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Base methylation is a significant contributor to intrinsic DNA damage. Human Mex- cells deficient in the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), are hypersensitive to killing by methylating agents. DNA O6-meGua-mediated killing requires active mismatch repair (MMR). This property has been exploited to select MMR-defective human cells in the laboratory. Endogenous DNA methylation is a possible contributor to the emergence of MMR defective colorectal tumours. Using cultured GM0637 human fibroblasts I demonstrated that chronic low dose exposure to a N-methyl-N'- nitrosourea can select for, or induce, MMR defects through defective hMSH6 expression. Since defective MMR is associated with some inflammation-associated tumours, I explored the possibility that nitric oxide produced by activated macrophages might promote the formation of a selective agent for methylation tolerance. Using azaserine, a potential contribution of DNA damage introduced by carboxymethylating agents to the emergence of MMR defective cells was investigated. Cytotoxic DNA damage introduced by azaserine was repaired by nucleotide excision repair (NER). Azaserine cytotoxicity was not influenced by MMR or MGMT to a detectable extent and DNA carboxymethylation is unlikely to provide a significant selection pressure for loss of the MMR pathway. The effects of MMR on the processing of persistent bulky DNA lesions were examined in MNU B4 a MMR deficient derivative of the NER defective XPA fibroblast cell line XP12RO. MNU B4 cells lacked detectable hMSH2 expression. The combined XPA and hMSH2 defects did not affect sensitivity to persistent DNA damage induced by UV, cross-linking agents (cisplatin, mitomycin C, nitrogen mustard), ionizing raciiation, and azaserine. MMR does not appear to act as a general DNA damage sensor. I also isolated several MMR defective derivatives of the XPC cell line GM2249. The MMR defects in these derivatives did not detectably alter their sensitivity to UV irradiation. These variants provide an important resource to investigate the proposed role of MMR in the transcription coupled DNA nucleotide excision repair pathway.

Tie-1 is an endothelial cell specific tyrosine kinase receptor that is essential for the stabilisation of newly developed vessels and maintenance of endothelial cell integrity in the latter stages of angiogenesis. There is presently no known ligand for the tie-1 receptor, little is known of the factors that regulate its expression and information is lacking on downstream signalling events that occur following its activation. This study demonstrates that tie-1 protein expression in endothelial cells is increased at the level of transcription by VEGF and low oxygen tension, factors which are known to initiate angiogenesis. The cell surface expression of the receptor is also regulated by a metalloprotease enzyme which results in tie-1 ectodomain cleavage at a region close to the transmembrane region. This event generates a tie-1 endodomain fragment that persists in the cell membrane for several hours. This results in downstream signalling events, which lead to further secretion of the metalloprotease enzyme that can cleave tie-1 ectodomain in surrounding endothelial cells. The cleavage event may well be protein kinase C dependent and can be activated by VEGF. The loss of the extracellular domain of tie-1 inhibits tie-1 ligand binding at the cell surface and probably results in destabilising the endothelial cell allowing it to undergo angiogenesis or vessel remodelling. A potential tie-1 ligand, produced by a malignant melanoma cell line, results m activation of the tie-1 receptor by autophosphorylation of tyrosine kinase residues. Attempts at isolation of this protein suggest that it is a glycoprotein with a molecular weight of approximately 60-90kDa and this protein has been visualised on a nitrocellulose membrane and is currently awaiting N-terminal sequencing. Work presented in this thesis demonstrates for the first time factors that regulate expression of tie-1, novel downstream signalling events and the possible isolation of the tie-1 ligand.

