A cell biological approach to studying lameness in the dairy cow

This thesis describes a cell biological approach to studying lameness in the dairy cow. Lameness has been associated with altered keratinisation of the epidermis of the bovine hoof. Claw tissue was obtained by an in vivo claw biopsy technique. The biopsies were cultured in the presence of L-[35S]-methionine and [3H]-thymidine for measurement of protein synthesis and cell proliferation respectively. The influence of nutritional and environmental stressors on keratinisation in the claw was investigated. Protein synthesis was found to be significantly higher after challenge with these factors. It was not clear, however, if this was an effect of challenge or the biopsy procedure itself. Physiological and endocrinological changes associated with pregnancy and lactation also appeared to have an effect on claw keratinisation. In a long-term developmental study of first-calving heifers changes in cell proliferation and protein synthesis were related to reproductive state. A dramatic seasonal effect on claw cell biology was also demonstrated. The keratinocytes were actively proliferating and keratinising during the summer months but were quiescent during the winter. A final study investigated the cell biological changes which occur in the claw during the onset, development and recovery stages of weight-bearing challenge and the effects of concrete flooring. During challenge cell proliferation increased significantly in claw subjected to weight-bearing. The tissue may have been responding to challenge, however, the biopsy procedure itself could also have had an effect due to the short-time period between samplings. To conclude, altered keratinisation in the claw may be related to physiological and endocrinological changes associated with season and reproduction. However, changes in management may also be important and further investigation is required

Collaboration of MYC and RUNX2 in lymphoma simulates T‐cell receptor signaling and attenuates p53 pathway activity

MYC and RUNX oncogenes each trigger p53‐mediated failsafe responses when overexpressed in vitro and collaborate with p53 deficiency in vivo. However, together they drive rapid onset lymphoma without mutational loss of p53. This phenomenon was investigated further by transcriptomic analysis of premalignant thymus from RUNX2/MYC transgenic mice. The distinctive contributions of MYC and RUNX to transcriptional control were illustrated by differential enrichment of canonical binding sites and gene ontology analyses. Pathway analysis revealed signatures of MYC, CD3, and CD28 regulation indicative of activation and proliferation, but also strong inhibition of cell death pathways. In silico analysis of discordantly expressed genes revealed Tnfsrf8/CD30, Cish, and Il13 among relevant targets for sustained proliferation and survival. Although TP53 mRNA and protein levels were upregulated, its downstream targets in growth suppression and apoptosis were largely unperturbed. Analysis of genes encoding p53 posttranslational modifiers showed significant upregulation of three genes, Smyd2, Set, and Prmt5. Overexpression of SMYD2 was validated in vivo but the functional analysis was constrained by in vitro loss of p53 in RUNX2/MYC lymphoma cell lines. However, an early role is suggested by the ability of SMYD2 to block senescence‐like growth arrest induced by RUNX overexpression in primary fibroblasts

Regulation and function of macrophage colony-stimulating factor (CSF1) in the chicken immune system

Macrophage colony-stimulating factor (CSF1) is an essential growth factor to control the proliferation, differentiation and survival of cells of the macrophage lineage in vertebrates. We have previously produced a recombinant chicken CSF1-Fc fusion protein and administrated it to birds which produced a substantial expansion of tissue macrophage populations. To further study the biology of CSF1 in the chicken, here we generated anti-chicken CSF1 antibodies (ROS-AV181 and 183) using CSF1-Fc as an immunogen. The specific binding of each monoclonal antibody was confirmed by ELISA, Western blotting and immunohistochemistry on tissue sections. Using the anti-CSF1 antibodies, we show that chicken bone marrow derived macrophages (BMDM) express CSF1 on their surface, and that the level appears to be regulated further by exogenous CSF1. By capture ELISA circulating CSF1 levels increased transiently in both layer and broiler embryos around the day of hatch. The levels of CSF1 in broilers was higher than in layers during the first week after hatch. Antibody ROS-AV183 was able to block CSF1 biological activity in vitro and treatment of hatchlings using this neutralising antibody in vivo impacted on some tissue macrophage populations, but not blood monocytes. After anti-CSF1 treatment, CSF1R-transgene reporter expressing cells were reduced in the bursa of Fabricius and cecal tonsil and TIM4 Kupffer cells in the liver were almost completely ablated. Anti-CSF1 treatment also produced a reduction in overall bone density, trabecular volume and TRAP osteoclasts. Our novel neutralising antibody provides a new tool to study the roles of CSF1 in birds

