The central feature of prion disease is the conversion of normal host‐encoded cellular prion protein to an abnormal isoform; designated PrPSc. Current research has shown that PrPSc is the principal, and possibly the sole, component of infectious prions. Despite a wealth of experimental data that supports this protein‐only hypothesis, the goal of systematically producing prions in vitro still remains. The failure to generate high‐titre synthetic mammalian prions from fully defined starting materials has underlined the need to establish the composition of ex vivo purified mammalian prions that retain high titre infectivity. Such characterisation of infectious prions necessitates their purification from brain and this requires an approximate 14,000‐fold enrichment (based upon protein content). The main aim of this thesis therefore, has been to address the problems of purification and to isolate infectious prions. An array of novel enzymatic and biochemical techniques have been used including precipitation with sodium phosphotungstic acid, systematic washing steps with ionic salts, digestion with pronase E, proteinase K, amyloglucosidase and sphingomyelinase. Rapid and accurate measurement of prion infectivity throughout purification has been made possible by the use of the scrapie cell assay. Pronase E has been demonstrated to selectively digest PrPC while preserving both proteinase K‐sensitive and resistant infectious prions. This discovery now facilitates the factionation, isolation and detailed charaterisation of prions with distinct physicochemical properties without being confounded by contaminating PrPC. Alongside purification, high resolution imaging of the infectious prion has been a key unaccomplished challenge in the prion field. The problems of imaging insoluble aggregated proteinaceous material and correlating observed structures with specific infectivity have so far frustrated meaningful studies. Work in this thesis has focused upon solubilising and disaggregating large amorphous protein aggregates obtained after purification for imaging by electron microscopy, and has defined key problems that remain to be addressed. Collectively this thesis describes novel methods for investigating mammalian prions that will improve our understanding and the diagnosis of prion disease.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:625590 |
| Date | January 2011 |
| Creators | D'Castro, L. M. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1310247/ |
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