrate degrading enzymes, which merit further investigation to determine the metabolic pathways associated with ISA degradation. In addition, some of isolated Alkaliphiles considered as rare strains and some of them identified as new strains; further investigation to find the possibility to introduce these isolates in the bioremediation and an industrial application.

The first chapter in this thesis describes research on the asymmetric [1,2]-sigmatropic rearrangement of benzylic ammonium ylids. Our group previousely developed method showing that DMSO as solvent, and BTPP as base, in the presence of 5Å molecular sieves, dramatically improves the yield of the reaction. Hence, we applied the developed the method using8-(–)-phenylmenthol and (2S)-camphorsultam as chiral auxiliaries. In the second chapter, a new application of Pd-catalysed allylation is reported. This enabled the synthesis of (30)of branched sp3-functionalised sulfonamides, a compound class for which few reported methods exist. By reacting benzyl sulfonamides with allylic acetates in the presence of Pd0 catalysts and a base, at room temperature, direct allylation was efficiently performed, yielding products that are analogues of structural motifs seen in biologically active small molecules. The reaction was performed under mild conditions and could be applied to nanomolar sigma-receptor binders, thus enabling a late-stage functionalisation and efficient expansion of drug-like chemical space. The third chapter described a synthesis of benzosultam without recourse to transition-metal catalysis, or stoichiometric amounts of organometallic building blocks. Iodomethane sulfonylamide adds to benzyne (generated using fluoride sources), and then the formed intermediate undergos an intramolecular cyclisation to afford sultam. Using this method that procceds under simple reaction conditions, (11) benzosultams were synthesised in modest yield.

Toxoplasma gondii is an obligate intracellular pathogen. Due to its experimental tractability it has acted as an excellent model system to understand the fundamental principles of pathogenic mechanisms within the group Apicomplexa, including Plasmodium spp. the causative agent of malaria. Work on T. gondii has provided the foundation to understanding how apicomplexan parasites power motility and invasion, which centres around the parasites gliding machinery. This movement depends on the parasite's acto-myosin system, which is thought to generate the force during gliding. However, recent evidence questions the exact molecular role of this system. Deletions of core components of the gliding machinery, such as parasite actin or subunits of the glideosome indicate that the parasites remain motile and invasive, albeit at significantly reduced efficiencies. These findings could be explained by different possibilities, such as functional redundancies or compensatory mechanisms for multiple components of the glideosome. Toxoplasma only encodes a single copy of ACT1, therefore redundancies for ACT1 are unlikely. Much of the research in to the role(s) of TgACT1 focuses on motility and invasion. Interestingly, while the conditional act1 KO shows a deficiency in gliding and invasion, severe defects affecting parasite survival were observed during intracellular replication and egress. The amount of actin remaining in the act1 KO parasites was disputed which led to alternate conclusions about actins role in the parasites. Therefore, this study provides a much more detailed characterisation of the conditional act1 KO and when the phenotypes are observed in relation to actin levels within the parasite. Furthermore, the study provides evidence of an alternative model for motility that is independent of the parasites acto-myosin system. Several studies assert that the polymerisation kinetics of TgACT1 is unusual, allowing the formation of only short, unstable actin filaments. However, to date, it has not been possible to study actin in vivo, therefore its physiological role has remained unclear. In order to investigate this, parasites expressing a chromobody that specifically binds to F-actin were generated and characterised. Importantly, TgACT1 forms a vast network during the intracellular life-stages that is important for parasite replication and egress. Moreover, these filaments allow vesicle exchange and produce F-actin connections between parasites in neighbouring vacuoles. This study also demonstrates that the formation of F-actin depends on a critical concentration of G-actin, implying a polymerisation mechanism akin to all other actins. This work is important for understanding the mechanisms used by Toxoplasma to move and invade with regards to the functions of the acto-myosin system. Moreover, it highlights a novel role of actin that is required to control the organisation of the parasitophorous vacuole during division. The role of actin during the lifecycle may have wider implications to other apicomplexan species, such as Plasmodium spp. and also much further in the field of parasitology where F-actin information is scarce.

