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Direct investigation of the autocatalytic effect i.. (FibCat)
Direct investigation of the autocatalytic effect in protein fibrillation – from molecular mechanism to macroscopic polymorphism
(FibCat)
Date du début: 1 sept. 2012,
Date de fin: 31 août 2014
PROJET
TERMINÉ
Protein fibrillation is an important research topic in different scientific fields. For example, increased knowledge on protein fibrillation may clarify different aspects related to pathologies like Alzheimer's and Parkinson's diseases, which are characterized by the build-up of amyloid fibrils in the involved tissues. An important aspect of fibril formation is the ability of fibril itself to catalyze the fibrillation process, known as secondary nucleation, with the fibril surface playing a still poorly understood role in this mechanism. The present proposal aims to investigate aspects of macroscopic fibril polymorphism in relation to the molecular structure of the fibrils and the molecular mechanism of aggregate growth. The focus will be on the surface properties of the fibrils and their role in determining both the evolution of the aggregate growth and the 3D arrangement. Fibril formation will be studied in vitro for two different systems: insulin and alpha synuclein (aSN). The temporal features of the aggregation process will be studied in different experimental conditions using an approach based on amyloid sensitive probes and dynamic light scattering. A morphological investigation on the macroscopic scale of the occurring species will be performed by means of imaging techniques (atomic force and optical microscopy) and the structural details of these species (both intermediate and mature aggregates) will be investigated by small angle X-ray scattering and X-ray fibre diffraction. Surface structural details will be investigated by a novel approach based on a combined use of SAXS and ultrasonic resonator technology, able to provide quantitative information on the compressibility of the solvent around the aggregate. The catalytic propensity of species with different surface properties will be quantified by fluorescence spectroscopy during seeding experiments, and visualized by confocal microscopy.
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