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

Adenovirus-mediated transfer of a gene encoding acyloxyacyl hydrolase (AOAH) into mice increases tissue and plasma AOAH activity

Although the host response to gram-negative bacterial infection follows largely from the interactions of bacterial lipopolysaccharides (LPS or endotoxin) with host cells, little information is available concerning the mechanisms by which the host eliminates or detoxifies LPS. Acyloxyacyl hydrolase (AOAH) is an enzyme, found in phagocytic cells, that catalyzes the enzymatic deacylation of the lipid A moiety of LPS. Enzymatically deacylated LPS is much less potent than LPS at inducing responses in human cells, and it can antagonize the ability of LPS to activate human macrophages, neutrophils, and endothelial cells. Despite these observations, the physiologic role of LPS deacylation remains undefined. To investigate the ability of AOAH to carry out LPS deacylation in vivo, we produced a recombinant adenovirus carrying a gene encoding AOAH (Ad.CMV-AOAH) and employed this vector to elicit transient overexpression of AOAH in mice. Mice infected with Ad.CMV-AOAH expressed high levels of the enzyme in plasma, liver, spleen, and kidney. Although adenovirus-induced hepatitis reduced hepatic uptake of intravenously injected [H-3]LPS, animals expressing the transgene deacylated a larger fraction of the [3H]LPS taken up by their livers than did mice infected with a control adenovirus. These studies indicate that AOAH can catalyze the deacylation of LPS in vivo, and they provide evidence that the rates of hepatic LPS uptake and deacylation are not closely linked

Predicting uncertainty in sediment transport and landscape evolution - the influence of initial surface conditions

© 2015. Numerical landscape evolution models were initially developed to examine natural catchment hydrology and geomorphology and have become a common tool to examine geomorphic behaviour over a range of time and space scales. These models all use a digital elevation model (DEM) as a representation of the landscape surface and a significant issue is the quality and resolution of this surface. Here we focus on how subtle perturbations or roughness on the DEM surface can produce alternative model results. This study is carried out by randomly varying the elevations of the DEM surface and examining the effect on sediment transport rates and geomorphology for a proposed rehabilitation design for a post-mining landscape using multiple landscape realisations with increasing magnitudes of random changes. We show that an increasing magnitude of random surface variability does not appear to have any significant effect on sediment transport over millennial time scales. However, the random surface variability greatly changes the temporal pattern or delivery of sediment output. A significant finding is that all simulations at the end of the 10,000 year modelled period are geomorphologically similar and present a geomorphological equifinality. However, the individual patterns of erosion and deposition were different for repeat simulations with a different sequence of random perturbations. The alternative positions of random perturbations strongly influence local patterns of hillslope erosion and evolution together with the pattern and behaviour of deposition. The findings demonstrate the complex feedbacks that occur even within a simple modelled system

Comparative efficacy of a secretory phospholipase A2 inhibitor with conventional anti-inflammatory agents in a rat model of antigen-induced arthritis

INTRODUCTION: Previously, secretory phospholipase A(2 )(sPLA(2)) inhibition has been used as an adjunct to conventional rheumatoid arthritis therapy in human clinical trials without significant improvement of arthritic pathology. In this study, we compared the efficacy of a potent and orally active group IIa secretory phospholipase A(2 )inhibitor (sPLA(2)I) to conventional anti-arthritic agents; infliximab, leflunomide and prednisolone, in a rat model of antigen-induced arthritis. METHODS: Initially, to establish efficacy and dose-response, rats were orally dosed with the sPLA(2)I (1 and 5 mg/kg) two days prior to arthritis induction, and then daily throughout the 14-day study period. In the second trial, rats were orally dosed with the sPLA(2)I (5 and 10 mg/kg/day) beginning two days after the induction of arthritis, at the peak of joint swelling. Separate groups of rats were also dosed with the tumour necrosis factor-alpha (TNF-α) inhibitor infliximab (single 3 mg/kg i.v. injection), leflunomide (10 mg/kg/day, oral) or prednisolone (1 mg/kg/day, oral) at this same time point and used as comparative treatments. RESULTS: In the pathology prevention trial, both 1 and 5 mg/kg dose groups of sPLA(2)I demonstrated a significant reduction in joint swelling and gait disturbances; however, only the higher 5 mg/kg dose resulted in significantly reduced histopathology scores. In the post-induction trial, rats dosed with sPLA(2)I showed a significant improvement in joint swelling and gait scoring, whereas none of the conventional therapeutics achieved a significant decrease in both of these two disease markers. Histopathological scoring at the end-point of the study demonstrated significantly reduced median scores in rats treated with 10 mg/kg sPLA(2)I and leflunomide. CONCLUSIONS: The results from this study suggest a pathogenic role for sPLA(2 )enzymes in this model of arthritis in rats, and the potential clinical utility of sPLA(2 )inhibition as a safer, and more effective, alternative to conventional anti-arthritic therapeutics

Non-linearity and spatial resolution in a cellular automaton model of a small upland basin

International audienceThe continuing development of computational fluid dynamics is allowing the high resolution study of hydraulic and sediment transport processes but, due to computational complexities, these are rarely applied to areas larger than a reach. Existing approaches, based upon linked cross sections, can give a quasi two-dimensional view, effectively simulating sediment transport for a single river reach. However, a basin represents a whole discrete dynamic system within which channel, floodplain and slope processes operate over a wide range of space and time scales. Here, a cellular automaton (CA) approach has been used to overcome some of these difficulties, in which the landscape is represented as a series of fixed size cells. For every model iteration, each cell acts only in relation to the influence of its immediate neighbours in accordance with appropriate rules. The model presented here takes approximations of existing flow and sediment transport equations, and integrates them, together with slope and floodplain approximations, within a cellular automaton framework. This method has been applied to the basin of Cam Gill Beck (4.2 km2 ) above Starbotton, upper Wharfedale, a tributary of the River Wharfe, North Yorkshire, UK. This approach provides, for the first time, a workable model of the whole basin at a 1 m resolution. Preliminary results show the evolution of bars, braids, terraces and alluvial fans which are similar to those observed in the field, and examples of large and small scale non-linear behaviour which may have considerable implications for future models

Religious Vehicle Stickers in Nigeria: a discourse of identity, faith and social vision

This study focuses on analysing the ways in which vehicle stickers construct individual and group identities, people’s religious faith and social vision in the context of religious assumptions and practices in Nigeria. Data comprise 73 vehicle stickers collected in Lagos and Ota, between 2006 and 2007 and are analysed within the framework of the post-structuralist model of discourse analysis which views discourse as a product of a complex system of social and institutional practices that sustain its continuous existence (Derrida, 1982; Fairclough, 1989, 1992, 1995; Foucault, 1972, 1981). Results show that through stickers people define their individual and group identities within religious institutional practices. And as a means of group identification, they guarantee social security and privileges. In constructing social vision the stickers help mould the individual aspiration about a future which transcends the present. Significantly, stickers in the data also reveal the tension between Islam and Christianity and the struggle to propagate one above the other. KEY WORDS: assumption, discourse, discursive, practices, religion, stickers