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Seward, Christopher M. P. "Stereoselective glycosylation chemistry." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270281.
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Rising, Thomas W. D. F. "Glycosylation using endohexosaminidases." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436989.
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Yassine, Hanane. "Caractérisation d’endocan murin : dualité fonctionnelle pro- ou anti-tumorale de l’endocan selon son statut de glycosylation. Etude des mécanismes d’action anti-tumorale." Thesis, Lille 2, 2014. http://www.theses.fr/2014LIL2S029/document.
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Une tumeur nécessite un approvisionnement en oxygène et en nutriments pour sa croissance mais aussi pour la dissémination à distance vers d’autres organes. L’angiogenèse tumorale est le phénomène exploité par la tumeur pour accomplir ses besoins. Les «Tip cells » situées à l’extrémité des capillaires en bourgeonnement initient et guident la croissance des néovaisseaux. Ces cellules sont considérées actuellement comme une cible thérapeutique pertinente pour les médicaments anti-angiogéniques. De nombreuses études ont permis d’identifier un cluster de marqueurs moléculaires exprimés de manière privilégiée au niveau des « Tip cells ». Un de ces marqueurs appelé endocan, a été identifié au laboratoire, et a fait objet du travail réalisé pendant la thèse. Endocan est un protéoglycane circulant surexprimé dans de nombreux cancers humains dont l’expression est fréquemment associée à un mauvais pronostic. Par son glycane, endocan intervient dans la croissance tumorale en augmentant l’effet des facteurs de croissance, mais aussi la migration des cellules endothéliales. Mon travail de thèse s’est orienté sur la caractérisation biochimique et fonctionnelle d’endocan murin afin d’avoir un modèle animal utile pour une meilleure compréhension de l’activité pro-tumorale d’endocan humain. Les travaux présentés dans ce manuscrit montrent qu’endocan murin est un protéoglycane de type chondroitine sulfate, mais partiellement glycosylé. Ce déficit de glycosylation est gouverné par des domaines peptiques distants codés par l’exon 1 et l’exon 2 et qui distinguent l’endocan murin de son homologue humain. Dans un modèle de xénogreffe tumorale chez la souris SCID, nous avons démontré qu’endocan murin ne présente aucun pouvoir pro-tumoral. Contrairement à l’endocan humain, il ralentit la vitesse de croissance tumorale. Cette propriété anti-tumorale est liée à la présence d’une forme non glycosylée. Nous avons pu montrer à travers plusieurs modèles de xénogreffes tumorales que cette propriété de freinage de la croissance tumorale s’étend aussi au core protéique d’endocan humain. De plus, nous avons pu démonter qu’une administration systémique d’endocan non glycosylé est significativement associée à un ralentissement de la croissance tumorale. Ceci établit la relation de causalité entre le polypeptide d’endocan et la propriété anti-tumorale observée dans les différents modèles animaux. Le polypeptide d’endocan ne modifie pas in vitro la prolifération ni la viabilité des cellules HT-29 ce qui laisse penser à un mécanisme d’action indirect. Sur le plan pathologique, nous avons montré que les formes non glycosylée d’endocan humain et murin sont associées à une réaction inflammatoire stromale constituée d’une infiltration pan-leucocytaire. La déplétion des leucocytes CD122+ (essentiellement les cellules NK murines) abolit partiellement l’effet anti-tumoral induit par l’endocan non glycosylé. Nos résultats ajoutent endocan au concert des molécules endothéliales tumorales qui participent au contrôle de la réaction inflammatoire stromale<br>Solid tumor requires a supply of oxygen and nutrients for growth but also for metastasizing to another organ. Tumor angiogenesis is the phenomenon exploited by tumor to fulfill these needs. The"Tip cells" located at the end of sprouting capillaries initiate and guide the growth of neovessels. These cells are currently considered as an important therapeutic target for anti-angiogenic drugs. Many studies have identified a cluster of molecular markers selectively expressed by the \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\"Tip cells.\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\" One of these markers called “endocan”, has represented the subject of the thesis work.Endocan is a circulating proteoglycan overexpressed in many human carcinomas, and expression is often associated with poor prognosis. It is suspected to play an important role in tumor development. Through its glycan chain, endocan modulates the effect of growth factors, and also the migration of endothelial cells. My thesis work has focused on the biochemical and functional characterization of mouse endocan in order to obtain a useful animal model for better understanding of the pro tumoral activity of human endocan. The work presented in this manuscript shows that mouse endocan is a chondroitin sulfate proteoglycan but much less glycanated than human endocan. Our data indicate that combinatorial distant domains from the O-glycanation site, located within exons 1 and 2 determine the glycanation pattern of endocan. In SCID mouse model of tumor xenograft we demonstrated that mouse endocan does not exhibit a pro tumoral activity. In opposite to the human homologue, overexpression of mouse endocan in HT-29 cells delayed the tumor appearance and reduced the tumor growth rate. This tumor growth inhibition was mostly supported by non glycanated mouse endocan. Unexpectedly, human non glycanated endocan overexpressed in HT-29, A549, or K1000 cells also delayed the tumor appearance and reduced the tumor growth. Moreover, systemic delivery of human non glycanated endocan also reproduced HT-29 tumor delay. In vitro, endocan polypeptide did not affect HT-29 cell proliferation, nor cell viability.Interestingly, a stromal inflammatory reaction was observed only in tumors overexpressing endocan polypeptide. In addition, depletion of CD122+ cells was able to delete partially the tumor delaying effect of endocan polypeptide. These results reveal a novel pathway for endocan in the control of tumor growth, which involves innate immune cells
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Rivinoja, A. (Antti). "Golgi pH and glycosylation." Doctoral thesis, University of Oulu, 2009. http://urn.fi/urn:isbn:9789514292699.
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Abstract
Glycans, as a part of glycoproteins, glycolipids and other glycoconjugates, are involved in many vital intra- and inter-cellular tasks, such as protein folding and sorting, protein quality
control, vesicular trafficking, cell signalling, immunological defence, cell motility and adhesion. Therefore, their correct construction is crucial for the normal functioning of eukaryotic cells and
organisms they form. Most cellular glycans are constructed in the Golgi, and abnormalities in their structure may derive, for instance, from alkalinization of the Golgi lumen.
In this work we show that Golgi pH is generally higher and more variable in abnormally glycosylating, i.e. strongly T-antigen (Gal-β1,3-GalNAc-ser/thr) expressing cancer cells, than in
non-T-antigen expressing cells. We also confirmed that the Golgi pH alterations detected in cancer cells have the potential to induce glycosylation changes. A mere 0.2 pH unit increase in Golgi pH is
able to induce T-antigen expression and inhibit terminal N-glycosylation in normally glycosylating cells. The mechanism of inhibition involves mislocalization of the corresponding
glycosyltransferases.
We also studied potential factors that can promote Golgi pH misregulation in health and disease, and found that cultured cancer cells, despite variation and elevation in Golgi pH, are fully
capable of acidifying the Golgi lumen under the normal Golgi pH. Moreover, we introduce a Golgi localized Cl-/HCO3- exchanger, AE2a, that participates in Golgi pH regulation by
altering luminal bicarbonate concentration and thus also buffering capacity. Participation of AE2a in Golgi pH regulation is especially intriguing, because it also provides a novel mechanism for
expelling protons from the Golgi lumen.
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Anttonen, Katri Pauliina. "Protein glycosylation in actinomycetes." Thesis, University of Aberdeen, 2010. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=92515.
