This dissertation describes the analysis of the carbohydrate portion of glycosphingolipids from colorectal carcinoma cells, SW1116, by metabolically labelling the cells with radioactive monosaccharide precursors. SW1116 cells (1 x 10⁶) metabolically labelled with 222 μCi/ml of either 6-[³H]-D-galactose (25 Ci/mMol) or 6-[³H]-D-glucosamine (38 Ci/mMol) for 30 hours, incorporated 1%-3% of the radioactivity into their glycoconjugates. Approximately 63% of the radioactivity recovered in the glycoconjugates corresponded to glycolipids when cells were labelled with 6-[³H]-D-galactose, and about 12% when cells were radiolabelled with 6- [³H]-D-glucosamine. Metabolically radiolabelled glycolipids were separated into neutral (88-91% of the radioactivity recovered in glycolipids) and acidic (9-12% of the radioactivity in glycolipids) fractions by ion exchange chromatography. Glycolipids in these fractions were subjected to ozonolysis and alkali fragmentation to release the oligosaccharide chains from the ceramide portion. Oligosaccharides obtained from the neutral glycolipids were separated into single components by a combination of high performance liquid chromatography (HPLC) and Ricinus communis agglutinin I (RCA-I)-agarose affinity chromatography. Oligosaccharides were identified based on the monosaccharide composition, methylation analysis, and exoglycosidase digestions. Major glycolipid components present in the neutral fraction were, glucosylceramide (Glc-Cer), galactosylceramide(Gal-Cer), galabiosylceramide (Galαl-4Gal-Cer), lacto-N-tetraosylceramide (Galβ1-3GIcNAcβ1-3Galβ1-4Glc-Cer), Le^a- pentaglycosylceramide (Galβ1-3[Fucal-4]GlcNAcβ1-3Galβ1-4Glc-Cer), HIpentaglycosylceramide (Fucal-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Cer), a difucosylated lacto-N-tetraosylceramide, and a fucosylated lacto-Nnorhexaglycosylceramide. Minor components detected in this fraction corresponded to lactosylceramide (Galfp1-4Glc-Cer), lacto-Nneotetraosylceramide (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Cer), and fucosylated and difucosylated lacto-N-neotetraosylceramides. The acidic fraction was separated into monosialylgangliosides and _ disialylgangliosides by ion exchange chromatography. Monosialyloligosaccharides were further purified on HPLC, and biochemically characterized by methylation analysis, exoglycosidase digestions, and monosaccharide composition. The major component of this fraction corresponded to the sialyl-Le^a glycolipid (NeuAcα2-3Galβ1-3[Fucαl-4]GlcNAcβ1-3Galβ1-4Glc-Cer) as previously reported by Magnani et al. [183]. GM3 (NeuAcα2-3Galβ1-4Glc-Cer) (0.42% of radioactivity recovered in glycolipids), sialyltetraosylceramide a (NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Cer) (0.46% of radioactivity in glycolipids), sialyltetraosylceramide b (Galβ1-3[NeuAcα2-6]GIcNAcβ1-3Galβ1- 4Glc-Cer) (0.21% of radioactivity in glycolipids), and sialyllated fucosylhexaglycosylceramide, were present in minor quantities. Results from this study demonstrate that metabolic radiolabelling provides a method for the structural analysis of glycolipids, as sensitive as the immunostaining procedures, as unmistakable as physical techniques (Mass Spectrometry, and Nuclear Magnetic Resonance), and that permits the identification of the majority of glycolipids expressed by a cell line, using relatively small number of cells in culture (6 x 10⁶). Application of this method could be extended to the study of changes in glycolipid accompanying oncogenic transformation and differentiation, glycolipid biosynthesis, intracellular sorting of glycolipids, recycling and turnover. / Ph. D.

