Phospholipases C (PLCs) are a large family of enzymes that regulate the cleavage of phosphatidylinositol-4,5- bisphosphate into Inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This project included research on PLCζ and PLCβ1 on male infertility and thyroid cancer respectively. Human PLCζ (hPLCζ) is the key protein for egg activation following sperm-egg fusion and triggers the release of Ca2+ producing a series of oscillations which initiate embryogenesis. Defects in hPLCζ lead to infertility, thus the project aimed at its expression and enrichment suitable for biomedical applications. The results included 1) Comparison of hPLCζ bacterial expression from twenty four plasmid constructs with varying protein tags. 2) Expression of active soluble hPLCζ, free from its bacterial solubility partners. The PLCβ1 project involved a novel InDel (1076 bp deletion, ATAA junction insertion) identified in the 3rd intron of PLCβ1 (Chr 20) by genome-wide linkage analysis (GWLA, LOD score 3.01) of a large kindred with multinodular goitre (MNG) progressing to papillary thyroid cancer (PTC). Affected individuals exhibited increased PLCβ1 transcript levels in their thyroids and the InDel has a putative ERα binding site. The project aimed to, 1) Develop a genotyping technique for high-throughput PLCβ1 InDel screening in large cohorts 2) Determine the InDel’s mechanism of action in modifying thyrocyte proliferation. 3) Next generation sequencing (NGS) of the chr20 region identified by GWLA to identify potential variants responsible for MNG. 1) A QPCR genotyping method was established and the InDel identified in patients with benign thyroid disease. 2) Reporter gene assays suggested silencer elements within the InDel. 3) PLCβ1 knockdown using siRNA exhibited a trend in inhibiting proliferation of a thyroid cell-line. 4) NGS of the 20cM-Chr20 GWLA identified region found no additional disease loci confirming a role for the PLCβ1 InDel. The InDel provides a biomarker for MNG patients most likely to develop PTC.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:665873 |
| Date | January 2015 |
| Creators | Ladas, Ioannis |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/75723/ |
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