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The aims of this project were to study the glycoproteome in two mycobacteria; <i>M. marinum</i> and <i>M. smegmatis</i>, and to assess the role of protein glycosylation by knocking out the Pmt homologues. Due to technical difficulties in the construction of knock out strains in mycobacteria, the aims were changed to further elucidate the protein <i>O</i>-glycosylation pathway identified in <i>S. coelicolor. </i>Reverse transcriptase PCR was used to show that <i>pmt, ppm1 </i>and the putative <i>ppm2 </i>(SCO1014) are expressed throughout the complex life cycle of <i>S. coelicolor. </i>The putative <i>ppm2<sup>-</sup></i> strain AV301, which can not be complemented with a wild type copy of SCO1014, was shown to harbour a point mutation in<i> ppm1</i>. In Western blots, soluble Ppm1 localised to both the cytosolic and membrane fractions whereas Ppm2 was only seen in the membrane fraction. Two bands at different molecular masses for Ppm2 were seen suggesting that this enzyme might be processed in <i>Streptomyces. </i>Using the bacterial two hybrid system, it was shown that unlike in mycobacteria, Ppm1 does not interact with Ppm2 <i>in vivo</i>. Furthermore, unlike the yeast Pmt enzymes, <i>Streptomyces </i>Pmt does not dimerise <i>in vivo</i>, suggesting that bacterial Pmt homologues might have an alternative mode of action from the eukaryote enzymes. To study the role of GDP-Mannose (GDP-Man) in protein glycosylation, three putative GDP-Man synthases were identified and disrupted; disruption in SCO1388 caused no obvious phenotypes whereas the SCO3039 and SCO4238 disruption strains had an earlier onset of pigment production as a sign of stress. In attempts to disrupt all three GDP-Man synthases, it was discovered that the disruption of both SCO3039 and SCO4238 was lethal.
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Green, L. "Studies in glycosylation methodology." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599657.
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This thesis is divided into two sections. The first section covers techniques, strategies and mechanisms of oligosaccharide formation, illustrated with both literature and the author's results, highlighting the many subtleties involved in performing a glycosylation. These include attempts at <I>trans</I>-glycoside formation on a polymer support without the use of an acetyl directing group and the curious failure of an acetyl group to direct a glycosylation in the synthesis of a lactosamine derivative. The second section describes studies towards the development of stereocontrolled glycosylation protocol, based upon a intramolecular aglycon delivery strategy. Various tethers ranging from anomeric carbonates to anomeric phosphoranes were tried to realise this strategy but a solution remained elusive.
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Karg, Saskia Ruth. "N-glycosylation engineering in tobacco /." Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17989.
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Kameh, Homa. "Aberrant glycosylation in HEMPAS patients." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ28786.pdf.
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Harthill, Jean Elizabeth. "N-glycosylation of horseradish peroxidase." Thesis, University of Oxford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292612.
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Kaloo, Sara. "Glycosylation of Carbohydrates by Glycosyltranferases." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.508556.
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Allen, Alice. "IgA glycosylation in IgA nephropathy." Thesis, University of Leicester, 1995. http://hdl.handle.net/2381/35028.
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IgA nephropathy (IgAN) is a common glomerulonephritis characterised by deposition of IgA1 in the glomerular mesangium The underlying abnormality lies within the IgA system rather than the kidney, and modest irregularities of IgA biology have been described, but the mechanisms involved in IgA1 deposition and glomerular injury remain elusive. A few reports have suggested an abnormality of the carbohydrate component of IgA1 in IgAN. These studies sought to confirm and further characterise the putative glycosylation defect and to identify its biochemical basis. Lectin binding assays were developed and used to analyse the N- and O-linked glycans of IgA1 in IgAN and controls. No gross abnormality of N-glycosylation was detected in IgAN, though these studies were subject to technical limitations. IgA1 in IgAN displayed significantly increased binding to lectins with affinity for O-linked N-acetylgalactosamine (Ga1NAc) as compared to controls. One explanation for this finding is reduced terminal galactosylation of the O-linked sugars of IgA1. A novel assay was developed to measure the functional activity of alpha1,3 galactosyltransferase (alpha1,3GT), the enzyme responsible for O-galactosylation, in cell lysates. In IgAN, peripheral blood B cells appeared to show significantly lower alpha1,3GT activity than controls, and this was inversely proportional to Ga1NAc expression of serum IgA1 as measured by lectin binding. These studies confirm an abnormality of O-linked glycosylation of serum IgA1 in IgAN, which may be attributed to low B cell alpha1,3GT activity. Altered O-glycosylation of IgA1 may be relevant to the pathogenesis of IgAN.
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Keenan, Tessa. "Protein O-glycosylation in Streptomyces." Thesis, University of York, 2016. http://etheses.whiterose.ac.uk/16889/.
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Previously, a protein O-mannosyl transferase (Pmt, SCO3154) and a polyprenol phosphate mannose synthase (Ppm1, SCO1423) were found to be required for the glycosylation of PstS, a phosphate binding protein, in the bacterium Streptomyces coelicolor. Bacteria in this genus are prolific producers of antibiotics and are often phenotypically resistant to multiple antibiotics. S. coelicolor pmt and ppm1 deficient mutants were hypersusceptible to cell-wall active antibiotics, suggesting that the protein modification could be required for cell wall and membrane homeostasis. The aim of this project was to investigate the S. coelicolor glycoproteome in order to better understand the physiological role of protein O-glycosylation in this model actinobacterium. Glycoproteins were detected in, and enriched from the membrane and culture filtrate proteomes of the S. coelicolor parent strain, J1929 and these were absent from the glycosylation deficient pmt (DT1025) and ppm1 (DT3017) mutants. Liquid chromatography coupled to mass spectrometry was used to characterise the membrane glycoproteome from the S. coelicolor parent strain, J1929 and 37 new glycoproteins were identified. Glycopeptides were modified on Ser/Thr residues with up to 3 hexoses; consistent with previous observations that the glycoprotein PstS is modified with a trihexose. The S. coelicolor glycoprotein glycans were shown to consist of Hex₂ and Hex₃ oligosaccharides. A carbohydrate linkage analysis led to the observation of 2-substituted, 4-substituted and terminal mannose residues, suggesting presence of (1->2) and (1->4) linkages in S. coelicolor glycoprotein glycans. The S. coelicolor glycoproteome comprises glycoproteins with various biological roles including solute binding, transport and cell wall biosynthesis. The genes encoding two S. coelicolor glycoproteins with putative roles in cell wall biosynthesis, an L, D transpeptidase (SCO4934) and a D-Ala-D-Ala, carboxypeptidase (SCO4847) were disrupted. Both mutants were hypersusceptible to β-lactam antibiotics, while the sco4847 mutant was hypersusceptible to lysozyme. These findings suggest that both proteins could be required for cell wall biosynthesis. As the phenotypes of the knockout mutants are reminiscent of the glycosylation deficient strains, we propose that glycosylation might be required for enzyme function.
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Cox, Daniel. "New approaches to stereocontrolled glycosylation." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:b3eb90c9-3149-4761-930f-391607ee134c.
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The conceptually simple process of linking carbohydrate units by glycosylation has proven to be one of the most difficult synthetic processes to control from a stereochemical perspective. In particular it is the stereocontrolled synthesis of 1,2-cis glycosyl linkages (e.g. α-glucosides, β-mannosides) which poses the most difficult challenge. The research presented in this thesis describes new ways in which stereocontrol in glycosylation reactions can be achieved. New methods of neighbouring group participation have been explored, utilising novel protecting groups at the 2-postion of a series of glycosyl donors. In particular the use of glycosyl donors bearing a (thiophen-2-yl)methyl protecting group at the 2-hydroxyl have shown exceptional α-selectivity especially when used in conjunction with a sterically hindered glycosyl acceptor. Work within this thesis also describes the first use of chiral Brønsted acid catalysts in the activation of glycosyl donors. It has been clearly demonstrated that not only can such catalysts be used in glycosylation reactions, but also that the chirality of the catalyst can dictate the stereochemical outcome of the reaction. The preliminary studies presented demonstrate that this methodology warrants further investigation.
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Singh, Govind Pratap. "New approaches to stereocontrolled glycosylation." Thesis, University of Canterbury. Chemistry, 2015. http://hdl.handle.net/10092/11126.
