'Psedomonas aeruginosa' is a prevalent nosocmial pathogen predominantly associated with infections in immune compromised individuals and long term colonisation and pathogenesis in the lungs of Cystic Fibrosis patients. With multi-drug resistant strains increasingly common, the discovery of novel targets for antimicrobial chemotherapy is of utmost importance and expansion of data on 'P. aeruginosa's' complex genome could facilitate this. Azoreductases are a group of enzymes mainly noted for their reductive capacity against azo and quinone compounds. Ubiquitous amongst many classes of organism including prokaryotes and eukaryotes, the primary physiological role of azoreductases remians unclear. This study characterises azoreductase-like enzymes from 'P. aeruginosa' in terms of biochemical properties, substrate specificity and structural analysis. The effect of these enzymes on bacterial physiology in 'P. aeruginosa' is also explored in relation to antibiotic susceptibility. Three azoreductase-like genes from 'P. aeruginosa' (pa1224, pa1225 and pa4975) were overexpressed in 'E. coli' strains following molecular cloning. Recombinant proteins were biochemically characterised by means of Thin Layer Chromatography, Differential Scanning Fluorimetry and ezymatic assays. All enzymes were noted to be selective for FAD as the flavin cofactor and NADPH as the preferred reductant. All three enzymes were confirmed as NAD(P)H dependent quinone oxidoreductases (NQOs) with PA1224 also catalysing reduction of the azo substrate methyl red, albeit at a rate an order of magnitude lower than that observed for the quinone compounds. The preferred flavin cofactor for four previously characterised azoreductase and NQO enzymes (PA2280, PA2580, PA1204 and PA0949) was also explored and PA2280 and PA0949 were observed to select for FMN while PA2580 and PA1204 were selective for FAD. The crystal structure of PA2580 was solved with the nicotinamide group of NADPH bound and was noted to form a homodimer with the same short flavodoxin-like fold as previously described for other members of this enzyme family. Complemented strains of azoreductase-like gene deletion mutants of 'P. aeruginosa' PAO1 were generated via molecular cloning and used to monitor the effects of these enzymes on antibiotic susceptibility. Antimicrobial sensitivity assays were carried out and although the knockout strains displayed increased sensitivity to fluroquinolones, they did not revert to the wild type phenotype upon reinsertion of the genes of interest. This study has for the first time characterised three new NQO's from 'P. aeruginosa' PAO1 and solved the crystal structure of an azoreductase/NQO with nicotinamide bound. With these findings and a library of complemented strains generated, this original study offers a platform for the continued research into the physiological role of these enzymes.

Cardiovascular complications are the leading cause of death in patients with chronic kidney disease (CKD). Uraemic cardiomyopathy (UCM) is characterised by structural and cellular remodelling including left ventricular hypertrophy (LVH), metabolic remodelling and mitochondrial dysfunction. Although ex vivo studies have highlighted evidence of enhanced glucose utilisation in the hypertrophied heart, cardiac glucose metabolism in uraemia has yet to be established in vivo. In addition, little is known about mitochondrial morphology or the impact of iron therapy on cardiac mitochondrial function in CKD. The aims of this study were to (I) investigate cardiac glucose metabolism in vivo using 18F-flurodeoxyglucose positron emission tomography (18F-FDG PET) during the development of UCM and (II) characterise mitochondrial morphology and the impact of iron therapy on cardiac mitochondrial function in uraemia. Experimental uraemia was induced surgically in male Sprague-Dawley rats via a subtotal nephrectomy. Dynamic PET/CT scans were acquired at 5, 9 and 13 weeks post-surgery using 18F-FDG PET. The rate and distribution of 18F-FDG uptake were determined using Patlak and polar map analysis. In a separate series of experiments the iron complex, ferumoxytol, was administered 6 weeks post-surgery and mitochondrial respiratory rates and enzyme activities determined following sacrifice 6 weeks later. Cardiac mitochondrial morphology was characterised by probing the expression of key mitochondrial fusion and fission proteins and evaluating mitochondrial size and structure in left ventricular tissue and isolated mitochondria. Renal dysfunction was prominent in uraemic animals by 12 weeks as evidenced by elevated serum creatinine, urea and the presence of anaemia. LVH was associated with moderately increased 18F-FDG uptake in the uraemic heart at 5, 9 and 13 weeks. This was paralleled at the cellular level by altered mitochondrial morphology, characterised by a more sparsely packed cristae, and increased mitochondrial state 4 respiration, indicative of reduced efficiency. However, ferumoxytol treatment did not impact on cardiac mitochondrial function at this stage of uraemia. Collectively these data suggest there is evidence of enhanced glucose utilisation in the uraemic heart in vivo and these changes are associated with altered mitochondrial structure and bioenergetics.

