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Title: Understanding the initial activation of Toll-like receptor 5 and Toll-like receptor 8 by their ligands
Author: Gibbard, R. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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The precise mechanism by which TLRs recognise their ligands and lead to downstream signalling is currently largely unclear. The major aim of this work was to begin to understand the nature of the interaction between human toll-like receptors and their ligands, using biochemical, structural, cell biological and fluorescence imaging techniques. This research aimed to elucidate the mechanism by which two TLRs recognise their ligands; TLR5 and its bacterial protein ligand flagellin and TLR8 and its viral ligand ssRNA and a synthetic ligand, resiquimod. Initial studies were to express and purify the extracellular domain of the receptors to generate purified protein to study the receptor/ligand interactions by biophysical and structural methods. Creating sufficiently pure protein in the baculovirus expression system was unachievable due to the fairly low levels of expression and the inability of insect cells to secrete the recombinant proteins. Alternative approaches to study the receptor/ligand interactions were pursued using cell based systems. This included the use of NF-κB reporter assays to study receptor activation in cells and fluorescent protein fusion receptors with confocal microscopy to determine the cellular localisation of the receptors in transiently transfected cells. Mutagenesis studies of TLR8 were also performed to determine residues that were critical for NF-κB activation and protein expression of these mutants was shown to be similar to wild-type receptors by microscopy. Förster resonance energy transfer (FRET) was performed to look at possible receptor/receptor interactions before and after stimulation with ligand in live cells. Fluorescent ligands were able to show the likely interaction site of receptor and ligand in transiently transfected cells. All together these techniques lead to insights as to how and where these two TLRs interact with their ligands and possible models for receptor/ligand interaction can now be proposed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available