This study presents findings on the effect of domestic laundry detergents on the fastness to light of selected fibre reactive dyes applied to cotton. The study was carried out to elucidate the reasons for the accelerated colour loss of cotton garments washed under Australian domestic laundering conditions. Cotton fabric dyed with commonly used reactive dyes were laundered with water only, several domestic detergents and a laboratory formulated neutral detergent, and then exposed to light for two hours in the wet state. Quantities of detergent used were in accordance with the manufacturers recommendations. Exposures were repeated fifteen times equivalent to 30 hours exposure. Colour loss and colour difference were measured after 5, 10 and 15 wash cycles and 10, 20 and 30 hours exposure. When the fabric was exposed wet the colour faded more rapidly than when exposed dry to light. Detergents increased the colour loss even when the fabric was not exposed to light. The presence of an oxidizing bleach (sodium perborate) in the detergent increased colour loss during washing and wet exposure to light. Ultraviolet radiation from the light source, heat, moisture, alkali and oxidising bleach during exposure resulted in hydrolysis of the dye-fibre bond causing dye desorption during washing and rinsing. Water alone increased the fading of the dyes most likely due to presence of dissolved oxygen held within the fibre. The combination of ultraviolet radiation and oxidizing bleaches altered the chemistry of the dye and hence its shade. This was particularly evident on the black dye and one of the navy blue dyes.

Membrane proteins are vital components in the cell and crucial for the proliferation of all living organisms. Unfortunately our collective knowledge of structures of membrane proteins is very limited, as compared to the information available on soluble proteins. This is to a large extent due to the outstanding challenge of working with membrane proteins and the relatively high cost associated with determining a membrane protein structure.  Therefore, the establishment of efficient methods and means for the production and crystallization of membrane proteins is urgently needed. The two methods explored in this thesis  are aimed to achieve rapid optimization of expression and purification conditions of membrane proteins, thereby allowing for the rapid production of more suitable samples for crystallization trials. Despite the challenges in membrane protein structure determination two structures are presented in the thesis: The first structure determined is of the CorA magnesium transporter from Thermotoga maritima will be the focus of this thesis. The CorA revealed a pentameric protein in a closed state. The presence of two regulatory metal binding sites is suggested, as well as a putative magnesium ion bound in the ion conductive pathway. The second structure is of the human enzyme LTC4-synthase, which catalyzes the pivotal step in eicosanoid synthesis by the conjugation of glutathione to LTA4, a reactive epoxide-containing derivative from arachidonic acid. The products of this step, the so-called cysteinyl leukotrienes are potent inflammatory mediators making this enzyme a potential drug target. The structure reveals a charged binding pocket for a horseshoe-shaped glutathione, and a hydrophobic binding pocket for a lipophilic LTA4 molecule. Based on the structure a key residue for catalysis has been identified, Arg 104, which is proposed to play a critical role in activating the thiol group of glutathione for the nucleophilic attack on LTA4.

