No description available.

Introduction: The focus of this thesis is set on how green innovation can be facilitated through supplier relationships. Early supplier integration (ESI) can be defined as a collaboration between a purchasing firm and its supplier at an early stage of the product conceptualization and green innovation can be defined as the implementation of several management activities aimed at reducing environmental impact. Throughout the years researchers have examined the advantages and impact of green innovation, however, how manufacturing companies can manage early supplier integration to foster green innovation has not been explicitly studied. Purpose: The purpose of this research is to explore how early supplier integration can foster green innovation between manufacturer and supplier. Furthermore, this research proposes a conceptual model that addresses how early supplier integration can foster green innovation. Research Questions: Which are the current corporate practices for green innovation related to supplier relationships? What are the main prerequisites for green innovation in ESI? How can manufacturers manage their early supplier integration to foster green innovation?  Methodology: Based on the purpose and research questions in this thesis the authors deemed a qualitative research approach to be the most applicable research design. To gather in-depth data for the thesis, semi-structured interviews were chosen as the most appropriate data collection method. Respondents that were included based on their extensive knowledge regarding internal green integration, supplier selection, early supplier integration, co-development, and green innovation. Conclusion: This study provides knowledge regarding how early supplier integration can foster green innovation between manufacturer and supplier. It can be concluded that in order for early supplier integration to actively foster green innovation it is necessary to consider a holistic perspective by adapting four chronological concepts that are managed in the order: internal green integration, supplier selection, early supplier integration, and co-development.

No / This study shows that prostaglandins in human FM55 melanoma cells and epidermal melanocytes are produced by COX-1. Prostaglandin production in FM55 melanoma cells was unrelated to that of melanin suggesting that the two processes can occur independently. ¿-Melanocyte stimulating hormone (¿-MSH), which had no effect on melanin production in FM55 cells, stimulated PGD2 production in these cells without affecting PGE2. While cAMP pathways may be involved in regulating PGD2 production, our results suggest that ¿-MSH acts independently of cAMP, possibly by regulating the activity of lipocalin-type PGD synthase. This ¿-MSH-mediated effect may be associated with its role as an immune modulator. / The Wellcome Trust

No / The Flinders Sensitive Line (FSL) rat is a genetic animal model of depression. Following recent findings that the brain fatty acid composition of FSL is characterised by increased arachidonic acid (AA), we used electrospray tandem mass spectrometry and 1H-NMR to examine lipid species in different brain areas. Cholesterol and sphingolipids were increased in the hypothalamus of the FSL rats. Furthermore, arachidonic acid-containing phosphatidylcholine species (AA-PC) were elevated with PC16:0/20:4, PC18:1/20:4 and PC18:0/20:4 (p<0.003) increased in the hypothalamus and striatum. In contrast, there was a decrease in some docosahexaenoic acid (DHA)-containing species, specifically PC18:1/22:6 (p<0.003) in the striatum and PE18:1/22:6 (p<0.004) in the prefrontal cortex. Since no significant differences were observed in the erythrocyte fatty acid concentrations, dietary or environmental causes for these observations are unlikely. The increase in AA-PC species which in this animal model may be associated with altered neuropathy target esterase activity, an enzyme involved in membrane PC homeostasis, may contribute to the depressive phenotype of the FSL rats.

No / Prostanoids modulate the activity of human pregnant myometrium and their functional role can be appreciated through characterisation of prostanoid receptors and tissue concentration of prostanoids. We have applied a lipidomic approach to elucidate the profile of prostanoids in human non-labouring and labouring myometrium. We have identified a total of nineteen prostanoids including prostacyclin, thromboxanes, prostaglandins and dihydro-prostaglandins. Prostacyclin was the predominant prostanoid in both non-labouring and labouring myometria, with PGD2 and PGF2¿ being the second most abundant. Although the total amount of prostanoids was increased in the labouring tissue, PGE2 and 13,14-dihydro-15-keto-PGE2 were the only prostanoids to increase significantly at early and late labour (p¿0.001). Our data suggest that PGF2¿ plays an important role in parturition, whilst the increase in PGE2 could occur to facilitate cervical dilation and relaxation of the lower myometrium during labour. Although the elevation in TXA2 was less marked than expected, in terms of translation to function even a relatively small increase in the level of this potent spasmogen may have significant effects.