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The conceptually simple process of linking carbohydrate units by glycosylation has proven to be one of the most difficult synthetic processes to control from a stereochemical perspective. In particular it is the stereocontrolled synthesis of 1,2-cis glycosyl linkages (e.g. α-glucosides, β-mannosides) which poses the most difficult challenge. The research presented in this thesis describes new ways in which stereocontrol in glycosylation reactions can be achieved.
New methods of neighbouring group participation have been explored, utilising novel protecting groups at the 2-postion of a series of glycosyl donors.
In particular the use of glucosyl donors bearing a 2-O-(2-(2,4,6- trimethoxyphenyl)thio)ethyl protecting group at the 2-hydroxyl, have shown exceptional α-selectivity especially when a completely armed donor was used.
Work within this thesis also describes the use of chiral Brønsted acid catalysts in stereoselective glycosylation reactions. However the yields and stereoselectivity obtained were not very encouraging.
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Lige, Bao. "Role of glycosylation in peanut peroxidase." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0006/NQ42539.pdf.
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Leaney, Sarah J. "GIT : glycosylation 'via' inter/intramolecular transfer." Thesis, University of Oxford, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409812.
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Warren, C. E. "N-glycosylation in mice and rats." Thesis, University of Oxford, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315795.
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McAulay, Kathrine. "Glycosylation of Staphylococcus aureus surface proteins." Thesis, University of Sheffield, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.555122.
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Staphylococcus aureus is a major human pathogen, causing a wide spectrum of superficial and systemic infections. With the advent of methicillin resistant Staphylococcus aureus (M RSA) and the increasing spread of antibiotic resistance it has become clear that novel approaches must be taken to control S. aureus. Glycosylation, the modification of proteins with carbohydrate moieties, was long considered a process confined to eukaryotes; however, the last 3 decades have seen increasing awareness and characterisation of glycosylation systems in bacteria, including many pathogens In this study S. aureus strains were found to express several glycoproteins, covalently anchored to the cell wall peptidoglycan, including Cif A, Pis and putatively SdrC, SdrD and Clfb. CIf A glycosylation was readily detected and the glycosyltransferase enzymes responsible for Cif A modification (GtfA and GtID) were found to be encoded in a separate chromosomal location from cl(A. MALDI-MS/MS and GC-MS experiments revealed the presence of hexose and terminal HexNAc residues, consistent with an observed affinity for concanavalin A (ConA) and wheatgerm agglutinin (WGA) lectins. ConA affinity was then utilised to localise CIf A on the surface of S. aureus cells using a lectin-fluorophore conjugate, revealing a punctate presence of glycosylated protein around the cell wall. Glycosylation of CIf A was found to have no effect on fibrinogen (Fg) binding, however a role has been revealed in infection in a murine model of septic arthritis. A strain deficient in c/fA was highly attenuated in this model, whereas a strain deficient in both c/fA and gtfAB resulted in infection similar to the wild type. This data suggests a role for glycosylation in anti-virulence and thus has provided a novel insight into the nature of the host-pathogen interaction. Also, glycosylated CIf A was found to react with human sera at a significantly higher level than recombinant protein. The results highlight potential new avenues concerning immunological approaches to the control of S. aureus.
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Day-Williams, Michaela Joanne. "Polar flagellum glycosylation in aeromonas caviae." Thesis, University of Sheffield, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.515262.
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Hong, Sung You. "Surface glycosylation and filling of nanotubes." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.509960.
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Kong, Leopold. "HIV-1 gp120 : flexibility and glycosylation." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.533859.
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Hills, Anna E. "Control of monoclonal antibody N-glycosylation." Thesis, University of Kent, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.344101.
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Huffman, Jennifer Elizabeth. "Genetic analysis of protein N-glycosylation." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/10038.
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The majority of human proteins are post-translationally modified by covalent addition of one or more complex oligosaccharides (glycans). Alterations in glycosylation processing are associated with numerous diseases and glycans are attracting increasing attention both as disease biomarkers and as targets for novel therapeutic approaches. Using a recently developed high performance liquid chromatography (HPLC) method for high-throughput glycan analysis, genome-wide association studies (GWAS) of 33 directly measured and 13 derived N-glycan features were performed in 3533 individuals from four European isolated populations. Polymorphisms at six loci were found to show genome-wide significant association with plasma concentrations of N-glycans. Several of these gene products have well characterised roles in glycosylation, however, SLC9A9 and HNF1A were two of the novel findings. Subsequent work performed by collaborators found HNF1A to be a “master regulator” of genes involved in the fucosylation of plasma N-glycans. Additionally, this work led to the discovery that N-glycans could act as biomarkers to discriminate HNF1A-MODY from type 1 and type 2 diabetes mellitus (T1D, T2D) patients. After the success of the total plasma N-glycan GWAS, it was thought that stronger and more biologically interpretable associations may be found from the investigation of N-glycans isolated from a single protein. Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic networks that govern IgG glycosylation, N-linked IgG glycans were quantitated using ultra performance liquid chromatography (UPLC) in 2247 individuals from the same four European populations from the previous study. GWAS of the 77 N-glycan measures identified 15 loci with a p-value < 5x10-08. Four loci contained genes encoding glycosyltransferases, while the remaining loci contained genes that have not previously been implicated in protein glycosylation. However, most have been associated with autoimmune and inflammatory conditions and/or hematological cancers. Several high-throughput methods for the analysis of N-glycans have been developed in the past few years but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. To this end, four of these methods were compared, UPLC, multiplexed capillary gel electrophoresis (xCGE), and two mass spectrometric (MS) methods, for quantitative profiling of N-glycosylation of plasma IgG in a subset of 1201 individuals recruited from two of the cohorts used in the previous GWAS studies. A “minimal” dataset was compiled of N-glycan structures able to be measured by all four methods. To evaluate their accuracy, correlations were calculated for each structure in the minimal dataset. Additionally, GWAS was performed to test if the same associations would be observed across methodologies. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and xCGE, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should aid in the selection of the most appropriate method for future studies. This work shows that it is possible to identify new loci that control glycosylation of plasma proteins using GWAS and the potential of N-glycans for biomarker development. It also provides some guidelines for methodology selection for future studies of N-glycans.
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Gorbea, Carlos M. "Expression and glycosylation of meprin isoforms." Diss., This resource online, 1992. http://scholar.lib.vt.edu/theses/available/etd-07282008-134351/.
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Taylor, Thomas Alex. "Investigations into novel enzymatic glycosylation methods." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:3bfd1078-79d8-4c68-83a0-0bb5343583eb.
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Glycosylation is one of the most prevalent post-translational modifications found on human proteins. There is a great interest in developing new methodologies for the synthesis of glycoproteins, both to elucidate the functions of glycans on proteins and to exploit the beneficial properties they can confer on biotherapeutics. Here, research was carried out investigating enzymatic techniques to achieve the formation of homogenous glycoproteins. EndoS was the first enzyme investigated. EndoS natural activity is for the hydrolysis of glycans specifically from IgG, yet there has been research into its use for the opposite reaction and for glycan extension reactions. Here, a number of EndoS mutants were formed and investigated for this reaction, with a number of novel constructs giving a moderate yield of the glycosylated product. Structural investigations of EndoS were also conducted. In addition, research was conducted into the use of PglB in vitro for the direct glycosylation of an asparagine reside on a peptide with GlcNAc. Studies here demonstrate that it is possible to use far simpler glycan donor substrates with PglB. Additional studies showed that it was possible to conduct further enzymatic glycosylation reactions with GalT and EndoA after the initial PglB reaction. Further research was undertaken with EndoA, with it being desired to investigate whether it could catalyse the formation of a thioglycosidic linkage between two glycans, with the thiol bearing glycan acceptor to be attached to a peptide using PglB. After the successful synthesis of all the substrates, disappointingly, neither of the two enzymatic reactions were successful. Finally, the use of a glycal donor in an EndoA catalysed glycosylation was investigated. Unfortunately, no consumption of the glycan was observed in this reaction.