Under genotoxic stress conditions, the genome of any organism may become compromised thus undermining cellular functions and the high fidelity transmission of the genome. Should integrity become compromised, cells have evolved a plethora of pathways to monitor, assess and direct the removal or bypass of genomic lesions. Collectively, this response is known as the DNA damage response (DDR). At the forefront of the DDR are specialised enzymes known as protein kinases (PKs), which act to co-ordinate many aspects of this response. In the kinetoplastid parasite Trypanosoma brucei, the role of PKs in other processes, such as the control of the cell cycle and during differentiation between the mammalian and insect lifecycle stages, have already been investigated using mutant cells lines, RNA interference (RNAi) and genome wide and kinome focused screens. However, virtually no work has examined the role of PKs in the context of genome repair. To this end, two RNAi targeted screens (RITseqs), one to examine the genome as a whole and the other to focus directly on the kinome compliment, were performed by others in the mammalian infection stage of T. brucei, searching for genes whose loss sensitises the cells to the presence of the DNA alkylating agent Methyl methanesulfonate (MMS). To validate both screens here, putative DNA damage associated PKs were examined by RNA interference (RNAi). Across both screens, a total of eleven PKs, whose loss sensitised cells to MMS, were validated in this study by monitoring their proliferation. Amongst these novel PKs were a pseudokinase (Tb6560) and an aurora kinase (TbAUK2), whose functions were investigated using disruption mutants generated in BSF T. brucei cells combined with immunolocalisaton. Analysis of the Tb6560 mutants revealed the pseudokinase to be non-essential in vitro and uncovered a potential role for Tb6560 during endocytosis or internal trafficking. Analysis of the TbAUK2 mutants also revealed this PK to be non-essential in vitro. However, when exposed to a variety of genotoxic agents, the growth of the mutants was significantly reduced suggesting TbAUK2 is required for parasite survival under DNA damage conditions. Further phenotypic analysis revealed a potential role for TbAUK2 in the maintenance of the parasite’s nuclear genome. Additionally, the roles of two PKs central to the DDR in other organisms were examined by RNAi. The atypical PKs Ataxia Telangiectasia Mutated (ATM) and the related PK ATM and Rad3 related (ATR) are activated in response to double stranded break (DSB) lesions or replicative lesions, respectively, in other eukaryotes. RNAi of TbATR was associated with perturbation of proliferation, extensive nuclear defects and sensitivity to genotoxic agents. Further analysis also revealed a potential role for this kinase in maintaining transcriptional silencing of bloodstream VSG expression sites (BESs). Conversely, RNAi of TbATM did not disrupt cellular proliferation in vitro and nor were TbATM deficient cells sensitised to alkylating or replication blocking agents, suggesting TbATM is not required for T. brucei survival or for maintenance of growth in these conditions. Collectively, these data reveal a diverse array of PKs required for genome maintainance, in BSF parasites, under genotoxic stress conditions. An indepth characterisation of three DNA damage associated PKs uncovered a variety of putative roles ranging from nuclear and chromosomal segregation to cell cycle regulation, proliferation and endocytosis. These data in particular highlight the complexity underpinning a cell’s response when threatened with genomic instability. Furthermore, depletion of one kinase, TbATR, was also associated with loss of transcriptional silencing within BESs suggesting that PK activity could operate during antigenic variation, a process vital for the persistence of this parasite within a host.

