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Title: Investigating type 2 diabetes related biosensing and amyloid peptide fibrillation
Author: Wang, Shih-Ting
ISNI:       0000 0004 7969 8172
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Type II diabetes (T2D) is an epidemic and chronic disease that accounts for 80-90 % of the diabetic individuals and associates with various micro- and macro-complications. It is important to understand the underlying disease mechanisms and pathways, and develop simple and robust platforms for early diagnosis, monitoring and treatment of T2D. In this thesis, two T2D related pathogeneses, amyloidosis of T2D-related islet amyloid polypeptide (IAPP or amylin) and methylglyoxal (MGO, an α-dicarbonyl metabolite in T2D)-induced glycation were investigated by multiple techniques including chemical, structural, microscopic characterisations and computer simulations to probe the interactions at the bio-bio and bio-nano interfaces. Specifically, Chapter 3 discussed the roles of peptide structures on amyloid self-assembly and morphological diversity by the design of amyloidogenic peptides derived from IAPP. Among the segments, distinct fibril morphologies, including twisted and planar (untwisted) ribbons were observed with varied diameters, thicknesses, and lengths. The transformation from twisted ribbons into untwisted structures with increased aggregation propensity was especially triggered by substitution of the C-terminal serine with threonine, where the side chain methyl group was responsible for the distinct assembly properties. This effect was ascribed to the restriction of inter-sheet torsional strain through increased hydrophobic interactions and hydrogen bonding, which was further confirmed by serine substitution with alanine and valine, N-terminal capping, and sequence elongation. Chapter 4 presented a new assay for the early detection of amylin fibrillation using the biarsenical dye, 4,5-bis(1,3,2-dithiarsolan-2-yl)fluorescein (FlAsH), which could recognise the tetracysteine motifs and transform into a strong fluorescent complex. Due to the close proximity of two cysteine residues within the hydrophilic domain of amylin, a non-contiguous tetracysteine motif formed upon amylin dimerisation or oligomerisation, which was recognised by FlAsH and emitted strong fluorescence. The developed assay not only allowed the tracking of initial nucleation events without modification of the amylin sequence, but also enabled imaging of amyloid fibrils and investigating the effects of amyloid inhibitor/modulator toward amylin fibrillation. Chapter 5 presented a systematic study of the mechanism of the interaction between citrate-capped gold nanoparticles (AuNPs) with IAPP by a combination of chemical characterisations, electron microscopic imaging and molecular dynamics simulations. Because of the Au-binding sequence motif at the hydrophilic peptide domain, IAPP strongly interacts with the AuNP surface in both the monomeric and fibrillar states. AuNPs were observed to trigger IAPP conformational transition from random coil to ordered structures (α-helix and β-sheet), resulting in the acceleration of IAPP fibrillation. Additionally, MD simulations revealed IAPP conformational change in a Au facet dependent manner, in which IAPP was unfolded and adsorbed directly onto the Au(111) surface, while it interacted predominantly with the citrate adlayer on the Au(100) surface and retained some helical conformation. The observed IAPP-AuNP affinity was further applied to reduce peptide-induced lipid membrane disruption. Finally, Chapter 6 presented a simple and selective colorimetric biosensor for MGO by employing a bio-inspired chemical reaction to mediate the aggregation states of AuNPs. The bidentate-amino ligand, o-phenylenediamine (OPD) was found to cause AuNP aggregation by increasing the surface hydrophobicity of the NPs, which could be inhibited by the formation of a stable adduct, 2-methylquinoxaline by the MGO-OPD reaction. Depending on the level of target MGO and the remaining OPD in solution, AuNP solution displaying different solution colour and plasmonic properties could be achieved in 30 min reaction time. MGO at a level as low as 1 μM was detected by the naked eye and 0.05 μM by UV/vis spectrometry, which were within the clinical range marking oxidative stress in diabetes. Additionally, the assay enabled discrimination of MGO from other biologically relevant ketones and aldehydes based on the different reaction rates of these compounds with OPD. The research works reported in this thesis are anticipated to shed light on the structural interpretation of amyloidogenesis in T2D and to assist the development of nanomaterial based amyloid modulators and new diagnostic/therapeutic tools.
Supervisor: Stevens, Molly Sponsor: Imperial College London ; United States Department of Energy ; Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral