Lysostaphin (EC.3.4.24.75) is an extracellular glycylglycine endopeptidase produced exclusively by Staphylococcus simulans biovar staphylolyticus (ATCC 1362, NRRL B-2628). The zinc-containing endopeptidase demonstrates specific and potent staphylolytic activity therefore shows great promise for the treatment of blood-borne and biofilm-associated staphylococcal infections. The gene encoding mature lysostaphin has therefore been cloned and expressed using Escherichia coli, the most widely used expression host for recombinant protein production. Lysostaphin (EC.3.4.24.75) is an extracellular glycylglycine endopeptidase produced exclusively by Staphylococcus simulans biovar staphylolyticus (ATCC 1362, NRRL B-2628). The zinc-containing endopeptidase demonstrates specific and potent staphylolytic activity therefore shows great promise for the treatment of blood-borne and biofilm-associated staphylococcal infections. The gene encoding mature lysostaphin has therefore been cloned and expressed using Escherichia coli, the most widely used expression host for recombinant protein production. Lysostaphin (EC.3.4.24.75) is an extracellular glycylglycine endopeptidase produced exclusively by Staphylococcus simulans biovar staphylolyticus (ATCC 1362, NRRL B-2628). The zinc-containing endopeptidase demonstrates specific and potent staphylolytic activity therefore shows great promise for the treatment of blood-borne and biofilm-associated staphylococcal infections. The gene encoding mature lysostaphin has therefore been cloned and expressed using Escherichia coli, the most widely used expression host for recombinant protein production. Overall these findings are of concern for the production of recombinant protein production using E. coli. However this research suggested that chromatography can be used to sensitively detect and monitor product heterogeneity, so that a more homogeneous product could be produced by careful adjustment of expression, harvest and formulation conditions.

Streptococcus equi subspecies equi (S. equi) is the pathogen responsible for the prevalent and highly contagious equine disease called strangles. Strangles has been reported worldwide as a cause of a high level of animal suffering and economic loss. S. equi is susceptible to many antibiotics in vitro, but relapse due to insufficient vascularity often renders such treatment ineffective. Getting effective and universally accepted vaccines against S. equi have been slow mainly because of safety concerns. It was previously reported that colonization of air interface organ cultures, after inoculation with a mutant strain (ΔprtM138-213) deficient in the putative maturase lipoprotein (PrtM, a homologue of the pneumococcal PpmA) was less than that seen in cultures which were infected with wild-type S. equi strain 4047 (Hamilton et al, 2006), indicating that PrtM is a major virulence factor in strangles. It has also been demonstrated by in vitro and in vivo studies that many streptococcal adhesins, for example, serve as colonization or virulence factors and this makes them attractive targets for therapeutic and preventive strategies against streptococcal infections (Nobbs et al., 2009). S. equi adhesins or other colonization factors may be substrates for PrtM. Understanding PrtM is key to designing drugs or vaccines against the equine S. equi infection and strangles. In this research, advanced biomolecular techniques were systematically applied to investigate and characterise PrtM, and to evaluate its potential as a therapeutic or vaccine target. Bioinformatics, microbiological, biochemical and molecular biology techniques were used in screening the S. equi WT 4047 and Mutant (prtM138-213) strains to evaluate the immunogenicity and conservation of PrtM. Proteomics techniques: two-dimensional gel electrophoresis and mass spectrometry were employed in evaluating the cell-associated and secreted protein extracts of both the S. equi WT 4047 and mutant (prtM138-213) strains. In this study, genetic engineering technology involving targeted domain knock-out and/or knock-in, was employed in producing the central domain recombinant protein and mutant (prtM138-213) revertants. Following cloning, over-expression and purification of the full length and central domain, biochemical data on the PrtM protein (kcat/KM for S. equi 4047 PrtM full length recombinant protein = 5.84 x 106 /M/s) were derived via enzyme(peptydylprolyl isomerase - PPIase) assay; and crystallography was applied in an attempt to derive structural data on the PrtM protein. Advanced biomolecular techniques (including Western blots and Proteomics) were employed in screening the complemented mutants. It has been proven from this research that the PrtM of S. equi 4047 is involved in adaptation to NaCl stress and in regulating sensitivity to antibiotics; PrtM may have roles in speeding up the synthesis of hyaluronic acid and in the folding or remodeling of HPr Kinase. The parvulin-type structure of PrtM elucidated by bioinformatics analysis, the cross reactions of the WT and mutant with a number of antisera, the observation that PrtM may be a multisubstrate foldase due to the detectable and significant differences in the proteomes of the WT, mutant and complemented mutants, the dimeric protein formed by the full length recombinant protein of S. equi 4047 WT, and the PPIase-Chaperonine activities of PrtM, all observed from this study, validate PrtM of S. equi 4047 as a viable and novel therapeutic target which pharmaceutical industries should extensively evaluate for the prevention and treatment of S. equi infection and strangles.

