Strategies for the Mitigation of Oxysterol-Induced Cytotoxicity

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dc.contributor.advisorAlvarez, Pedro J.en_US
dc.creatorMathieu, Jacques M.en_US
dc.date.accessioned2013-03-08T00:36:20Zen_US
dc.date.available2013-03-08T00:36:20Zen_US
dc.date.issued2011en_US
dc.description.abstractChronic exposure to some oxysterols might contribute to deterioration of human or environmental health. Oxysterols are both biomarkers of oxidative stress as well as mediators of its damage, and play a central role in many independent, but converging, disease processes, such as atherosclerosis, Alzheimer's disease, and age-related macular degeneration. Therefore, the aim of this thesis was to identify enzymes capable of transforming oxysterols to either reduce their toxicity or facilitate their metabolism or excretion. 7-ketocholesterol (7KC), being amongst the most cytotoxic and recalcitrant of these compounds, was the main focus of this work. We isolated various bacteria capable of utilizing 7KC as a sole carbon and energy source. One of these, Rhodococcus jostii RHAl , was subjected to rigorous transcriptomic and mutational analysis to elucidate its 7KC degradation pathway, which was similar, but not identical, to that of cholesterol. Metabolite screening revealed the reduction and subsequent removal of the 7-keto moiety prior to the step catalyzed by HsaC, the enzyme responsible for cleavage of sterol ring A. Furthermore, cloning and expression of a number of reductases from two gene clusters that were highly up-regulated during growth on 7KC identified three reductases that are active against several closely related structural analogs, though not 7KC itself. 7KC and a number of analogs were assayed for toxicity against human fibroblasts Several enzymes were overexpressed in these fibroblasts by transient transfection with mammalian expression vectors to screen for their ability to mitigate 7KC-induced cytotoxicity. A LAMP1/cholesterol oxidase chimera was found to be significantly cytoprotective to exposure to up to 50 μM 7KC compared to mock transfection as well as 7KC-transforming enzymes targeted to either the mitochondria or cytosol. Additionally, transfection with LAMP1 alone and treatment with 0.9% hydroxypropyl β-cyclodextrin also reduced toxicity. Therefore, it seems likely that addressing 7KC toxicity within the lysosome is critical for cytoprotection. This work provides preliminary evidence to support this approach, and may have implications for the treatment of oxysterol-associated diseases. However, further research is needed to evaluate the effects and safety of heterologous gene expression within the lysosome, both in vitro and in vivo.en_US
dc.format.extent190 p.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.callnoTHESIS C.E. 2011 MATHIEUen_US
dc.identifier.citationMathieu, Jacques M.. "Strategies for the Mitigation of Oxysterol-Induced Cytotoxicity." (2011) Diss., Rice University. <a href="https://hdl.handle.net/1911/70340">https://hdl.handle.net/1911/70340</a>.en_US
dc.identifier.digitalMathieuJen_US
dc.identifier.urihttps://hdl.handle.net/1911/70340en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectHealth sciencesen_US
dc.subjectEnvironmental scienceen_US
dc.subjectApplied sciencesen_US
dc.subjectOxysterolen_US
dc.subjectCytotoxicityen_US
dc.subjectKetocholesterolen_US
dc.subjectLysosomesen_US
dc.subjectAgingen_US
dc.subjectBiomedical engineeringen_US
dc.subjectEnvironmental engineeringen_US
dc.titleStrategies for the Mitigation of Oxysterol-Induced Cytotoxicityen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentChemical and Biomolecular Engineeringen_US
thesis.degree.disciplineEngineeringen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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