The crystal impedance technique was used to study, in situ, electroactive polymer film dynamics. An equivalent circuit transmission line model was applied to extract physical characteristics of polymer films in solution in terms of their shear storage (G') and loss (G'') moduli. This model comprises three components that describe the surface roughness features of the quartz oscillator, the overlaying viscoelastic polymer film and the contacting electrolyte solution. Crystal impedance measurements were made on poly(3-hexylthiophene) (PHT) films, exposed to propylene carbonate electrolyte solutions, that were electrochemically maintained at different fixed potentials (corresponding to a range of film oxidation states) and at different temperatures. The p-doped film is substantially softer than the undoped film and G' and G" can show maxima at partial p-doping. Spectra recorded at a range of frequencies, corresponding to the fundamental (10 MHz) and higher harmonics (30 MHz to 110 MHz), indicate that PHT shear moduli are frequency dependent substantial increases in shear modulus are found upon increasing perturbation frequency. Film shear modulus values also exhibited temperature dependence, increased temperature corresponding to a decrease in shear modulus value. The role of solvent upon electroactive polymer film dynamics was explored by exposing PHT films to polar (propylene carbonate) and non- polar (dichloromethane) media. p-Doped films were highly swollen in the polar solvent and relatively compact in the non-polar medium, these trends being reversed for undoped films. Thin PHT films were also found to exhibit mechanical resonance effects. This is a special situation in which the mechanical shear deformation across the polymer film corresponds to one quarter of the acoustic wavelength in that medium. The new phenomenon of peak splitting was also observed at film resonance. This data indicates spatial variation of the PHT film shear modulus.

The transport and separation of oil and water is a vital process to the oil and chemical industries. Fluids exiting from oil wells usually consist of gas, oil and water and these three phases need to be transported and separated before they can be processed further. Operation of the primary separators has often proved to be problematic due to the change in composition of the fluids as the well matures, often accompanied by the build up of sand or asphaltenes. These vessels are very expensive to install so there is motivation to improve their design and performance. One major factor affecting separator performance is the phase distribution of the inlet flow, as reflected in the flow pattern and droplet size. In this work, flow pattern boundaries and drop sizes of liquid-liquid dispersions were measured for vertical and horizontal flow of a kerosene and water mixture in a 0.063m tube. Drop size was investigated by using two different laser optical techniques. A laser backscatter technique was employed for concentrated dispersions and a diffraction technique was used at low concentrations. In order to develop a greater understanding of separator performance, a 1/5th-scale model was constructed of diameter 0.6m and length 205m. Residence Time Distributions were obtained for a range of different internal configurations and flow rates using a colorimetric tracer technique. Flow rates of 1.5-4 kg/s oil and 1-4 kg/s water were used and the vessel was equipped with a perforated flow-spreading baffle at the inlet and an overflow weir. Experiments were performed with no internals and with dip or side baffles. The side baffles acted to create quiescent zones within the vessel while the dip baffle caused a local acceleration of both phases. These situations are similar to those that can be caused by blocked internals or existing baffling or structured packing within field separators. A Residence Time Distribution model of a primary separator, the Alternative Path Model, was developed using transfer functions. This model has the ability to reproduce features of the experimental data by representing the flow as a series of continuous stirred tanks in series or in parallel. The model was used to develop parameters that could be used to obtain information about the performance of the separator. This model was also applied to Residence Time Distribution data obtained from field separators by BP Exploration, to relate features of the pilot scale separator to the field vessels.

