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dc.contributor.advisorRandall, R. E.
dc.contributor.authorDominguez Palao, Francisco
dc.coverage.spatialxxiii, 216 p.en_US
dc.date.accessioned2017-05-11T14:53:43Z
dc.date.available2017-05-11T14:53:43Z
dc.date.issued2017-06-21
dc.identifier.urihttp://hdl.handle.net/10023/10750
dc.description.abstractRNA:protein interactions are central in many cellular processes, including activation of innate immune responses against microbial infection. Their study is essential to better understand the diverse biological events that occur within cells. However, isolation of RNA:protein complexes is often laborious and requires specialized techniques. This thesis is concerned with attempts to develop an improved purification protocol to isolate specific RNA:protein complexes. Taking advantage of the specific interaction of the Pseudomonas aeruginosa PP7 protein with its cognate RNA binding site, termed the PP7 recognition sequence (PRS), the aim was to identify cellular proteins involved in activating cell-signalling pathways, including the interferon-induction cascade, following viral infection with stocks of parainfluenza virus 5 (PIV5) rich in copyback defective interfering (DI) particles. Copyback DI genomes are powerful inducers of IFN and, here, I show they also activate the induction of IL-6, IL-8 and TNFα; cytokines that also have antiviral properties. Following the successful cloning of the PRS into a copyback DI genome, we investigated conditions for optimal in vitro capture of DI-PRS:protein complexes by PP7 on Dynabeads. When tested, the protocol led to the successful capture of ILF3 and PKR, two dsRNA binding proteins induced by IFN. We further developed a tap-tagging system to minimize the presence of non-specifically bound proteins to Dynabeads that may interfere with future mass spectrometry analysis. To isolate DI-PRS RNA:protein complexes from infected cells, attempts were made to rescue replicating DI-PRS genomes in the context of wild type PIV5. Similarly, efforts were made to isolate influenza A virus RNPs that contained the PRS in the neuraminidase (NA) gene from infected cells using the PP7-based protocol developed. However, for reasons discussed, unfortunately RNA:proteins complexes were not successfully purified from infected cells in either case.en_US
dc.language.isoenen_US
dc.publisherUniversity of St Andrews
dc.rightsAttribution-ShareAlike 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by-sa/4.0/*
dc.subjectRIG-Ien_US
dc.subjectParamyxovirusen_US
dc.subjectDefective interferingen_US
dc.subjectInterferonen_US
dc.subjectPP7en_US
dc.subject.lccQP623.8P75D7
dc.subject.lcshRNA-protein interactionsen
dc.subject.lcshParamyxovirusesen
dc.subject.lcshInterferonen
dc.subject.lcshPseudomonas aeruginosaen
dc.titleInterferon induction by paramyxoviruses : investigations into specific RNA:protein interactionsen_US
dc.typeThesisen_US
dc.type.qualificationlevelDoctoralen_US
dc.type.qualificationnamePhD Doctor of Philosophyen_US
dc.publisher.institutionThe University of St Andrewsen_US


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