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dc.contributor.advisorPohl, C. H.
dc.contributor.advisorKock, J. L. F.
dc.contributor.authorElls, Ruan.
dc.date.accessioned2017-06-22T06:07:16Z
dc.date.available2017-06-22T06:07:16Z
dc.date.issued2008-11
dc.identifier.urihttp://hdl.handle.net/11660/6392
dc.description.abstractEnglish: Candida albicans and C. dubliniensis are two closely related pathogenic yeast species, sharing many phenotypic characteristics which make it difficult to differentiate them, especially in clinical samples. As a result of the similarities between these species, identification techniques, based on phenotypic characteristics, have been developed. In this study some of these techniques and virulence factors were used to characterise strains belonging to these species and to select phenotypically dissimilar strains for further study. This was performed to evaluate if the effect of arachidonic acid (20:4) on these strains were the same, even though they are phenotypically different. Candida albicans and C. dubliniensis can form biofilms which play an important role during infection. During C. albicans infection, 20:4, a long-chain polyunsaturated fatty acid (PUFA), derived from the phospholipids (PLs) of the infected host cell membrane, serves as carbon source and precursor for eicosanoid production. Conflicting results are presented in literature regarding the effect of 20:4 on morphogenesis in C. albicans. In addition, the effect of 20:4 on growth and morphology of C. dubliniensis is unknown. Microscopic examination and enzyme activity assay indicated that 1 mM 20:4 had little to no effect on growth and metabolic activity of planktonic cells and biofilms, as well as on the morphology and viability of the cells in the biofilms. The uptake of PUFAs by yeasts is necessary for utilisation as metabolic fuels, cellular building blocks and the production of signalling molecules. However, there are no definitive studies regarding the uptake and cellular metabolism of 20:4 by these pathogenic yeasts. The uptake and incorporation of 20:4 by planktonic cells and biofilms of selected strains of C. albicans and C. dubliniensis were evaluated by subjecting residual and cellular lipids from planktonic cells and biofilms, grown in the presence and absence of 20:4, to gas chromatography and gas chromatography-mass spectrometry. Strain specific variation in 20:4 uptake and incorporation into different lipid fractions of planktonic cells and biofilms were found. In addition, eicosanoids produced by biofilms in the presence of 20:4 were extracted and it was found that biofilms of these strains were capable of producing 3-hydroxy fatty acids from 20:4. Arachidonic acid can be incorporated into the PLs of yeasts, influencing saturation in cell membranes. It is suggested that the effectiveness of antifungals may depend upon the level of unsaturation and ergosterol content of the membrane, therefore the effect of 20:4 on the cell membrane and susceptibility of C. albicans and C. dubliniensis biofilms towards amphotericin Band clotrimazole were also determined. This was performed by confocal laser scanning microscopy, determination of mitochondrial metabolism, unsaturation index of the PL fractions and ergosterol content of the membranes of biofilms grown in the presence and absence of 20:4. The results indicated that 20:4 influences PL unsaturation and ergosterol content of both C. albicans and C. dubliniensis type strains, increasing susceptibility to the antifungals. Pre-treatment of biofilms with PUFAs may result in the reduction in antifungal dose needed to inhibit biofilms.en_ZA
