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dc.contributor.advisorRossi, Adriano
dc.contributor.advisorBradley, Mark
dc.contributor.advisorDorward, David
dc.contributor.authorPotey, Philippe Marios Daniel
dc.date.accessioned2022-07-01T12:12:16Z
dc.date.available2022-07-01T12:12:16Z
dc.date.issued2022-07-01
dc.identifier.urihttps://hdl.handle.net/1842/39269
dc.identifier.urihttp://dx.doi.org/10.7488/era/2520
dc.description.abstractThe body is exposed to harmful stimuli, such as bacterial or cellular debris, on a daily basis. Inflammatory cells travelling to the site of damage are responsible in removing the harmful stimulus, a process known as a proinflammatory response. Once the stimulus is neutralised, the inflammatory cells are no longer required and are removed in a controlled process, known as the resolution of inflammation. In inflammatory diseases, such as acute respiratory distress syndrome, the pro-inflammatory response is excessive and prolonged without a resolution phase, resulting in tissue damage. The formyl peptide receptors (FPR) are a family of G-protein coupled receptors that play a role in not only the initiation, mainly through FPR1, but also resolution of inflammation, mainly through FPR2. The production of FPR1 and FPR2 specific optical probes can provide an insight about the inflammatory stages either in an in vitro or in vivo setting. As FPR2 probes have not been extensively investigated in the literature, a fluorescent peptide library of seven probes was produced using Trp-Lys-Tyr- Met-Val-Met (WKYMVM) and its variation Trp-Lys-Tyr-Met-Val-Met (WKYNleVNle) as the FPR2 agonist and the green fluorophores fluorescein, NBD and BODIPY. The FPR1 probe was constructed by coupling the FPR1 antagonist cinnamoylPhe-D-Leu-Phe -D-Leu-Phe-Lys (cFlFlFK) with the farred fluorophore Cy5. Fluorescent imaging and flow cytometry was carried out on primary cells and immortal cell lines expressing varying levels of FPR2 in 3 order to assess their fluorescent capabilities. Furthermore, competition binding assays with FPR ligands confirmed the binding of the fluorescent probes to their respective receptors. Prior data was used to select the best FPR2 probe candidate and along with the FPR1 probe, FPR levels were investigated in dexamethasone primed macrophages and platelet-activating factor (PAF) treated neutrophils. When the probes were tested as a single stain, dexamethasone increased FPR2 surface expression on human-derived macrophages whereas PAF increased FPR1 surface expression on granulocytes. However, when the experiments were repeated as a dual stain, the FPR2 probe showed to induce neutrophil shape change and increase FPR1 expression. However, fixing the cells prior to staining with probes inhibited the pro-inflammatory action of the FPR2 probe. The work carried out showed that FPR probes can be successfully produced without interfering with their structural integrity and specificity towards the receptor. Furthermore, they can be used to identify changes in FPR levels after exposure to various stimuli. The fluorescent probes could ultimately be used with optical endomicroscopic imaging systems in order to provide an insight of the role that FPR has in lung pathologies and how that may progress, but also how the levels may change upon treatment.en
dc.language.isoenen
dc.publisherThe University of Edinburghen
dc.titleDevelopment of novel imaging probes to investigate FPR1 and FPR2 mediated pathways in inflammationen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen
dc.rights.embargodate2025-07-01en
dcterms.accessRightsRestricted Accessen


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