Identification, mutagenesis, and transcriptional analysis of the methanesulfonate transport operon of methylosulfonomonas methylovora

Recently identified genes located downstream (3') of the msmEF (transport encoding) gene cluster, msmGH, and located 5' of the structural genes for methanesulfonate monooxygenase (MSAMO) are described from Methylosulfonomonas methylovora. Sequence analysis of the derived polypeptide sequences encoded by these genes revealed a high degree of identity to ABC-type transporters. MsmE showed similarity to a putative periplasmic substrate binding protein, MsmF resembled an integral membraneassociated protein, and MsmG was a putative ATP-binding enzyme. MsmH was thought to be the cognate permease component of the sulfonate transport system. The close association of these putative transport genes to the MSAMO structural genes msmABCD suggested a role for these genes in transport of methanesulfonic acid (MSA) into M. methylovora. msmEFGH and msmABCD constituted two operons for the coordinated expression of MSAMO and the MSA transporter systems. Reverse-transcription-PCR analysis of msmABCD and msmEFGH revealed differential expression of these genes during growth on MSA and methanol. The msmEFGH operon was constitutively expressed, whereas MSA induced expression of msmABCD. A mutant defective in msmE had considerably slower growth rates than the wild type, thus supporting the proposed role of MsmE in the transport of MSA into M. methylovora.

Conditional volatility and firm size:an empirical analysis of UK equity portfolios

The techniques and insights from two distinct areas of financial economic modelling are combined to provide evidence of the influence of firm size on the volatility of stock portfolio returns. Portfolio returns are characterized by positive serial correlation induced by the varying levels of non-synchronous trading among the component stocks. This serial correlation is greatest for portfolios of small firms. The conditional volatility of stock returns has been shown to be well represented by the GARCH family of statistical processes. Using a GARCH model of the variance of capitalization-based portfolio returns, conditioned on the autocorrelation structure in the conditional mean, striking differences related to firm size are uncovered.

Analysis of plasmid profiles of Aeromonas salmonicida isolates by pulsed field gel electrophoresis

Plasmid constitutions of Aeromonas salmonicida isolates were characterised by flat-bed and pulsed field gel electrophoresis. Resolution of plasmids by pulsed field gel electrophoresis was greater and more consistent than that achieved by flat-bed gel electrophoresis. The number of plasmids separated by pulsed field gel electrophoresis varied between A. salmonicida isolates, with five being the most common number present in the isolates used in this study. Plasmid profiles were diverse and the reproducibility of the distances migrated facilitated the use of principal components analysis for the characterisation of the isolates. Isolates were grouped according to the number of plasmids supported. Further principal components analysis of groups of isolates supporting five and seven plasmids showed a spatial separation of plasmids based upon distance migrated. Principal components analysis of plasmid profiles and antimicrobial minimum inhibitory concentrations could not be correlated suggesting that resistance to antimicrobial agents is not associated with either one plasmid or a particular plasmid constitution.

The dialectics of corporate reporting:a semiotic analysis of corporate financial and environmental reporting

The central argument to this thesis is that the nature and purpose of corporate reporting has changed over time to become a more outward looking and forward looking document designed to promote the company and its performance to a wide range of shareholders, rather than merely to report to its owners upon past performance. it is argued that the discourse of environmental accounting and reporting is one driver for this change but that this discourse has been set up as in conflicting with the discourse of traditional accounting and performance measurement. The effect of this opposition between the discourses is that the two have been interpreted to be different and incompatible dimensions of performance with good performance along one dimension only being achievable through a sacrifice of performance along the other dimension. Thus a perceived dialectic in performance is believed to exist. One of the principal purposes of this thesis is to explore this perceived dialectic and, through analysis, to show that it does not exist and that there is not incompatibility. This exploration and analysis is based upon an investigation of the inherent inconsistencies in such corporate reports and the analysis makes use of both a statistical analysis and a semiotic analysis of corporate reports and the reported performance of companies along these dimensions. Thus the development of a semiology of corporate reporting is one of the significant outcomes of this thesis. A further outcome is a consideration of the implications of the analysis for corporate performance and its measurement. The thesis concludes with a consideration of the way in which the advent of electronic reporting may affect the ability of organisations to maintain the dialectic and the implications for corporate reporting.

