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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.

No description available.

The lipid bilayer membrane of enveloped viruses, such as human immunodeficiency virus type 1 (HIV-1), plays an important role in key steps of the infection, including cell binding and uptake. Phosphatidylserine (PS) and monosialotetrahexosylganglioside (GM1) are examples of two host-derived lipids in the membrane of enveloped virus particles that are known to contribute to virus attachment, uptake, and ultimately dissemination. A quantitative characterization of their contribution to the functionality of the virus requires information about their relative concentrations in the viral membrane. In this dissertation, a gold nanoparticle (NP) binding assay for probing relative PS and GM1 lipid concentrations in the outer leaflet of different virus-like particles (VLPs) using small sample sizes is introduced. The assay evaluates both scattering intensity and resonance wavelength and determines relative NP densities through plasmon coupling as a measure for the target lipid concentrations in the NP-labeled VLP membrane. The performed studies reveal significant differences in the membrane of HIV-1 and Ebola VLPs that assemble at different intracellular sites and pave the way to an optical quantification of lipid concentration in virus particles at physiological titers. In addition, this technique was used in another application to improve the understanding of the relationship between the membrane PS lipid and the infectivity of HIV-2 and murine leukemia virus (MLV). The composition of the membrane, in particular the cholesterol (chol) content, determines its fluidity. As differences in the membrane composition of individual virus particles can lead to different intracellular fates, biophysical tools capable of probing the membrane fluidity on the single-virus level are required. In this dissertation, we demonstrate that fluctuations in the polarization of light scattered off gold or silver nanoparticle (NP)-labeled virus-like-particles (VLPs) encode information about the membrane fluidity of individual VLPs. We developed a plasmonic polarization fluctuation tracking microscopy (PFTM) which facilitated, for the first time, the investigation of the effect of chol content on the membrane fluidity and its dependence on temperature on the single-VLP level. Chol extraction studies with different methyl-β-cyclodextrin (MβCD) concentrations yielded a gradual decrease in polarization fluctuations as function of time. The PFTM revealed chol content and fluidity heterogeneities of an HIV-1 VLP population.

A irradiação de carnes e seus derivados têm sido empregada em alguns países como medida de tratamento para garantir a segurança microbiológica destes produtos evitando infecções alimentares. A irradiação pode provocar algumas mudanças indesejáveis na composição química dos alimentos tal como a oxidação lipídica. Na produção de derivados de carnes, o perfil lipídico está diretamente relacionado com a qualidade sensorial e nutricional do produto. Para retardar o processo da oxidação lipídica são utilizados antioxidantes, que podem ser sintéticos ou naturais. Atualmente, maior atenção tem sido dada aos antioxidantes naturais derivados de ervas e especiarias, tais como o alecrim e o orégano. O objetivo deste trabalho foi avaliar o potencial antioxidante dos extratos destas duas especiarias em hambúrgueres bovinos submetidos à irradiação por fonte de 60CO com a dose de 6, 7 e 8 kGy e aceleradores de elétrons com dose de 3,5 e 7 kGy e estocados sob congelamento por períodos pré-estabelecidos de 0, 45 e 90 dias. Os resultados demonstraram que o alecrim apresentou maior proteção antioxidante em uma matriz alimentícia heterogênea como o hambúrguer, porém o orégano quando utilizado em sinergismo com o alecrim ou com o BHT/BHA conseguiu retardar a oxidação lipídica durante todo o período de análise. Apesar de terem ocorrido alterações no perfil de ácidos graxos não foi possível demonstrar uma linearidade dependente da dose de irradiação e/ou do tempo de estocagem. A análise sensorial mostrou que dentre as amostras formuladas com antioxidantes naturais, o orégano recebeu maiores notas entre os provadores. A amostra irradiada formulada com alecrim recebeu maior nota em relação a não-irradiada. O uso de especiarias com atividade antioxidante com o objetivo de inibir a degragação oxidativa em alimentos gordurosos mostra-se promissor para a aplicação industrial. / Radiation processing has been employed in some countries as a mean of treatment to assure microbiological safety of meat and meat products, avoiding the occurrence of food-borne disease. The ionizing radiation may cause some undesirable changes on chemistry composition of food and the lipid oxidation is one of the main reactions. In meat products processing industry, the lipid composition is directly related to nutritional and sensory quality of the product. For preventing oxidation, use of antioxidants which can be synthetic or natural, has been practically applied in some products. Currently, most attention has been given to natural antioxidants from herbs and spices like rosemary and oregano. The aim this study was to assess the antioxidant effects of either rosemary and oregano extract in beef burgers submitted to irradiation in 60Co source with dose 6, 7 e 8 kGy, electron beams with dose 3,5 e 7 kGy and storage under freeze along 0, 45 e 90 days. The results showed that rosemary extract has the major antioxidant effects when it is used on heterogeneous food matrix like beef burger, but oregano extract was better efficient to delay lipid oxidation along storage time when it is used in synergism with rosemary and/or BHT/BHA. Although to have occurred changes in the fatty acids composition it was not possible to demonstrate a straight dependence of irradiation dose and/or storage time. Sensory analysis showed that between the samples prepared with natural antioxidants, the beef burger prepared with oregano has received better scores by panelists. Irradiated beef burger prepared with rosemary has received better scores when compared to non-irradiated one. The use of spices with antioxidant activity to avoid the oxidative damage in foods that contain fats in their formulation is thought to be promising to application in food facilities.