Trypanosoma brucei is the protozoan parasite causing African trypanosomiasis, a neurological disease that affects humans and farm-stock in the impoverished sub-Saharan areas where tsetse fly transmission vector is endemic. Although it has great impact on public health and local economies, it has been neglected in drug discovery for almost a century. Current treatments are either toxic or of difficult administration, besides having serious risks of inducing resistance. Protein kinases are the primary set of signaling proteins in eukaryotes, including Trypanosoma brucei. Their druggability has been widely exploited in cancer research, and has been established in the parasite too. A recent kinome-wide RNAi screen with 176 individual cell lines of mammalian infective bloodstream forms of Trypanosoma brucei identified protein kinases required for proliferation in vitro. In order to investigate which protein kinases are also essential virulence factors in vivo, lines were pooled, inoculated into mice and screened for loss of fitness after 48 hours RNAi compared to uninduced controls. The presence of trypanosomes in the bloodstream was assessed using RNAi target sequencing (RITseq) and compared to an in vitro control. This revealed 49 protein kinases with a significant loss of fitness in vivo in two independent experiments, and a strong correlation between in vitro and in vivo loss of fitness for the majority. However, depletion of nine protein kinases affected more pronouncedly the growth in vivo than in vitro. Amongst these protein kinases were several with putative functions related with stress responses mediated through the PI3K/TOR or MAPK signaling cascades including CK2A2, a promiscuous protein kinase whose activity can be stress-induced; two MAP3Ks, involved in cell integrity upon osmotic shock; VPS15, component of the PI3K complex with roles in autophagosome formation and vesicular trafficking; BUD32, transducer of the PI3K/TOR pathway involved in translational regulation; and FAZ20, a parasite-specific pseudo-kinase localizing to the flagellum attachment zone. The other three have been implicated in repair of alkylation-induced cellular damage: SRPK1, a stress response RNA splicing regulator; AUK2, which acts during mitosis; and CAMKL, an AMPK with calcium-binding domains putatively involved in metabolic regulation. Identification of these virulence-associated protein kinases provides new insights in T. brucei-host interaction and reveals novel potential drug targets for protein kinase inhibitors. This RNAi screen revealed that the evolutionary divergent NEK kinase Repressor of Differentiation Kinase 2 (RDK2) has severe loss of fitness both in vitro and in vivo. Depletion of RDK2 had been shown previously to promote differentiation from bloodstream to procyclic-like forms causing the parasite’s death. Further investigation showed RDK2 to be an active protein kinase capable both of phosphorylating a substrate and to autophosphorylate. Protein kinase activity could be ablated by mutation of lysine 70 to methionine. Mutation of both serine residues (195 and 197), identified as sites of phosphorylation by phosphoproteomics, to alanine or glutamic acid, preventing and mimicking phosphorylation respectively, had no effect on protein kinase activity, suggesting they do not have a direct regulatory role on protein kinase activity. Introducing in the RNAi line a recoded RDK2 whose transcript eluded interference, permitted to some extent the rescue of the induced phenotype, while introducing a recoded inactive mutant did not. This may suggest that the lack of kinase activity was responsible for the RNAi phenotype and not depletion of the protein alone. RDK2 RNAi-differentiated cells could be maintained in conditioned procyclic form media for more than a week. However, they were unable to proliferate. Overexpression of RDK2 blocked the differentiation mediated by sequential treatment with 8-pCPT-cAMP and citrate/cis-aconitate (CCA). RNAi experiments in combination with known differentiation cues, suggested that when differentiation is triggered by the CCA signalling pathway, RDK2 inactivation happens downstream of the phosphatase TbPTP1. Differentiation caused by RDK2 inactivation could be traced in flow cytometry by the detection of EP procyclin expression. This was exploited in a cell-based mechanism-directed phenotypic screen for RDK2 inhibitors. A preliminary run with 518 drug-like molecules that had shown protein kinase inhibition, trypanocidal activity and/or activation of the EP procyclin promoter, unveiled 6 compounds triggering EP procyclin expression and parasite death in the low micromolar range. These compounds can be investigated further to assess whether RDK2 is their in vivo target.