This study analyses the purchasing behaviour of households and briefly on industrial consumers with regard to their detergent purchases. Following from this analysis, the scope for a new detergent business venture will be investigated. The local industry has a plethora of detergent manufacturing companies supplying the retail and industrial markets, thus an appropriate analysis and strategy developed from this study will enable a new detergent business venture to have an improved understanding of the detergent industry in Pietermaritzburg leading to some minimisation of the risks for potential detergent entrepreneurs. The objectives for the study is detailed below. Primary Research Objectives : 1. To examine whether mcome accounts for household purchasing preferences between branded and non-branded detergents. 2. To determine whether location is a factor that must be considered m the marketing of detergents. 3. To examine whether there are gender differences in the purchasing behaviour of detergents among households. 4. To determine which are the major factors that influence detergent buying behaviour among industries. Secondary Research Objective: To determine whether there is scope for the introduction of a new detergent venture among households in Pietermaritzburg. This study found that income accounts for differences in purchasing of detergents. The manufacturer of detergents needs to be aware that location is a factor that must be considered in the marketing of detergents. Branded detergents carry a premium price whereas non-branded detergents are cheaper. Correspondingly, the marketer must match the type of detergent product to the consumer profile of the location in which the business is operating. It was found that gender does not account for differences in monthly expenditure on detergents. Price and quality of detergents were found to be important characteristics for both the household and business user. In addition, it was found that consumers were willing to support a new detergent business venture in Pietermaritzburg. The major variables influencing detergent brand choice in the Pietermaritzburg market seem to be attitudinal variables such as perception regarding the efficacy (QUALITY) of the brand, closely followed by the perception on the valuefor- money (PRICE). Field level promotional activities such as price-offs, freebies associated with different pack sizes of the same brand also seem to impact the choice of the brand, although the impact was low. Base price reduction is generally resorted to quite infrequently and hence a price elasticity measure may be found to be statistically insignificant. A critical result drawn from this study is the importance of attitudes in affecting behavior in the purchase of detergents. In the South African market, it is generally believed that brand attitudes are primarily influenced by advertising. If this assertion is assumed to be true, this research makes a case for better management of consumer attitudes through the deployment of appropriate advertisements using the correct media (Banerjee, 2004: 3). The major recommendations from this study found that manufacturers of household detergents need to dedicate resources to print media when advertising their products. Targeting the most appropriate consumer and capturing potential consumers by television should be looked at by marketers (Dutta-Bergman, 2006: 103). Liquid detergent manufacturers need to major more on the price of the product and the quality. These two factors are critical in ensuring a new detergent business venture is successful and sustainable . Davis (1993: 19) punts 'green marketing' as one of the strategies that may shift consumers to purchase products. Accordingly, companies that can market a 'green' detergent product would have a greater chance of penetrating the market, and gaining market share from the dominant and larger multi-national detergent companies. / Thesis (MBA)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Membrane rafts are small plasma membrane domains that contain high levels of cholesterol and sphingolipids. They have been implicated in processes as diverse as signal transduction, endocytosis and cholesterol trafficking. Traditional methods for the biochemical preparation of lipid rafts involve the extraction of membranes with nonionic detergents followed by the separation of a low-density, detergent-resistant membrane fraction on density gradients. Because of concerns regarding the possible introduction of artifacts through the use of detergents, several methods were developed for the isolation of lipid rafts that do not involve detergent extraction. / In this study, we compared three different biochemical methodologies of membrane rafts preparation from purified rat liver PM. Only detergent-resistant membranes (DRMs) fulfill the requirements of membrane rafts. We subsequently found using the low dose of EGF (1 ug/100 g BW); the content of EGFR in PM-DRMs did not changed significantly following EGF administration. When a higher dose of EGF (5 ug/100 g BW) is administrated we observed a rapid and almost complete disappearance of EGFR (around 80%) from both PM and DRMs fractions. Interestingly, following the administration of a low or high dose of EGF, the pool of EGFR in the PM-DRMs fraction becomes highly Tyr-phosphorylated. In accordance with the higher level of EGFR Tyr-Phosphorylation, EGF induced an augmented recruitment of Grb2 and Shc proteins to PM-DRMs compared with whole PM. / Furthermore neither high nor low dose of EGF affects the caveolin content in DRMs and PM. These observations suggest that EGFR located in DRM are competent for signaling and non-caveolae PM rafts are involved in the compartmentalization and presumably internalization of the EGFR.

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

Membrane proteins are essential cellular components, responsible for a wide variety of biological functions. In order to better understand such aspects of cell activity, researchers have pursued detailed structural analysis of this class of proteins. Because of the complexities in isolating and studying membrane proteins in their native environment, detergents are often employed as a membrane mimetic media. This thesis examines several features of transmembrane (TM) protein structure and folding in detergents through which we are able to gain insights into membrane protein folding, as well as explore the suitability of detergents as membrane-mimetic environments. We first compare the helix-helix association of a series of model TM sequences in a native bilayer to the corresponding association in a detergent environment. We find that while various classes of helix-helix interaction motifs are preserved in detergents, alterations in detergent solvation may, in turn, lead to altered association affinity. We further explore this phenomenon through investigation of the consequences of the insertion of a strongly polar residue into a TM segment. In these studies we find a correlation between sequence-dependent alterations in detergent solvation and predicted in vivo folding. We also extend such analyses to a variety of detergents and native TM segments, finding that native secondary structure, as it occurs in the context of a full-length protein, is generally well preserved in a variety of detergents. Finally, we assess the determinants of membrane protein folding using two-transmembrane segment constructs, in the process optimizing expression, production and characterization techniques for a diverse range of transmembrane protein sequences. Overall this thesis finds that, detergents are capable of solubilizing membrane proteins in a form suitable for in-depth structural characterization that may not be feasible in other environments. Thus, as an approximation of a native membrane, detergents are able to preserve certain features of membrane proteins such as helix-helix association and native secondary structure.

