Phenotypic and genotypic characterisation of Clostrum Difficile

Clostridium difficile is at present one of the most common nosocomial infections in the developed world. Hypervirulent strains (PCR ribotype 027) of C. difficile which produce enhanced levels of toxins have also been associated with other characteristics such as a greater rate of sporulation and resistance to fluoroquinolones. Infection due to C. difficile PCR ribotype 027 has also been associated with greater rates of morbidity and mortality. The aim of this thesis was to investigate both the phenotypic and genotypic characteristics of two populations of toxigenic clinical isolates of C. difficile which were recovered from two separate hospital trusts within the UK. Phenotypic characterisation of the isolates was undertaken using analytical profile indexes (APIs), minimum inhibitory concentrations(MICs) and S-layer protein typing. In addition to this, isolates were also investigated for the production of a range of extracellular enzymes as potential virulence factors. Genotypic characterisation was performed using a random amplification of polymorphic DNA(RAPD) PCR protocol which was fully optimised in this study, and the gold standard method, PCR ribotyping. The discriminatory power of both methods was compared and the similarity between the different isolates also analysed. Associations between the phenotypic and genotypic characteristics and the recovery location of the isolate were then investigated. Extracellular enzyme production and API testing revealed little variation between the isolates; with S-layer typing demonstrating low discrimination. Minimum inhibitory concentrations did not identify any resistance towards either vancomycin or metronidazole; there were however significant differences in the distribution of antibiogram profiles of isolates recovered from the two different trusts. The RAPD PCR protocol was successfully optimised and alongside PCR ribotyping, effectively typed all of the clinical isolates and also identified differences in the number of types defined between the two locations. Both PCR ribotyping and RAPD demonstrated similar discriminatory power; however, the two genotyping methods did not generate amplicons that mapped directly onto each other and therefore clearly characterised isolates based on different genomic markers. The RAPD protocol also identified different subtypes within PCR ribotypes, therefore demonstrating that all isolates defined as a particular PCR ribotype were not the same strain. No associations could be demonstrated between the phenotypic and genotypic characteristics observed; however, the location from which an isolate was recovered did appear to influence antibiotic resistance and genotypic characteristics. The phenotypic and genotypic characteristics observed amongst the C. difficile isolates in this study, may provide a basis for the identification of further targets which may be potentially incorporated into future methods for the characterisation of C. difficile isolates.

Mathematical modelling and control of agitated extraction columns

Liquid-liquid extraction has long been known as a unit operation that plays an important role in industry. This process is well known for its complexity and sensitivity to operation conditions. This thesis presents an attempt to explore the dynamics and control of this process using a systematic approach and state of the art control system design techniques. The process was studied first experimentally under carefully selected. operation conditions, which resembles the ranges employed practically under stable and efficient conditions. Data were collected at steady state conditions using adequate sampling techniques for the dispersed and continuous phases as well as during the transients of the column with the aid of a computer-based online data logging system and online concentration analysis. A stagewise single stage backflow model was improved to mimic the dynamic operation of the column. The developed model accounts for the variation in hydrodynamics, mass transfer, and physical properties throughout the length of the column. End effects were treated by addition of stages at the column entrances. Two parameters were incorporated in the model namely; mass transfer weight factor to correct for the assumption of no mass transfer in the. settling zones at each stage and the backmixing coefficients to handle the axial dispersion phenomena encountered in the course of column operation. The parameters were estimated by minimizing the differences between the experimental and the model predicted concentration profiles at steady state conditions using non-linear optimisation technique. The estimated values were then correlated as functions of operating parameters and were incorporated in·the model equations. The model equations comprise a stiff differential~algebraic system. This system was solved using the GEAR ODE solver. The calculated concentration profiles were compared to those experimentally measured. A very good agreement of the two profiles was achieved within a percent relative error of ±2.S%. The developed rigorous dynamic model of the extraction column was used to derive linear time-invariant reduced-order models that relate the input variables (agitator speed, solvent feed flowrate and concentration, feed concentration and flowrate) to the output variables (raffinate concentration and extract concentration) using the asymptotic method of system identification. The reduced-order models were shown to be accurate in capturing the dynamic behaviour of the process with a maximum modelling prediction error of I %. The simplicity and accuracy of the derived reduced-order models allow for control system design and analysis of such complicated processes. The extraction column is a typical multivariable process with agitator speed and solvent feed flowrate considered as manipulative variables; raffinate concentration and extract concentration as controlled variables and the feeds concentration and feed flowrate as disturbance variables. The control system design of the extraction process was tackled as multi-loop decentralised SISO (Single Input Single Output) as well as centralised MIMO (Multi-Input Multi-Output) system using both conventional and model-based control techniques such as IMC (Internal Model Control) and MPC (Model Predictive Control). Control performance of each control scheme was. studied in terms of stability, speed of response, sensitivity to modelling errors (robustness), setpoint tracking capabilities and load rejection. For decentralised control, multiple loops were assigned to pair.each manipulated variable with each controlled variable according to the interaction analysis and other pairing criteria such as relative gain array (RGA), singular value analysis (SVD). Loops namely Rotor speed-Raffinate concentration and Solvent flowrate Extract concentration showed weak interaction. Multivariable MPC has shown more effective performance compared to other conventional techniques since it accounts for loops interaction, time delays, and input-output variables constraints.