The three dimensional structure and dynamics of three biologically important oligosaccharides were probed using a range of NMR techniques. The solution structure of oligosaccharide moiety of was solved using conventional 1H - 1H ROESY data in conjunction with molecular dynamics simulations. It was evident from this study that the dominant conformation in free solution is similar to the structure of oligo-GM1 in complex with the B-subunit of the enteropathogenic cholera toxin. The solution dynamics of an analogue of globoside (Gb4-OEtTMS), a cellular receptor for an E. coli verotoxin, was also elucidated using conventional 1H - 1H ROESY data. However, it was not clear that these data alone could adequately model the structure. Additional distance restraints were derived from 1H - 1H NOE contacts between exchangeable and non-exchangeable protons measured in H2O/acetone-d6 solutions at low temperature (-15 °C). It was concluded on the basis of these studies that Gb4-OEtTMS is an extremely flexible sugar, however, the time- averaged conformation was similar to the proposed conformation of Gb4 in complex with a verotoxin (VT2e). In order to increase the structural information which may be measured from NMR spectroscopy, carbon-13 enriched carbohydrate moieties, commonly found in glycolipids and glycoproteins, were synthesised. Isotopically labelled N-acetyl neuraminic acid, a2,3 sialyl N-acetyl lactosamine (required for the synthesis of sialyl Lewis antigen) and a2,3 sialyl lactose (oligo-GM3), the core ganglioside which is required for the synthesis of more complex gangliosides (e.g. GM1), was accomplished using a mixture of enzymatic and synthetic methods. To test the applicability of heteronuclear NMR spectroscopy in the study of oligosaccharides, the solution structure and dynamics of [U-13C] oligo- GM3 was probed by recording three dimensional 13C-edited ROESY spectra, and long-range carbon-carbon and carbon-proton spin-coupling constants. Inter-residue ROEs could be unambiguously assigned from the 3D experiment, and these assignments contradict previous inter-residue ROE contacts published in the literature (based only on homonuclear data). Two additional inter-residue ROE contacts could be measured across the Gaibeta1-4Glc linkage. The solution conformation of [U-13C] oligo-GM3 in complex with wheat germ agglutinin was solved on the basis of 3D TRNOESY-HSQC data. Theoretical TRNOE intensities back-calculated from full relaxation matrix calculations performed on the X-ray crystal structures of the complex were consistent with experimentally measured values and confirmed the flexible nature of the Galbeta1-4Glc linkage when bound to the protein. Finally, carbon-13 enriched N-acetyl neuraminic acid and galactose were introduced into the glycan chains of an intact glycoprotein FcREA. 13C-1H HSQC data suggest that the two arms of the biantennary glycan experience two distinct magnetic environments.

Cloned antibody binding sites (known as Fv antibody fragments) are attractive to the fine chemicals industry for several applications: in particular, as ligands in affinity chromatography. However, optimised systems for producing Fv fragments are not currently available. This thesis aims to establish new methods for the analysis and recovery of Fv antibody fragments that have been expressed in Escherichia coli cultures by recombinant DNA technology, thereby enabling improved control of the processes required for Fv production. An important objective was to prepare and test generic analytical reagents that bind all Fv fragments regardless of their specificity. Tracer antibodies were raised against a common peptide motif and used to design a generally applicable immunoassay for analysing Fv protein in microbial culture. The immunoassay was used as a component part in a new analytical system that can determine total Fv and active Fv independently, thus providing the fermentation scientist with a complete picture of the immunoreactivity or "authenticity" of the Fv throughout the process. Using an Fv specific for human gonadotropin as an example, the immunoreactivity of the Fv in the fermenter was found to approach 100%. A new process for recovering active Fv fragments from microbial cultures was designed and tested, using an Fv specific for estrone-3-glucuronide as an example. The new process was based on antigen affinity chromatography and used two antigen analogues: one as a column ligand, the other as a specific eluant. The recovery efficiency of active Fv was several-fold superior to that obtained with conventional methods. Overall, the results show that the analysis and recovery of Fv fragments are related in analytical and production systems: both require the preparation and synthesis of reagents that can discriminate between Fv and non-specific biomass and between active Fv and inactive Fv.

