Although the primary defect in cystic fibrosis is the dysfunctional CFTR protein, the major pathology of the disease is due to aberrant mucus properties which give rise to repeated and prolonged infections of the lungs. Electron tomography was used to generate a three-dimensional model of a mucin network in its native, hydrated state. This has allowed the examination of the physical and organisational properties of healthy mucus which could potentially lead to a better understanding of how mucus gels behave in vivo. Human sublingual and submaxillary mucous secretions were extracted and 90% of the water was removed using 14 kDa MWCO dialysis tubing immersed in Aquacide II followed by centrifugation through a 1 MDa MWCO filter to remove water and concentrate the sample. The resultant partially concentrated mucus was then frozen in vitrified ice and imaged using an FEI Polara 300kV FEG transmission electron microscope. The major glycoprotein of mucus was immunolabelled with gold-conjugated antibodies and cryo-electron tomography was performed. Tomograms were rendered using IMOD; eTomo and Chimera were used for image analysis. This work was carried out to examine the physical properties of mucus by using cyro-electron microscopy in an effort to better understand how mucin networks are organised in their native, hydrated state.
|Date of Award||1 Aug 2014|
- The University of Manchester
|Supervisor||Robert Ford (Supervisor)|