Osteoarthritis (OA) is a disease characterised by changes in the structure and function of articular joints, leading to pain and loss of mobility. Bone Morphogenetic protein 7 (BMP-7), a member of the transforming growth factor β superfamily, has been shown to promote anabolic events within articular cartilage, and confer protection from OA associated destruction in a number of animal models. It has been shown that a disease-associated loss of BMP-7 in OA may contribute to the joint destruction. The mechanism associated with the loss of BMP-7 has yet to be fully elucidated. Recently, small non-coding RNAs (microRNAs) that participate in post-transcriptional gene regulation, have been identified as a potential dysregulated mechanisms in OA. It was therefore hypothesised that disease-associated alterations in these microRNAs could lead to subsequent changes in the expression of BMP-7 and its signalling family. The aims of this thesis were to investigate the expression of BMP-7 and other associated BMP signalling molecules and identify any microRNAs that may regulate these transcripts. Furthermore, the study aimed to elucidate the molecular mechanisms by which BMP-7 is able to confer protection in cartilage. The studies presented in this thesis show that both articular chondrocytes and the synovial membrane can express very low levels of BMP-7 transcript in a subset of patients. In juxtaposition, protein can be clearly detected in both articular chondrocytes and synovial membrane. Interrogation of the BMP-7 signalling family transcripts revealed that all members are detectable in OA cartilage. This expression was independent of the eroded nature of the cartilage. Evaluation of the circulating microRNAs that were predicted to target the BMP-7 pathway revealed that several miRNAs (including miR24-3p) were altered in the plasma of OA patients. Interestingly, miR24-3p was able to target BMP receptors ALK2 and BMPR1B. Moreover, there was a significant negative correlation between the expression of miR24-3p and the ALK2 receptor in OA patients. Thus suggesting there is a role for this miRNA in the negative regulation of BMP-7 signalling in OA cartilage. To complement the work evaluating the endogenous signalling pathway, studies were also undertaken to investigate the impact of exogenous BMP-7 stimulation on chondrocytes. BMP-7 was able to promote its own transcriptional expression in a patient specific manner and induce expression of IL-1β in all of the donors investigated. In addition to the induction of IL-1β, BMP-7 was also able to up-regulated the IL-1β antagonist, IL-1Ra. Taken together this data suggests a role for BMP-7 in the regulation of the inflammatory mediator IL-1β. Finally, BMP-7 was able to up-regulate several pro-inflammatory cytokines and chemokines in both primary OA chondrocytes and in vitro differentiated macrophages. In summation, the work presented in this thesis suggests that BMP-7 may be contributing to the promotion of inflammation and subsequent repair as part of the cartilage homeostatic mechanisms. Further to this, miR24 has been highlighted as a regulator of cartilage homeostasis via the direct targeting of ALK2. Changes in the expression of this miRNA over the course of OA disease progression may be involved in driving disease pathogenesis. Therefore understanding the targets for this disease-associated miRNA may help in the development of disease modifying therapies.

Nowadays, the quality of air in the indoor environment is of increasing concern as it is estimated that the majority of people in developed countries spend about 85% of their time in various indoor micro-environments. The biological fraction of airborne particulate matter has become of increasing interest owing to the potential allergenic and pathogenic nature of airborne micro-organisms. Biological aerosols are ubiquitous and abundant in the air we breathe in the built environment and despite the recognition of the importance of bioaerosol exposure on human health, relatively little is known about the microbial agents in indoor air and there are still critical gaps in our understanding of the airborne microbial communities. Molecular biology methods demonstrate a great potential for improving our comprehension of the significance of the indoor aerosol microbial load in the context of human exposure, providing in-depth characterisation of microbial communities. However, the number of bioaerosol studies which have applied culture-independent techniques is still limited and the data on the airborne microbial particles in indoor environments remain scant and need to be further examined. The aim of the present study is to investigate the microbial abundance and diversity of bioaerosols in various different types of indoor environments using molecular techniques. Several research questions are being addressed by application of high throughput next generation sequencing technology, providing insights into the aerosol microbiome in micro-environments where people spend considerable amount of their time on a daily basis, including educational, residential and transportation settings. The results of this project provide novel information on a previously largely unexplored microbiome which could improve and expand our comprehension of the specific characteristics of bioaerosols in the indoor environment. Moreover, the data generated by this study could make a significant contribution to improving air quality by facilitating the development of methods for bioaerosol exposure monitoring.