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Title: Development of molecular sensors for the measurement of glucose in airway secretions
Author: Bearham, Jade Marie
ISNI:       0000 0004 7962 5221
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2019
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The mammalian airway is lined with a thin layer of liquid (the airway surface liquid) which is important for protecting the airway against infection. In a healthy individual, airway surface liquid (ASL) glucose concentration is typically 0.4 mM and in individuals with inflamed airways and/or hyperglycaemia, this value rises. An increase of glucose concentration in the ASL correlates with an increase in the presence of pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. Therefore, creating and utilising a sensor as a method for measuring ASL glucose concentrations in situ could be used to predict susceptibility to airway infection. These sensors could also be used as a tool to investigate glucose concentrations in the ASL and glucose metabolism throughout the airways. Glucose binding protein (GBP) was encoded on a plasmid. GBP was genetically manipulated, expressed and purified in Escherichia coli and labelled with environmentally sensitive fluorophores. Fluorescent GBPs were assessed for their affinity for glucose and dynamic fluorescence range. GBP was applied to the apical surface of airway epithelial cells and to murine bronchoalveolar lavage samples to measure ASL glucose concentration. Membrane-tethered glucose sensor Sweetie was genetically manipulated to include an apical target signal to direct the sensor to the apical membrane to measure ASL glucose concentrations in situ. GBP based FRET sensor was transfected into airway epithelial cell to investigate intracellular glucose and glucose metabolism. Of the multiple fluorescent GBP glucose sensors, the E149C/A213R GBP labelled with IANBD was the most successful glucose sensor candidate. It produced a large fluorescence range and an affinity suitable for measuring glucose in the ASL. Sweetie was not solely trafficked to the apical membrane. FRET data showed that intracellular glucose concentrations fluctuated and that this was under the control of hexokinases. In hyperglycaemia, intracellular glucose was shown to be lower than in normoglycaemia.
Supervisor: Baines, D. L. ; Baker, E. H. ; Taylor, J. D. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available