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Mas, Eric. "Glycosylation de la lipase sels biliaires dépendante du pancréas : Glycosylation de la lipase sels biliaires dépendante du pancréas." Aix-Marseille 2, 1995. http://www.theses.fr/1995AIX22028.
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La lipase sels biliaires-dependante (bsdl) est une enzyme lypolitique de la secretion pancreatique. Dans un premier temps, nous avons montre que la n-glycosylation de la bsdl, issue d'un sujet sain, est de type biantennaire complexe et elle presente 12 a 14 structures o-glycosylees de type (gal betal-3 galnac)-ser/thr plus ou moins sialylees et/ou fucosylees. La n-glycosylation est aussi essentielle a l'expression d'une bsdl totalement active et a sa secretion. Cependant, les inhibiteurs de la maturation de la n-glycosylation n'influent ni sur l'activite ni sur la secretion de l'enzyme. La structure oligosaccharidique n-liee participerait donc a l'etablissement de la conformation correcte du site actif de la bsdl. Un glycovariant oncofoetal de la bsdl, la proteine foeto-acinaire pancreatique (fap) identifiee par l'epitope oncofoetal j28 a ete caracterise. Leur principale difference reside dans leurs structures glycosidiques: la fap montre une n-glycosylation immature de type oligomannosidique avec une diminution des motifs oligo-saccharidiques o-lies. La caracterisation de la structure de l'epitope j28 montre l'implication de residus de fucoses portes par la o-glycosylation. Nous avons aussi montre que, lors des pancreatites chroniques, seule la bsdl montre une alteration de la n-glycosylation qui reste immature. Enfin, nous avons mis au point un dosage elisa de la bsdl presente dans le serum. Ce dosage, couple a celui de l'antigene ca 19/9, permet de diagnostiquer les cancers pancreatiques et de les distinguer des pathologies pancreatiques
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Zerbib, Marie. "Etude de la glycosylation de flavanols dans le raisin et incidence dans les vins." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTG051/document.
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Les flavan-3-ols appartiennent à la famille des polyphénols retrouvés dans diverses plantes et majoritairement dans le raisin. Ils jouent un rôle primordial dans les mécanismes de défense des plantes, influent sur les propriétés organoleptiques du vin et sont potentiellement bénéfiques au niveau de la santé humaine. La voie de biosynthèse des flavanols monomères est décrite dans la littérature. Cependant, les mécanismes de formation des proanthocyanidines (polymères) sont inconnus à ce jour. Des études ont montré que les flavanols glycosylés sont des intermédiaires potentiels dans la biosynthèse des PA et permettent le transport des unités de flavanols du cytoplasme vers la vacuole de la cellule, où a lieu la polymérisation. Un criblage global des flavanols glycosylés présents dans des raisins à trois stades de développement et dans des vins de différents cépages a été réalisé en analysant séparément les pellicules et les pépins de raisin par une méthode d’UPLC-DAD-ESI-MS. La teneur de ces composés dépend de paramètres de type variété du raisin, tissu biologique étudié et stade de maturité. La présence de dimères de flavanol glycosylés a été montrée pour la première fois dans le raisin. Grâce à l’hémisynthèse de la (+)-catéchine 4’-O--glucoside et 7-O--glucoside, certains monomères ont été caractérisés comme appartenant à la classe des -glucosides. Une étude quantitative a montré l’évolution des flavanols glycosylés au cours du développement de la baie de raisin (pépins et pellicules séparés) provenant de trois cépages différents. Les monomères et dimères d’ (épi) catéchine diglycosylés ont été découverts pour la première fois et uniquement dans les pépins. Une diminution en monomères d’ (épi) catéchine monoglycosylés a été observée dans la pellicule au cours de la maturation du raisin. Les dimères d’ (épi) catéchine monoglycosylés s’accumulent un peu après le stade de véraison et diminuent ensuite à maturité. Les monomères et dimères d’ (épi) catéchine diglycosylés s’accumulent dans les pépins au cours de la maturation. Finalement, l’évolution des teneurs en flavanols mono et diglycosylés au cours de microvinifications a été étudiée. On observe des profils d’extraction similaires pour les deux variétés utilisées (Grenache et Syrah). La quantité totale des différentes familles de composés augmente au cours de la vinification, et ensuite diminue en fin de FA. Certains composés sont dégradés préférentiellement, suggérant la présence d’activités glycosidases spécifiques du raisin ou de la levure<br>Flavan-3-ols belong to a group of polyphenols present in a wide variety of plants, and particularly in grapeberries. They play an important role in defense mechanisms in plants, have a significant impact on wine organoleptic properties; and their beneficial effects on human health may help to protect against chronic diseases such as atherosclerosis. The sequence of common flavanol monomer biosynthesis is widely described in the literature, but the formation mechanisms of proanthocyanidins (PA) remain unknown. Studies show that flavanol glycosides are potential intermediates in PA polymerization and have transporter roles of monomeric units from cytoplasm to vacuole in cell, where polymerization takes place. Global screening of grapeberry flavanol glycosides were carried out at three stages of grape development and in wines of different varieties; skin and seeds were measured separately using an UPLC-DAD-ESI-MS method. The composition of the target isomers depends on different parameters such as tissue type or stage of development. The presence epi catechin monoglycoside is reported here for the first time in grapes. Using (+)-catechin 4’-O--glucoside and 7-O--glucoside hemisynthesis, several monomers were shown to -glucosides. Quantitative analysis demonstrates the evolution of flavanol glycosides in both skin and seeds during the development of three grapevine varieties. For the first time monomeric and dimeric (epi) catechin diglycosides were revealed and shown accumulate only in grape seeds during ripening. A reduction in the concentration of monomeric (epi) catechin monoglycoside was observed during grape skin development. Dimeric (epi) catechin monoglycosides accumulate after veraison and then decrease at the end of grape ripening. The extraction profiles of flavanol glycosides during red grape fermentation showed similar evolution patterns for both varieties used. The total concentration of different compound families increases during winemaking, and then decreases at the end of fermentation. Degradation of specific compounds was observed at the end of fermentation which may be explained by the activity of glycosidases from grape extracts released during fermentation and pressing
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Luz, Johanna Da. "Aspects of N-glycosylation in human IgE /." Stockholm : [Karolinska institutets bibl.], 2002. http://diss.kib.ki.se/2002/91-7349-130-6.
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Ho, Ming-Kai 1978. "Characterization of glycosylation of prolactin in galliformes." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98724.
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Prolactin (PRL) is a highly versatile hormone in terms of its biological functions. In Galliformes, high levels of PRL are associated with incubation behaviour and hatching. It has been shown that these high levels of PRL were associated with an increased ratio of glycosylated PRL (G-PRL) versus non-glycosylated PRL (NG-PRL) in turkey (Bedecarrats et al., 1999a). This suggests that glycosylation of PRL is related to its proper function during those stages of life. However, the mechanism(s) controlling post-translational modification of PRL are unknown. In order to investigate genes associated with the glycosylation of PRL, an in vitro study was undertaken. The pituitary glands of day 24 turkey embryos (n=60) were collected and pooled into two groups which were incubated in medium 199 for 4 hours in the absence or presence of 10-7 M vasoactive intestinal peptide (VIP). Western blot analysis of PRL was used to assess the response of the pituitary glands to the VIP stimulation. As expected, the absolute level of PRL as well as the percentage of glycosylated PRL isoform increased following stimulation with VIP. The mRNA of both stimulated and non-stimulated samples were extracted and reverse transcribed into cDNA. Using suppression subtractive hybridization (SSH), a cDNA subtractive library which only contained differentially expressed cDNAs between VIP stimulated and non-VIP stimulated turkey pituitary glands was constructed. Seventeen percent of the genes from the library are related to cell proliferation and apoptosis. Ten genes were selected for real-time PCR analysis. The functions of these genes and their potential roles in response to the stimulation by VIP and glycosylation of PRL are discussed.
