Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.639625
Title: Molecular characterisation of human central retina
Author: Vevis, K.
ISNI:       0000 0004 5364 6024
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2015
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Abstract:
In this study we aimed to identify gene expression differences between macula and periphery within the human neuroretina. To this end we used qPCR and RNA sequencing to analyse post-mortem tissue. Equal sized punches, one within the macula and 4 extramacular areas were isolated and RNA was extracted from 7 donor eyes. We then tested specific genes for differential expression by qPCR. This was done firstly to establish whether mRNA analysis was feasible on human post-mortem tissue, and secondly to validate the dissection method by focusing on genes with known distributions. Next, specific genes with unknown distribution were tested to interrogate whether various key molecular pathways in the retina were differentially active in the macula versus the periphery. This approach revealed many differences but was ultimately limited as each gene had to be measured individually. We therefore decided to use RNA sequencing (RNAseq) in order to obtain expression levels of all genes active in the retina. This method allowed us to identify and quantify transcripts from around 20,000 different genes. We then developed a method to assess the confidence levels in the quantification of individual genes. We also used genes with known distribution for method validation. Focusing on the largest differences between the macula and the periphery revealed marked differential expression components from retinoic acid signalling pathway. Furthermore, roughly ¾ of the genes with the most reliable quantification was used for global pathway analysis. This showed that lipid metabolism and energy production pathways were also differentially used in the macula. 5 In order to further query the role of lipid metabolism in the retina we studied knockout mice lacking the very low density lipoprotein receptor (VLDLR) by using 3D Scanning Electron Microscopy to map mitochondria size and distribution in photoreceptors. This showed marked changes in the mutant animals suggesting abnormal lipid metabolism can directly affect photoreceptors and may play a role in macular pathologies in humans.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.639625  DOI: Not available
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