Nanoparticles (NPs) are progressively being investigated for use in biomedical applications, including biological agents delivery like gene delivery, drug and protein delivery. Activation of complement pathways and interactions with immune recognition subcomponents can modulate the clearance of the NPs and subsequent inflammatory response. Such modulation could affect the intended translational applications either in the development as tissue-specific drug delivery platform or in the treatment of pulmonary diseases such as tuberculosis and lung cancer thus, poses challenges to develop them for in vivo applications. Here, we set out to study the interaction between innate immune components such as properdin, a small fragment of properdin, TSR4+5, a key lung pattern recognition molecule, surfactant protein D (SP-D), and carbon nanotubes (CNTs), and potential downstream effects on the immune response via macrophages. We report, that human properdin, an up-regulator of the complement alternative pathway and stabilizer of C3 convertase can opsonize CNTs via its thrombospondin type I repeat (TSR) 4 and 5. Uptake of properdin bound CNTs was enhanced by a macrophage cell line, THP-1, surging a robust pro-inflammatory immune response. In addition, recombinant TSR4+5 on CNTs, inhibited complement consumption, suggesting that TSR4+5, can be potentially used as a complement inhibitor in a number of pathological circumstances arising due to unintentional complement activation. Similarly, a recombinant fragment of human SP-D (rfhSP-D) bound to CNTs via its C-type lectin domain and augmented phagocytosis by THP-1 monocytic cell lines, together with an increased pro-inflammatory response. Furthermore, rfhSP-D opsozined CNTs continued to activate complement pathway via the classical pathway. Complement deposition on the rfhSP-D opsonised CNTs led to dampening of the pro-inflammatory immune response. Furthermore, like CNTs, Iron oxide nanoparticles are also recognized by complement pathway, but mainly by alternative complement pathway. Complement deposition enhanced their uptake by activated THP-1 macrophages and dampened the pro-inflammatory responses. These studies emphasise the significance of understanding the interaction between innate immune humoral factors including complement in developing nanoparticle-based drug delivery strategies.

Varicella-zoster virus causes chicken pox (Varicella) primary infection, which becomes latent in the dorsal root ganglia and trigeminal ganglia, and may reactivate to cause shingles, the most serious complication of which is post-herpetic neuralgia occurring in 50% of individuals over 60 years. Severity of lesions depends on host's immune response. Like many viruses, varicella-zoster virus appears to have evolved escape mechanisms from host immune surveillance by downregulating cell surface major histocompatibility complex class 1 expression and may delay of resolution of infection. Major histocompatibility complex class 1 is processed and transported to the cell surface through the Golgi apparatus. Varicella-zoster virus genome encodes a membrane gene, open reading frame type 1, which is localised in the enoplasmic reticulum and Golgi apparatus. Given the cellular localisation of open reading frame type 1 in the Golgi apparatus, this study investigated the expression of major histocompatability complex class 1 in human immortalised keratinocytes transfected with only empty vector. As a control, the expression of major histocompatability complex class 1 human immortalised keratinocytes parental cells were also examined using current molecular biology techniques. The results of this thesis demonstrate that the expression of varicella-zoster virus-open reading frame type 1 prevents the transport of major histocompatibility complex class 1 complexes to the cell surface and causes its retention in the Golgi apparatus, which is compensated by treatment with IFN-[alpha]. Varicella-zoster virus (VZV)-open reading frame type 1 does not affect the synthesis of human leukocyte antigen class 1 heavy chains or the expression of the transporter associated with antigen processing. Additionally, we determined that varicella-zoster virus-open reading frame type 1 impedes the surface expression of human leukocyte antigen class-A and human leukocyte antigen class-B, which present viral peptides to major histocompatibility complex class I-restricted cytotoxic T lymphocytes, but not the natural killer cell inhibitory ligands human leukocyte antigen class-C and non-classical human leukocyte antigen class-E. This selective downregulation of cell surface human leukocyte antigen class 1 molecules may allow the virus to establish infection by avoiding immune clearance of virus-infected cells by both cytotoxic T lymphocytes and natural killer cells. However, it remains to be seen if open reading frame type 1 expressing cells evade cytotoxic T lymphocytes killing, through downregulation of classical major histocompatibility complex class I, and natural killer killing, through lack of downregulation of non-classical major histocompatibility complex class I. The study outcome will be a valuable attempt to elucidate factors involved in varicella-zoster virus-related lesion progression, contribute to existing knowledge, and importantly allude to further investigations on the pathogenesis of this virus on human disease. The data obtained may also offer novel means of therapeutic intervention.