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Critchley, Alison. "Congenital disorders of glycosylation and disease pathogenesis." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424758.
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Al-Ghouleh, Abeer. "The role of glycosylation in allergen recognition." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12103/.
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This project is an attempt to have a better understanding of the role of carbohydrates in recognition and uptake of allergens by the innate immune system. Glycosylation analysis of different allergens like Der p 1, Fel d 1, Ara h 1, Ber e 1, Der p 2, Bla g 2, Can f 1, Bromelain and Papain was made using labelled lectins that can detect specific carbohydrate moieties on proteins to reveal their pattern of glycosylation. These experiments showed that all major allergens are glycosylated and this represented a major first step towards demonstrating a link between glycosylation and allergen recognition by the innate immune system. N- and O-glycosylation patterns were predicted in different allergens and a difference in mannosylation and fucosylation was detected between allergens and non-allergenic proteins. We found that the main dominant sugars on allergens are 1,2 1,3 and 1,6 mannose, as detected by GNA lectin. We have also showed that Der p 1 and Der p 2 possess 1,3 fucose in their natural forms, thus concluding that Der p1 and Der p 2 have part of the CCDs which are epitope structures for IgE. O-glycosylation in allergens was also studied giving a better understanding of the whole glycan structure in allergens. The role of mannosylation in allergen recognition by the immune system was investigated further. Different methods, including recombinant expression, enzymatic and chemical deglycosylation, were optimised to produce glycoforms of Der p 1. A recombinant preparation of Der p 1 produced in Pichia pastoris was used as a hypermannosylated form of the allergen. These glycoforms served as useful tools in addressing the nature of glycoallergen recognition by looking at the uptake of hyper- and hypo-glycosylated preparations by DCs, with confocal microscopy, ELISA and FACS as readouts. Results indicate that deglycosylated forms of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant allergen. Comparative analysis of the hypermannosylated preparation of Der p 1 and its natural counterpart, possessing less mannan, showed that the recombinant form was taken up more readily by DCs at 37°C and at 4°C. We also showed that these glycoforms bind to the MR subfragment CTLD 4-7-FC, the C-type lectin carbohydrate recognition domain. This binding significantly decreased when the Der p 1 allergen was deglycosylated. These results were confirmed further using confocal microscopy imaging which also showed that recombinant Der p 1 uptake is immediate, starting at 5 mins of incubation and that a higher quantity accumulates inside the DC compared to natural allergen. Recombinant and natural Der p 1 both co-localised with MR, DC-SIGN and LAMP-2 lysosomal marker, suggesting a key role for these receptors in allergen uptake and a common fate for these preparations inside the DC. Further experiments were done to show the effect of Der p 1 and Der p 1 glycoforms on TSLP secretion by epithelial cells, which is known to induce Th2 driven immune responses. The results show that TSLP secretion decreases significantly when epithelial cells are challenged with deglycosylated preparations of the same allergen. This may indicate a change in the outcome of adaptive immune responses when a deglycosylated allergen challenges epithelial cells. In conclusion, this work has demonstrated a link between allergenicity and glycosylation patterns in allergens. It therefore appears that mannosylation is the dominant sugar moiety associated with allergen uptake and recognition by humans DCs, and this is in line with MR being the main receptor involved in allergen binding by these cells.
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McKenzie, Samuel Noel. "Asymmetric Control of the Diastereoselectivity of Glycosylation." Thesis, University of Canterbury. Chemistry, 2011. http://hdl.handle.net/10092/5931.
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Diastereoselective control of glycosylation still remains a difficult task. Therefore, new glycosylation methods using asymmetric catalysis were developed to control the diastereoselectivity. Two systems were developed and each focused on a separate type of glycosyl donor. In the first system, glycosyl halides were subjected to reaction conditions inspired by Hamilton et al., who effectively had controlled the substitution of a racemic chloroamine by an alcohol. Asymmetric control of glycosylation was achieved through this adapted catalytic system. Both enantiomers of the catalyst ((R) and (S) TRIP) generally displayed b-selectivity with tertiary butyl methyl ether (TBME) as the solvent allowing almost exclusive formation of the β-anomer. However, low and inconsistent yields
were obtained.
The second system proposed the use of the same phosphoric acid catalyst (TRIP) to catalyse the glycosylation of glycals. However, this was ineffective as the catalyst was not a strong enough Brønsted acid. These studies then led to the development of two new chiral catalysts which then promoted the glycosylation of glycals, along with the formation of an undesired side product. Attempts were made to reduce the formation of the side product but unfortunately this proved unsuccessful. The diastereoselective outcome displayed between the different catalysts in separate trials was negligible, but the principles developed in this study should lead to the further development of new chiral catalysts for the glycosylation of glycals.
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Mann, Alexander Charles. "Serum protein glycosylation in alcoholic liver disease." Thesis, University of Newcastle Upon Tyne, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323479.
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Chen, Qiushi. "Mass spectrometric investigation of biomedically important glycosylation." Thesis, Imperial College London, 2015. http://hdl.handle.net/10044/1/56202.
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Glycobiology is the comprehensive study of the structure, biosynthesis, function and evolution of saccharides which are also named sugars or glycans. Glycosylation is a type of modification in which sugars are added to another molecule, such as a protein molecule or a ceramide. Abnormal glycosylation is frequently associated with diseases such as cancer and immune responses. Defining glycan structures is therefore important for understanding glycan function in health and disease. In addition, identification of glycan populations can provide essential information for further research on glycoproteins and glycolipids. In this thesis, glycomic experimental approaches were employed to characterize the structures and populations of glycans of glycoconjugates from HeLa cells, normal human dermal fibroblast (NHDF) cells, myoblasts, myotubes and trophoblasts. These approaches include sample preparation methodologies which were followed by the application of highly sensitive mass spectrometry, particularly MALDI-TOF MS, MALDI-TOF/TOF MS/MS and GC-MS. Ribosome inactivating proteins (RIPs) and lectins from elderberry are more toxic to HeLa cells than to NHDF cells. The difference in the cytotoxicity was hypothesized to be caused by the difference in the glycome patterns of HeLa and NHDF cells. To test the hypothesis, glycome patterns on both glycoproteins and glycolipids of HeLa and NHDF cells were investigated. Glycomic results have revealed that glycome patterns in HeLa cells and NHDF are different, and this gives a possible explanation for the difference observed in the cytotoxicity assay. Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway which yields uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an essential substrate for protein glycosylation. N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. Mutations in the gene GFPT1 can result in “limb-girdle CMS with tubular aggregates” which is a subtype of congenital myasthenic syndromes (CMS). To investigate whether protein glycosylation at the neuromuscular junction might be involved in this impairment, the N-glycomes of myoblasts and myotubes derived from healthy controls and patients were investigated. My result showed that global glycosylation is not significantly impaired in the muscle cells from the CMS patients caused by GFPT1 mutations. The human fetoembryonic defense system hypothesis (hu-FEDS) is a hypothetical model depicting a way via which the human immune system is able to recognize foreign substances as "own species" as has been observed with maternal immune tolerance in pregnancy. The fundamental idea of this hypothesis is that glycoproteins existing in the reproductive system and exposed on gametes can either inhibit immune responses or prevent rejection of the foetus. This model has not been tested in human trophoblasts. My glycomic analyses of three trophoblast populations (CTB, STB and evCTB) revealed that functional glycan structures that are present on human gametes are also expressed on trophoblasts, and this provides further evidence for the hu-FEDS hypothesis.
