The devastating failure of Alzheimer’s disease (AD) therapies indicates a lack of mechanistic understanding of its pathogenesis. While major hallmarks of AD include aggregates of the amyloid beta protein (A-beta) and dysregulation of vital cellular contents, key gaps in our knowledge remain. This highly interdisciplinary project will develop and apply cutting-edge single-molecule microscopy and advanced biophysical techniques for the observation of A-beta nanostructures conferring toxicity in live brain cells, rendering key quantitative details that are unobtainable by conventional methods and urgently required for the effective and efficient design of next-generation therapeutics. Specifically, this project will involve:
1. The design and implementation of new optical microscopy tools to enable the real-time nanoscale localization and 3D tracking of single fluorescently labelled A-beta nanostructures across cell compartments with sub-millisecond time-resolution, to elucidate the environmental factors which regulate A-beta diffusion.
2. The development of high-precision ratiometric assays compatible with single-molecule imaging techniques for the quantification of (i) A-beta induced perturbation of the cell membrane via a Förster resonance energy transfer approach and (ii) A-beta induced dysregulation of ionic strength and pH via sensitive reporter dyes.
3. The development of novel microfluidic flowcells to facilitate and enhance single-molecule imaging by enabling the handling of precise liquids, the manipulation of small microlitre volumes, environmental exchange, rapid cellular/biomolecular immobilization and controlled temperature gradients.
4. The development of high-throughput advanced data analysis methods based on Bayesian and Hidden Markov Modelling approaches to extract enormously detailed data regarding A-beta morphologies, A-beta diffusion, conformational changes, kinetics and local environmental changes.
3 years tuition fees plus stipend (£15,009 for 2019/20).