Characterisation of the iron uptake mechanisms of Aeromonas Salmonicida : role in virulence and protective immunity

The ability of the bacterial fish pathogen Aeromonas Salmonicida to grow under conditions of iron-restriction In vitro was examined in an attempt to identify and characterise the iron uptake mechanisms of the pathogen. Both typical and atypical strains of A. Salmonicida grew and multiplied in the presence of the synthetic high affinity iron chelators, ethylene diamine di(o-hydroxyphenylacetic acid) (EDDA) and 2,2'dipyridyl (Op) and possessed one or more functional iron uptake mechanisms. The first mechanism involved the inducib1e production of a phenolate siderophore which was detected using chrome azurol S (CAS) agar. This inducible siderophore-mediated mechanism was possessed only by typical strains of A. Salmonicida. A second mechanism was identified which enabled both typical and atypical strains to utilise a number of mammalian sources of transferrins (Tf). Typical strains were able to utilise Tf via a siderophore-mediated mechanism; atypical strains were able to utilise Tf via a mechanism involving the proteolytic degradation of Tf by an extracellular metalloprotease. A third mechanism was identified which enabled virulent strains of A. Salmonicida to utilise haem sources of iron via a constitutive, siderophore-independant mechanism. The mechanism involved a common cell-surface associated haem-binding protein, thought to be the 49-k1lodalton (kOa) A-layer protein capable of binding the sulphonated diazo dye, congo red. Growth under conditions of Iron-restriction resulted In the Increased synthesis of a number of extracellular virulence factors Involved In the pathogenesis of furunculosis, Including haemolysin and protease. Iron-restricted growth also resulted in the expression of four Iron-regulated outer membrane proteins (IROMPs) of molecular weight 82, 77, 72 and 70k0a which were present and immunologically cross-reactive in eighteen strains of A. Salmonicida grown In the presence of an iron chelator. The IROMPs were shown by Immunoblotting techniques to be expressed In vivo during Infection, since A. Salmonicida isolated directly without subculture from the furuncle material of an Infected Atlantic salmon expressed IROMPs. Immunoblotting techniques and an enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of anti-IROMP immunoglobulin (Ig) found the IROMPs to be immunogenic In Atlantic salmon, and in addition, convalescent sera of Atlantic salmon surviving an A. Salmonicida challenge contained antibodies against the IROMPs. Outer membrane proteins ((3MPs) of A. Salmonicida were then evaluated for their ability to Induce protective immunity In Atlantic salmon. Compared with OMP prepared from A. Salmonicida grown under iron-replete conditions, IROMPs conferred protection against both natural and experimental heterologous A. Salmonicida bath challenge. In addition, passive immunisation of Atlantic salmon with an antisera containing antibodies to the IROMPs, and a rabbit anti-IROMP antisera or affinity-purified immunoglobulin 0 (IgO) also conferred protection against heterologous bath challenge. In addition, an Iron-restricted bacterin of A. Salmonicida containing IROMPs was found to be capable of protecting Atlantic salmon against A. Salmonicida bath challenge. Both the Atlantic salmon antisera raised against the IROMPs, and the rabbit anti-IROMP antisera were bactericidal against virulent strains of A. Salmonicida In the presence of complement. Results presented in this thesis Indicate that the IROMPs of A. Salmonicida represent important protective antigens in the vaccination of Atlantic salmon against furunculosis

Automated BioPart characterisation for synthetic biology

Synthetic Biology is an approach to the development of biological systems based on engineering principles. By using concepts from other engineering disciplines such as abstraction it is possible to break down complicated biological functions into components termed ‘BioParts’. BioParts can be assembled collectively into modules and systems to carry out advanced functions, designed from the bottom up. A key part of this approach is the standardisation of BioParts and practices to aid design, testing and implementation. An automated characterisation methodology focused primarily on promoter BioParts has been developed which is potentially scalable to other BioPart families. The standardised workflow is optimised to enable BioPart characterisation under highly reproducible growth conditions, reliably producing high quality data. It has been designed around automation equipment which should ensure accurate reproduction of experiments at other sites. The automated characterisation workflow has been demonstrated to produce high quality data for both constitutive and inducible promoters. The entire Anderson promoter collection has been characterised and high details results for all library members are available for the first time. A pair of inducible promoter BioPart were characterised to obtain a deep data level regarding their activity in response to inducer over time. To allow the characterisation of more inducible BioParts in a shorter period of time, promoter engineering was also used to generate novel promoters which are induced by xylose. The development of the automated workflow should be a step towards the standardisation of characterisation protocols and production of large numbers of BioParts with associated high quality, reproducible characterisations. Standardisation will further aid the comparison of the data sets produced, potentially shining light on unknown interactions between BioParts and their environment and improving the ability of Synthetic Biologists to design novel biological systems from the ground up.Open Acces

