Loss-of-function mutations in the filaggrin gene(FLG) have recently been shown to be strongly associated with atopic dermatitis (AD). The overall aim of this study was to explore the role of filaggrin in skin barrier function and AD. There were two main focuses in this study. The first was a functional study whose primary objective was to determine if FLG mutations correlated with skin barrier dysfunction in AD. Fifty-five mild to moderate AD individuals were recruited, genotyped and had their skin barrier assessed using three different measures - transepidermal water loss (TEWL), skin capacitance and the number of tape strips required to abrogate skin barrier. A secondary aim of this functional study was to test the hypothesis that corneocytes were less adherent to one another in filaggrin-related AD compared to wild-type AD skin. The second main focus of this thesis was a structural study aimed at interrogating the structure-function relationship of filaggrin. Filaggrin protein was extracted and purified from a total of 21 AD and non-AD subjects and analysed using mass spectrometric techniques. Specifically, matrix assisted laser desorption/ionisation time-of-flight(MALDI-TOF)mass spectrometry (MS) and nano liquid chromatography tandem MS(LC-MS/MS) were utilised. Part of this structural study also involved developing and optimising the extraction and purification of filaggrin protein, including a novel way of extracting filaggrin from skin using tape stripping. In addition, novel filaggrin-specific enzyme-linked immunosorbant assay (ELISA) was also developed, which could serve as a useful screening test for the protein. In this study, FLG mutations were found to correlate with higher TEWL and fewer number of tape strips required to abrogate skin barrier, but not with skin capacitance. FLG mutations were also not shown to correlate with AD severity. The mean amount of protein extracted from filaggrin-related AD skin was also significantly higher compared to wild-type AD skin, supporting the hypothesis that corneocytes were less adherent to one another (and therefore, densely packed) in filaggrin-related AD skin. MS analysis of filaggrin confirmed the heterogeneic nature of filaggrin protein, even within a single individual. Interestingly, this structural study also showed that filaggrin was only minimally expressed in the skin of all the AD individuals studied, whether or not they possessed any FLG mutation. Due to the limited amount of filaggrin extracted from AD skin, it was not possible to conduct comparative structural analysis between filaggrin from AD and non-AD skin.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:685740 |
| Date | January 2012 |
| Creators | Chu, Roland Poh Cheong |
| Contributors | Weller, Richard ; Rees, Jonathan |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/9964 |
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