الفهرس 
General IntroductionOnly 14 pages are availabe for public view
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Abstract
Tumour necrosis factor receptor associated periodic syndrome (TRAPS) is an auto-inflammatory disease that is caused by mutations in the tumour necrosis factor receptor 1 (TNFR1) gene (TNFRSF1A). To date, despite knowing the mutated gene, the pathogenesis of the disease is not fully clear. This work was conducted to study the effects of TNFR1 associated mutations upon important intracellular signalling pathways that are involved in inflammation and associated with TNF induced signalling. One type of TNFR1 mutation has been studied in this project, TNFR1-C33Y (cysteine 33 is replaced with tyrosine) as cysteine mutation phenotypes are characterised by aggressive forms of TRAPS disease.
SK-Hep1 cells were stably transfected with full length C33Y or wild-type (WT) TNFRSF1A-containing plasmid constructs and were used as an in-vitro model to study the C33Y TNFR1 mutation effects on different cell signalling pathways in comparison to WT TNFR1. PBMC from TRAPS patients with C33Y mutation and matched controls from healthy individuals were also included in this study. At the beginning of this study, western blotting was used to evaluate different regulatory molecules of different signalling pathways in C33Y transfectants compared to WT transfectants. Reverse Phase Protein Microarray (RPPA) was optimised and applied to studying signalling pathways. The results showed that the presence of the C33Y mutation is associated with general evidence of subtle up-regulation of many pro-inflammatory and pro-survival components of the studied pathways, and decreased pro-apoptotic signalling, when compared to cells expressing WT TNFR1 at similar low levels. This was demonstrated in both cultured SK-Hep1 transfectants and in isolated peripheral blood mononuclear cells of TRAPS patients with the C33Y mutation. The results describe, for the first time, the extent to which the TNFR1-associated signalling network is perturbed by the influence of the mutated receptor protein. Both canonical and non-canonical NF-B pathways are activated, as are other important signalling pathways that are involved in inflammatory processes such as Mitogen Activated Protein Kinase (MAPK), Phosphoinositide (PtdIns) 3 kinase (PI3K), Signal Transducers and Activators of Transcription (STAT3), Janus family of tyrosine kinases (JAK2)/c-Src and activated proteins such as phospho ATF, phospho June and phospho ELK. A key observation was that these changes are seen at both the level of the active (often phosphorylated) form and of the total protein for many of these pathways components, which suggests that the continuous low level activation resulting from the presence of the mutant TNFR1 leads to unexpected compensations in the pathways.
In conclusion, the results of this work indicate a mechanism by which cells expressing the C33Y-mutant form of TNFR1 are potentially more readily triggered into a sustained inflammatory response. Even in the absence of external triggers, such cells are in a heightened state of pro-inflammatory readiness.
This work offers insight into the molecular pathophysiology of the TRAPS disease and expands the range of potential target pathways for future specific therapeutic interventions.