Base modified peptidic nucleic acids: synthesis and crystallographic analysis of intermediates

Peptidic Nucleic Acids (PNAs) are achiral, uncharged nucleic add mimetics, with a novel backbone composed of N-(2-aminoethyl)glycine units attached to the DNA bases through carboxymethylene linkers. With the aim of extending and improving upon the molecular recognition properties of PNAs, the aim of this work was to synthesjse PNA building block intermediates containing a series of substituted purine bases for subsequent use in automated PNA synthesis. Four purine bases: 2,6~diaminopurine (D), isoGuanine (isoG), xanthine (X) and hypoxanthine (H) were identified for incorporation into PNAs targeted to DNA, with the promise of increased hybrid stability over extended pH ranges together with improvements over the use of adenine (A) in duplex formation, and cytosine (C) in triplex formation. A reliable, high-yielding synthesis of the PNA backbone component N -('2- butyloxycarbonyl-aminoethyl)glycinate ethyl ester was establishecl. The precursor N~(2-butyloxycarbonyl)amino acetonitrile was crystallised and analysed by X-ray crystallography for the first time. An excellent refinement (R = 0.0276) was attained for this structure, allowing comparisons with known analogues. Although chemical synthesis of pure, fully-characterised PNA monomers was not achieved, chemical synthesis of PNA building blocks composed of diaminopurine, xanthine and hypoxanthine was completely successful. In parallel, a second objective of this work was to characterise and evaluate novel crystalline intermediates, which formed a new series of substituted purine bases, generated by attaching alkyl substituents at the N9 or N7 sites of purine bases. Crystallographic analysis was undertaken to probe the regiochemistry of isomers, and to reveal interesting structural features of the new series of similarly-substituted purine bases. The attainment of the versatile synthetic intermediate 2,6-dichloro~9- (carboxymethyl)purine ethyl ester, and its homologous regioisomers 6-chloro~9- (carboxymethyl)purine ethyl ester and 6-chloro-7-(carboxymethyl)purine ethyl ester, necessitated the use of X-ray crystallographic analysis for unambiguous structural assignment. Successful refinement of the disordered 2,6-diamino-9-(carboxymethyl) purine ethyl ester allowed comparison with the reported structure of the adenine analogue, ethyl adenin-9-yl acetate. Replacement of the chloro moieties with amino, azido and methoxy groups expanded the internal angles at their point of attachment to the purine ring. Crystallographic analysis played a pivotal role towards confirming the identity of the peralkylated hypoxanthine derivative diethyl 6-oxo-6,7-dihydro-3H-purlne~3,7~djacetate, where two ethyl side chains were found to attach at N3 and N7,

Meta-analysis of the population and phenotypic expression of CYP2C9/2C19 polymorphism on drug metabolism in different ethnicities