La glycochimie est un domaine qui prend de plus en plus d'expansion en chimie bioorganique et médicinale. Les glucides, étant la classe de biomolécules la plus répandue, sont des composants de plusieurs molécules bioactives naturelles et synthétiques. Ils présentent notamment un intérêt dans l'industrie pharmaceutique, puisqu'ils peuvent améliorer différentes propriétés pharmacocinétiques de certains composés. À ce jour, plus de 170 médicaments approuvés par les différentes organisations de la santé possèdent une unité glucidique dans leur structure. Ces molécules actives biologiquement ont une grande variété d'applications comme agents thérapeutiques : elles peuvent notamment être utilisées comme antibiotique ou encore comme médicament contre plusieurs types de cancers. Parmi ceux-ci, l'étoposide est un dérivé glycosylé semi-synthétique de la podophyllotoxine, un composé naturel isolé des espèces Podophyllum, et est employé pour le traitement de divers cancers. L'utilisation de ces deux molécules comme agents thérapeutiques est compromise par différents facteurs comme leur toxicité, les effets secondaires engendrés et leur faible solubilité dans l'eau. C'est pour ces raisons que plusieurs groupes de recherche travaillent sur le développement de nouveaux analogues du produit naturel qui seraient potentiellement moins toxiques, plus hydrophiles et dont l'activité biologique est améliorée, ou, du moins, non affectée. Ce mémoire présente la synthèse d'un dérivé de la glucosamine comportant un acide carboxylique et une amine dans sa structure. À partir de cette molécule, une nouvelle méthodologie synthétique d'oligosaccharides linéaires portant un groupement alcyne a été développée. Ces derniers ont pu être liés à la podophyllotoxine grâce à une réaction dite réaction click afin de générer de nouveaux dérivés glycoconjugués de la toxine. Les divers analogues obtenus diffèrent par le groupement retrouvé à la position C-2 du glucide terminal. Une voie de synthèse d'oligosaccharides utilisant les principes de couplage peptidique a donc été développée afin d'accéder à une librairie de nouveaux composés potentiellement actifs biologiquement. / There is a growing interest for the use of glycochemistry in the bioorganic and medicinal chemistry fields. Being the most abundant class of biomolecules, carbohydrates are components of various natural and synthetic bioactive products. They are of particular interest in the pharmaceutical industry since they can improve various pharmacokinetic properties of certain compounds. Nowadays, more than 170 commercially available drugs contain a carbohydrate unit in their structure. These biologically active molecules present a wide range of applications as therapeutic agents: they can be used as antibiotics or even as anticancer drugs. Among these, etoposide is a semi-synthetic glycosylated derivative of podophyllotoxin, a naturally occurring product in Podophyllum species, used as treatment of various cancers. The use of etoposide and podophyllotoxin as therapeutic agents is compromised by various problematics such as high toxicity, undesirable side effects and low water solubility. For these reasons, research groups are working on the development of new podophyllotoxin analogues that would potentially be less toxic, more hydrophilic and whose biological activity is either improved, or not affected. This thesis presents the synthesis of a glucosamine derivative bearing both a carboxyl and an amine group in its structure. From this molecule, a new synthetic methodology to access linear oligosaccharides carrying an alkyne group has been developed. These glycomolecules could be linked to podophyllotoxin by click reaction to generate new glycoconjugate derivatives of podophyllotoxin. Each analogue differs by the group found at the C-2 position on the terminal carbohydrate. Therefore, a new synthetic approach of oligosaccharides using the principles of peptide coupling has been developed to access a library of new potentially biologically active compounds.

