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Title: Genesis, phototoxicity and degradation of retinal lipofuscin
Author: Davies, Matthew W.
ISNI:       0000 0004 2751 8045
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2008
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Retinal lipofuscin (RLF) is an intra-lysosomal accumulating waste product thought to be composed of oxidatively modified lipids and proteins. The substrates are proposed to be derived from old or damaged organelles and phagocytosed photoreceptor outer segments (POS). The material that is not degradable by retinal pigment epithelium (RPE) lysosomes accumulates within the cell and has the capacity to photogenerate reactive oxygen species. Consequently, the accumulation of RLF within RPE cells has been linked to cell dysfunction and the onset of age-related macular degeneration (AMD) and other retinal degenerative diseases. This study sought to gain important insights into RLF composition and formation, to analyse the efficacy of optical filters in reducing photoexcitation of RLF and develop a nanomedicine with the potential to prevent RLF accumulating and/or degrade RLF accrued within RPE cells. Ex vivo RLF granules were isolated and analysed by mass spectrometry to determine their protein content, and Western blotting to probe for oxidatively modified components. RLF was found to contain a number of molecules adducted to lipid peroxidation products and minimal protein. In vitro models of RLF formation were designed and their physio-chemical characteristics analysed using: spectrophotometry/fluorometry, SDS-PAGE, Western blotting, HPLC and singlet oxygen production measurements. The in vitro models shared a number of characteristics with ex vivo RLF including: the presence of species adducted to lipid peroxidation products, a protein smear on an SDS-PAGE gel and the ability to photogenerate singlet oxygen. The capability of yellow-tinted optical filters to reduce the blue-light hazard of RLF towards RPE cells was analysed using cell viability assays and fluorescence microscopy. Results showed yellow-tinted filters provided significant protection to RLF-loaded RPE cells when irradiated with blue-light compared to a UV blocking filter or no filter. The suitability of a potential nanomedicine for use in abolishing RLF from RPE cells was assessed using: fluorescence and transmission electron microscopy, cell viability assays and flow cytometry. The study succeeded in identifying a carrier molecule that was localised to the lysosomes of RPE cells and an active component capable of degrading POS. When conjugated, the nanomedicine displayed the ability to degrade accumulated RLF. The findings presented within this study have enhanced our understanding of RLF composition and the key substrates involved in its formation. This has lead to proposals of how lipofuscin is associated with the onset of AMD. Studies conducted using UV and blue-light blocking optical filters may assist in the future design of intraocular lenses and aid ophthalmologists in advising cataract patients of the most appropriate lens for surgery. Preliminary work has been undertaken to develop a nanomedicine which has been demonstrated to eliminate RLF from the RPE in vitro. providing the basis for further development of a therapeutic strategy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available