dc.description.abstractAfrikaans: Candida albicans en C. dubliniensis is twee nabyverwante patogeniese gisspesies, wat verskeie fenotipiese eienskappe in gemeen het. Dit maak dit moeilik om tussen die twee spesies te onderskei, veral in kliniese monsters. As gevolg hiervan is identifikasietegnieke, gebaseer op fenotipiese eienskappe, ontwikkel. In hierdie studie is daar van die tegnieke en virulensiefaktore gebruik gemaak om stamme wat aan die spesies behoort, te karakteriseer en sodoende fenotipies verskillende stamme te selekteer vir gebruik in verdere studies. Dit was gedoen om te bepaal of die effek van arachidoonsuur (20:4) op hierdie stamme dieselfde is, alhoewel hulle fenotipies verskil. Candida albicans en C. dubliniensis is in staat om biofilms, wat 'n belangrike rol speel tydens infeksies, te vorm. Tydens C. albicans infeksies word 20:4, 'n lang-ketting poli-onversadigde vetsuur uit die fosfolipiede van die geïnfekteerde gasheer se sel membraan vrygestel. Dit dien dan as koolstofbron en as voorloper vir eikosanoïedproduksie. In literatuur is daar teenstrydige resultate in verband met die effek van 20:4 op die groei en morfogenese in C. albicans. Boonop is die effek van 20:4 op die groei en morfologie van C. dubliniensis nog onbekend. Deur mikroskopiese ondersoek en ensiemaktiwiteitstoetse is bevind dat 1 mM 20:4 min tot geen effek op die groei en metaboliese aktiwiteit van planktoniese selle en biofilms asook op die morfologie en lewensvatbaarheid van selle in die biofilm het nie. Die opname van lang-ketting poli-onversadigde vetsure deur giste is noodsaaklik vir gebruik as metaboliese energiebronne, sellulêre boustene en vir die produksie van seinmolekules. Daar is egter geen bepaalde studies wat die opname en sellulêre metabolisme van 20:4 deur die patogeniese giste aandui nie. Die opname en inkorporasie van 20:4 deur planktoniese selle en biofilms van die geselekteerde C. albicans en C. dubliniensis stamme is dus bestudeer. Dit is gedoen deur die oorblywende en sellulêre lipiede van planktoniese selle en biofilms, gegroei in die teenwoordigheid en afwesigheid van 20:4, te analiseer m.b.v. gaschromatografie en gaschromatografiemassaspektrometrie. Die resultate het variasie in die opname en inkorporasie van 20:4 in die veskillende lipied fraksies tussen stamme van planktoniese selle en biofilms aangedui. Daarby is eikosanoïede, geproduseer deur biofilms in die teenwoordigheid van 20:4, ontrek en is dit bevind dat al die stamme in staat is om 3-hidroksievetsure vanaf 20:4 te produseer. Arachidoonsuur word in die fosfolipiede van giste ingebou en beïnvloed daardeur die versadiging in die selmembrane. Daar is aangedui dat die effektiwiteit van antifungale kan afhang van die vlak van onversadiging en die ergosterolinhoud van die membrane. Dus is die effek van 20:4 op die selmembrane en die vatbaarheid van C. albicans en C. dubliniensis biofilms teenoor amfoterisien B en klotrimazool bepaal. Dit is gedoen deur konfokale laserskandeermikroskopie, bepaling van die mitochondriale metabolisme, onversadiging van die fosfolipiedfraksies en die ergosterolinhoud in die membrane van biofilms gegroei in die teenwoordigheid en afwesigheid van 20:4. Die resultate het aangedui dat 20:4 die onversadiging van die fosfolipiede en die ergosterolinhoud van die tipestamme van C. albicans en C. dubliniensis beïnvloed, asook lei tot 'n verhoging in die vatbaarheid vir die antifungale. Voor-behandeling van biofilms met lang-ketting poli-onversadigde vetsure mag dus lei tot die vermindering in die dosis antifungaal benodig om die biofilms te inhibeer.af
dc.description.sponsorshipNational Research Foundation (NRF)en_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectAmphotericin Ben_ZA
dc.subjectAntifungalen_ZA
dc.subjectArachidonic aciden_ZA
dc.subjectBiofilmsen_ZA
dc.subjectClotrimazoleen_ZA
dc.subjectFatty aciden_ZA
dc.subjectMetabolic activityen_ZA
dc.subjectPhenotypic characteristicsen_ZA
dc.subjectPhospholipidsen_ZA
dc.subjectPlanktonic cellsen_ZA
dc.subjectEicosanoidsen_ZA
dc.subjectCandidaen_ZA
dc.subjectDissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2008en_ZA
dc.titleThe effect of arachidonic acid on lipid metabolism and biofilm formation of two closely related Candida speciesen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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