Analysis of Campylobacter jejuni infection in the gnotobiotic piglet and genome-wide identification of bacterial factors required for infection

To investigate how Campylobacter jejuni causes the clinical symptoms of diarrhoeal disease in humans,use of a relevant animal model is essential. Such a model should mimic the human disease closely in terms of host physiology, incubation period before onset of disease, clinical signs and a comparable outcome of disease. In this study, we used a gnotobiotic piglet model to study determinants of pathogenicity of C. jejuni. In this model, C. jejuni successfully established infection and piglets developed an increased temperature with watery diarrhoea, which was caused by a leaky epithelium and reduced bile re-absorption in the intestines. Further, we assessed the C. jejuni genes required for infection of the porcine gastrointestinal tract utilising a transposon (Tn) mutant library screen. A total of 123 genes of which Tn mutants showed attenuated piglet infection were identified. Our screen highlighted a crucial role for motility and chemotaxis, as well as central metabolism. In addition, Tn mutants of 14 genes displayed enhanced piglet infection. This study gives a unique insight into themechanisms of C. jejuni disease in terms of host physiology and contributing bacterial factors

Development of a T-cell Receptor Mimic Antibody against Wild-Type p53 for Cancer Immunotherapy

The tumor suppressor p53 is widely dysregulated in cancer and represents an attractive target for immunotherapy. Due to its intracellular localization, p53 is inaccessible to classical therapeutic monoclonal antibodies, an increasingly successful class of anti-cancer drugs. However, peptides derived from intracellular antigens are presented on the cell surface in the context of major histocompatibility class I (MHC I), and can be bound by T cell receptors (TCRs). Here, we report the development of a novel antibody, T1-116C, that acts as a TCR mimic to recognize an HLA-A*0201-presented wild-type p53 T cell epitope, p5365-73(RMPEAAPPV). The antibody recognizes a wide range of cancers, does not bind normal peripheral blood mononuclear cells, and can activate immune effector functions to kill cancer cells in vitro. In vivo, the antibody targets p5365-73 peptide-expressing breast cancer xenografts, significantly inhibiting tumor growth. This represents a promising new agent for future cancer immunotherapy

Combination of novel and public RNA-seq datasets to generate an mRNA expression atlas for the domestic chicken

Background: The domestic chicken (Gallus gallus) is widely used as a model in developmental biology and is also an important livestock species. We describe a novel approach to data integration to generate an mRNA expression atlas for the chicken spanning major tissue types and developmental stages, using a diverse range of publicly-archived RNA-seq datasets and new data derived from immune cells and tissues. Results: Randomly down-sampling RNA-seq datasets to a common depth and quantifying expression against a reference transcriptome using the mRNA quantitation tool Kallisto ensured that disparate datasets explored comparable transcriptomic space. The network analysis tool Graphia was used to extract clusters of co-expressed genes from the resulting expression atlas, many of which were tissue or cell-type restricted, contained transcription factors that have previously been implicated in their regulation, or were otherwise associated with biological processes, such as the cell cycle. The atlas provides a resource for the functional annotation of genes that currently have only a locus ID. We cross-referenced the RNA-seq atlas to a publicly available embryonic Cap Analysis of Gene Expression (CAGE) dataset to infer the developmental time course of organ systems, and to identify a signature of the expansion of tissue macrophage populations during development. Conclusion: Expression profiles obtained from public RNA-seq datasets - despite being generated by different laboratories using different methodologies - can be made comparable to each other. This meta-analytic approach to RNA-seq can be extended with new datasets from novel tissues, and is applicable to any species

A normative and structural analysis of the HGSHS: a with a large Australian sample

Australian norms and structural analysis for the Harvard Group Scale of Hypnotic Susceptibility, Form A (HGSHS:A) are presented. Results relating to score distributions, item difficulty level, and reliability were considered for a large sample of Australian students (N = 4,752) obtained over eight years of testing at Macquarie University. The aggregated sample, which represents the largest normative study of the HGSHS:A undertaken to date, was compared to recent normative studies conducted in Australia, Canada, Germany, and Spain, using both English and non-English versions of the test. In general, the aggregated sample was consistent with other reference samples, and results indicated that the HGSHS:A continues to function well as an instrument for the initial screening of hypnotisability. Further, the emergence of a three-factor solution from the principal components analysis was also consistent with previous factor-analytic studies, and suggested that performance on this scale reflects three dimensions of hypnotic responding