The NF-κB transcription factor is expressed in the majority of mammalian cells and regulates a large number of genes with important functions in a variety of cellular processes including cell growth, division, apoptosis and inflammatory responses. Perturbation of NF-κB response has been implicated in a variety of diseases such as asthma and inflammatory bowel disease, in addition to various forms of cancer. Through experiments at the single cell level it has been shown that NF-κB displays complex temporal activation, notably including nucleo-cytoplasmic oscillations. It has been observed that these oscillations occur in a heterogeneous manner; as such they are masked when measured at the population level. In contrast, pulsed TNFα treatment at 100 min intervals produces regular and synchronous nuclear peaks of NF-κB. Such pulsatile stimulation may reflect more accurately physiological conditions. The work in this project uses a Systems Biology approach consisting of bioinformatic, mathematical, and experimental methodologies to investigate how NF-κB can regulate such a diverse set of gene responses. Previously published studies have proposed that target gene expression levels following NF-κB activation (continuous TNFα) can be explained by a combination of key parameters, including transcript degradation rate, transcript structure, and transcription initiation rate. Initial work in this project highlighted that these explanatory factors are not sufficient to describe the observed temporal order of gene transcription. The roles of miRNAs and NF-κB subunit phosphorylation in regulation were additionally explored. A large set of genes was identified that are activated more strongly by pulsed TNFα than by continuous TNFα treatment. This suggests a new unreported mechanism of gene regulation, the possible causes of which are examined in this thesis. The gene list was refined by altering pulse frequency, which revealed an enrichment of NF-κB targets correlated with the regularity of these pulses. Temperature shift and anti-inflammatory drug treatment (Diclofenac) were shown to have a profound effect on NF-κB oscillation frequency. These perturbations provide an alternative method to study the effects of NF-κB oscillation frequency on specific target genes, independent of a pulse regime. Integration and analysis of these datasets suggested that a core, frequency-encoded set of genes regulated by NF-κB might exist. It is proposed that such genes may respond optimally to specific frequencies of NF-κB activation, implying a potential frequency threshold. The presence of such genes may explain the need for the complex systems that control NF-κB timing. It was noted that there was an enrichment of genes encoding transcription factors within the frequency encoding set, in addition to proteins which are known to be involved in the control of inflammation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764391 |
| Date | January 2015 |
| Creators | Daniels, Damon |
| Contributors | Jackson, Dean |
| 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/nfkappabregulated-differential-gene-transcription-a-systems-biology-analysis(f25d85c4-5463-4d41-bbb0-d23e6265fda8).html |
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