The purpose of this thesis is to explore the methodological and empirical issues relating to the economic evaluation of minimal access surgery (MAS). Given the likely increase in the utilisation of economic analysis in this area, it is crucial to explore whether the methods of economic evaluation have limitations in the face of the particular characteristics of MAS. The comparison of abdominal hysterectomy (AH) and transcervical endometrial resection (TCRE), for the treatment of menorrhagia, is used as a vehicle to develop methods in relation to MAS. Having reviewed the literature and issues relating to the economic analysis of this group of technologies, the empirical starting point of the thesis is the assessment of the limitations of economic evaluations alongside clinical trials, using a trial comparing AH and TCRE. Three major areas of weakness are identified, and alternative ways of addressing these weaknesses are explored in the remainder of the thesis. The first area of methodological development relates to the measure of benefit used in economic evaluation of MAS. In this clinical context, it is argued that the trade-offs that exist between MAS and conventional surgery, in terms of process characteristics and outcomes, should result in an important role for patients' preferences in the construction of a benefit measure. A cost-utility analysis using the standard quality-adjusted life year (GALY) is undertaken, using trial data augmented with valuation data from a further study. The lack of consistency between individual preferences and standard QALYs suggests a major weakness with this measure of benefit. The strengths and weaknesses of an alternative measure of benefit in cost-utility analysis - the ex ante healthy years equivalent (HYE) - are assessed based on a further valuation study. It is shown that it is feasible to elicit ex ante HYEs from patients and that this measure of benefit exhibits some consistency with other expressions of patients' preferences. However, the HYE is likely to impose a greater measurement burden than the standard QALY. The second area of methodological development in the thesis is the analysis of the generalisability of trial-based economic evaluation, given the limitations that often exist with the external validity of trials. A framework is developed within which trial and observational data can be synthesised. This facilitates the use of sensitivity analysis to explore the robustness of base-case (trial-generated) results to alternative sources of data, which may be more representative of routine practice. The third area of methodological development stems from the importance of patients' preferences in relation to MAS. This element relates to the use of methods to model and to evaluate management strategies which use patients' preferences to determine treatment allocation. It is concluded that preference-based decision making has the potential to be cost effective in relation to TCRE and AH, and MAS applications more generally. The thesis demonstrates the importance of continued development in the detailed methods of economic evaluation.

A number of broad questions were addressed (a) Is counselling effective?, (b) Are post-counselling gains maintained at follow-up? (c) Does the sgape of change across counselling sessions adhere to the 'dose-effect' model?, (d) Do measures of distress and interpersonal problems differ in the extent of pre-post change? and (e) Are there any within-group differences in the extent of pre-post change on measures?, (f) A further aim of the study was to collect qualitative accounts of the intervention from clients, to build up a 'picture' of clients experiences of service use: To obtain a consumers point of view. The study took place at two sites, one in London, the second in the Midlands. A prepost-follow-up design was adopted. In addition, measures were completed for each session of counselling. Finally, clients also completed an evaluation questionnaire. Hypotheses were, generally, supported by analyses. There were substantial pre-post reductions on measures of distress and interpersonal problems, which were maintained at follow-up. Significant reductions on measures across sessions of counselling were observed, with change curves adhering to the 'dose-effect' model. Qualitative analyses built-up a picture of the rationale for service use and the costs and benefits that clients perceived from counselling. Discussion focused on a number of issues: The first, the difference between the reported study and the bulk of psychotherapeutic studies, secondly, the methodological and practical issues that arose during the study and, thirdly, the need to approach applied counselling research from a new perspective, that is less dependent on the techniques developed by efficacy studies.

The proposal is made that very small changes in gravitational field could be detected by monitoring a cell of heated fluid. Variations in gravity would be observed by their effect on the convection in the fluid cell. It is proposed that the convection patterns be observed by using an interferometer to image the temperature gradient in the fluid by utilising the effect of temperature variations on the refractive index of the fluid. It was proposed that this system may be able to detect changes in the Earth's gravitational field from orbit. The possibility of using the NASA GAS programme to perform a space flight test of this proposal was suggested. Activities in this programme were therefore surveyed and a proposal made. The primary experimental results were recorded in the form of convection images which were later processed on the ground to extract information. Research has been carried out in this area as investigation has shown that existing image processing techniques are not suitable to process the anticipated fringe images. Meanwhile design and development of a GAS experiment was performed. This was undertaken in liaison with NASA in order to achieve the required safety approvals for flight. Subsequently the flight experiment was performed aboard the Space Shuttle Endeavour during the STS-77 mission launched on the 19th May 1996. As a result of this project an image processing system for the analysis of interferogram images of convection has been developed and an experiment to image convection in microgravity, with a view to analysing its use for the detection of changes in gravity has been performed.