The chemical structure and mechanical properties of phenolic resins and related polymers

Paper-based phenolic laminates are used extensively in the electrical industry. Many small components are fabricated from these materials by the process known as punching. Recently an investigation was carried out to study the effect of processing variables on the punching properties. It was concluded that further work would be justified and that this should include a critical examination of the resin properties in a more controlled and systematic manner. In this investigation an attempt has been made to assess certain features of the resin structure in terms of thermomechanical properties. The number of crosslinks in the system was controlled using resins based on phenol and para-cresol formulations. Intramolecular hydrogen bonding effects were examined using substituted resins and a synthetically derived phenol based on 1,3-di-(o-hydroxyphenyl) propane.. A resin system was developed using the Friedel Crafts reaction to examine inter-molecular hydrogen bonding at the resin-paper interface. The punching properties of certain selected resins were assessed on a qualitative basis. In addition flexural and dynamic mechanical properties were determined in a general study of the structure-property relationships of these materials. It has been shown that certain features of the resin structure significantly influenced mechanical properties. :F'urther, it was noted that there is a close relationship between punching properties, mechanical damping and flexural strain. This work includes a critical examination of the curing mechanism and views are postulated in an attempt to extend knowledge in this area of the work. Finally, it is argued that future work should be based on a synthetic approach and that dynamic mechanical testing would provide a powerful tool In developing a deeper understanding of the resin fine structure.

Control of soundness and mechanical properties in aluminium-silicon-magnesium alloys

Previously, specifications for mechanical properties of casting alloys were based on separately cast test bars. This practice provided consistently reproducible results; thus, any change in conditions was reflected in changes in the mechanical properties of the test coupons. These test specimens, however, did not necessarily reflect the actual mechanical properties of the castings they were supposed to represent'. Factors such as section thickness and casting configuration affect the solidification rate and soundness of the casting thereby raising or lowering its mechanical properties in comparison with separately cast test specimens. In the work now reported, casting shapes were developed to investigate the variations of section thickness, chemical analysis and heat treatment on the mechanical properties of a high strength Aluminium alloy under varying chilling conditions. In addition, an insight was sought into the behaviour of chills under more practical conditions. Finally, it was demonstrated that additional information could be derived from the radiographs which form an essential part of the quality control of premium quality castings. As a result of the work, it is now possible to select analysis and chilling conditions to optimize the as cast and the heat treated mechanical properties of Aluminum 7% Silicon 0.3% Magnesium alloy.