Submitted in partial fulfillment for the Degree of Master of Applied Sciences in Biotechnology, Durban University of Technology, Durban, South Africa, 2016. / Prebiotics are defined as non-digestible oligosaccharides (NDOs) or polysaccharides (NDPs), which promote the growth of beneficial lactic acid bacteria in the colon. Algae are rich in polysaccharides and can be exploited as prebiotics for functional food ingredients to improve human and animal health. Currently, inulin is the most widely used ingredient in the prebiotics market, which is produced from live plants and requires expensive production processing. There is a vast repository of marine life with algae as a major source of nutrients. Therefore, this study provides an alternative source for prebiotic production and examines marine and freshwater algae that promote the growth of two strains of Lactobacillus delbrueckii subs. (Lactobacillus lactis and Lactobacillus bulgaricus) and one strain of Bifidobacterium spp. (Bifidobacterium longum). Monosaccharides of the oligosaccharide fraction of marine and freshwater algal extracts were investigated with the use of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) after acidic hydrolysis of cell matrix polysaccharides. A total of fifty-five marine and freshwater aqueous algal extracts were assessed for their effect on the growth of L. lactis, B. longum and L. bulgaricus over a 96 hour period. Relative to the negative control, 34.5% algal extracts showed improved growth on one or more probiotic bacteria. The optimum time for maximum bacterial growth was noted at 48 h for all the tested aqueous algal extracts. Five marine and freshwater algal cultures (Spirulina platensis, Chlorococcum spp., Dunaliella salina, Scenedesmus magnus, Chlorella spp. and algal extract no. 48) from various aquatic environments in Kwa-Zulu Natal showed the best growth dynamics and demonstrated the greatest potential as sources of biomass for prebiotic production. These algal extracts were able to significantly increase the growth of at least one of the three probiotic bacteria (p < 0.05). Aqueous algal extract from S. platensis was regarded as the best algal source for prebiotics as it demonstrated a greater stimulatory effect on the growth of all three probiotic bacteria (L. lactis, B. longum and L. bulgaricus) compared to tested aqueous algal extracts and the inulin used as a positive control. The results obtained by HPLC for characterization confirmed TLC data, as xylose and galactose were detected by both chromatograms. These data indicated that xylose and galactose were present in aqueous algal extracts from S. magnus and S. platensis and galactose in aqueous algal extract no. 48. Xylose was most abundant in aqueous algal extracts from S. platensis (3mg/ml) and S. magnus (2.3mg/ml). In conclusion aqueous algal extracts from S. platensis, Chlorococcum, D. salina, S. magnus, Chlorella and algal extract no. 48 are potential sources for prebiotic production. Spirulina platensis extract was regarded as the best algal source. Xyose and galactose characterized by HPLC in algal extracts make up oligosaccharides that function as prebiotic compounds for stimulation of probiotic bacteria. There is a great scope for successful production of prebiotics from algal sources in South Africa. / M

Title from title page of PDF (University of Missouri--St. Louis, viewed Mar. 2, 2010). Includes bibliographical references.

Thesis (Academic dissertation)--University of Helsinki, 1998. / Includes bibliographical references. Also available on the World Wide Web.

Résumé français confidentiel / Résumé anglais confidentiel

Phloem transport is along hydrostatic pressure gradients generated by differences in solute concentration between source and sink tissues. Numerous species accumulate raffinose-family oligosaccharides (RFOs) in the phloem of mature leaves to accentuate the pressure gradient between source and sinks. In this study, metabolic engineering was used to generate RFOs at the inception of the translocation stream of Arabidopsis thaliana, which transports predominantly sucrose. To do this, three genes, GALACTINOL SYNTHASE, RAFFINOSE SYNTHASE and STACHYOSE SYNTHASE, were expressed from promoters specific to the companion cells of minor veins. Two transgenic lines homozygous for all three genes (GRS63 and GRS47) were selected for further analysis. Sugars were extracted and quantified by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and 21-day old plants of both lines had levels of galactinol, raffinose, and stachyose approaching 50% of total soluble sugar. All three exotic sugars were also identified in phloem exudates from excised leaves of transgenic plants whereas levels were negligible in exudates from wild type leaves. Differences in starch accumulation or degradation between wild type and GRS63 and GRS47 lines were not observed. Similarly, there were no differences in vegetative growth between wild type and engineered plants, but engineered plants flowered earlier. Finally, since the sugar composition of the phloem translocation stream is altered in these plants, we tested for aphid feeding. When green peach aphids were given a choice between WT and transgenic plants, WT plants were preferred. When aphids were reared on only WT or only transgenic plants, aphid fecundity was reduced on the transgenic plants. When aphids were fed on artificial media with and without RFOs, aphid reproduction did not show differences, suggesting the aphid resistance is not a direct effect of the exotic sugars.

