Background: The NKG2D ligands are a set of cell surface proteins, the expression of which can make cells susceptible to immunity mediated by NKG2D receptor expressing cells, which include NK cells, CD8<sup>+</sup> αβ T cells and γδ T cells. The NKG2D ligands are known to be expressed in distinct settings, including viral infection, cancer, T cell activation, and cellular proliferation, settings also tightly associated with Warburg metabolism. The molecular events which determine NKG2D ligand expression status are unknown. Aims: We aim to enhance understanding of the deterministic molecular events that control NKG2D ligand expression. Specifically, to explore the relationship between Warburg metabolism and NKG2D ligand expression in cell line and physiological models, and second, to identify open chromatin elements at NKG2D ligand loci, and develop computational methods to analyse this data. Methods: We use a range of molecular biology techniques to delineate the role of glucose metabolism in NKG2D ligand expression in a HEK293T cell model. We develop a physiological CMV-primary fibroblast model of NKG2D ligand induction to validate our key findings. We adapt, optimise and validate a DNaseI-seq protocol, to define open chromatin sites at the NKG2D ligand loci. We develop a data analysis `pipeline', including our own peak-finding software (“PeakHunter"), to identify open chromatin sites in the data. Key results: Glucose drives NKG2D ligand expression. This effect requires cellular uptake and metabolism of glucose. Purine nucleotides are a key glucose metabolite for this effect, and purine nucleosides are sufficient to induce NKG2D ligand expression in our HEK293T model. We have identified the open chromatin sites at the NKG2D loci in MCF7 breast cancer cells, and optimised and validated this protocol. Finally we have developed “PeakHunter" a multifunctional software tool for mapped DNaseI-seq data analysis. Conclusions: Glucose and its contribution to purine metabolism play a central role in the induction of NKG2D ligand expression in physiological settings. The influence of glucose leads to significant alterations in cellular NKG2D-dependent immunogenicity. PeakHunter is a useful tool for analysis of mapped DNaseI-seq data.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:581344 |
| Date | January 2013 |
| Creators | McCarthy, Michael Thomas |
| Contributors | O'Callaghan, Christopher A. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:69de3276-0b1e-4174-9309-988242468682 |
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