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Yuk, Inn Huam Yvonne. "Protein expression and glycosylation in CHO cells." Thesis, Massachusetts Institute of Technology, 2001. http://hdl.handle.net/1721.1/8201.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2001.<br>Includes bibliographical references (p. 193-207).<br>A successful mammalian cell culture process depends on sufficient expression and correct glycosylation of the recombinant product. Low product titer and inconsistent protein glycosylation constitute two major problems frequently encountered in biopharmaceutical production. Using Chinese Hamster Ovary (CHO) cells expressing recombinant human interferon-gamma (IFN-y) as the model system, this thesis investigated strategies that can potentially enhance heterologous protein expression or control the extent of protein glycosylation. There has been considerable interest in developing culture strategies to improve productivity in mammalian cell lines by limiting the cellular growth rate. An initial investigation demonstrated the difficulty in obtaining growth-arrested cells that are robust and productive. In the following study, a method to rapidly generate and isolate CHO cells that exhibit enhanced potential for use in proliferation-controlled bioprocesses was developed. By combining bicistronic retroviral technology with an appropriate selection strategy, a subpopulation was isolated from a heterogeneous cell population. To evaluate the effectiveness of this screening process, the performance of this selected subpopulation was compared with that of the original population under identical growth-arresting conditions. Important differences were observed: by contrast to the original population, the selected cells maintained consistently high viabilities and continued to stably express recombinant proteins after being growth-arrested for two weeks. Asparagine-linked (N-linked) glycosylation can significantly impact critical properties of human therapeutic proteins.<br>(cont.) An essential step in N-linked glycosylation is the transfer of an oligosaccharide from dolichol phosphate (Dol-P) to a potential glycosylation site on a polypeptide. Variability in the success of this reaction affects the extent of glycosylation on proteins. Radiolabeling studies showed that over the course of CHO batch culture, glycosylation precursor concentrations remained within a two-fold range, and overall protein glycosylation increased by 15-25%. Given the key role of Dol-P in glycosylation, its availability was postulated to limit glycosylation by controlling the abundance of glycosylation precursors. To test this hypothesis, the impact of Dol-P feeding on CHO cells was investigated. Although exogenous Dol-P was incorporated by CHO cells and processed into glycosylation precursors in a dose-dependent manner, Dol-P supplementation had no marked effects on the level of glycosylation precursors or on the extent of glycosylation.<br>by Inn Huam Yvonne Yuk.<br>Ph.D.
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Mérit, Xavier. "Protéines G et glycosylation chez Aspergillus niger." Lyon 1, 1991. http://www.theses.fr/1991LYO10086.
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Nous avons mis en evidence chez aspergillus niger, une activite gtpase qui est souvent une des fonctions des proteines g, connues pour fixer et hydrolyser le gtp. L'utilisation d'analogues non hydrolysables du gtp et du gdp a permis d'etudier les modalites de leur fixation et la localisation des proteines impliquees. Un fractionnement cellulaire met en evidence des localisations distinctes pour ces proteines particulieres. En effet, apres electrotransfert sur nitrocellulose, les anticorps anti-site de fixation du gtp revelent l'existence de 6 proteines g dans le cytosol. Une separation des fractions membranaires montre l'existence d'une seule proteine g dans les vesicules legeres. Trois msont localisees dans le rel, deux dans le rer et une seule dans la membrane plasmique. Les proteines de la moisissure different des proteines g classiques de la membrane plasmique. Les proteines de la moisissure different des proteines g classiques de la membrane plasmique par leur masse moleculaire plus faible (moins de 20 kda) ce qui les rangent dans la famille des petites proteines g. La fraction contenant les membranes du rel hydrolyse non seulement le gtp, mais aussi le gdp, ce qui n'est pas classique. Cette fraction presente la plus forte activite dolp-mannose synthetase. Cet enzyme intervient lors de la biosynthese des glycoproteines (a partir de gdp-mannose) qui est un processus membranaire mettant en jeu un intermediaire lipidique: le dolp-mannose. Le gdp, produit de la reaction, inhibe totalement la synthese du mannolipide. Ainsi, toute proteine modulant la quantite de gdp au niveau du site de biosynthese des glycoproteines possede un role de regulation, qui du reste, est souvent associe aux proteines g
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Fernandes, Elisabete Cristina Nunes. "Humoral responses against esophageal cancer-associated glycosylation." Master's thesis, Universidade de Aveiro, 2012. http://hdl.handle.net/10773/11301.
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Mestrado em Bioquímica Clínica<br>O cancro de esófago (CE) tem um mau prognóstico e uma baixa taxa de sobrevivência. É diagnosticado tardiamente, por endoscopia, um método invasivo que carece de especificidade e sensibilidade. Atualmente, não existem biomarcadores para melhorar a precisão do diagnóstico.
A transformação maligna é acompanhada por alterações no padrão de glicosilação das células, que são utilizadas no diagnóstico não-invasivo, monitorização da doença, bem como terapêutica. Além disso, as proteínas exibindo glicosilação alterada são capazes de induzir respostas humorais. Os auto-anticorpos são uma nova geração de biomarcadores tumorais, com capacidade de amplificar alterações moleculares nos tumores. São ainda mais resistentes a proteólise o que os correspondentes epitopos. No entanto, pouco se sabe sobre a glicosilação em CE e a sua imunogenicidade.
Assim, a primeira parte deste trabalho teve como objetivo identificar padrões de glicosilação no soro de doentes com CE por slot blotting. Foram considerados para este estudo os cinco antigénios tumorais Tn, sTn, T, sLea, e sLex. Verificou-se que os níveis de Tn, sTn, e do sLea estavam aumentados no soro de pacientes com EC, em comparação com um grupo de controlo, comparável em idade e género, sem doença maligna conhecida. A combinação dos antígenos Tn e sTn permitiu uma melhor discriminação entre CE e os controlos (sensibilidade de 93,8% e especificidade de 100%). A expressão destes antigénios nos tecidos de CE correspondentes, estimada por imuno-histoquímica, mostrou uma falta de correlação com as observações feitas no soro. Os dados sugerem que os padrões de glicosilação do soro são maioritariamente influenciados por proteínas não directamente secretadas ou libertadas por células tumorais, apesar de a sua contribuição não poder ser excluída.
A segunda parte do trabalho visou identificar respostas humorais contra proteínas que transportam o sLea, um biomarcador associado à glicosilação com maior potencial de migração celular e metástase. A análise do perfil de IgG das amostras apresentou uma expressão aumentada de uma subclasse (IgG1) nos doentes com CE. As IgG1 produzidas de novo por indução tumoral demonstraram possuir sLea na sua estrutura, contribuindo assim para o aumento dos níveis deste antigénio no soro de doentes com EC. Ainda que os mecanismos biológicos por detrás deste evento não sejam ainda conhecidos, isso poderá permitir melhorar a sensibilidade e especificidade do teste sorológico de sLea (teste de CA19-9). Usando uma combinação de técnicas de immunoprecipiações e de Western blotting, foi ainda demonstrado, pela primeira vez, que as proteínas tumorais que transportam sLea podem induzir a produção de IgG1. A remoção do ácido siálico confirmou que a expressão de sLea era determinante para o reconhecimento de IgG1. Estas observações, bem como a identificação futura de proteínas imunogénicas transportadoras de sLea, prmitirão determinar o seu valor clínico e poderão ser um ponto de partida para o desenvolvimento de testes serológicos baseados em autoanticorpos.
Em suma, eviências importantes foram encontradas com este estudo, que permitirão o desenvolvimento de testes serológicos não invasivos para a detecção de CE<br>Esophageal cancer (EC) has an extremely poor prognosis and decreased overall survival. It is generally diagnosed at a late stage, by endoscopy, which is invasive and lacks both specificity and sensitivity. At the moment, there are no biomarkers to improve the accuracy of diagnosis.
The modification of cell glycosylation patterns is a recognized hallmark of cancer, explored in non-invasive diagnostic, therapeutic decision, disease monitoring as well as therapeutics. Moreover, abnormally glycosylated proteins have been proven capable of eliciting humoral responses. Autoantibodies are regarded as the new generation of tumor biomarkers, cable to amplifying events occurring in tumors and showing higher stability to proteolysis. Nevertheless, little is known about EC associated glycosylation nor and its immunogenicity.