The term "pharmacogenetics" has been defined as the scientific study of inherited factors that affect the human drug response. Many pharmacogenetie studies have been published since 1995 and have focussed on the principal enzyme family involved in drug metabolism, the cytochrome P450 family, particularly cytochrome P4502C9 and 2C19. In order to investigate the pharmacogenetic aspect of pharmacotherapy, the relevant studies describing the association of pharmacogenetic factor(s) in drug responses must be retrieved from existing literature using a systematic review approach. In addition, the estimation of variant allele prevalence for the gene under study between different ethnic populations is important for pharmacogenetic studies. In this thesis, the prevalence of CYP2C9/2C19 alleles between different ethnicities has been estimated through meta-analysis and the population genetic principle. The clinical outcome of CYP2C9/2C19 allelic variation on the pharmacotherapy of epilepsy has been investigated; although many new antiepileptic drugs have been launched into the market, carbamazepine, phenobarbital and phenytoin are still the major agents in the pharmacotherapy of epilepsy. Therefore, phenytoin was chosen as a model AED and the effect of CYP2C9/2C19 genetic polymorphism on phenytoin metabolism was further examined.An estimation of the allele prevalence was undertaken for three CYP2C9/2C19 alleles respectively using a meta-analysis of studies that fit the Hardy-Weinberg equilibrium. The prevalence of CYP2C9*1 is approximately 81%, 96%, 97% and 94% in Caucasian, Chinese, Japanese, African populations respectively; the pooled prevalence of CYP2C19*1 is about 86%, 57%, 58% and 85% in these ethnic populations respectively. However, the studies of association between CYP2C9/2C19 polymorphism and phenytoin metabolism failed to achieve any qualitative or quantitative conclusion. Therefore, mephenytoin metabolism was examined as a probe drug for association between CYP2C19 polymorphism and mephenytoin metabolic ratio. Similarly, analysis of association between CYP2C9 polymorphism and warfarin dose requirement was undertaken.It was confirmed that subjects carrying two mutated CYP2C19 alleles have higher S/R mephenytoin ratio due to deficient CYP2C19 enzyme activity. The studies of warfarin and CYP2C9 polymorphism did not provide a conclusive result due to poor comparability between studies.The genetic polymorphism of drug metabolism enzymes has been studied extensively, however other genetic factors, such as multiple drug resistance genes (MDR) and genes encoding ion channels, which may contribute to variability in function of drug transporters and targets, require more attention in future pharmacogenetic studies of antiepileptic drugs.

IN VIVO analysis of ocular morphological changes during phakic accommodation

The principal theme of this thesis is the in vivo examination of ocular morphological changes during phakic accommodation, with particular attention paid to the ciliary muscle and crystalline lens. The investigations detailed involved the application of high-resolution imaging techniques to facilitate the acquisition of new data to assist in the clarification of aspects of the accommodative system that were poorly understood. A clinical evaluation of the newly available Grand Seiko Auto Ref/ Keratometer WAM-5500 optometer was undertaken to assess its value in the field of accommodation research. The device was found to be accurate and repeatable compared to subjective refraction, and has the added advantage of allowing dynamic data collection at a frequency of around 5 Hz. All of the subsequent investigations applied the WAM-5500 for determination of refractive error and objective accommodative responses. Anterior segment optical coherence tomography (AS-OCT) based studies examined the morphology and contractile response of youthful and ageing ciliary muscle. Nasal versus temporal asymmetry was identified, with the temporal aspect being both thicker and demonstrating a greater contractile response. The ciliary muscle was longer in terms of both its anterior (r = 0.49, P <0.001) and overall length (r = 0.45, P = 0.02) characteristics, in myopes. The myopic ciliary muscle does not appear to be merely stretched during axial elongation, as no significant relationship between thickness and refractive error was identified. The main contractile responses observed were a thickening of the anterior region and a shortening of the muscle, particularly anteriorly. Similar patterns of response were observed in subjects aged up to 70 years, supporting a lensocentric theory of presbyopia development. Following the discovery of nasal/ temporal asymmetry in ciliary muscle morphology and response, an investigation was conducted to explore whether the regional variations in muscle contractility impacted on lens stability during accommodation. A bespoke programme was developed to analyse AS-OCT images and determine whether lens tilt and decentration varied between the relaxed and accommodated states. No significant accommodative difference in these parameters was identified, implying that any changes in lens stability with accommodation are very slight, as a possible consequence of vitreous support. Novel three-dimensional magnetic resonance imaging (MRI) and analysis techniques were used to investigate changes in lens morphology and ocular conformation during accommodation. An accommodative reduction in lens equatorial diameter provides further evidence to support the Helmholtzian mechanism of accommodation, whilst the observed increase in lens volume challenges the widespread assertion that this structure is incompressible due to its high water content. Wholeeye MRI indicated that the volume of the vitreous chamber remains constant during accommodation. No significant changes in ocular conformation were detected using MRI. The investigations detailed provide further insight into the mechanisms of accommodation and presbyopia, and represent a platform for future work in this field.