Estuaries are often challenging to manage, as management must strike a balance between the needs of the users and the ecological and economic values within the context of multiple legislative drivers. To help facilitate integrated management, a novel Estuarine Planning Support System (EPSS) framework, using the Humber Estuary in Eastern England as a case study, has been developed. This integrated EPSS framework goes beyond previous approaches as it brings together the legislative drivers, management tools and other mechanisms for controlling plans (formal/legal management, action or work plans (e.g. shoreline management plans) and projects (a new structure or activity such as extending a port). It thereby enables managers and users to assess and address both the current environmental state, and the way in which the new project could impact an estuary in an accessible and understandable tool. This study has been primarily completed by desk based research using peer reviewed literature, technical and research reports, marine licence applications and legislation, with correspondence to several sources to determine the baseline information and existing knowledge gaps. Further to the framework, an EPSS tool was developed to provide a practical application of these requirements. The GIS-based tool ensures that the information is accessible for regulators, managers, the scientific community, developers and the public. Whilst the tool is adaptable for regions within and outside the United Kingdom (UK), the research presented in this thesis focussed on the Humber Estuary. The successful application of the tool for a complex socio-economic and environmental system such as the Humber Estuary shows that the tool can efficiently guide users through the complex administrative requirements needed to implement a management plan, and therefore support sustainable development. In addition, the tool can be used as a scoping mechanism to identify potential stressors which are to be addressed in an environmental impact assessment (EIA). The tool was validated against four case studies and was also tested by a number of stakeholders to determine the utility and accuracy of the tool. The tool was subsequently updated to reflect feedback from the stakeholders. This project should be viewed as a ‘proof of concept’ in that its primary purpose is to demonstrate the potential for developing and operationalizing an approach in the field. The method has the potential to integrate highly technical knowledge from scientists, and the views of non-scientists, to make better-informed management and planning decisions and to provide reasonable assurance to justify those decisions. The tool can be used to prevent conflicts among stakeholders and/or between uses and users and the environment, and makes it possible to integrate all the existing background data in thematic maps and identifies the human activities that use the areas, resources and services. The EPSS tool can save time and resources, aid in the decision-making process and make the decision process more transparent and consistent. It has the potential to make the governance of the marine area more logical, simple, fast and therefore more cost effective. The tool has been developed to be flexible in its approach, this means that it can be easily adapted to be used internationally and to allow for it to be adaptable to future changes. It combines the many aspects required for a holistic approach to marine management, from the inclusion of governance and stakeholder views, to the need for, and use of, monitoring information. In marine management, there will always be a need for robust and scientifically and legally defendable science to inform management. The increasingly large spatial scales that are addressed by policy makers, and the reduction in funding, means that new methods are needed to provide the evidence base which this tool helps to provide, and can be applied worldwide. The developed toolbox is an important step towards such an integrated, holistic ecosystem based approach for marine management, demonstrating that a single tool can bring together the legislative, environmental and economic considerations. The tool is a method to undertake the assessments that are currently being carried out by separate organisations, to combine in a single process that is consistent and transparent and on a quicker timescale helping to reduce costs.

Tissue factor (TF) is the main initiator of blood coagulation. In addition to its procoagulant property, TF has the ability to regulate various functions within cells including proliferation, angiogenesis and apoptosis. These outcomes appear to depend on the amount of TF with which the cell comes into contact with. In this study, human dermal blood endothelial cells (HDBEC) were transfected to express wild-type TF which is released following the activation of PAR2 in a normal physiological response. In addition, a model for the accumulation of TF in vascular disease and cancer was used by expressing a mutant form of TF (TFAla253-tGFP) which although expressed is not released by the cells and therefore it accumulates intracellularly. Initially, the phosphorylation of Src1 and Rac1 were monitored in order to determine any difference in phosphorylation patterns following PAR2 activation of cells. Phosphorylation of Src1, but not Rac1 was prolonged on expression of TF and was further enhanced on intracellular accumulation of TF. Therefore, the role of Src1 as a mediator of TF-induced apoptosis was examined next. Either inhibition of Src using pp60c-srcpeptide, or suppression of Src1 expression using siRNA prevented the TF-induced p38 MAPK activation and subsequent cellular apoptosis. Following confirmation of the role of Src1 in this process, an attempt was then made to delineate upstream intermediaries involved in this pathway. By using an inhibitory antibody (AIIB2), β1-integrin was shown to participate in TF-induced Src1 activation. In contrast, prevention of Src1-FAK complex formation using FAK inhibitor-14 did not interfere with the TF-mediated Src1 activation, despite a clear reduction in Src1 phosphorylation. Furthermore, TF-induced apoptosis did not appear to require Src1-FAK binding. In conclusion, this study has established further steps in the pathway by which TF can induce cellular apoptosis, and suggests a mechanism by which the increased amount of TF during inflammation can have detrimental outcome on the vascular system.