Social networking, social harassment and social policy

This paper reports on the misuse of social networking sites (SNS). It was based on a study of 226 students in UK, Sweden, Turkey and France and a panel survey of 1068 Australian adults. Although only a minority of people experienced social harassment and abuse, the distressing nature of the bad experiences suggested that social marketing was needed on several fronts -self-regulation, regulation, education and personal responsibility - in order to minimise these behaviours.<br /

Locating and quantifying gas emission sources using remotely obtained concentration data

We describe a method for detecting, locating and quantifying sources of gas emissions to the atmosphere using remotely obtained gas concentration data; the method is applicable to gases of environmental concern. We demonstrate its performance using methane data collected from aircraft. Atmospheric point concentration measurements are modelled as the sum of a spatially and temporally smooth atmospheric background concentration, augmented by concentrations due to local sources. We model source emission rates with a Gaussian mixture model and use a Markov random field to represent the atmospheric background concentration component of the measurements. A Gaussian plume atmospheric eddy dispersion model represents gas dispersion between sources and measurement locations. Initial point estimates of background concentrations and source emission rates are obtained using mixed L2-L1 optimisation over a discretised grid of potential source locations. Subsequent reversible jump Markov chain Monte Carlo inference provides estimated values and uncertainties for the number, emission rates and locations of sources unconstrained by a grid. Source area, atmospheric background concentrations and other model parameters are also estimated. We investigate the performance of the approach first using a synthetic problem, then apply the method to real data collected from an aircraft flying over: a 1600 km^2 area containing two landfills, then a 225 km^2 area containing a gas flare stack

Transcriptional Targeting in Cancer Gene Therapy

Cancer gene therapy has been one of the most exciting areas of therapeutic research in the past decade. In this review, we discuss strategies to restrict transcription of transgenes to tumour cells. A range of promoters which are tissue-specific, tumour-specific, or inducible by exogenous agents are presented. Transcriptional targeting should prevent normal tissue toxicities associated with other cancer treatments, such as radiation and chemotherapy. In addition, the specificity of these strategies should provide improved targeting of metastatic tumours following systemic gene delivery. Rapid progress in the ability to specifically control transgenes will allow systemic gene delivery for cancer therapy to become a real possibility in the near future

When too much entertainment is barely enough : current affairs television in the 1990s

The article examines the state of current affairs journalism and looks at news values versus entertainment values

Uncertainty in Discriminant Analysis

The aim of this thesis is to review and develop theory in discriminant analysis. In chapter one an example of medical diagnosis is considered, and two types of uncertainty are illustrated. Firstly, the log odds ratio can be close to zero, and secondly there can be considerable uncertainty about its true value. In chapter two we review existing methodology for constructing Interval estimates for the log odds when the two populations are normal. Five different methods are considered for distributions with equal covariances, and three are generalised to the unequal covariance situation. In chapter three these methods are Investigated by simulation. It is seen that only two methods in the equal covariance case give intervals of reliable empirical confidence, and only one generalises successfully to the unequal covariance case. In chapter four we go on to use the interval estimation methodology to assess a discriminant rule, suggesting some new ways of displaying the information available. In chapter five we develop the methods of chapter four to construct an accurate error rate estimator, which is compared with standard techniques by simulation. In chapter six the error rate estimator developed in chapter five is extended to the situation where there are more than two groups, and it is compared by simulation with generalisations of other standard techniques. The different methods are applied to a data set. In chapter seven the limitations of the work are discussed, and possible developments suggested