The goal of the present study is to elucidate the modulatory effect of lipid compositions on phospholipase A2 (PLA2) activity. Sphingomyelin (SM) incorporation inhibited catalytic activity and membrane-damaging activity of native and mutated PLA2 toward egg yolk phosphatidylcholine (EYPC) vesicles. The inhibitory effects were through the reduction of membrane fluidity and modulation of the mode of membrane binding of PLA2 at water/lipid interface. The modulated effect of SM depended on inherent structural elements of PLA2. Moreover, cholesterol (Chol) incorporation into EYPC/egg yolk sphingomyelin (EYSM) vesicles relieved the inhibitory effect of sphingomyelin on PLA2 activity via lipid domain formation by SM and Chol. The effects on the interactive mode of PLA2 with phospholipids induced by the physical state changes of membrane bilayers abolished the inhibition of SM on catalytic activity and membrane-damaging activity of PLA2. Additionally, quercetin incorporation increased PLA2 activity and membrane-damaging activity toward EYPC/SM vesicles via its raft-making effect. Quercetin incorporation reduced PLA2 activity and membrane-damaging activity toward EYPC/SM/Chol vesicles via its raft-breaking effect. Membrane-inserted quercetin affected on membrane structure and membrane-bound mode of PLA2 to modulate PLA2 interfacial activity and membrane-damaging activity. Finally, studies on the effects of phosphatidylserine (PS) content on the sensitivity of lipid vesicles mimicking inner and outer plasma membrane toward PLA2 activity revealed that the membrane-binding mode adopted by PLA2 depended on the lipid composition. The effects of PS content on the extent of lipid domain formation and the conformation of PLA2 adopted at water-lipid interface modulate PLA2 catalytic activity. Collectively, these results indicate that lipid composition modulates PLA2 activity via its effects on membrane structure and membrane-bound mode of PLA2

The monotopic membrane protein alMGS, a glycosyltransferase catalyzing glucolipid synthesis in Acholeplasma laidlawii, was overexpressed in Escherichia coli. Optimization of basic growth parameters was performed, and a novel method for detergent and buffer screening using a small size-exclusion chromatography was developed. This resulted in a tremendous increase in protein yields, as well as the unexpected discovery that the protein induces intracellular vesicle formation in E. coli. This was confirmed by sucrose density separation and Cryo-TEM of membranes, and the properties of the vesicles were analyzed using SDS-PAGE, western blot and lipid composition analysis. It is concluded that both alMGS and alDGS, the next enzyme in glucolipid pathway, have the ability to make the membrane bend and eventually form vesicles. This is likely due to structural and electrostatic properties, such as the way the proteins penetrate the membrane interface and thereby expand one monolayer. The highly positively charged binding surfaces of the glycosyltransferases may bind negatively charged lipids, such as Phosphatidylglycerol (PG), in the membrane and withdraw it from the general pool of lipids. This would increase the overall lipid synthesis, since PG is a pace-keeper, and the local concentration of nonbilayer prone lipids, such as Phosphatidylethanolamine, can increase and also induce bending of the membrane. The formation of surplus membrane inside the E. coli cell was used to develop a generic method for overexpression of membrane proteins. A proof-of-principle experiment with a test set of twenty membrane proteins from E. coli resulted in elevated expression levels for about half of the set. Thus, we believe that this method will be a useful tool for overexpression of many membrane proteins. By engineering E. coli mutants with different lipid compositions, fine-tuning membrane properties for different proteins is also possible. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 3: Manuscript.