Analysis of magnetoencephaoographic data as a nonlinear dynamical system

This thesis presents the results from an investigation into the merits of analysing Magnetoencephalographic (MEG) data in the context of dynamical systems theory. MEG is the study of both the methods for the measurement of minute magnetic flux variations at the scalp, resulting from neuro-electric activity in the neocortex, as well as the techniques required to process and extract useful information from these measurements. As a result of its unique mode of action - by directly measuring neuronal activity via the resulting magnetic field fluctuations - MEG possesses a number of useful qualities which could potentially make it a powerful addition to any brain researcher's arsenal. Unfortunately, MEG research has so far failed to fulfil its early promise, being hindered in its progress by a variety of factors. Conventionally, the analysis of MEG has been dominated by the search for activity in certain spectral bands - the so-called alpha, delta, beta, etc that are commonly referred to in both academic and lay publications. Other efforts have centred upon generating optimal fits of "equivalent current dipoles" that best explain the observed field distribution. Many of these approaches carry the implicit assumption that the dynamics which result in the observed time series are linear. This is despite a variety of reasons which suggest that nonlinearity might be present in MEG recordings. By using methods that allow for nonlinear dynamics, the research described in this thesis avoids these restrictive linearity assumptions. A crucial concept underpinning this project is the belief that MEG recordings are mere observations of the evolution of the true underlying state, which is unobservable and is assumed to reflect some abstract brain cognitive state. Further, we maintain that it is unreasonable to expect these processes to be adequately described in the traditional way: as a linear sum of a large number of frequency generators. One of the main objectives of this thesis will be to prove that much more effective and powerful analysis of MEG can be achieved if one were to assume the presence of both linear and nonlinear characteristics from the outset. Our position is that the combined action of a relatively small number of these generators, coupled with external and dynamic noise sources, is more than sufficient to account for the complexity observed in the MEG recordings. Another problem that has plagued MEG researchers is the extremely low signal to noise ratios that are obtained. As the magnetic flux variations resulting from actual cortical processes can be extremely minute, the measuring devices used in MEG are, necessarily, extremely sensitive. The unfortunate side-effect of this is that even commonplace phenomena such as the earth's geomagnetic field can easily swamp signals of interest. This problem is commonly addressed by averaging over a large number of recordings. However, this has a number of notable drawbacks. In particular, it is difficult to synchronise high frequency activity which might be of interest, and often these signals will be cancelled out by the averaging process. Other problems that have been encountered are high costs and low portability of state-of-the- art multichannel machines. The result of this is that the use of MEG has, hitherto, been restricted to large institutions which are able to afford the high costs associated with the procurement and maintenance of these machines. In this project, we seek to address these issues by working almost exclusively with single channel, unaveraged MEG data. We demonstrate the applicability of a variety of methods originating from the fields of signal processing, dynamical systems, information theory and neural networks, to the analysis of MEG data. It is noteworthy that while modern signal processing tools such as independent component analysis, topographic maps and latent variable modelling have enjoyed extensive success in a variety of research areas from financial time series modelling to the analysis of sun spot activity, their use in MEG analysis has thus far been extremely limited. It is hoped that this work will help to remedy this oversight.

