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An investigation into effects of in vitro glycation on type I collagen fibrillar structure and related biochemistry

Goodson, Simon John 2013. An investigation into effects of in vitro glycation on type I collagen fibrillar structure and related biochemistry. PhD Thesis, Cardiff University.
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Abstract

Overall, this project aimed to evaluate and advance the use of two techniques in the study of structural changes brought about by in vitro glycation of mouse tail tendon type I collagen. Firstly, using transmission electron microscopy with positive staining, study of collagen glycation by glucose, galactose, fructose and ribose was conducted. Site specificity of glycation along the fibril was seen and it was demonstrated for the first time using this method that there was variation in the progression of the process between the monosaccharides, suggesting different reaction mechanisms and determinants of site specificity for each sugar. From the early changes after 1 week galactose treatment, early-preferred glycation sites for this sugar were indicated. Using sorbitol, broad non-glycation-related sugar binding along the fibril was identified and this was also seen following galactose, fructose and ribose treatment. Secondly, using X-ray diffraction with isomorphous replacement, a 1D electron density map for collagen fibrils in mouse tail was constructed for the first time as a baseline for this and future intended glycation studies in mice. Use of this technique to study tendon incubated for 2 weeks in ribose and galactose showed electron density changes that appeared consistent with the corresponding electron microscopy glycation data. Using HPLC with fluorescence detection, it was shown that the four sugars produced different “fingerprint” profiles of products of glycation. Galactose produced a similar profile to glucose as hypothesised. However despite its chemical similarity to glucose, galactose also produced glycation products that were absent from the glucose profile. The biochemical information produced was invaluable for interpretation of the data from the structural techniques, inasmuch as providing representations of the relative extents of glycation occurring during each monosaccharide and control treatment. In conclusion, this thesis has demonstrated for the first time that transmission electron microscopy with positive staining, and X-ray diffraction with isomorphous replacement techniques together are useful complementary methods for studying glycation-related changes in type I collagen structure. It is anticipated that these techniques together will be of value in further in vitro and in vivo studies of type I collagen glycation, contributing to a better understanding of the structural bases of the deleterious effects of this process during diabetes and ageing.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Optometry and Vision Sciences
Subjects: R Medicine > RE Ophthalmology
Uncontrolled Keywords: Type I collagen; fibril; D-period; mouse tail tendon; in vitro; glycation; ageing; aging; diabetes; glucose; galactose; fructose; ribose; sorbitol; 3-(N-Morpholino)propanesulphonic acid; MOPS; buffer effects; lysine; arginine; advanced glycation end-products; AGEs; AGE cross-links; pentosidine; HPLC; fluorescent detection; transmission electron microscopy; TEM, positive staining; phosphotungstic acid; PTA, X-ray diffraction; synchrotron; heavy atom isomorphous replacement; gold; iodine; phase determination; Patterson plot; electron density plot; sugar binding effects.
Funders: BBSRC
Date of First Compliant Deposit: 30 March 2016
Last Modified: 23 Mar 2017 04:48
URI: http://orca.cf.ac.uk/id/eprint/58030

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