Chronic, noncommunicable diseases such as chronic fatigue syndrome (also known as myalgic encephalomyelitis) are rapidly becoming a worldwide epidemic that profoundly affects public health and productivity. Chronic fatigue syndrome (CFS) is characterised by severe and debilitating fatigue and although its etiology is still unknown, recent studies have found considerable evidence that mitochondrial dysfunction and oxidative stress might be responsible for the underlying energy deficit in these patients. Adenine and pyridine nucleotides could be used as potential biomarkers for energy related disorders such as chronic fatigue syndrome because of their various functions in the energy and redox pathways. The first part of this study focussed on developing a liquid chromatography electrosprayionisation tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of these nucleotides in blood samples. Due to the instability of nucleotides in biological matrices it was also necessary to find a suitable extraction method that would be able to stop enzymatic activity via protein precipitation. Out of the four extraction methods investigated during this study, deproteinisation of whole blood samples with perchloric acid produced the highest nucleotide abundances. Although nucleotide standards were found to be stable in perchloric acid, nucleotide levels in blood samples were not stabilised by addition of perchloric acid. The second part of this study consisted of measuring the nucleotide levels in blood samples of controls and possible CFS patients in order to test the proof of concept of the new LCESI- MS/MS method. Despite changes in the nucleotide levels due to perchloric acid and problems with nucleotide instability, it was still possible to distinguish between the two groups based on the results obtained with the new LC-ESI-MS/MS method. The newly developed LC-ESI-MS/MS method proved to be reliable and adequate for nucleotide quantification in whole blood samples, thus the aim of this study was achieved. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

The application of ICP-MS to the fields of proteomics and genomics has arisen in part due to its ability to detect and quantify trace levels of S and P, which are major constituents in proteins and nucleic acids respectively. The development of collision/reaction cell technology and high resolution instruments has enabled these biologically important elements to be measured and quantified at the pg - ng ml-1 level. Despite these advances, the detection limits of P and S are still inferior compared to other elements. Oligonucleotides containing biotin functionality were labelled with Au nano-particles attached to a streptavidin protein to achieve site specific labelling, with 100% labelling efficiency. Each nano-particle contained ~86 Au atoms, resulting in an 882 fold signal enhancement for 24 base length oligonucleotides. However, this enhancement factor was only observed when one oligonucleotide bound to one nano-particle in a 1:1 ratio. Much lower Au labelling efficiencies and signal enhancements were observed when thiolated oligonucleotides were labelled with maleimide functionalised gold nano-particles. This was attributed to the extensive and difficult sample preparation steps that were required prior to labelling. The detection and quantification of adducts formed between DNA and the Pt anti-cancer drugs cisplatin and oxaliplatin were also investigated with ICP-MS. Acid digestion of the carbon based DNA matrix enabled Pt adducts to be quantified at low dose rates of 1 Pt atom per 1 500 000 nucleotides in ~12 μg DNA. Such sensitive mass spectrometric determinations could be employed in clinical tests to detect and quantify low level adducts formed in patients in-vivo. To complement ICP-MS analysis, electrospray ionisation linear ion trap mass spectrometry was employed to study the interaction of oxaliplatin with the four DNA nucleobases. Multiple stage mass spectrometry enabled detailed Pt-nucleobase adduct fragmentation pathways to be established. The method of DNA detection using P in conjunction with the collision cell, or cool plasma to form PO+ was also demonstrated and the limitations of the method, namely, polyatomic interferences and severe matrix effects were highlighted.

