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Expression of tumour necrosis factors during chick lens development

Williams, Llinos 2008. Expression of tumour necrosis factors during chick lens development. PhD Thesis, Cardiff University.

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During development of the lens, epithelial cells at the lens equator begin a differentiation process to become secondary fibre cells. The differentiating cells elongate and migrate towards the centre of the lens where they envelop the older, central fibre cells. Differentiation into fibre cells is accompanied by the breakdown of all organelles, such as the mitochondria. All organelle degradation is completed and denucleation occurs at the border of the organelle free zone (OFZ) which contains the central, terminally differentiated, fibre cells. The differentiation pathway is not well characterised, though it is believed to have similarities to an attenuated form of apoptosis supported by the identification of apoptosis related genes, such as TNF, in the lens. This study continues the search for and characterisation of apoptosis related genes expressed during lens development, focusing on TNFs and their extended family. Reverse Transcriptase-(RT-) PCR was carried out, identifying a number of TNF and extended family member genes in the chick lens, expression studies established novel, statistically significant differential expression for TRAF2 and TRAF3. TRAF2 protein expression from western blotting, similar to RT-PCR expression was found to decline as the lens developed. TRAF2 localisation studies showed limited expression in the equatorial region but there was extensive signalling found in the developing iris, a region in the corneal-scleral boundary and some staining was also detected in the ciliary body. TRAF3 protein and RT-PCR expression were similar, with increasing expression as the lens developed. Western blotting identified two bands and subcellular fractionation confirmed different localisation for the two isoforms. Immunofluorescence identified increasing TRAF3 staining in the cortical fibre cells, this staining was found to be similar to proteins that were reported to be involved in lens fibre cell remodelling and maintenance, suggesting a possibly similar role for TRAF3. Following interest in TRAIL as a gene therapy for Posterior Capsule Opacification (PCO) its expression was examined using RT-PCR and Western blotting which showed low, similar levels of expression throughout the stages of lens development studied. Peroxidase staining showed interesting staining in the equatorial epithelial cells and those just beginning to differentiate at the transition zone. Novel nuclear staining was identified at all time points in both epithelial and fibre cells containing nuclei. Characterisation of whole lens culture was undertaken to discover the optimum culture system for the whole chick lens. Of the published research using whole chick lens culture none stated the basic morphology of the developing lens in organ culture, though each lab had their preferred methodology. The characterisation resulted in the preference of E10 chick lenses being grown with vitreous attached in medium containing glucose. Understanding the morphology of lenses in culture will be invaluable when undertaking the functional studies required to clarify the roles in the lens of the newly identified genes, specifically TRAF2 and TRAF3.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Optometry and Vision Sciences
ISBN: 9781303214936
Date of First Compliant Deposit: 30 March 2016
Last Modified: 19 Mar 2016 23:31

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