No description available.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The raffinose family of oligosaccharides (RFOs) are α1,6-galactosyl extensions of sucrose (Suc-Galn) unique to the plant kingdom. Their biosynthesis is mediated via α1,6-galactosyltransferases which catalyse the formation of raffinose (Raf, Suc-Gal1), stachyose (Sta, Suc-Gal2) and higher oligomers (Suc-Galn, n ≥ 13) in a stepwise manner. RFOs are well known for their historical roles as phloem translocates and general carbon storage reserves. In recent years their physiological roles have expanded to include potential functions in global plant stress-responses, where correlative mass increases are associated with abiotic stresses such as desiccation, salinity and low temperatures and, to a lesser extent biotic stress (pathogen infection). This study focused on (i) the functional characterisation of a putatively annotated stachyose synthase from Arabidopsis seeds (RS4, At4g01970), (ii) dissection of the proposed functional role of the RFO precursor galactinol in biotic stress tolerance using the Arabidopsis/Botrytis cinerea pathosystem and, (iii) an attempt to engineer long-chain RFOs into Arabidopsis by constitutive over-expression of the unique RFO chain elongation enzyme galactan:galactan galactosyltransferase (ArGGT) from Ajuga reptans. In Arabidopsis Raf is the only RFO known to accumulate in leaves, strictly during conditions of abiotic stress. However, seeds accumulate substantial amounts of both Raf and Sta. While RFO physiology in Arabidopsis leaves and roots is quite well characterised, little is known about the RFO physiology in the seeds. Apart from a single enzyme being described to partially contribute to seed Raf accumulation (RS5, At5g40390), no other RFO biosynthetic genes are known. In this work we functionally characterised an α1,6-galactosyltransferase putatively annotated as a stachyose synthase (RS4, At4g01970) in the Arabidopsis database. Using two insertion mutants (atrs4-1 and 4-2) we demonstrated Sta deficiency in mature seeds. A double mutant with the recently characterised RS5, shown to partially be responsible for Raf accumulation in mature seeds was completely deficient in seed RFOs. This provided the first hint that RS4 could potentially also be involved in Raf biosynthesis. Seed specific expression of RS4 was deregulated by constitutive over-expression in wild-type (Col-0) and the atrs5 mutant background (RS and Raf deficient). Both Raf and Sta unusually accumulated in Col-0 leaves over-expressing RS4, under normal growth conditions. Further, leaf crude extracts from atrs5 insertion mutants (RS and Raf deficient) over-expressing RS4 showed enzyme activities for both RS and SS, in vitro. Collectively our findings have physiologically characterised RS4 as a RFO synthase responsible for Sta and, partially Raf (along with RS5) accumulation during Arabidopsis seed development. The galactosyl donor in RFO biosynthesis, galactinol (Gol) has recently been implicated in biotic stress signalling (pathogen response) in cucumber, tobacco and Arabidopsis. Those studies focused exclusively on Gol in their experimental approaches using both over-expression (tobacco, Arabidopsis) and loss-of-function (Arabidopsis) strategies. However, they did not address the invariable accumulation of Raf that is routinely obtained from such over-expression strategies. We therefore investigated if Raf could play a functional role in induced systemic resistance (ISR), a well-studied mechanism employed by plants to combat necrotrophic pathogens such as Botrytis cinerea. To this end we looked to the RS5 mutant backgrounds (Raf deficient but Gol hyper-accumulating) reasoning that the Gol accumulating mutants should be resistant to B. cinerea (as previously described for transgenic over-expression of GolS1 isoforms in tobacco and Arabidopsis). Such findings would then preclude a role for Raf, since the system would be Raf deficient. Surprisingly, two independent T-DNA insertion mutants for