Studying the Dynamics and Inhibition of Vimentin Assembly by Small-Angle X-ray Scattering

by Gerrit Brehm
Doctoral thesis
Date of Examination:2021-03-04
Date of issue:2022-03-01
Advisor:Prof. Dr. Sarah Köster
Referee:Prof. Dr. Tim Salditt
Referee:Prof. Dr. Thomas Burg
Persistent Address: http://dx.doi.org/10.53846/goediss-9083

 

 

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Abstract

English

In this work, various methods are employed to investigate different aspects of the vimentin intermediate filament assembly process. Small-angle X-ray scattering provides insights on the occurring lateral assembly on the nanometer scale, and optical fluorescence microscopy provides insight on subsequent longitudinal assembly on the micrometer scale. Microfluidic techniques enable the precise control of chemical conditions and thus reaction times. Combining these techniques enable investigation of ion-induced assembly and small molecules induced assembly inhibition of vimentin intermediate filament. In the first part of this thesis, early time-points of vimentin intermediate filament assembly are measured by combining microfluidics and small-angle X-ray scattering. Finite element method simulations are performed to determine suitable flow rates, geometries and ion concentrations for vimentin intermediate filament assembly inside a microfluidic device. Simulation efficiency and results are improved by subdividing the finite element mesh based on theoretical considerations. The mechanical stability and geometry of the microfluidic device is improved by developing manufacturing protocols. In the second part of this thesis the inhibition of vimentin intermediate filament assembly due to N-Acetyl-L-cysteine and sodium phenylbutyrate is measured by combining optical fluorescence microscopy and small-angle X-ray scattering. A structure finding algorithm is developed to analyse the length of assembled vimentin intermediate filaments. Various N-Acetyl-L-cysteine and sodium phenylbutyrate concentrations and assembly times are investigated, to characterize their effect on assembling vimentin intermediate filaments.
Keywords: microfluidic devices; small-angle x-ray scattering; vimentin; intermediate filaments; protein self-assembly
 
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