Nitric oxide (NO), being a free radical, has a unique biological chemistry that, since its elucidation as a signaling molecule in the cardiovascular system, has been implicated in a range of diverse biological functions. Both cardiovascular disease, exacerbated by hypertension, and antibiotic-resistant bacterial infections are major yet unavoidable public health concerns. Atypical NO synthesis may be a factor these pathologies and exogenous NO may provide an alternative therapeutic strategy when endogenous NO synthesis is atypically low. Furoxans are an old, yet largely untapped resource of nitric oxide donor compounds. A panel of twelve such compounds were developed through heterocyclic synthetic chemistry and assessed for their potential biological activity. The compounds have had their NO release characterised via the Griess assay and have been investigated for both novel antibacterial and vasodilatory effects. Direct toxicity was explored using an optimised MTT assay to assess cell viability post incubation in three bacterial species: Staphylococcus epidermis, Escherichia coli, and Pseudomonas aeruginosa. The lead compound RJP06A was then exposed to bacterial biofilms, investigating its ability to prevent initial biofilm formation and promote its dispersal before determining its mechanism. Vasodilatory potential of the compounds was studied using rat thoracic aorta, pulmonary artery and renal artery. Results demonstrate that these compounds elicit a toxic effect against all three bacterial species with IC50 values ranging from 31-745 uM, which was enhanced in the presence of S-nitrosoglutathione, despite having negligible toxicity itself. Biofilm investigations of RJP06A demonstrate its ability to disperse mature single-species bacterial biofilms in a dose-dependent manner. Isometric tension data show that three quarters of the compounds produced strong, sustained relaxation of contracted aorta, and two in particular caused almost complete reversal of contraction at concentrations of 250 and 350 nM respectively. Mechanism studies suggest that the lead furoxan, RJP06A exerts its effects primarily through a cGMP-mediated NO-activated pathway, but may also have a small component whereby thiols can bypass this step and activate calcium channels directly. RJP06A has been shown to be better at inducing relaxation of resistance vessels, such as aorta, rather than pulmonary or renal artery. In both investigations, nitric oxide from RJP06A appears to be the key molecule involved in the observed effects, with scavenging evolved NO using haemoglobin attenuating its effect, and importantly the two mechanisms identified in very different models imply an evolutionarily conserved pathway of nitric oxide biology.

Promiscuous mutant EcoRI endonucleases produce lethal to sub-lethal effects because they cleave E. coli DNA despite the presence of the EcoRI methylase. Three promiscuous mutant forms, Ala138Thr, Glu192Lys and His114Tyr, have been characterized with respect to their binding affinities and first-order cleavage rate constants towards the three classes of DNA sites: specific, miscognate (EcoRI*) and nonspecific. We have made the unanticipated and counterintuitive observations that the mutant endonucleases that exhibit relaxed specificity in vivo nevertheless bind more tightly than the wild-type enzyme to the specific recognition sequence in vitro and show even greater preference for binding to the cognate GAATTC site over miscognate sites. Binding preference for EcoRI* over nonspecific DNA is also improved. The mutant enzymes cleave the cognate site GAATTC at a normal rate, but cleave EcoRI* sites faster than does the wild-type enzyme. Thus, the mutant enzymes use two mechanisms to partially bypass the multiple fail-safe mechanisms that protect against cleavage of genomic DNA in cells carrying the wild-type EcoRI restriction-modification system: (a) Binding to EcoRI* sites is more probable than for wild-type enzyme because nonspecific DNA is less effective as a competitive inhibitor; (b) The combination of increased affinity and faster cleavage at EcoRI* sites makes double-strand cleavage of these sites a more probable outcome than it is for the wild-type enzyme. The crystal structure of the A138T "promiscuous" mutant enzyme in complex with specific DNA shows reveals no changes in protein contacts to the bases of the GAATTC site relative to the wild-type complex; however, there are changes in water-mediated contacts between the enzyme and flanking bases, and changes in protein-phosphate contacts. These observations lead us to hypothesize that the improved specific DNA binding of the A138T enzyme relative to the wild-type enzyme is not attributable to a single new protein DNA contact, but rather the distributed effect of the improved complementarity between the mutant and the flanking bases, as well as the optimization of several phosphate contacts. The aggregate of biochemical data presented in this thesis leads us to propose a model where the A138T miscognate DNA binding ensemble (for a subset of miscognate sites) is partitioned more towards complexes that are on the path to the transition state than wild-type miscognate DNA binding ensembles; the mutant accomplishes this by forming 'specific-like' phosphate contacts to these sites, which in turn stabilize the DNA distortion that is critical for efficient cleavage. Given the proximity of amino acid 138 to the residues which make contacts to the DNA phosphates in the specific complex, we hypothesize that the A138T mutation has an effect on the structure and/or dynamics of the "arm" protein segment (contains residues contacting the DNA backbone) such that it is more adaptable, resulting in formation of functional phosphate contacts to a broader range of DNA substrates.

