Characterisation, genomic organisation, expression and function of the mEphA1 receptor Tyrosine Kinase

The Eph receptor tyrosine kinases and their ephrin ligands are cell surface molecules with a wide range of biological functions. Specifically, the Eph/ephrin receptor-ligand family influences cell behaviour during both embryogenesis and adult life, principally through modification of cytoskeletal organisation and cell adhesion. EphA1 (previously referred to as eph) was isolated during a search for novel tyrosine kinases with oncogenic potential. The murine homologue of hEphA1, formerly Esk and now mEphA1, was cloned by reverse transcriptase PCR using degenerate oligonucleotide primers and RNA prepared from embryonic stem cells in culture. Northern blot analysis revealed expression in day 12 mouse embryo, and adult mouse thymus, liver, kidney, lung and placenta. The work in this thesis investigates the expression and function of EphA1 in mutant mouse and other animal models. This has been achieved by a number of different techniques including:- (1) the classical techniques of molecular biology; (2) a search for the zebrafish homologue of mEphA1; (3) generation and phenotypic analysis of the hPLAP EphA1 reporter knockout mouse and (4) generation of the EphA1 conditional knockout mouse. The chromosomal localisation and Southern blotting of genomic digests confirmed that Esk (mEphA1) is the murine homologue of eph (hEphA1). The binding of soluble mEphA1 to a panel of ephrin ligands analysed by surface plasmon resonance (BIACore), and the binding of various ephrin-Fc molecules to cell surface expressed EphA1, confirmed that EphA1 is the cognate receptor for the ephrin-A1 ligand. The mEphA1 genomic sequence was isolated, sequenced and the exon-intron boundaries mapped. Interestingly, Exon 3, which includes the ligand binding domain, is split into two smaller exons (Exon 3a and Exon 3b). This pattern was also found in hEphA1; however, it is a novel finding compared with the other Ephs, and the reason underlying this difference remains speculative. In situ hybridisation analysis confirmed epithelial expression of mEphA1 in the basal layer of the epidermis, developing hair follicles, thymic epithelial cells and adult kidney. At the commencement of the zebrafish (ZF) library screening project in 1997, it seemed likely that there was an ZF orthologue of EphA1. However, over 50 clones were isolated by degenerate PCR of zebrafish cDNA and genomic libraries, and although some of the sequences had homology to known Ephs, none matched EphA1. The ZF genome has now been sequenced completely [http://wwwmap.tuebingen.mpg.de/ ; http://zfin.org/] and has confirmed that there is indeed no zebrafish orthologue of EphA1. The hPLAP EphA1 reporter knockout mouse was generated with the technical assistance of Dr Graham Kay (Queensland Transgenic Laboratory). The homozygous null mice have a kinky tail in two separate embryonic stem cell lines with a high degree of penetrance. A proportion of female null mice display the imperforate vagina phenotype. The null mice are otherwise grossly normal, with equal sex ratios and normal growth, health and life expectancy. The microscopic examination of haematoxylin and eosin stained sections of all the major organs revealed no histological abnormalities. The expression of hPLAP, (hence mEphA1), analysed in frozen sections confirmed the previous work which defined the epithelial expression of mEphA1 to the basal epidermis and hair follicle. There was also previously undescribed hPLAP (mEphA1) expression in the uterus, vagina and small intestine. The EphA1 conditional knockout mouse was also generated with the assistance of the Queensland Transgenic Facility. The homozygous null mice were grossly normal with equal sex ratio and normal health and life expectancy. The kinky tail phenotype was observed infrequently and has not yet been fully characterised in these mice. Similarly the imperforate vagina phenotype has not been observed in this strain of mice. This strain of genetically modified EphA1 knockout mice can be mated with various strains of Cre-deleter mice to achieve tissue specific silencing of EphA1 and consequently allow more precise analysis of EphA1 function. In summary, the studies described in this thesis have confirmed the importance of the Eph/ephrin receptor-ligands in both embryonic development and the maintenance of adult tissues, and have generated several new findings which add to our knowledge of the biology of EphA1. The generation of the hPLAP EphA1 reporter mice and EphA1 conditional knockout mice has provided us with very useful tools. These knockout mice will allow further analysis of the role of EphA1 in mouse models of human diseases, including skin and colon cancer, severe sepsis and post-traumatic injury

Isotonic versus hypotonic solutions for maintenance intravenous fluid administration in children

Backgroun

Over-expression of Eph and ephrin genes in advanced ovarian cancer: ephrin gene expression correlates with shortened survival