Membrane proteins are essential cellular components, responsible for a wide variety of biological functions. In order to better understand such aspects of cell activity, researchers have pursued detailed structural analysis of this class of proteins. Because of the complexities in isolating and studying membrane proteins in their native environment, detergents are often employed as a membrane mimetic media. This thesis examines several features of transmembrane (TM) protein structure and folding in detergents through which we are able to gain insights into membrane protein folding, as well as explore the suitability of detergents as membrane-mimetic environments. We first compare the helix-helix association of a series of model TM sequences in a native bilayer to the corresponding association in a detergent environment. We find that while various classes of helix-helix interaction motifs are preserved in detergents, alterations in detergent solvation may, in turn, lead to altered association affinity. We further explore this phenomenon through investigation of the consequences of the insertion of a strongly polar residue into a TM segment. In these studies we find a correlation between sequence-dependent alterations in detergent solvation and predicted in vivo folding. We also extend such analyses to a variety of detergents and native TM segments, finding that native secondary structure, as it occurs in the context of a full-length protein, is generally well preserved in a variety of detergents. Finally, we assess the determinants of membrane protein folding using two-transmembrane segment constructs, in the process optimizing expression, production and characterization techniques for a diverse range of transmembrane protein sequences. Overall this thesis finds that, detergents are capable of solubilizing membrane proteins in a form suitable for in-depth structural characterization that may not be feasible in other environments. Thus, as an approximation of a native membrane, detergents are able to preserve certain features of membrane proteins such as helix-helix association and native secondary structure.

L’obtention de la structure tridimensionnelle des protéines membranaires (PMs) est cruciale pour la chimie médicinale et la biochimie. Afin de maintenir les PMs en solution, et donc d’éviter leur agrégation, pour les études structurales et fonctionnelles, l’utilisation de détergents est indispensable. Cependant, la conformation des PMs en complexe avec les détergents pourrait être très différente de celle dans la membrane. Nous avons synthétisé une nouvelle famille de détergents, dans lesquels des carboxylates incorporés pourraient générer un réseau de ponts salins avec les acides aminés basiques des PMs, entraînant des interactions plus étroites qui pourraient préserver leur structure native. Les détergents dans lesquels la glycoconjugation a été réalisée par la réaction de cycloaddition d'azide-alkyne (CuAAC) catalysée par le Cu (I) ont montré une capacité remarquable à extraire, à stabiliser et à cristalliser les PMs étudiées. D'autre part, de nouveaux inhibiteurs efficaces d’ABCC2, une PM non cristallisée à ce jour, ont été identifiés par criblage : les 2-indolylméthylènebenzofuranones. De tels composés pourraient être considérés comme des outils pour étudier le rôle d’ABCC2 dans la pharmacocinétique des médicaments. / Understanding of tridimensional structure of membrane proteins (MPs) is crucial in medicinal chemistry and biochemistry. To maintain them in solution and consequently to prevent them from aggregation for structural and functional studies, utilization of detergents is indispensable. However, the conformation of MPs in complex with detergents could be very different from its membrane-embedded one. We synthetized new family of detergents, in which the incorporated carboxylates could generate a network of salt-bridges with basic-residue-enriched region of MPs, confer tighter interactions which could preserve their structural integrity. The detergents in which the glycoconjugation was achieved by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction showed remarkable capacity to extract, stabilize and crystallize studied PMs. On the other hand, novel inhibitors of ABCC2, a non-crystallized MP to date, were identified by screening: the 2-indolylmethylenebenzofuranones. Such compounds could be considered as useful tools to further investigate the role of ABCC2 in the pharmacokinetics of drugs.

