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Title: Regulation of gene expression in the immune system and in virally-transformed cells
Author: Bassano, Irene
ISNI:       0000 0004 5346 671X
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2014
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The correct development and functioning of the immune system is critical for the defence of the host organism against pathogens and cancers. V(D)J recombination generates diversity of immunoglobulin (Ig) and T cell receptor (TCR) genes by the regulated joining of variable (V), diversity (D) and joining (J) gene segments. Tissue-specific enhancers in the DNA genome activate these genes to undergo recombina-tion by triggering non-coding transcription through the recombining gene segments, following interaction with the respective promoters. How this is achieved is un-known. The specificity of enhancer/promoter interactions was examined using the murine Igλ chain locus. The transcription factors that bind to the three main promoters were identified by DNase I footprinting. Of these, a factor termed E47 was shown to interact with IRF4 by co-immunoprecipitation experiments. The importance of these interactions was confirmed by mutagenesis where it was shown that mutations of any of the binding sites in DNA for the transcription factors or mutations in the amino acids involved in protein-protein interactions decreased the rate of transcrip-tion. Together, these studies suggest that IRF4/E47 interactions may play a key role in triggering locus activation. RNA-Seq data from HPV-positive samples and cell lines were analysed to identify putative biomarkers for cervical cancer. Infection with HPVs is the main cause for cervical cancer accounting for 10-15% of cancer-related deaths in women world-wide. It is established that HPVs escape the immune response over decades to es-tablish tumorigenesis but the specific mechanism is unknown. Virus integration into the host genome and deregulation of several genes may play a key role in promot-ing cancer; of particular interest are those transcripts that form the “surfacesome”. Among these, particular interest was given to connexin 26 (Cx26), which is classified as cancer-predisposition gene and was found to be commonly down regulated in all samples analysed. Recombinant adenoviruses expressing the two HPV16 oncogenes were generated and employed to transduce HaCaT cells to analyse Cx26 mRNA and protein levels coupled with dye transfer assays to study the structural behaviour of connexins. The data presented showed that E6 and E7 alter Cx26 protein expression by relocating Cx26 within the cytoplasm from the membrane-bound form. This was confirmed in the dye transfer assay where cell-cell communications were lost.
Supervisor: Eric, Blair ; Cook, Graham ; Stead, Lucy Sponsor: Lady Tata Memorial Trust ; University of Leeds
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available