RS5 (atrs5-1 and 5-2) were equally hypersensitive to B. cinerea infection as two independent T-DNA insertion mutants for GolS1 (atgols1-1 and 1-2). The hyper-sensitivity of the GolS1 mutant background has previously been demonstrated. The RS5 mutant backgrounds accumulate substantial amounts of Gol, comparable to those reported for transgenic plants (tobacco and Arabidopsis) where pathogen resistance was reported. Further, during the course of our investigations we discovered that both AtGolS1 mutants also accumulated substantial amounts of both Gol and Raf under normal growing conditions. This was not reported in previous studies. Collectively our findings argue against a role for either Gol or Raf being responsible for the induction/signalling of ISR. However, we do not preclude that the RFO pathway is somehow involved, given the previous reports citing pathogen resistance when GolS1 genes are over-expressed. We are further investigating a potential role for the GolS transcript and/or protein being the component of the suggested signalling function in ISR. The unique enzyme from A. reptans (galactan:galactan galactosyltransferase, ArGGT) is able to catalyse the formation of higher oligomers in the RFO pathway without the use of Gol as a galactosyl donor but rather, using RFOs themselves as galactose donors and acceptors (Gol-independent biosynthesis). We constitutively over-expressed ArGGT in Arabidopsis as a way to engineer long-chain RFO accumulation to further dissect a role for them in improving freezing tolerance. To this end we have been unsuccessful in obtaining RFOs higher than Sta (which occurred in extremely low abundance) in the leaves. Since ArGGT would appear to show substrate preference for Sta, and Arabidopsis seeds accumulate substantial quantities of Sta, we further analysed the seed water soluble carbohydrate (WSC) profiles of three independent transgenic lines but detected no additional RFO oligomers beyond the normally accumulating Raf and Sta. We suggest further strategies to improve this approach (Chapter 4). Collectively this work represents case studies of RFOs in seed physiology, their abilities/requirement in biotic stress and the use of unique enzymes to engineer long-chain RFO accumulation using the Arabidopsis model. At the time of submission of this dissertation the following contributions have been made to the general scientific community: (i) Presentation of chapter 2 at the 26th International Conference for Arabidopsis Research (26th ICAR, 2015, Paris, France) and, (ii) Submission of chapter 2 as a manuscript presently under peer review for possible publication in Plant and Cell Physiology. / AFRIKAANSE OPSOMMING: Die raffinose familie van oligosakkariede (RFO) is α1,6-galactosyl uitbreidings van sukrose (Suc-Galn) uniek aan die plante koningryk. Hul biosintese word bemiddel deur α1,6-galactosyltransferases wat in 'n stapsgewyse manier die vorming van raffinose (Raf, Suc-Gal1), stachyose (Sta, Suc-Gal2) en hoër oligomere (Suc-Galn, n ≥ 13) kataliseer. RFOs is bekend vir hul historiese rol as floëem translokate en algemene koolstof reserwes. Meer onlangs was hul fisiologiese rolle uitgebrei om potensiële funksies te vervul in globale plant stres-reaksies, waar korrelatiewe massa toenames geassosieer word met abiotiese stresfaktore soos uitdroging, soutgehalte en lae temperature en tot 'n mindere mate biotiese stres (patogeen infeksie). Hierdie studie fokus op (i) die funksionele karakterisering van 'n tentatief ge-annoteerde stachyose sintase van Arabidopsis sade (RS4, At4g01970), (ii) disseksie van die voorgestelde funksionele rol van die RFO voorloper galactinol in biotiese stres verdraagsaamheid, met behulp van die Arabidopsis/Botrytis cinerea patogeen sisteem en (iii) 'n poging om 'n lang-ketting RFOs in Arabidopsis te inisieer deur konstitutiewe oor-uitdrukking van die unieke RFO ketting-verlengings ensiem galactan:galactan galactosyltransferase (ArGGT) afkomstig van Ajuga reptans. In Arabidopsis is Raf die enigste RFO