The design, synthesis and characterisation of novel liquid crystal materials for emerging applications presents many challenges. The physical property parameters become ever more stringent and corresponding exceptional property liquid crystalline compounds are required. Two topics have been researched; high birefringence (Δn), high photo and chemical stability nematic materials for PDLC/AMD use and chiral nematic (N*) photopolymerisable compounds for polymer network formation. Many materials were designed, synthesised and characterised for each application and materials were found that were very suitable for each application. Materials for PDLC/AMD use, should be low melting, high Δn nematogens. Additionally, these materials should possess a high degree of chemical and photostability. The synthesis and characterisation of compounds containing N-heterocyclic rings, naphthalene rings or lateral fluorosubstituted p-terphenyl moieties in combination with different endgroups is described. The study demonstrated that N-heterocyclic rings produce a high Δn but are not sufficiently photochemically stable. 2,6-naphthalene containing terminal chloro substituted compounds were shown to have high Δn and high stability but also had high melting points. Lateral fluoro substituted, alkyl p-terphenyls with fluoro, chloro, thiomethyl and methoxy had low melting points, broad nematic mesophases and high stability and were very suitable for PDLC/AMD use. The study of N* photopolymerisable compounds for polymer network formation examines the design, synthesis and characterisation of both mono and diacrylates, monoepoxides and monovinylethers in combination with a range of chiral centres. The (S)-(+)-2-methylbutyl group when in combination with various mesogenic cores was found to be very useful as the chiral portion in low melting point enantiotropic N* monoacrylates, monoepoxides and monovinylethers. These compounds were also found to be eminently suitable for the formation of binary mixtures with nematic diacrylates, divinylethers and diepoxides. These mixtures showed visible wavelength selective reflection and could be photopolymerised into coloured chiral polymer network films.

A study of gull management was made at a large colony of Lesser Black-backed Gulls Ljxrus fuscus on Tambrook Fell, Lancashire. Approximately 18,000 gulls presently breed at the site, and the area utilised by the gulls extends over 6 km2 on three private estates. The main studies were conducted on the Abbeystead Estate between 1992 and 1994. The reasons for increases in the numbers of several gull species in many parts of the world during the 20th century are presented, as well as the conflicts with humans caused by these increases. The practical and moral aspects of managing gulls are evaluated, as well as the conflicts likely to result from recent increases in the urbanisation of several Larid species. The need for management strategies to incorporate up-to-date knowledge of gull demography, density dependence, breeding biology and behaviour are discussed in the light of the limited success of past attempts at managing gulls. Experiments to entirely clear specific areas of the Tambrook Fell Gullery by disturbing breeding gulls are described. The aim was to ameliorate local problems by reducing the extent of the colony using non-lethal management techniques. Disturbance was carried out in a series of 2.25ha experimental plots. Audio, visual and physical disturbance methods, presented singly or in combination, were used to investigate their ability to exclude gulls and prevent breeding. The number of gulls using the plots and the number of nests built were compared with numbers on control plots. Gulls showed habituation to all disturbance methods, although the number of gulls using a plot was reduced during disturbance and a proportion of the original number of gulls were totally excluded. Only where two disturbance methods were utilised on the same plot was breeding completely prevented. It was demonstrated that disturbance was more effective when initiated prior to the start of nest building and when conducted at the edge of the colony, and that disturbance by human presence is an effective method of preventing breeding. In the year following disturbance, only a very few gulls attempted to utilise the disturbed areas, and the need to establish a 'sink' area for birds displaced by disturbance is discussed. In two seasons, a total of 75ha were cleared of breeding gulls. This was 23% of the Abbeystead Estate and 11 % of the total gullery area. This was the first time in over thirty years of management efforts at the colony, that the extent of the gullery was successfully reduced. Models are presented to show the effects of the 1978- 1988 culls on adult survivorship and recruitment into the breeding group at Tambrook Fell. The relative effects of management strategies aimed at survivorship and productivity are discussed. A study was made to quantify the percentage of nests built by gulls at the colony that are not subsequently laid in. Behavioural differences between pairs that failed to lay eggs and pairs that successfully bred are presented. In the past, calculations of the number of breeding gulls at the colony assumed that one nest represented one pair. This was shown not to be true and a correction factor (multiplier) of 0.61 was calculated to allow counts of nests at the colony to be converted to the number of breeding pairs.