NF-κB signalling induces transcriptional upregulation of a wide array of genes in response to inflammatory signalling caused by, for example, TNFα cytokine. In addition to inducing the expression of factors which mediate an intracellular response, such stimuli also cause the expression of further signalling factors, including TNFα itself, to propagate and refine an initial stimulus. However, while such positive feedback signalling can be seen to be beneficial in amplifying potentially small initial stimuli, excessive production can cause hyper-inflammatory responses; an occurrence linked to several autoimmune diseases. Therefore, correct regulation – in regards to both too little and too much TNFα signal production – is essential for a balanced immune response. In this thesis I have focussed on the effects of the IκB protein family member BCL-3 on TNFΑ transcription: demonstrating NF-κB dependent induction of both TNFΑ and BCL3 genes and a subsequent negative role for BCL-3 in regulating TNFΑ transcription in the human fibrosarcoma HT1080 cell line – forming an Incoherent Feed Forward Loop (I-FFL) motif. Notably, I have shown a differential rate of induction of TNFΑ (rapid) and BCL3 (delayed) transcript levels; demonstrating that while the TNFΑ gene has a pre-stimulus RNA polymerase II bound and poised for a rapid response, the BCL3 promoter requires histone modification and chromatin remodelling for binding of NF-κB and RNA polymerase II. Extensive characterisation of the temporal sequence of events constituting BCL3 promoter remodelling, mRNA plus protein levels and NF-κB nuclear localisation through live cell microscopy allowed the construction of a mathematical model which has been tested to ensure it can accurately recreate biological behaviour. This model has been utilised to show that the delayed production of inhibitory BCL-3 produces distinct TNFΑ transcript dynamics: (i.) initially allowing a high magnitude response but coupled to later strong repression of TNFΑ expression and (ii.) producing a non-monotonic response to pulsed stimuli. This behaviour cannot be quantitatively recreated with models in which BCL3 transcription is induced simultaneously with TNFΑ and proposed physiological benefits are outlined. Based on this work, time delays in I-FFLs are proposed as a novel mechanism to produce varied output dynamics. Future research tools have also been developed in this work - including generation of an expression vector to visualise BCL-3 protein in live cells (utilising a BAC recombinant engineering approach) - plus further research questions and predictions regarding TNFα signalling have been raised by additional modelling work.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:564350 |
| Date | January 2012 |
| Creators | Walker, Thomas |
| Contributors | Jackson, Dean; White, Michael |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-role-of-bcl3-feedback-loops-in-regulating-nfkappab-signalling(ed8214ca-e5f5-4edd-9754-59692ffbda9d).html |
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