In this project, the eggs of tench (Tinca tinca) were used for the evaluation of changes in fatty acid (FA) composition and lipid classes during in vitro ova ageing until the occurrence of over-ripening. Stripped ova of 6 females were collected separately and stored in sterile cell culture plates in an incubator at 20 °C for up to 24 hours post stripping (HPS). Stored ova were fertilized at 0, 2, 4, 6, 8, 10 and 24 HPS. Egg eyeing and hatching rates were assessed as indices of egg quality. Upon fertilization at each HPS, samples were taken separately frozen in liquid nitrogen and stored at -80°C till further analyses. Differences in the FA and lipid composition and embryo survival rates at the different fertilization times post-stripping were evaluated. The lipid fraction was extracted from the oocytes according to Hara and Radin (1978). The lipid content of the samples was determined gravimetrically from total extracted lipid. The FA were then analysed with a gas chromatograph equipped with a flame ionisation detector and PVT injector. The peaks were identified by comparing their retention times with those of authentic standards. Major lipid classes and the phospholipid classes were separated according to (Olsen and Henderson, 1989). Quantitative analysis of the separated lipid- and phospholipid classes was done by scanning the plates after derivatisation with a CAMAG TLC Scanner 3 (Camag, Switzerland). Identification of the lipid classes was performed by comparison with authentic standards applied on the same plate. In present study no significant changes were observed in lipid content and composition during storage of eggs until over-ripening. As well FA composition did not change over time indicating another reason of deterioration of oocyte quality than the proposed lipid oxidation. However individual fishes in this study showed much different fertilization rates that corresponded with their levels of cholesterol and triacylglycerols in minor degree.

A irradiação de carnes e seus derivados têm sido empregada em alguns países como medida de tratamento para garantir a segurança microbiológica destes produtos evitando infecções alimentares. A irradiação pode provocar algumas mudanças indesejáveis na composição química dos alimentos tal como a oxidação lipídica. Na produção de derivados de carnes, o perfil lipídico está diretamente relacionado com a qualidade sensorial e nutricional do produto. Para retardar o processo da oxidação lipídica são utilizados antioxidantes, que podem ser sintéticos ou naturais. Atualmente, maior atenção tem sido dada aos antioxidantes naturais derivados de ervas e especiarias, tais como o alecrim e o orégano. O objetivo deste trabalho foi avaliar o potencial antioxidante dos extratos destas duas especiarias em hambúrgueres bovinos submetidos à irradiação por fonte de 60CO com a dose de 6, 7 e 8 kGy e aceleradores de elétrons com dose de 3,5 e 7 kGy e estocados sob congelamento por períodos pré-estabelecidos de 0, 45 e 90 dias. Os resultados demonstraram que o alecrim apresentou maior proteção antioxidante em uma matriz alimentícia heterogênea como o hambúrguer, porém o orégano quando utilizado em sinergismo com o alecrim ou com o BHT/BHA conseguiu retardar a oxidação lipídica durante todo o período de análise. Apesar de terem ocorrido alterações no perfil de ácidos graxos não foi possível demonstrar uma linearidade dependente da dose de irradiação e/ou do tempo de estocagem. A análise sensorial mostrou que dentre as amostras formuladas com antioxidantes naturais, o orégano recebeu maiores notas entre os provadores. A amostra irradiada formulada com alecrim recebeu maior nota em relação a não-irradiada. O uso de especiarias com atividade antioxidante com o objetivo de inibir a degragação oxidativa em alimentos gordurosos mostra-se promissor para a aplicação industrial. / Radiation processing has been employed in some countries as a mean of treatment to assure microbiological safety of meat and meat products, avoiding the occurrence of food-borne disease. The ionizing radiation may cause some undesirable changes on chemistry composition of food and the lipid oxidation is one of the main reactions. In meat products processing industry, the lipid composition is directly related to nutritional and sensory quality of the product. For preventing oxidation, use of antioxidants which can be synthetic or natural, has been practically applied in some products. Currently, most attention has been given to natural antioxidants from herbs and spices like rosemary and oregano. The aim this study was to assess the antioxidant effects of either rosemary and oregano extract in beef burgers submitted to irradiation in 60Co source with dose 6, 7 e 8 kGy, electron beams with dose 3,5 e 7 kGy and storage under freeze along 0, 45 e 90 days. The results showed that rosemary extract has the major antioxidant effects when it is used on heterogeneous food matrix like beef burger, but oregano extract was better efficient to delay lipid oxidation along storage time when it is used in synergism with rosemary and/or BHT/BHA. Although to have occurred changes in the fatty acids composition it was not possible to demonstrate a straight dependence of irradiation dose and/or storage time. Sensory analysis showed that between the samples prepared with natural antioxidants, the beef burger prepared with oregano has received better scores by panelists. Irradiated beef burger prepared with rosemary has received better scores when compared to non-irradiated one. The use of spices with antioxidant activity to avoid the oxidative damage in foods that contain fats in their formulation is thought to be promising to application in food facilities.