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Title: The regulation and role of the inositol 1,4,5-trisphosphate receptor in Caenorhabditis elegans
Author: Ford, K. L.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2011
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IP3Rs in Caenorhabditis elegans are encoded by a single gene, itr-1. This thesis explores how itr-1 is regulated in vivo by both genetic and physical interactions, and the central role this regulation has to play in the phenotype of the animal. ITR-1 has numerous predicted and confirmed protein binding sites conserved with mammalian IP3Rs, but the importance of many of these to receptor function in vivo is largely unclear. Using a homologous recombination system, the predicted binding site for an FKBP12 homologue was mutated, and the altered construct introduced into an itr-1 reduction-of-function background. Behavioural analysis showed that this construct was able to rescue a subset of itr-1 phenotypes, implying tissue-specific regulation of the protein. A novel defecation expulsion defect of itr-1 reduction-of-function was also observed, suggesting that itr-1 has a previously unidentified role in contraction coupling. Further genetic analysis and calcium imaging were used to elucidate the mechanisms by which the putative ITR-1-FKBP interaction contributes to the described expulsion phenotype. This targeted strategy was complemented by a forward genetics approach to determine novel interactions with itr-1. A previously-identified suppressor of itr-1(sa73) was mapped using snip-SNP markers, followed by an RNAi screen to establish potential candidates. Although the suppressor remains uncloned, a number of genes were identified that potentially interact with IP3-mediated calcium signalling. Tissue-specific RNAi and tissue-specific rescue were used to identify the sites of action of itr-1 in the control of three parameters of the defecation cycle; timing, rhythmicity and contraction coupling. Calcium imaging using a genetically-encoded sensor was used to analyse how these genetic perturbations affect calcium dynamics within the intestine.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available