bekend daarvoor om te versamel in die blare, ekslusief tydens toestande van abiotiese stres. Maar, sade versamel aansienlike konsentrasies van beide Raf en Sta. Terwyl RFO fisiologie in Arabidopsis (blare en wortels) baie goed gekenmerk is, is min bekend oor die RFO fisiologie in die saad. Afgesien van 'n enkele ensiem wat beskryf word om gedeeltelik by te dra tot Raf versameling (RS5, At5g40390), is geen ander RFO biosintetiese gene bekend in saad nie. In hierdie werk beskryf ons die funksionele karakterisering van ‘n α1,6-galactosyltransferase wat tenetatief ge-annoteer word as 'n stachyose sintase (RS4, At4g01970) in die Arabidopsis databasis. Met die gebruik van twee invoegings mutante (atrs4-1 en 4-2) het ons die verlies van Sta in volwasse sade gedemonstreer. RFOs was heeltemal absent in sade van 'n dubbele mutant met die onlangs gekarakteriseerde RS5 (verantwoordelik vir gedeeltelike Raf versameling in volwasse sade). Dit het die eerste aanduiding daargestel dat RS4 potensieel ook betrokke kan wees in Raf biosintese. Saad-spesifieke uitdrukking van RS4 was gedereguleer deur konstitutiewe oor-uitdrukking in wilde-tipe (Col-0) en die atrs5 mutant agtergrond (RS en Raf gebrekkig). Oor-uitdrukking van RS4 in Col-0 blare het gelei tot beide buitengewone Raf en Sta konsentrasies, onder normale groeitoestande. Verder, oor-uitdrukkingvan RS4 in atrs5 invoeg mutante (waar beide RS en Raf absent is) het in vitro ensiemaktiwiteite vir beide RS en SS getoon. Gesamentlik beskryf ons bevindinge die fisiologies karakterisering van RS4 as 'n RFO sintase, verantwoordelik vir Sta en gedeeltelik Raf (saam met RS5) sintese tydens Arabidopsis saad ontwikkeling. Die galactosyl skenker in RFO biosintese, galactinol (Gol), was onlangs beskryf om ‘n rol te speel in biotiese stres (patogeen reaksie) in komkommer, tabak en Arabidopsis. Daardie studies het uitsluitlik gefokus op Gol in hul eksperimentele benaderings deur die gebruik van beide oor-uitdrukking (tabak, Arabidopsis) en die verlies-van-funksie (Arabidopsis) strategieë. Maar hulle het nie die onveranderlike opeenhoping van Raf, wat gereeld verky word uit sulke oor-uitdrukking strategieë, aangespreek nie. Ons het dus ondersoek of daar 'n funksionele rol vir Raf in geïnduseerde sistemiese weerstand (ISR) kan wees. ISR is 'n goed-bestudeerde meganisme wat deur plante ge-implementeer word om nekrotrofiese patogene soos Botrytis cinerea te beveg. Vir hierdie doel het ons gekyk na die RS5 mutant agtergronde (absent in Raf, maar hiper-akkumulasie van Gol) met die redenasie dat die Gol akkumulerende mutante weerstandbiedig teen B. cinerea moet wees (soos voorheen beskryf vir transgeniese oor-uitdrukking van GolS1 in tabak en Arabidopsis). Sulke bevindings verhinder dan 'n rol vir Raf, aangesien die stelsel geen Raf akkumuleer nie. Verbasend, twee onafhanklike T-DNA invoeg mutante vir RS5 (atrs5-1 en 5-2) was ewe hiper-sensitief vir B. cinerea infeksie as twee onafhanklike T-DNA invoeg mutante vir GolS1 (atgols1-1 en 1-2). Die hiper-sensitiwiteit van die GolS1 mutant agtergrond was reeds voorheen gedemonstreer. Die RS5 mutant agtergronde versamel aansienlike konsentrasies van Gol, vergelykbaar met dié berig vir transgeniese plante (tabak en Arabidopsis) waar patogeen-weerstandbiedigheid aangemeld is. Verder, in die loop van ons ondersoeke het ons ontdek dat beide AtGolS1 mutante ook aansienlike konsentrasies van beide Gol en Raf onder normale groei-toestande akkumuleer. Dit was nie aangemeld in die vorige studies nie. Gesamentlik argumenteer ons bevindinge teen 'n rol vir óf Gol, of Raf, tydens die induksie van ISR. Alhoewel, ons elimineer nie ‘n rol vir die RFO padweg nie, gegewe dat oor-uitdrukking van GolS1 gene tydens patogeen-weerstandbiedigheid in vorige verslae verwysig was. Ons ondersoek verder 'n moontlike rol vir die aanwesigheid van die GolS transkrip en/of proteïen as ‘n moontlike komponent van die voorgestelde funksie in ISR. Die unieke ensiem van A. reptans (galactan:galactan galactosyltransferase, ArGGT) is in staat