The inflammatory response is critical to healing outcome after cutaneous injury. Our current understanding of the response to injury has been compiled from targeted studies on components expected to play a role. It was hypothesised that an unbiased approach to characterisation of soluble mediators within the injured tissue might identify additional components, thereby increasing our understanding of the cellular processes involved. The aim of this research was to develop and characterise a model of injury with the potential to identify novel mediators of the early response. Microdialysis was chosen as both the sampling method and the means by which the tissue was injured as probe insertion causes a single injury that can be sampled continuously without further damaging the tissue. Early injury responses were initially characterised in terms of changes in blood flow and known markers of the inflammatory response, using Laser Doppler Imaging and a bead-based cytokine flow cytometric assay, respectively. A shotgun proteomic analysis was then undertaken to characterise of the protein content of the fluid obtained using microdialysis, dialysate. The phosphorylation status of proteins was also characterised following the implementation and optimisation of a recently reported method that uses dendrimer conjugation chemistry to capture phosphopeptides. Blood flow and cytokine measurements in the dialysate confirmed the occurrence of a reproducible inflammatory response to the microdialysis injury. Proteomic analyses of dialysate suggests that it has a relatively simple protein composition and is dominated by highly abundant components. The identified proteins originate from both intra- and extracellular locations and play a range of roles, including regulation of coagulation, cellular ii communication, and the immune response. Several are likely to undergo post-translational phosphorylation and hence their phosphorylation status was also investigated. Using the phosphopeptide capture method, potentially novel phosphorylation sites were identified in two abundant proteins, albumin and apolipoprotein L1 at positions S603 and S314, respectively. Collectively, the data obtained in this investigation increase our knowledge of the proteins and processes involved in responses to injury, and suggest that microdialysis may be of some use for studies in this area. Further, analysis of protein phosphorylation in dialysate suggests that this is an informative approach that could shed light on extracellular signalling events that occur during the progression of the response to injury.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:509532 |
| Date | January 2009 |
| Creators | Gill, Carolyn Anne |
| Contributors | Clough, Geraldine ; O'Connor, C. D. |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/72793/ |
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