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Title: Localised dosing and nanodetection using a novel scanning ion conductance microscope and its application to Alzheimer's disease
Author: Chen, Wei-Hsin Chen
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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Scanning ion conductance microscopy (SICM) is a technique for non-contact topographic imaging. In this thesis, a biophysical investigation into Alzheimer's Disease (AD) was carried, with toxic oligomers dosed locally and quantitatively on to single astrocytes using SICM and simultaneously monitoring the response of the target cell. Examination of the effectiveness of antibodies that bind to Abeta or alpha-synuclein (Asyn)peptides depends on the measurement of oligomer-induced abnormal calcium homeostasis in single astrocytes. The method was shown to work at physiological concentrations of oligomers. A series of experiments measuring the reduction in calcium inux in mixtures of antibodies and cerebrospinal fluid (CSF) of AD patients suggested that the binding to co-oligomers composed of Abeta and Asyn may be crucial in the treatment of AD. Furthermore, it may be beneficial to test antibodies before the clinical trial using this assay. The mechanism of this entry of calcium is hypothesised to be the result of the formation of oligomer-induced transient pores in the cell membrane. To verify this hypothesis, a new SICM instrument was built with two nanopipettes; one for dosing and one for detection of the adenosine triphosphate (ATP) release from these pores. A variety of different ATP sensors were made. The best had a sensitivity of 10 micro molar and works as a hexokinase-cofunctioned electrolyte-gated organic field-effect-transistor. However no statistically significant results for ATP release have been obtained in the experiments performed to date. Overall this thesis describes new biophysical methods to study the effect of protein aggregates on live cells and the effectiveness of potential therapies, such as antibodies and nanobodies, to reduce these aggregate induced effects. It can be applied to synthetic aggregates of Abeta or the aggregates present in human CSF.
Supervisor: Klenerman, David Sponsor: Taiwan-Cambridge Scholarship
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Alzheimer's Disease ; Scanning ion conductance microscopy ; cerebrospinal fluid (CSF) ; Abeta ; alpha-synuclein (Asyn) ; astrocyte ; ATP sensor ; oligomer-induced transient pores ; biophysical ; aggregates ; antibody ; nanobody ; assay