An analysis of the effect of process parameters on the formability of sheet metal

This investigation is in two parts, theory and experimental verification. (1) Theoretical Study In this study it is, for obvious reasons, necessary to analyse the concept of formability first. For the purpose of the present investigation it is sufficient to define the four aspects of formability as follows: (a) the formability of the material at a critical section, (b) the formability of the material in general, (c) process efficiency, (d) proportional increase in surface area. A method of quantitative assessment is proposed for each of the four aspects of formability. The theoretical study also includes the distinction between coaxial and non-coaxial strains which occur, respectively, in axisymmetrical and unsymmetrical forming processes and the inadequacy of the circular grid system for the assessment of formability is explained in the light of this distinction. (2) Experimental Study As one of the bases of the experimental work, the determination of the end point of a forming process, which sets the limit to the formability of the work material, is discussed. The effects of three process parameters on draw-in are shown graphically. Then the delay of fracture in sheet metal forming resulting from draw-in is analysed in kinematical terms, namely, through the radial displacements, the radial and the circumferential strains, and the projected thickness of the workpiece. Through the equilibrium equation of the membrane stresses, the effect on the shape of the unsupported region of the workpiece, and hence the position of the critical section is explained. Then, the effect of draw-in on the four aspects of formability is discussed throughout this investigation. The triangular coordinate system is used to present and analyse the triaxial strains involved. This coordinate system has the advantage of showing all the three principal strains in a material simultaneously, as well as representing clearly the many types of strains involved in sheet metal work.

Analysis of the intraocular pressure pulse

This thesis was concerned with investigating methods of improving the IOP pulse’s potential as a measure of clinical utility. There were three principal sections to the work. 1. Optimisation of measurement and analysis of the IOP pulse. A literature review, covering the years 1960 – 2002 and other relevant scientific publications, provided a knowledge base on the IOP pulse. Initial studies investigated suitable instrumentation and measurement techniques. Fourier transformation was identified as a promising method of analysing the IOP pulse and this technique was developed. 2. Investigation of ocular and systemic variables that affect IOP pulse measurements In order to recognise clinically important changes in IOP pulse measurement, studies were performed to identify influencing factors. Fourier analysis was tested against traditional parameters in order to assess its ability to detect differences in IOP pulse. In addition, it had been speculated that the waveform components of the IOP pulse contained vascular characteristic analogous to those components found in arterial pulse waves. Validation studies to test this hypothesis were attempted. 3. The nature of the intraocular pressure pulse in health and disease and its relation to systemic cardiovascular variables. Fourier analysis and traditional parameters were applied to the IOP pulse measurements taken on diseased and healthy eyes. Only the derived parameter, pulsatile ocular blood flow (POBF) detected differences in diseased groups. The use of an ocular pressure-volume relationship may have improved the POBF measure’s variance in comparison to the measurement of the pulse’s amplitude or Fourier components. Finally, the importance of the driving force of pulsatile blood flow, the arterial pressure pulse, is highlighted. A method of combining the measurements of pulsatile blood flow and pulsatile blood pressure to create a measure of ocular vascular impedance is described along with its advantages for future studies.

Measurement of ocular component contributions to residual astigmatism in adult human eyes

The aim of this study was to determine whether an ophthalmophakometric technique could offer a feasible means of investigating ocular component contributions to residual astigmatism in human eyes. Current opinion was gathered on the prevalence, magnitude and source of residual astigmatism. It emerged that a comprehensive evaluation of the astigmatic contributions of the eye's internal ocular surfaces and their respective axial separations (effectivity) had not been carried out to date. An ophthalmophakometric technique was developed to measure astigmatism arising from the internal ocular components. Procedures included the measurement of refractive error (infra-red autorefractometry), anterior corneal surface power (computerised video keratography), axial distances (A-scan ultrasonography) and the powers of the posterior corneal surface in addition to both surfaces of the crystalline lens (multi-meridional still flash ophthalmophakometry). Computing schemes were developed to yield the required biometric data. These included (1) calculation of crystalline lens surface powers in the absence of Purkinje images arising from its anterior surface, (2) application of meridional analysis to derive spherocylindrical surface powers from notional powers calculated along four pre-selected meridians, (3) application of astigmatic decomposition and vergence analysis to calculate contributions to residual astigmatism of ocular components with obliquely related cylinder axes, (4) calculation of the effect of random experimental errors on the calculated ocular component data. A complete set of biometric measurements were taken from both eyes of 66 undergraduate students. Effectivity due to corneal thickness made the smallest cylinder power contribution (up to 0.25DC) to residual astigmatism followed by contributions of the anterior chamber depth (up to 0.50DC) and crystalline lens thickness (up to 1.00DC). In each case astigmatic contributions were predominantly direct. More astigmatism arose from the posterior corneal surface (up to 1.00DC) and both crystalline lens surfaces (up to 2.50DC). The astigmatic contributions of the posterior corneal and lens surfaces were found to be predominantly inverse whilst direct astigmatism arose from the anterior lens surface. Very similar results were found for right versus left eyes and males versus females. Repeatability was assessed on 20 individuals. The ophthalmophakometric method was found to be prone to considerable accumulated experimental errors. However, these errors are random in nature so that group averaged data were found to be reasonably repeatable. A further confirmatory study was carried out on 10 individuals which demonstrated that biometric measurements made with and without cycloplegia did not differ significantly.

Kineto-elastodynamic analysis of high-speed four-bar mechanism

This thesis addresses the kineto-elastodynamic analysis of a four-bar mechanism running at high-speed where all links are assumed to be flexible. First, the mechanism, at static configurations, is considered as structure. Two methods are used to model the system, namely the finite element method (FEM) and the dynamic stiffness method. The natural frequencies and mode shapes at different positions from both methods are calculated and compared. The FEM is used to model the mechanism running at high-speed. The governing equations of motion are derived using Hamilton's principle. The equations obtained are a set of stiff ordinary differential equations with periodic coefficients. A model is developed whereby the FEM and the dynamic stiffness method are used conjointly to provide high-precision results with only one element per link. The principal concern of the mechanism designer is the behaviour of the mechanism at steady-state. Few algorithms have been developed to deliver the steady-state solution without resorting to costly time marching simulation. In this study two algorithms are developed to overcome the limitations of the existing algorithms. The superiority of the new algorithms is demonstrated. The notion of critical speeds is clarified and a distinction is drawn between "critical speeds", where stresses are at a local maximum, and "unstable bands" where the mechanism deflections will grow boundlessly. Floquet theory is used to assess the stability of the system. A simple method to locate the critical speeds is derived. It is shown that the critical speeds of the mechanism coincide with the local maxima of the eigenvalues of the transition matrix with respect to the rotational speed of the mechanism.

Genetic analysis of dyslexia candidate genes in the European cross-linguistic NeuroDys cohort

Dyslexia is one of the most common childhood disorders with a prevalence of around 5-10% in school-age children. Although an important genetic component is known to have a role in the aetiology of dyslexia, we are far from understanding the molecular mechanisms leading to the disorder. Several candidate genes have been implicated in dyslexia, including DYX1C1, DCDC2, KIAA0319, and the MRPL19/C2ORF3 locus, each with reports of both positive and no replications. We generated a European cross-linguistic sample of school-age children-the NeuroDys cohort-that includes more than 900 individuals with dyslexia, sampled with homogenous inclusion criteria across eight European countries, and a comparable number of controls. Here, we describe association analysis of the dyslexia candidate genes/locus in the NeuroDys cohort. We performed both case-control and quantitative association analyses of single markers and haplotypes previously reported to be dyslexia-associated. Although we observed association signals in samples from single countries, we did not find any marker or haplotype that was significantly associated with either case-control status or quantitative measurements of word-reading or spelling in the meta-analysis of all eight countries combined. Like in other neurocognitive disorders, our findings underline the need for larger sample sizes to validate possibly weak genetic effects. © 2014 Macmillan Publishers Limited All rights reserved.