BACKGROUND: Increased expression of Eph receptor tyrosine kinases and their ephrin ligands has been implicated in tumor progression in a number of malignancies. This report describes aberrant expression of these genes in ovarian cancer, the commonest cause of death amongst gynaecological malignancies. METHODS: Eph and ephrin expression was determined using quantitative real time RT-PCR. Correlation of gene expression was measured using Spearman's rho statistic. Survival was analysed using log-rank analysis and (was visualised by) Kaplan-Meier survival curves. RESULTS: Greater than 10 fold over-expression of EphA1 and a more modest over-expression of EphA2 were observed in partially overlapping subsets of tumors. Over-expression of EphA1 strongly correlated (r = 0.801; p < 0.01) with the high affinity ligand ephrin A1. A similar trend was observed between EphA2 and ephrin A1 (r = 0.387; p = 0.06). A striking correlation of both ephrin A1 and ephrin A5 expression with poor survival (r = -0.470; p = 0.02 and r = -0.562; p < 0.01) was observed. Intriguingly, there was no correlation between survival and other clinical parameters or Eph expression. CONCLUSION: These data imply that increased levels of ephrins A1 and A5 in the presence of high expression of Ephs A1 and A2 lead to a more aggressive tumor phenotype. The known functions of Eph/ephrin signalling in cell de-adhesion and movement may explain the observed correlation of ephrin expression with poor prognosis

Attention in neglect and extinction: Assessing the degree of correspondence between visual and auditory impairments using matched tasks

Claims have been made for associated degrees of impairment on both visual and auditory performance in unilateral neglect and extinction. Since this evidence is primarily based on different tests in each modality, it is difficult to properly quantify the degree of association between performance in vision and audition. The current study compares visual and auditory extinction and temporal order judgments (TOJs) in two cases with clinical visual neglect. Stimuli in both modalities were precisely matched in their temporal and spatial parameters. The results reveal a mixed pattern of association between different auditory tests and their visual counterparts. This suggests that associations between visual and auditory neglect can occur but these are neither obligatory nor pervasive. Instead, our data support models of spatial impairment in neglect and extinction that acknowledge differences in the contribution of spatial information to performance in each modality in responses to changing task demands

Improving Diabetes care through Examining, Advising, and prescribing (IDEA): protocol for a theory-based cluster randomised controlled trial of a multiple behaviour change intervention aimed at primary healthcare professionals

Background: New clinical research findings may require clinicians to change their behaviour to provide high-quality care to people with type 2 diabetes, likely requiring them to change multiple different clinical behaviours. The present study builds on findings from a UK-wide study of theory-based behavioural and organisational factors associated with prescribing, advising, and examining consistent with high-quality diabetes care. Aim: To develop and evaluate the effectiveness and cost of an intervention to improve multiple behaviours in clinicians involved in delivering high-quality care for type 2 diabetes. Design/methods: We will conduct a two-armed cluster randomised controlled trial in 44 general practices in the North East of England to evaluate a theory-based behaviour change intervention. We will target improvement in six underperformed clinical behaviours highlighted in quality standards for type 2 diabetes: prescribing for hypertension; prescribing for glycaemic control; providing physical activity advice; providing nutrition advice; providing on-going education; and ensuring that feet have been examined. The primary outcome will be the proportion of patients appropriately prescribed and examined (using anonymised computer records), and advised (using anonymous patient surveys) at 12 months. We will use behaviour change techniques targeting motivational, volitional, and impulsive factors that we have previously demonstrated to be predictive of multiple health professional behaviours involved in high-quality type 2 diabetes care. We will also investigate whether the intervention was delivered as designed (fidelity) by coding audiotaped workshops and interventionist delivery reports, and operated as hypothesised (process evaluation) by analysing responses to theory-based postal questionnaires. In addition, we will conduct post-trial qualitative interviews with practice teams to further inform the process evaluation, and a post-trial economic analysis to estimate the costs of the intervention and cost of service use. Discussion: Consistent with UK Medical Research Council guidance and building on previous development research, this pragmatic cluster randomised trial will evaluate the effectiveness of a theory-based complex intervention focusing on changing multiple clinical behaviours to improve quality of diabetes care

Common variants at ABCA7, MS4A6A/MS4A4E, EPHA1, CD33 and CD2AP are associated with Alzheimer's disease

We sought to identify new susceptibility loci for Alzheimer's disease through a staged association study (GERAD+) and by testing suggestive loci reported by the Alzheimer's Disease Genetic Consortium (ADGC) in a companion paper. We undertook a combined analysis of four genome-wide association datasets (stage 1) and identified ten newly associated variants with P ≤ 1 × 10−5. We tested these variants for association in an independent sample (stage 2). Three SNPs at two loci replicated and showed evidence for association in a further sample (stage 3). Meta-analyses of all data provided compelling evidence that ABCA7 (rs3764650, meta P = 4.5 × 10−17; including ADGC data, meta P = 5.0 × 10−21) and the MS4A gene cluster (rs610932, meta P = 1.8 × 10−14; including ADGC data, meta P = 1.2 × 10−16) are new Alzheimer's disease susceptibility loci. We also found independent evidence for association for three loci reported by the ADGC, which, when combined, showed genome-wide significance: CD2AP (GERAD+, P = 8.0 × 10−4; including ADGC data, meta P = 8.6 × 10−9), CD33 (GERAD+, P = 2.2 × 10−4; including ADGC data, meta P = 1.6 × 10−9) and EPHA1 (GERAD+, P = 3.4 × 10−4; including ADGC data, meta P = 6.0 × 10−10)

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Does probiotic milk prevent infections in children attending daycare centres?

Objective: To examine whether long term consumption of a probiotic milk could reduce gastrointestinal and respiratory infections in children in daycare centres