Based on these considerations, the first part of this work aimed to identify glycosylation patterns in serum associated with EC. Tn, sTn, T, sale, and sLex, the most studied tumors-associated carbohydrate antigens, were screened by slot blotting. The levels of Tn, sTn, and sLea antigens were found significantly increased in the serum of EC patients, when compared to a control group of matched age and gender, without known malignancy. Moreover, the combination of the Tn and sTn antigens allowed the best discrimination between EC and controls (93.8% sensitivity and 100% specificity). Still, the expression of these antigens in the corresponding EC tissues, estimated by immunohistochemistry, showed a lack of correlation with the observations made in serum. Data suggests that glycosylation patterns of serum are mostly influenced by proteins that are not directly secreted or released from tumor cells, even though their contribution cannot be excluded.
The second part of the work aimed to identify humoral responses against proteins carrying the sLea, a glycosylated biomarker associated with increased potential of cellular migration and metastasis. The analysis of the IgG profile of the samples showed increased expression of IgG subclasse 1 (IgG1) in EC patients. De novo produced IgG1 were found to carry sLea, accounting for the increase in the levels of this glycan in the serum of EC patients. Even though the biological events underlying these observations remain to be clarified, this may allow improving the sensibility and specificity of the serological test for sLea (CA19-9 test). Using a combination of immunoprecipiations and western blotting techniques it was further demonstrated, for the first time, that tumor proteins carrying sLea could elicit IgG1 production. Furthermore, experiments using desialylated proteins confirmed that the expression of sLea expression was determinant for IgG1 recognition.
These observations and the future identification of the immunogenic proteins carrying sLea will allow determining the clinical value of this explorative work and guiding the development of autoantibody-based serological tests.
Altogether, important insights have been provided to guide the development of non-invasive serological tests for the detection of EC.
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Borentain, Patrick. "Voies de la glycosylation et carcinome hépatocellulaire." Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM5047.
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La glycosylation est un processus enzymatique permettant l'ajout de sucres à des composés (sucres, lipides ou protides), modifiant ainsi leurs propriétés. La glycosylation est impliquée dans la détoxification des xénobiotiques et des variations d'activité des enzymes responsables ont été identifiées comme facteur de risque de cancer en particulier dans les organes exposés aux xénobiotiques. Dans la première partie de notre travail nous étudions l'impact des polymorphismes génétiques de certaines enzymes responsables de la détoxification (UGT1A7, GST et XRCC1) sur le risque de carcinome hépatocellulaire. Nous montrons que la combinaison de certains polymorphismes génétiques peut entraîner une augmentation du risque de CHC. Des modifications d'expression des glycoprotéines de surface ont été observées dans les cellules cancéreuses jouant un rôle dans leurs interactions avec le microenvironnement. Dans la seconde partie, nous étudions l'effet de l'inhibition des interactions des cellules de CHC/cellules endothéliales par le blocage du couple sialyl Lewis x/E-sélectine sur la croissance tumorale. Ce blocage est obtenu, d'une part par transfert du gène de la Fucosyl-transferase I, inhibant l'expression de sLex à la surface des cellules de CHC, et d'autre part, par utilisation de cimétidine ou d'amiloride permettant une inhibition de l'expression de la E-sélectine par les cellules endothéliales. Nous obtenons une inhibition de la croissance tumorale in vivo par blocage de la néoangiogénèse. Ces travaux permettent donc d'identifier des facteurs de risque génétiques de CHC et d'envisager une autre voie de traitement du CHC<br>Glycosylation is an enzymatic process that consists of the addition of glycosyl groups to compounds (sugars, lipids or proteins), thus modifying their properties. Glycosylation is involved in the detoxification of xenobiotics and variations in activity of enzymes responsible have been identified as a potential risk factor for cancer in particular in organs in contact with the external environment. In the first part of our work we study the impact of polymorphisms of detoxification enzyme (UGT1A7, GST and XRCC1) on the risk of hepatocellular carcinoma. We show that the combination of genetic polymorphisms of such enzymes may increase the risk of HCC. Modifications in the expression of surface glycoproteins have been observed in cancer cells and play a role in their interactions with the tumoral microenvironment. In the second part, we study the effect of inhibition of interactions of HCC cells / endothelial cells on tumor growth by blocking the interaction between sialyl Lewis x and E-selectin. First, we achieved the inhibition of the expression of sLex on the surface of HCC cells by introducing fucosyl transferase- I gene in HCC cells. In a second part of our work we used cimetidine and amiloride to inhibit the expression of E-selectin by endothelial cells. This approach resulted in inhibition of HCC cells / endothelial cells interaction and thereby tumor growth inhibition in vivo. This effect is mediated by an inhibition of tumor neoangiogenesis. This work therefore identifies genetic risk factors for HCC and allows considering another way of treatment of HCC
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Wood, Alison. "N-linked protein glycosylation in Helicobacter species." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/nlinked-protein-glycosylation-in-helicobacter-species(ef97ffdd-aca4-40ff-b52b-7b8ca030bc82).html.
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N-linked protein glycosylation involves the transfer of a glycan onto an Asparagine residue (N) of a polypeptide chain. It is common in Eukaryotes and has recently been observed in Prokaryotes, most notably in Campylobacter jejuni. The C. jejuni N-linked glycosylation system is encoded on a single pgl gene locus that also functions when expressed in Escherichia coli. The key enzyme involved in N-linked protein glycosylation is encoded by the pglB gene and transfers lipid-linked glycan onto N residues of glycoproteins in the periplasm. It is clear from accumulating genome sequence data that pglB orthologues are present in all Campylobacter species and in related species such as Wolinella succinogenes, Desulfovibrio vulgaris and Desulfovibrio desulfuricans. Most Helicobacter species, including Helicobacter pylori, lack the pglB gene but three related Helicobacter species Helicobacter pullorum, Helicobacter canadensis and Helicobacter winghamensis have two distinct pglB genes. These and other orthologues of C. jejuni pgl genes are located not within a single locus but rather at five distinct loci. One of the two pglB genes, termed pglB1, is required for in vitro N-glycosylation of peptides (Jervis et al., 2010). In this thesis I present data on the role of further pgl gene orthologues and previously uncharacterized genes in H. pullorum N-glycosylation. Furthermore I have also identified a number of H. pullorum glycoproteins and provide data comparing N-glycosylation processes in C. jejuni and H. pullorum. These data expand our preliminary observations on the first Helicobacter N-linked glycosylation system, and provide important information on the similarities and differences between the well characterised C. jejuni system and these more recently identified pathways.
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Lowry, Rebecca. "Glycosylation of the Aeromonas caviae polar flagellum." Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/9503/.
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The bacterial flagellum is an important appendage at the bacterial cell surface, not only for motility, but for adherence to host cells, and therefore has an extensive role in bacterial colonisation and virulence. A number of pathogenic bacteria modify their flagellins with nonulosonic acids, such as Aeromonas, Campylobacter and Helicobacter species, via an O-linked glycosylation process. This modification is essential for their ability to form flagella, thus having implications in pathogen virulence. However, the role of flagellin glycosylation is currently undetermined. The mesophilic aeromonad, Aeromonas caviae, forms a constitutively expressed polar flagellum necessary for motility in liquid environments. It is thought to be a good model for glycosylation as it decorates its flagellins solely with pseudaminic acid, and contains a genetically simple system for this process to occur. The work in this thesis has explored the pathway, and role of flagellin glycosylation in this microorganism. The function of a putative deglycosylation enzyme (AHA0618) and its possible role in the fine tuning of flagellin glycosylation was examined, where it was concluded that this protein is likely to be involved in peptidoglycan crosslinking at the cell wall, and not the flagellin glycosylation pathway. This demonstrates that subtle changes to bacterial cellular morphology are able to affect bacterial behaviour. Additionally, investigations into the sites of flagellin glycosylation via mass spectrometric methods concluded that the sites of modification on A. caviae flagellins can vary. However, certain residues were found to be predominantly glycosylated, suggesting partial selectivity to the glycosylation process via the putative glycosyltransferase, Maf1. Finally, protein interaction studies have provided evidence that glycosylation is likely to occur in the cytoplasm before binding of the flagellin-specific chaperone and flagellin export. Moreover, Maf specificity investigations, together with these interaction studies have suggested that the Maf proteins may be recognising and docking to the N-terminal region of the flagellins.