om die vorming van hoër oligomere in die RFO pad te kataliseer sonder die gebruik van Gol as 'n skenker galactosyl, maar eerder, met behulp van die RFO's hulself as galaktose skenkers en aanvaarders (Gol-onafhanklike biosintese). Ons het ArGGT konstitutief ooruitgedruk in Arabidopsis as 'n manier om 'n lang-ketting RFO akkumulasie daar te stel met die doel om 'n rol vir hulle in die verbetering van vriestoleransie verder te ontleed. Ons was tot dusver onsuksesvol in die verkryging van RFOs hoër as Sta in die blare (wat akkumuleer het in 'n baie lae konsentrasie). Sedert ArGGT ‘n affiniteit vir Sta as substraat toon, en Arabidopsis sade versamel aansienlike hoeveelhede Sta, het ons verder die saad water oplosbare koolhidraat (WSC) profiele van drie onafhanklike transgeniese lyne ontleed, maar bespeur geen bykomende RFO oligomere buite die normale Raf en Sta konsentrasie nie. Ons stel verdere strategieë voor om hierdie benadering (Hoofstuk 4) te verbeter. Gesamentlik verteenwoordig hierdie werk gevallestudies van RFOs in saadfisiologie, hul vermoëns/vereiste in biotiese stres en die gebruik van unieke ensieme om lang-ketting RFO akkumulasie daar te stel met behulp van die Arabidopsis model. Teen die tyd van die indiening van hierdie tesis was die volgende bydraes gemaak aan die algemene wetenskaplike gemeenskap: (i) Aanbieding van hoofstuk 2 op die 26ste Internasionale Konferensie vir Arabidopsis Navorsing (26ste ICAR, 2015, Parys, Frankryk), en (ii) indiening van hoofstuk 2 as 'n manuskrip tans onder nasiening vir moontlike publikasie in die joernaal ‘Plant and Cell Physiology’.

The possibilities of differentiating linkage positions and anomeric configurations of small oligosaccharides by negative ion mode MALDI using anion attachment followed by PSD are investigated. By careful initial adjustment of the focusing mirror ratios allowing acquisition of the peaks of interest within the same PSD segment, it is possible to obtain highly reproducible relative ion abundances. Discrimination of different linkage types is achieved by analysis of structurally-informative diagnostic peaks offered by PSD spectra of chloride adducts of oligosaccharides, whereas the relative peak intensities of selected diagnostic fragment pairs make differentiation of anomeric configuration possible. F- and Ac- cannot form anionic adducts with the oligosaccharides in significant yields. However, Br-, I- and NO3- anionic adducts consistently appear in higher abundances relative to [M - H]-, just like Cl-. Mildly acidic saccharides form both deprotonated molecules and anionic adducts, making it possible to simultaneously detect neutral and acidic oligosaccharides via anion attachment. PSD of [oligosaccharide + Cl]- yields structurally-informative fragment ions that retain the charge on the sugar molecule rather than solely forming Cl-, whereas PSD of Br-, I- and NO3- adducts of oligosaccharides yield the respective anions as the main product ions without offering structural information concerning the sugar. PSD of the chloride adduct of saccharides containing 1-2 linkages also yields chlorine-containing fragment ions. MALDI-TOF-MS and LDI-TOF-MS are shown to be useful for characterization of ultra-small titania nanoparticles. Peak maxima in MALDI-TOF mass spectra are found to correlate with nanoparticle size. The size distributions of TiO2 nanoparticles, obtained from MALDI- and LDI-TOF-MS are in good agreement with parallel TEM observations. PSD analysis of inorganic x nanomaterials is performed and valuable information about the structure of analytes has been obtained. A group of inorganic nitrate and perchlorate salts of forensic and health interest are investigated by LDI- and MALDI-TOF MS. In each case, a series of characteristic cluster ions are predominant in the negative-ion mode. The number and identity of metal atoms and anions in the recorded cluster ions can be positively identified by their m/z values, distinctive isotopic patterns and characteristic PSD fragmentation patterns.