Chromosome instability (CIN), a continuous change in the structure or number of chromosomes, is proposed to be a key mechanism driving the genomic changes associated with tumorigenesis. One major cause of CIN in cells is chromosome segregational defects occurring during mitosis. Two such examples are anaphase bridges and multipolar spindles, which are common in most cancer cells and many tumor tissues. Anaphase bridges are chromatin bridges in between separating chromosome masses during anaphase, which may result in gene amplification or loss when breaking. We have found that cigarette smoke condensate (CSC) induced anaphase bridges in cultured primary human cells, which in a short time led to genomic imbalances. The frequency of the induced bridges within the entire population decreased with time, independent of the p53-mediated apoptotic pathway. We also showed that CSC induced DNA double-stranded breaks (DSBs) in cultured cells as well as purified DNA. The reactive oxygen species (ROS) scavenger, 2 deoxyguanosine 5-monophosphate (dGMP) prevented CSC-induced DSBs, anaphase bridge formation and genomic imbalances. Therefore, we propose that CSC induces bridges and genomic imbalances via DNA DSBs. Further analysis in live oral cancer cells shows that cells with anaphase bridges mostly survive and these bridges frequently result in micronuclei formation, indicating that anaphase bridges actively contribute to CIN. Multipolar spindles (MPS) are aberrant mitotic structures when cells divide with greater than two spindle poles, which may result in uneven chromosome segregation. Multipolarity is strongly linked to centrosomal amplification, the mechanism of which remains controversial. We have examined the origin and fate of cells with MPS in real time. In both human embryonic kidney and oral cancer cells, the vast majority of multipolar cells originated from multinucleated cells. The frequency of cytokinesis failure was similar to the frequency of MPS, and each observed bipolar division that ended in a cytokinesis failure led to MPS formation in the subsequent mitosis. While grossly abnormal, these cells are still capable of dividing, often giving rise to a mixed progeny of multinucleated and mononucleated cells. These observations support the model that failure of cytokinesis may be the most common mechanism by which cells form MPS.

Allometric scaling theory has previously been used to estimate the functional relationship between two biological variables. In addition to parameter estimation, deviations from the general scaling relationship can be used to create hypotheses. Here, I explore deviations from the allometric scaling pattern for plant and floral size within the genus Collinsia on three levels: among species, within species, and among populations of a single species. Collinsia species are self-compatible annual herbaceous plants that have been shown to vary in floral size, autonomous fruit production, and estimated mating system. I quantified the amount of variation in characteristics related to plant mating systems: floral size and autonomous fruit production in a pollinator-free environment and used variation and scaling deviations to generate expectations about environmental selection pressures. I found that the scaling relationships differed on each of the three levels and that deviation from the general floral size-plant size relationship is common within this genus. The among-species regression explained only 20% of the variation in floral size, and species- and population-level regressions explained even less. The four species for which I obtained controlled environment estimates of vegetative and floral trait in this study differed significantly in autonomous fruit production, floral size, and plant size, while populations of C. heterophylla differed in floral and plant characteristics, but not autonomous fruit production. In addition, variation in plant size characteristics was 50-66% greater than variation in floral size characteristics suggesting selection to reduce variation in floral size and flexibility in plant size. Autonomous fruit production was correlated with floral size in C. tinctoria, with floral number in C. verna, and uncorrelated in C. heterophylla suggesting that the ability to self-fertilize varies among species. Using a comparative method and investigating factors correlated with plant mating system, such as floral traits, across a group of closely related species provides new insights into factors affecting their variation.