La dégradation protéolytique du collagène de type II est considérée comme étant un facteur majeur dans le processus irréversible de dégradation de la matrice cartilagineuse lors d’ostéoarthrose. Outre les collagénases de la famille des métaloprotéinases de la matrice (MMP-1, -8, -13), la cathepsine K est parmi les seules enzymes susceptibles de dégrader la triple hélice intacte du collagène de type II, devenant ainsi un élément pertinent pour les recherches sur l’ostéoarthrose. L’objectif à court terme de notre étude consiste en l’identification et la caractérisation de sites de clivage spécifiques de la cathepsine K sur le collagène de type II équin. La technique d’électrophorèse SDS-PAGE 1D permet la visualisation des produits de digestion et la validation des résultats de la caractérisation moléculaire des fragments protéolytiques. La caractérisation est réalisée en combinant la digestion trypsique précédant l’analyse HPLC-ESI/MS. Les résultats ont permis d’établir les sites, présents sur la carte peptidique de la molécule de collagène de type II équin, des 48 résidus prolines (P) et 5 résidus lysines (K) supportant une modification post-traductionnelle. De plus, 6 fragments majeurs, différents de ceux produits par les MMPs, sont observés par SDS-PAGE 1D puis confirmés par HPLC-ESI/MS, correspondant aux sites suivants : F1 [G189-K190], F2 [G252-P253], F3 [P326-G327], F4 [P428-G429], F5 [P563-G564] et F6 [P824-G825]. Le fragment F1 nouvellement identifié suggère un site de clivage différent de l’étude antérieure sur le collagène de type II bovin et humain. L’objectif à long terme serait le développement d’anticorps spécifiques au site identifié, permettant de suivre l’activité protéolytique de la cathepsine K par immunohistochimie et ÉLISA, dans le cadre du diagnostic de l’ostéoarthrose. / The proteolytic degradation of type II collagen is believed to be mainly an irreversible event in the process of cartilage matrix degradation in osteoarthritis. Cathepsin K is the most active enzyme protease outside the matrix metalloproteinase (MMP) family (MMP 13, -8, -1) capable of degrading the intact triple helical type II collagen. The short term objective of our study was to characterize the specific cleavage sites of CK on type II collagen. Our long term goal is to develop antibodies specific to these sites to develop biomarkers to detect it’s cleavage, for the early diagnosis of OA. Thus, in order to achieve our first goal, Cathepsin K cleavage of equine type II collagen was first examined by SDS-PAGE electrophoresis. Molecular characterization of proteolytic fragments, and therefore cleavage sites, was performed using tryptic digestion followed by LC-ESI/MS analysis to establish a comprehensive peptide map which was used as a template to identify specific proteolytic cleavage by cathepsin K. Comprehensive peptide mapping provided information on post-translational modifications and permitted the identification of 48 proline (P) and 5 lysine (K) residues that were subject to post translational modification. Six major fragments were observed on 1D SDS-PAGE and confirmed by HPLC-ESI/MS including F1 [189-190], F2 [252-253], F3 [326-327], F4 [428-429], F5 [563-564] and F6 [824-825]. The observed F1 fragment showed that cleavage was three residues N-terminal to the site reported previously for bovine type II collagen. These new findings will be used to develop new analytical methods to quantify biomarkers associate to equine type II collagen degradation in osteoarthritis patient and/or to support the development of new treatments.

Fluorescent labeling is a method used for visualization of various types of biomolecules including proteins and protein complexes. However, the effect of protein labeling on protein structure and functions has not been investigated so far. The goal of the diploma thesis was to examine an influence of NHS-fluorescein binding on structure and function of human carbonic anhydrase I (hCA-I). The particular aims of this work were to prepare recombinant 15N-hCA-I which was used for NMR structure analysis of carbonic anhydrase upon fluorescent labeling. Furthermore, enzyme activity was measured in order to find out a correlation between the concentration of NHS- fluorescein and protein function. In addition, the reaction mixtures were systematically analyzed by ESI FT-ICR mass spectrometry. The analysis revealed experimental conditions for fluorescent labeling of human carbonic anhydrase I with minimal effect on protein structure and function. The results of this study show that the calculation of molar excess of NHS-fluorescein cannot rely on a simple procedure provided by manufacturer. However, due to decrease of enzyme activity upon fluorescent labeling, it is better to take into count the influence of NHS-fluorescein concentration on the relative enzymatic activity. Moreover, the calculation of molar...

Ce mémoire décrit des réactions pallado-catalysées de Heck déshydrogénantes d'hétéroarènes pouvant être dérivés de la biomasse avec des alcènes riches en électrons tels que les styrènes. Le premier chapitre, présente une méthodologie permettant le couplage croisé de Heck désydrogénant de furanes et thiophènes avec des styrènes dans des conditions douces et discute de l'influence de l'agent oxydant sur l'activité du catalyseur. Le second chapitre est consacré à l'étude mécanistique par SM-IES de réactions de Heck déshydrogénantes de furanes avec des acrylates en présence de benzoquinone comme agent oxydant et de DMSO comme solvant. La présentation d'une nouvelle méthodologie pour les réactions de Heck déshydrogénantes d'hétérocyles avec des styrènes dans des conditions aérobies et l'explication de l'effet négatif de co-oxydants métalliques représentent le dernier chapitre. / This thesis describes Pd-catalyzed dehydrogenative Heck reactions of heteroarenes that could be derived from the biomass with electron-rich alkenes such as styrenes. The first chapter presents a new methodology enabling cross coupling dehydrogenative Heck Reactions of furans and thiophenes with styrenes under Mild conditions and discusses the Influence of the oxidizing agent on the reaction rate. The second chapter focuses on ESI-MS studies of the dehydrogenative Heck reactions of furans with acrylates using benzoquinone as reoxidant and DMSO as solvent. The presentation of a new methodology for aerobic dehydrogenative Heck reactions of heterocycles with styrenes and the explanation about the negative effect of metallic co-oxidants represent the third chapter.