The luminescent properties of transition metal complexes of heavy metals such as platinum(II) and irdium(III) are often highly luminescent and therefore are interesting for use as phosphorescent dopants in organic light-emitting diodes (OLEDs) and as luminescent probes in bioimaging. The majority of complexes investigated to date contain only one metal centre with multimetallic complexes becoming more widely studied in recent decades. This work explores the synthesis of novel dinuclear emitters based on cyclometallated Pt(II) and Ir(III) centres. In particular, complexes in which the metal centres are rigidly-linked via cyclometallating bridging ligands. The complexes described in this work are highly luminescent with high quantum yields in degassed solution and relatively short luminescence lifetimes. Dinuclear platinum(II) complexes linked via pyrimidine-based bis-bidentate ligands incorporating mono- and bis-thiophene cyclometallating units have been prepared and their photophysical properties investigated. These complexes were observed to emit in the orange (610 nm) and near-infrared (730 nm) regions of the spectrum for the mono- and bis-thiophene respectively, with relatively high quantum yields (φ = 0.15–0.85). A further dinuclear platinum(II) complex, rigidly-linked via a bis-tetradentate bridging ligand offering O^N^C^N coordination, was successfully prepared. To the best of our knowledge, this is the first example of di-platinum(II) complex in which the two metal centres are linked via this type of bridging ligand. Dinuclear iridium(III) complexes linked via bis-bidentate bridging ligands based on the central heterocycle pyrimidine have been prepared. These pyrimidine-linked complexes exhibited high quantum yields in degassed dichloromethane solution at room temperature, in the range 0.88-1.0. A representative structure is shown below. Variation of the monodentate chloride ligands to alternative ligands such as cyanide and acetonitrile was also investigated. Incorporation of a cyanide ligand was observed to blue-shift the emission relative to the analogous chloro complex. The effect of varying the central heterocycle of the bis-bidentate bridging ligand was investigated. The identity of the central heterocycle of the bridging ligand has been shown to have significant effects on the properties of the resulting dinuclear Ir(III) complexes, as is discussed below. Di-iridium(III) complexes linked via 2,5-pyrazine based bridging ligands have been successfully prepared. However, photodegradation of the complexes was observed in chlorinated solvents. To resolve this problem, a bis-bidentate bridging ligand incorporating a thiazolo[5,4]thiazole core was employed and the resulting di-iridium complex showed improved photostability. A 2,3-pyrazine-linked dinuclear iridium complex was synthesised and was observed to have a twisted geometry at room temperature and therefore, is chiral. Through a high-temperature NMR experiment, it was shown that this complex racemises at 80 °C. Finally, incorporating pyridazine as the central heterocycle leads to the sharing of a monodentate chloride ligand between the two iridium centres. This leads to the formation of a cationic dinuclear iridium(III) complex. This complex was investigated as a luminescent probe in bioimaging.

The major economic and environmental consequences of unfettered growth of marine organisms (fouling) on any substrate immersed in the marine environment have a global reach. Currently biocidal antifouling coatings are the most widely employed method to control this growth on vessels. These coatings are under increasing legislative scrutiny because they release primarily metal-based biologically active agents (biocides) to deter the fouling, and hence have the potential to persist in some form in the environment. This thesis explores the potential use of organic salt-based biocides, quaternary ammonium compounds (quats), as new marine biocides which are considered to be environmentally benign due to their short environmental half-lives. Detailed are methodologies to control the release of the quats as well as a new liquid chromatography (LC) mass spectroscopy (MS) analytical method to quantify the quats in seawater. The primary focus of the controlled release research has been the development of novel quat-polymer systems, which hydrolyze in the presence of seawater to generate biocidal quats in-situ. The quat-polymers are synthesised via a free radical polymerisation from different monomers, which are a blend of commercial available film forming monomers and a novel quat-monomer. The preparation of the quat-monomer is also detailed within this thesis. The quat-polymers have been shown to be stable in a prototype antifouling paint formulation and several coatings have been developed which exhibit significantly better antifouling performance than a current commercial product, Intersmooth 460, even after over 1 year with the testing continuing. The developed LC-MS method utilizes a sample pre-treatment solid phase extraction (SPE) process and is capable of quantifying quats to low parts-per-trillion (ppt) levels in seawater. The method is exceptionally flexible and able to detect a range of commercially available quats simultaneously. The application of this method to the determination of the release rates of the quats from antifouling coatings under standard laboratory conditions is also outlined.

This work is concerned with the synthesis and property investigation of a relatively new class of cyclometallated Ir(III) and Pt(II) complexes in which two metal centres are coordinated to a common heterocycle resulting in a rigid polymetallic assembly. Highly luminescent materials which can emit and absorb in a red region of the spectrum were targeted. There are three main parts of the thesis. The first part investigates how luminescent properties of the diplatinum systems are affected by the bridging ligand. A series of novel mono- and dinuclear Pt(II) complexes has been prepared and their luminescent and redox properties investigated. The main observation is that the introduction of the second metal centre leads to a substantial red-shift in absorption and emission. In the second part the role of changing the ligand substituents in a cyclometallated complex has been investigated to determine the extent to which luminescence is affected by the nature of the substituents. A series of mono- and dinuclear Pt(II) complexes have been prepared using substituted pyrazine bridging ligands. It was found that electron donating substituents such as –OMe in the benzene cyclometallating ring cause a red-shift, while electron withdrawing substituents such as –F cause a blue-shift in emission. The final part of the work describes the synthesis of cyclometallated homometallic bis Ir(III) complexes. A series of bis- Ir(III) complexes have been prepared using a terdentate cyclometallating N^C^N coordinating 1,3-di(2-pyridyl)benzene derivative as an auxiliary ligand. It was found that the nature of the bridging ligand determines the overall stability of the complex. Pyrimidine-linked systems were found to be the most stable, while pyrazine analogues readily photodecompose/isomerise. Pyridazine-linked systems lead to ionic complexes where one chloride ligand is shared by two Iridium metal centres.

The background to this research program was the need to investigate novel technologies and their application to fired ceramic processes that would facilitate the return to profitability for a small size brick maker. The company, Gasser Brick Company. Ziegel Gasser Mattoni GmbH S.r.l., in Natz-Schabs (Naz-Sciaves) in the north of Italy, is a relatively small manufacturer producing < 20,000 metric tons of brick per year. In this thesis the economic, environmental and social advantages consequent to a complete review of the approach to the company’s manufacture of cored clay bricks are discussed, including energy saving measures and the use of novel fuel and clay body supplements. A number of wide ranging novel technical modifications to the production processes of the Gasser Brick company have been tested, evaluated, reviewed, compared, and critically evaluated. The approach includes the utilization of a substantial percentage of various wastes as part-substitution of, or as an addition to, quarried raw materials and also the use of renewable and alternative fuels as a substitute for fossil fuels. A number of these modifications have resulted in beneficial changes to the product and the efficiency of the production process and considerably reduced the environmental footprint of the operation. Process modifications and the technological improvements to production equipment are explained and discussed along with detailed information about the measures that enabled the Gasser Brick company to return the profitability. The modifications resulted in a substantial increase of income. Revenues from waste gate fees are about 30% of the total turnover of the company. Costs for thermal energy dropped by 2/3: A ton of boiler oil commanded a price of 220 - 245 €/ton versus the around 80 €/ton of rendering fat.

The Group B Streptococcus (GBS) is a Gram-positive opportunistic pathogen which is a leading cause of neonatal disease globally. In 2000-2001, the general incidence of neonatal GBS infection was 0.72 per 1000 live births in U.K. and the mortality rate is about 10%, because of which neonatal GBS disease is a significant burden on society. GBS is part of the commensal flora, colonising the vagina and gastrointestinal tract of women. Vertical transmission is the main cause of early onset GBS disease. During the process of GBS neonatal disease, GBS must be able to survive in several very different host environments, including the vagina, amniotic fluid, the neonate's lung and blood. The vagina is normally acidic, low oxygen and with limited nutrients while the neonate's lung and blood are neutral, high oxygen and with abundant nutrient. Proteomic investigations of GBS protein expression under conditions representing those associated with benign maternal colonisation and foetal exposure may help us understand the molecular basis of GBS virulence. GBS growth characteristics, long term survival, acid adaptation, viable but non-culturable state and biofilm formation were investigated to help us understand how GBS survives in different environments and also help us to develop an in vitro model to reflect in vivo conditions during GBS disease development. An in vitro model of GBS growth under conditions reflecting maternal vaginal carriage (low pH, low oxygen, nutrient stress) and exposure to body fluids during invasive disease (neutral pH, aeration, nutrient sufficient) was established. Proteins expressed under each growth conditions were separated by two dimensional electrophoresis. Individual proteins were subjected to in-gel trypsin digestion and identified using liquid chromatography-mass spectrometry with peptide mass fingerprinting followed with bioinformatic research. A total of 76 proteins were identified and 16 of these were expressed differentially. The putative virulence factor C protein β antigen and proteins involved in responses to oxidative stress were up-regulated under the conditions reflecting neonatal exposure. Another in vitro model of GBS growth on Todd Hewitt agar in the presence or absence of 10% human serum was established and followed by proteomic investigation of proteins differentially expressed under these two conditions, as this model reflects GBS neonatal septicaemia (exposure to serum). A total of 84 proteins were identified and 11 of which were expressed differentially. The putative virulence factor C protein β antigen, arginine deiminase, an ABC transporter substrate-binding protein and glyceraldehyde-3-phosphate dehydrogenase were up-regulated in the presence of human serum.

This research aimed to investigate the bioavailability of 9 metals (Cr, Mn, Fe, Ni, Cu, Zn, Mo, Cd, and Pb) to vegetable crops (spinach, lettuce, carrot and radish) cultivated in compost soils at different levels of metal contamination. The uptake and accumulation of these metals by the plants were examined. The elemental speciation using SEC-UV-ICP-MS and Nanospray Mass Spectrometry had been performed to characterize the metal containing species induced in the plants exposed to metal stress. In order to evaluate potential health risks arising from ingestion of the metal contaminated plants, the oral bioaccessibility i.e. the use of an in vitro physiologically based extraction test (PBET) simulating the transition of the metal pollutants in the plants into the human gastrointestinal system was undertaken. It was found that, with the exception of Cr, metal concentrations (Mn, Fe, Ni, Cu, Zn, Mo and Cd) in lettuce, spinach, carrot and radish depended on the concentrations of the total metal in the soils in which the plants were grown. For Pb, the amounts accumulated in the leafy vegetables also depended on their levels of contamination in the soils while the root vegetables had rather low uptake and the uptake levels did not increase when higher levels of contamination were applied. Mn, Fe and Zn were relatively easily mobilised from soils to plants; they tended to accumulate in all plants studied at high concentrations. The elements which were more enriched in leaves included Mn and Zn (in all plant types), and Fe and Cd (only in the root vegetables). In contrast, Fe, Ni, Cu, Mo and Pb were accumulated more in roots of the leafy vegetables. Among all plants studied, it was observed that carrot had low uptake for all elements (Cr, Ni, Cu, Mo, Cd and Pb), except for Mn, Fe, Zn which were found in all plants. The metal mobilised from soil to plant as indicated by the metal contents accumulated in the plants decreased in the order Mn >> Zn > Fe > Cu > Mo > Ni > Cd > Pb Cr. The metal bioavailability to plants was assessed by measuring transfer factor (TF) values of the metals based on total metal contents in the soils. It was found that the order of TF values was Mn > Zn >> Cd > Ni > Cu > Mo Pb > Cr Fe. The mean TF values of each element irrespective of plant types were 1.93, 1.77, 0.485, 0.194, 0.111, 0.052, 0.045, 0.037 and 0.036 for Mn, Zn, Cd, Ni, Cu, Mo, Pb, Cr, and Fe, respectively. Hence, Mn and Zn were most bioavailable to plants i.e. they can be transferred from soils to plants more easily than Ni, Cu, Mo, Pb, Cr and Fe. Whereas, the bioavailability of Cd was relatively moderate. In addition, the results enabled the development of statistical regression models that are suited to predict metal uptake by plants. It indicated that the relationship between the TF values and the extractable soil metals followed the power regression curve. However, there were some cases in which it did not follow the power regression curve but a linear model, these are; Mn (for carrot leaves and radish roots), Mo (for spinach roots and carrot roots), and Cd (for lettuce leaves, spinach roots and leaves and carrot roots). In the multi-elemental speciation study, it was found that a common association of the metals (Cd, Cu, Mo, Pb, and Zn) to the high molecular weight (MW) fractions (8160 Da) was observed in all plant extracts. The lower MW fractions of approximately 1000 — 3000 Da of Cd, Cu, Mo, Ni, Pb, and Zn containing compounds were found to be present in all plant extracts. Iron was not detected in the roots of carrot and radish, but present as both high MW (8200 Da) and low MW (2500 Da) compounds in the leaves of spinach and lettuce. To characterize the individual metal containing species present in the plant samples, the Nanospray Mass Spectrometry was employed. Unfortunately, no evidence from this analysis can confirm that these compounds are related to the phytochelatin family.

A former industrial site now used for recreational activities was investigated for total PTE content, uptake of the PTEs by foraged fruits and mobility of the PTEs using single extraction such as HOAc and EDTA. In order to evaluate the health risks arising from ingestion of the PTE contaminated soil, the oral bioaccessibility using in vitro physiologically based extraction test (PBET) and tolerable daily intake (TDI) or mean daily intake (MDI) was used. The PBET simulates the transition of the PTE pollutants in the soil into human gastrointestinal system while the TDI or MDI is the mass of soil that a child would require to take without posing any health risk. In addition to the former industrial site, an investigation of the urban road dust from Newcastle city centre and its environs was undertaken with the view to looking into the PTE content, oral bioaccessibility and the platinum group elements (PGEs). Optimized microwave procedure was applied to 19 samples obtained from a former industrial site (St Anthony's lead works) in Newcastle upon Tyne. Of the range of PTEs potentially present at the site as a consequence of former industrial activity (As, Cd, Cr, Cu, Ni, Pb and Zn), the majority of top soil samples indicated elevated concentrations of one or more of these PTEs. In particular, data obtained using either inductively coupled plasma mass spectrometry (ICP-MS) or flame atomic absorption spectroscopy (FAAS) indicates the high and wide concentration of Pb on the site (174 to 33,306 mg/kg). Comparing the resulting PTEs data with UK Soil Guidelines Values (SGVs) suggests at least parts of the site represent areas of potential human health risk. It was found that Pb soil values exceeded the SGV on 17 out of the 19 sampling sites; similarly for As 7 out of 19 sampling sites exceeded the SGV. While for Cd and Ni the soil levels were below the stated SGVs. Samples of foraged fruits collected from the same site were also analysed for the same PTEs. The foraged fruit was gathered over two seasons along with samples of soil from the same sampling areas, acid digested using a microwave oven, and then analysed by ICP-MS. The foraged fruits samples included blackberries, rosehips and sloes which were readily available on the site. The concentration levels of the selected elements in foraged samples varied between not detectable limits and 24.6 mg/kg (Zn). Finally, the soil-to plant transfer factor was assessed for the 7 elements. In all cases, the transfer values obtained were below 1.00,except Cd in 2007 which is 1.00, indicating that the majority of the PTE remains in the soil and that the uptake of PTE from soil to plant at this site is not significant. The determination of total or pseudo total PTE content of soil is often insufficient to assess the risk to humans. A range of extraction protocols were applied to the 19 samples urban topsoils, and report on the correlations between pseudo total PTE content and results obtained following a physiologically-based extraction procedure (oral bioaccessibility), EDTA and HOAc extraction protocols (reagent-specific available fraction), for a broad range of PTEs (As, Cd, Cu, Cr, Ni, Pb, Zn). Results of the single-reagent extraction procedures did not, in general, provide a good indication of oral bioaccessibility but shows positive correlation with the pseudo total PTE content. The bioaccessibility data shows that considerable variation exists both spatially across the site, and between the different PTEs, but correlates well with the pseudo-total concentrations for all elements (r2 exceeding 0.8). One of the main objectives of this work is to show the role of bioaccessibility in generic risk assessment. Comparison of the pseudo-total PTE concentrations with SGV or generic assessment criteria (GAC) indicated that all of the PTEs investigated need further action, such as receptor exposure modelling.

Agricultural soils are heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, the molecular ecology of the total bacterial and free-living nitrogen fixing communities in soils from the Nafferton factorial systems comparison (NFSC) study in North East England were examined. The field experiment is factorial in design with organic versus conventional crop rotation, crop protection and fertility management factors. Soils were sampled on three dates (March, June and September) in 2007. Total RNA was extracted from all soil samples and reverse transcribed. DGGE and qPCR were used to analyse nifH and 16S rRNA genes in order to study free-living diazotrophs and the total bacterial community respectively. Crop rotation was shown to have a significant effect on total bacterial diversity (and that of free-living N fixers) (P=<0.001). On all three dates nifH activity was higher in the conventional crop rotation. In contrast, qPCR analysis of free-living N fixers indicated significantly higher levels of activity in conventionally fertilised plots in June (P=0.0324) and in plots with organic crop protection in September (P=0.0143). To our knowledge the effects of organic and conventional farming systems on free-living diazotrophs have never been studied. An increased understanding of the impacts of management practices on free-living N fixers could allow modifications in soil management practices to optimize the activity of these organisms.