The phosphate and sulfate esters of carbohydrates are widespread in nature, where they partake in a variety of signalling roles. This thesis focuses on the involvement of trehalose-6-phosphate (Tre-6-P) in regulating plant growth, and the function of sulfated carbohydrates in the immune response. In plants, Tre-6-P is a critical messenger that conveys environmental information for the control of metabolic state. Beneficial natural conditions lead to an increase in the in vivo concentration of Tre-6-P, which in turn triggers biosynthetic processes facilitating plant growth. However, the mechanism by which this effect is exerted is largely unknown. A thorough understanding of this fundamental signalling pathway could allow considerable improvements in agriculture. Recent evidence has found an interaction between Tre 6 P and a protein kinase system (SnRK1), mediated via an intermediary protein whose identity is unknown. Our efforts to identify this elusive protein are presented. Methodology is developed to allow the synthesis of Tre-6-P analogues for defining structure-activity relationships, with particular attention focused towards the desymmetrization of trehalose and the control of phosphate migrations. Tre-6-P is tethered to a solid phase resin allowing affinity extraction of putative intermediary factors. The 14-3-3 class of regulatory proteins is identified as a potential binding partner of Tre-6-P. The mannose receptor (MR) is an essential element of the mammalian immune system. Expressed on macrophages, the protein can bind and internalize pathogens through their surface exposed carbohydrates. However, the receptor can also dimerize, upon which it is able to divalently bind to sulfated carbohydrates. The biological significance of this sulfate binding is poorly understood. Current hypotheses invoke a role for the sulfate mediated trafficking of MR dimers to areas of B-cell maturation for antigen presentation, thus linking the innate and adaptive defence mechanisms. In any case, the dimerization of the MR is an essential prerequisite for interaction with carbohydrate sulfates. Methodology is required to probe this dimerization on the surface of cells under their native conditions. In this thesis, we use fluorescence microscopy to study the binding of various probes to macrophage and endothelial liver derived cell lines. Results of our initial investigations are presented that show internalization of the probes. The use of FRET imaging to directly probe the presence of MR dimers is studied.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:580888 |
| Date | January 2011 |
| Creators | Patel, Mitul Kiran |
| Contributors | Davis, Benjamin G. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:6fb4de15-5585-4f62-ae56-4c6bf32593ab |
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