Formulation and the characterisation of cationic liposomal adjuvants for the delivery of a promising subunit tuberculosis vaccine

Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) incorporating the glycolipid trehalose 6,6-dibehenate (TDB) forms a promising liposomal vaccine adjuvant. To be exploited as effective subunit vaccine delivery systems, the physicochemical characteristics of liposomes were studied in detail and correlated with their effectiveness in vivo, in an attempt to elucidate key aspects controlling their efficacy. This research took the previously optimised DDA-TDB system as a foundation for a range of formulations incorporating additional lipids of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), by incrementally replacing the cationic content within DDA-TDB or reducing the total DDA-TDB dose upon its substitution, to ascertain the role of DDA and the effect of DDA-TDB concentration in influencing the resultant immunological performance upon delivery of the novel subunit TB vaccine, Ag85B–ESAT-6-Rv2660c (H56 vaccine). With the aim of using the DPPC based systems for pulmonary vaccine delivery and the DSPC systems for application via the intramuscular route, initial work focused on physicochemical characterisation of the systems with incorporation of DPPC or DSPC displaying comparable physical stability, morphological structure and levels of antigen retention to that of DDA-TDB. Thermodynamic analysis was also conducted to detect main phase transition temperatures and subsequent in vitro cell culture studies demonstrated a favourable reduction in cytotoxicity, stimulation of phagocytic activity and macrophage activation in response to the proposed liposomal immunoadjuvants. Immunisation of mice with H56 vaccine via the proposed liposomal adjuvants showed that DDA was an important factor in mediating resultant immune responses, with partial replacement or substitution of DDA-TDB stimulating Th1 type cellular immunity characterised by elevated levels of IgG2b antibodies and IFN-? and IL-2 cytokines, essential for providing protective efficacy against TB. Upon increased DSPC content within the formulation, either by DDA replacement or reduction of DDA and TDB, responses were skewed towards Th2 type immunity with reduced IgG2b antibody levels and elevated IL-5 and IL-10 cytokine production, as resultant immunological responses were independent of liposomal zeta potential. The role of the cationic DDA lipid and the effect of DDA-TDB concentration were appreciated as the proposed liposomal formulations elicited antigen specific antibody and cellular immune responses, demonstrating the potential of cationic liposomes to be utilised as adjuvants for subunit vaccine delivery. Furthermore, the promising capability of the novel H56 vaccine candidate in eliciting protection against TB was apparent in a mouse model.

Modelling and experimental verification of a solar-powered liquid desiccant cooling system for greenhouse food production in hot climates

Experiments and theoretical modelling have been carried out to predict the performance of a solar-powered liquid desiccant cooling system for greenhouses. We have tested two components of the system in the laboratory using MgCl2 desiccant: (i) a regenerator which was tested under a solar simulator and (ii) a desiccator which was installed in a test duct. Theoretical models have been developed for both regenerator and desiccator and gave good agreement with the experiments. The verified computer model is used to predict the performance of the whole system during the hot summer months in Mumbai, Chittagong, Muscat, Messina and Havana. Taking examples of temperate, sub-tropical, tropical and heat-tolerant tropical crops (lettuce, soya bean, tomato and cucumber respectively) we estimate the extensions in growing seasons enabled by the system. Compared to conventional evaporative cooling, the desiccant system lowers average daily maximum temperatures in the hot season by 5.5-7.5 °C, sufficient to maintain viable growing conditions for lettuce throughout the year. In the case of tomato, cucumber and soya bean the system enables optimal cultivation through most summer months. It is concluded that the concept is technically viable and deserves testing by means of a pilot installation at an appropriate location.

Selective localization of organoclay and effects on the morphology and mechanical properties of LDPE/PA11 blends with distributed and co-continuous morphology

A study was made on the effect of small amounts of organically modified clay on the morphology and mechanical properties of blends of low-density polyethylene and polyamide 11 at different compositions. The influence of the filler on the blend morphology was investigated using wide angle X-ray diffractometry, scanning and transmission electron microscopy and selective extraction experiments. The filler was found to locate predominantly in the more hydrophilic polyamide phase. Although such uneven distribution does not have a significant effect on the onset of phase co-continuity of the polymer components, it brings about a drastic refinement of the microstructure for the blends both with droplets/matrix and co-continuous morphologies. In addition to the expected reinforcing action of the filler, the resulting fine microstructure plays an important role in enhancing the mechanical properties of the blends. This is essentially because of a good quality of stress transfer across the interface between the constituents, which also seems to benefit for a good interfacial adhesion promoted by the filler. Our results provide the experimental evidence for the capabilities of nanoparticles added to multiphase polymer systems to act selectively as a reinforcing agent for specific domains of the material and as a medium able to assist the refinement of the polymer phases during mixing.

Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies

Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A-class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg(2.39), His(2.43) and Glu(3.46), which makes a polar lock with T(6.37). These alignments and models provide useful tools for understanding class B GPCR function.

Pyrolysis of contaminated energy crops and the characterisation of the gained biochar

The simultaneous use of willow as a vegetation filter and an energy crop can respond both to the increasing energy demand and to the problem of the soil and water contamination. Its characteristics guarantee that the resources are used economically. As a vegetation filter, willow uptakes organic and inorganic contaminants. As a fast growing energy crop it meets the requirements of rural areas without the exploitation of existing forestry. The aim of the research was to gather knowledge on the thermal behaviour of willow, uptaking contaminants and then used as an energy crop. For this reason pyrolysis experiments were performed in two different scales. In analytical scale metal-contaminated wood was investigated and bench scale pyrolysis experiments were performed with nitrogen-enriched willow, originated from a wastewater treatment plant. Results of the pyrolysis showed that 51-81 % of the wastewater derived nitrogen of willow was captured in the char product. Char had low surface area (1.4 to 5.4 m2/g), low bulk density (0.15–0.18 g/cm3), high pH values (7.8–9.4) and high water-holding capacity (1.8 to 4.3 cm3/g) while the bioavailability of char nutrients was low. Links were also established between the pyrolysis temperature and the product properties for maximising the biochar provided benefits for soil applications. Results also showed that the metal binding capacity of wood varied from one metal ion to another, char yield increased and levoglucosan yield decreased in their presence. While char yield was mainly affected by the concentration of the metal ions, levoglucosan yield was more dependent on the type of the ionic species. Combustion experiments were also carried out with metal-enriched char. The burnout temperatures, estimated ignition indices and the conversion indicate that the metal ions type and not the amount were the determining factors during the combustion. Results presented in the Thesis provide better understanding on the thermal behaviour of nitrogen-enriched and metal contaminated biomass which is crucial to design effective pyrolysis units and combustors. These findings are relevant for pyrolysis experiments, where the goal is to yield char for energetic or soil applications.

The definition, theoretical modelling and empirical research of front-line service employee corporate identity within a UK national grocery retailer:determining the FLE corporate brand identification construct

Over the past forty years the corporate identity literature has developed to a point of maturity where it currently contains many definitions and models of the corporate identity construct at the organisational level. The literature has evolved by developing models of corporate identity or in considering corporate identity in relation to new and developing themes, e.g. corporate social responsibility. It has evolved into a multidisciplinary domain recently incorporating constructs from other literature to further its development. However, the literature has a number of limitations. It remains that an overarching and universally accepted definition of corporate identity is elusive, potentially leaving the construct with a lack of clear definition. Only a few corporate identity definitions and models, at the corporate level, have been empirically tested. The corporate identity construct is overwhelmingly defined and theoretically constructed at the corporate level, leaving the literature without a detailed understanding of its influence at an individual stakeholder level. Front-line service employees (FLEs), form a component in a number of corporate identity models developed at the organisational level. FLEs deliver the services of an organisation to its customers, as well as represent the organisation by communicating and transporting its core defining characteristics to customers through continual customer contact and interaction. This person-to-person contact between an FLE and the customer is termed a service encounter, where service encounters influence a customer’s perception of both the service delivered and the associated level of service quality. Therefore this study for the first time defines, theoretically models and empirically tests corporate identity at the individual FLE level, termed FLE corporate identity. The study uses the services marketing literature to characterise an FLE’s operating environment, arriving at five potential dimensions to the FLE corporate identity construct. These are scrutinised against existing corporate identity definitions and models to arrive at a definition for the construct. In reviewing the corporate identity, services marketing, branding and organisational psychology literature, a theoretical model is developed for FLE corporate identity, which is empirically and quantitatively tested, with FLEs in seven stores of a major national retailer. Following rigorous construct reliability and validity testing, the 601 usable responses are used to estimate a confirmatory factor analysis and structural equation model for the study. The results for the individual hypotheses and the structural model are very encouraging, as they fit the data well and support a definition of FLE corporate identity. This study makes contributions to the branding, services marketing and organisational psychology literature, but its principal contribution is to extend the corporate identity literature into a new area of discourse and research, that of FLE corporate identity