Orientador: Eneida de Paula / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T15:46:25Z (GMT). No. of bitstreams: 1 Paula_Eneidade_D.pdf: 7752367 bytes, checksum: 4ac3295742d441a15e315a3d1c9c2c3d (MD5) Previous issue date: 2009 / Resumo: Detergentes constituem uma das ferramentas mais importantes no estudo de membranas biológicas. A eficiência de um detergente em solubilizar biomembranas e suas proteínas depende de suas propriedades físico-químicas e a solubilização parcial pode resultar em membranas resistentes a detergentes (DRMs). A resistência a detergentes dessas frações membranares constituídas principalmente de colesterol, esfingolipídios e proteínas específicas foi analisada nesse trabalho, usando membranas de eritrócitos humanos. Embora não haja evidências experimentais suficientes pra afirmar que DRMs correspondam aos microdomínios naturais existentes em biomembranas, conhecidos como lipid rafts e que também são enriquecidos em colesterol e esfingolipídios, DRMs são bons modelos para o estudo daqueles. A obtenção de DRMs com uso do detergente octaetilenoglicol mono lauril éter (C12E8) a baixa temperatura (4oC) é descrita pela primeira vez. Os detergentes zwiteriônicos ASB-14, ASB-16 e CHAPS também foram testados, mas falharam no isolamento de DRMs com baixa densidade. DRMs obtidas com C12E8 e com Triton X-100 apresentaram um aumento da razão colesterol/proteína de pelo menos três vezes em relação à membrana original. A proteína flotilina-2, considerada um marcador de lipid rafts, foi detectada em DRMs isoladas com Triton X-100 e em DRMs com C12E8 isoladas a partir de células depletadas de colesterol. Proteínas do citoesqueleto também foram encontradas em DRMs, exceto quando as células foram previamente depletadas de colesterol. Resultados de ressonância paramagnética eletrônica com uso de marcadores de spin do tipo doxil-estearato revelaram maior grau de organização das cadeias acila dos lipídios de DRMs em relação à membrana original, independentemente do detergente utilizado. Nossos resultados também mostraram que DRMs de eritrócitos podem ser também obtidos na temperatura fisiológica (37°C) como mesmo conteúdo de colesterol presente em DRMs isoladas a 4°C. A necessidade do uso de carbonato de sódio no protocolo para obtenção de DRMs sugere fortemente a existência de uma associação eletrostática entre DRMs e o citoesqueleto eritrocitário. / Abstract: Detergents constitute an important tool for the study of cell membranes. The solubilization efficiency of a specific detergent depends upon its physicochemical properties so that detergent-resistant membranes (DRMs) can be obtained as a result of the partial solubilization of biological membranes. In this study DRMs which are structures enriched in cholesterol, sphingolipids and specific proteins, were isolated and characterized from human erythrocyte membranes. Although there are no evidences to support that DRMs correspond to the natural existing microdomains of natural membranes, known as lipid rafts which are also cholesterol and sphingolipid-enriched structures, DRMs are good models for the study of rafts. DRMs from erythrocytes obtained with 8 polyoxyethylene lauryl ether (C12E8) at low temperature (4oC) are described here for the first time. The zwitterionic detergents ASB-14, ASB-16 and CHAPS failed to isolate these low buoyant density fractions. Triton X-100 and C12E8 DRMs presented a cholesterol/protein mass ratio 3 times higher than in the whole membrane. Flotillin-2, a marker of lipid rafts, was confined within the DRM fractions obtained with Triton X-100 and it was partially associated with C12E8 DRMs when erythrocyte cells were previously cholesterol-depleted. Association of membrane-skeleton proteins with DRMs was also observed, except when DRMs were prepared from cholesterol-depleted cells. Results of electron paramagnetic resonance through the use of doxyl stearate spin labels revealed that DRMs are highly ordered structures in respect to the original membrane and that their acyl chain packing is not different if prepared with either Triton X-100 or C12E8. We have also found that DRMs from erythrocytes can be isolated at physiological temperature (37°C), presenting the same cholesterol content as in DRMs prepared at 4°C. The fact that we were only able to prepare DRMs when sodium carbonate was used stronglysuggests the existence of an electrostatic association between DRMs and the membrane-skeleton. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular