Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Previous studies in yeast in this laboratory have found carnitine to be both protective against
oxidative stress induced by hydrogen peroxide and to increase the detrimental effect of
dithiothreitol. These phenotypes were found to be independent of the role of carnitine within the
carnitine shuttle. A screen for suppressor mutations for these carnitine-dependent phenotypes
identified, among others, Δcho2 and Δopi3. Cho2p and Opi3p catalyse the sequential
methylation reactions in the formation of phosphatidylcholine from phosphatidylethanolamine.
Therefore, this study aimed to investigate the relationship between choline, phosphatidylcholine
and the carnitine phenotypes. Liquid growth assays of Δcho2 and Δopi3 cultures revealed that
addition of choline can restore the protective effects of carnitine against hydrogen peroxide. The
connection between the cellular phospholipid composition and the carnitine-dependent shuttleindependent
phenotypes was also investigated. Analysis of the lipid composition of cells by
LCMS showed that Δcho2 and Δopi3 had a largely different lipid composition compared with the
wild type, most notably, a reduction in phosphatidylcholine and an increase in triacylglycerol
content were observed for both mutants. These changes were reversed by supplementation
with choline. However, no effects on the lipid composition of cells in response to carnitine
treatment were observed, either when supplemented alone or in combination with DTT and
hydrogen peroxide.
Carnitine has also been investigated in mammalian systems for its potential to protect cells from
oxidative stress, an effect which would be of benefit in various neurodegenerative disorders.
Several studies have documented the positive effects of carnitine against oxidative stress in
mammalian cells however the mechanism behind this action remains unknown. It is therefore
thought that, provided similar effects for carnitine can be shown in mammalian cells as was
observed in yeast, it would be beneficial to use yeast as a model system for the study of the
molecular changes induced by carnitine. In view of this, the effects of carnitine on toxicity
induced by oxidative stress in mammalian neural cells were compared to that which has been
observed in yeast. For this purpose the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide (MTT) assay, a measure of reductive capacity of cells, was used. However, no effects
for carnitine were observed in the MTT assay in combination with either dithiothreitol or
paraquat. / AFRIKAANSE OPSOMMING: Vorige studies op gis in hierdie laboratorium het bevind dat karnitien beskermend is teenoor
oksidatiewe stres wat deur waterstofperoksied geïnduseer word en ook die nadelige effek van
ditiotreitol verhoog. Hierdie fenotipes is gevind om onafhanklik te wees van die rol van karnitien
binne die karnitien-pendel. Die sifting vir onderdrukker-mutasies van hierdie karnitienafhanklike
fenotipes het onder andere Δcho2 en Δopi3 geïdentifiseer. Cho2p en Opi3p kataliseer die
opvolgende metileringsreaksies tydens die vorming van fosfatidielcholien vanaf
fosfatidieletanolamien.
Hierdie studie het dus gepoog om die verhouding tussen cholien, fosfatidielcholien en die
karnitienfenotipes te ondersoek. Vloeistofanalises van Δcho2- en Δopi3-kulture het aangedui
dat die byvoeging van cholien die beskermende effekte van karnitien teenoor
waterstofperoksied kan herstel. Die verband tussen die sellulêre fosfolipiedsamestelling en die
karnitienafhanklike pendel-onafhanklike fenotipes is ook ondersoek. Die analise van die
lipiedsamestelling van selle deur middel van LCMS het getoon dat Δcho2 en Δopi3 ‘n grootliks
verskillende samestelling het in vergelyking met die wilde tipe, en daar is veral ‘n afname in
fosfatidielcholien en ‘n verhoging in triasielgliserol-inhoud vir beide mutante waargeneem.
Hierdie veranderinge is omgekeer deur aanvulling met cholien. Geen effekte op die
lipiedsamestelling van die selle is egter in reaksie op die karnitienbehandelings waargeneem
nie, hetsy toe dit alleen aangevul is of in kombinasie met ditiotreitol en waterstofperoksied.
Karnitien is ook in soogdierstelsels ondersoek vir sy potensiaal om selle teen oksidatiewe stres
te beskerm, ‘n effek wat groot voordeel sal inhou vir verskeie neurodegeneratiewe steurings.
Verskeie studies het reeds die positiewe effekte van karnitien teen oksidatiewe stres in
soogdierselle opgeteken, hoewel die meganisme agter hierdie werking nog onbekend is. Daar
word dus vermoed dat, gegewe dat soortgelyke effekte vir karnitien in soogdierselle getoon kan
word as wat in gis waargeneem is, dit voordelig sou wees om gis as ‘n modelsisteem vir die
studie van die molekulêre veranderinge wat deur karnitien geïnduseer word, te gebruik. In die
lig hiervan is die effekte van karnitien op giftigheid wat deur oksidatiewe stres in
soogdiersenuselle geïnduseer is, vergelyk met dít wat in gis waargeneem is. Om hierdie rede is
die 3-[4,5-dimetieltiasool-2-iel]-2,5-difeniel tetrasoliumbromied (MTT) essaiëring, ‘n meting van
die verminderende kapasiteit van selle, gebruik. Geen effekte vir karnitien is egter met die MTT
essaiëring in kombinasie met óf ditiotreitol óf parakwat waargeneem nie.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/97946 |
| Date | 12 1900 |
| Creators | Du Plessis, Michelle |
| Contributors | Bauer, Florian, Franken, Jaco, Loos, Ben, Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 67 pages : illustrations |
| Rights | Stellenbosch University |
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