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Delannoy, Philippe. "Etude des modifications de la glycosylation de la fibronectine des cellules transformées : Conséquence sur l'affinité de la fibronectine pour le collagène." Lille 1, 1989. http://www.theses.fr/1989LIL10069.
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L'analyse comparée des glycanes de fibronectine de fibroblastes de rein de hamster normaux ou transformés par le virus h5v a montré que les glycanes majeurs de la fibronectine de fibroblaste de 1er type diffèrent du second type. Étude des activités sialyltransférases de ces cellules
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Lin, Chaoqi. "Synthesis and study of photoswitchable glycomacrocycles." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLN039.
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Grace à leur excellente propriété photochromique, les dérivés d’azobenzène trouvent de plus en plus d’applications pour le photocontrôle spatio-temporel de conformations moléculaires, de réactivités chimiques, des activités biologiques et pharmaceutiques. Dans ce contexte, introduire un motif photochromique dans les macrocycles contenant de glucides représente une approche excitante afin de moduler par la lumière les propriétés physicochimiques, chimiques et biologiques de cette unique classe de molécules qui attirent d’attention croissante en chimie médicinale et en science des matériaux.Deux séries de glycomacrocycles photocommutables ont été synthétisés dans cette thèse. La première est obtenue via une approche d’O-alkylation en one-pot, à partir 2,2’-dihydroxyazobenzène et di-O-bromoacétyl glycosides. Ces glycomacrocycles possèdent d’excellentes propriétés photochromiques, avec une haute résistance à la fatigue. Le transfert de chiralité de glucide à l’azobenzène a été observé. L’un des glycomacrocycles est capable de former d’organogels qui répond aux stimuli photo, thermique et mécanique. La seconde série de macrocycles est synthétisée via une glycosylation intramoléculaire, en utilisant dihydroxyazobenzène comme attache. Dans cette approche, l’efficacité et la stéréoselectivité de glycosylation sont étudiées en modulant la nature de liens et la configuration d’azobenzène<br>As excellent photochromic molecules, azobenzene derivatives have increasing applications in the spatial and temporal photocontrol of molecular conformation, chemical reactivity, biological and pharmacological activities. In this context, an exciting attempt is introducing a photochromic motif into carbohydrate-containing macrocycles, a unique class of products with growing attention in medicinal and material science, so as to modulate their physicochemical, chemical and biologic properties by light.Two series of photoswitchable glycomacrocycles have been prepared in this thesis. The first one is obtained from 2,2’-dihydroxyazobenzene and di-O-bromoacetyl glycosides via one-pot O-alkylation approach. These macrocycles show excellent photophromic properties with high fatigue resistance. Chirality transfer from sugar to azobenzene moiety has been observed. One of the macrocycles can form organogel that responses to photo-, thermal- and mechanical stimulus. The second series of glycomacrocycles were prepared through intramolecular glycosylation by using dihydroxyazobenzene as tether. The efficiency and the stereoselective outcome of glycosylation have been investigated by modulating the nature of the linkers and the configuration of the azobenzene tether
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Marchal, Ingrid. "Modification des capacités de glycosylation des cellules d'insectes." Phd thesis, Université des Sciences et Technologie de Lille - Lille I, 2001. http://tel.archives-ouvertes.fr/tel-00258437.
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L'utilisation thérapeutique de glycoprotéines recombinantes produites dans le système baculovirus-cellules d'insectes reste limitée par le potentiel de glycosylation de ces cellules, qui produisent essentiellement des structures O- et N-glycanniques courtes. Notre travail s'inscrit donc dans un effort global d'"humanisation" de la glycosylation des protéines produites dans les cellules de Lépidoptères.<br />Nous nous sommes d'abord attachés à comprendre la voie de maturation des N-glycannes dans des cellules Sf9. L'utilisation d'inhibiteurs de la maturation ou du trafic intracellulaire nous a permis d'identifier des intermédiaires clés et de confirmer l'hypothèse que la maturation des N-glycannes dans les cellules d'insectes et de mammifères suivent un chemin métabolique parallèle jusqu'à la formation de l'espèce GlcNAcMan3[Fuc]GlcNAc2. Chez les insectes, cette structure est ensuite substrat d'une β-N-acétylglucosaminidase qui produit l'espèce finale Man3[Fuc]GlcNAc2.<br />Cette voie de maturation peut néanmoins être déviée vers la synthèse de N-glycannes de type complexe par l'addition de glycosyltransférases absentes : ainsi, l'expression d'une β1,4-galactosyltransférase permet la synthèse de l'espèce GalGlcNAcMan3[Fuc]GlcNAc2.<br />Notre intérêt s'est ensuite porté sur l'ingénierie de la sialylation dans les cellules d'insectes, compliquée par l'absence du donneur d'acides sialiques, le CMP-Neu5Ac. Notre stratégie a été d'exprimer la trans-sialidase de Trypanosoma cruzi sur la membrane plasmique des cellules, afin qu'elle puisse sialyler les glycoprotéines sécrétées en utilisant des donneurs du milieu. La construction exprimée grâce à un vecteur baculovirus code une enzyme active, dont une partie est retrouvée sur la membrane plasmique et sur l'enveloppe des particules virales, tandis qu'une partie, croissante avec l'infection, est soluble. Néanmoins, le système permet une sialylation quantitative d'accepteurs exogènes.<br />Notre étude contribue à montrer que l'ingénierie de la glycosylation dans le système baculovirus-cellules d'insectes est envisageable. Pour résoudre le problème de la sialylation, la trans-sialidase est une alternative possible aux sialyltransférases.
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Alonzi, Dominic S. "Cellular Mechanism of Glycosylation Inhibition by Imino Sugars." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487138.
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Glycosylation is the most common posttranslational modification of proteins. It is a complex process involving many functional proteins and resulting'in a great diversity of structures. The retention of glucose residues on N-linked oligosaccharides following ER a-glucosidase inhibition by imino sugars such as N-butyl deoxynojirimycin, increases the protein misfolding and the amount of glycoprotein destined for degradation via the endoplasmic reticulum associated degradation (ERAD) pathway. Intracellular peptide N-glycosidases (PNGases) act generating free oligosaccharides (FOS) and the protein is targeted for digestion. Free oligosaccharides were extracted from cells treated with NB-DNJ and subjected to ion-exchange chromatography before fluorescence labelling with 2-AA (anthranilic acid) and lectin-affinity chromatography. Separation of labelled FOS by NP-HPLC provided a rapid and sensitive method for the detection of all FOS species resulting from the degradation of glycoprotein exported from the ER. A. robust, cellular-based assay for ER a-glucosidase activity in the presence of inhibitor was developed that provided meaningful kinetics for a-glucosidase-mediated hydrolysis ofN-linked oligosaccharides as proteins are folded in the ER. Furthermore, the origin of FOS generation was studied, to determine the relative amount of FOS produced as a result of ERAD (protein derived) relative to the amount produced by a possible hydrolytic activity of oligosaccharyltransferase (lipid linked derived). A dual localisation of PNGase activity and non-proteasomal and proteasomal ERAD were demonstrated. Analysis of FOS in endomannosidase negative/non-utilised cells revealed the extent of ER/Golgi recycling of glycoproteins and the role of this enzyme in qua~ity control pathways in cells. Oral administration of NB-DNJ results in the production of glucosylated free oligosaccharide in vivo. Further to cellular based assays these glucosylated free oligosaccharides were detected in murine and human samples. The observed differences in the free oligosaccharides produced in different tissues can be explained according to hypothesises generated from culture cell studies. The effects observed with NB-DNJ, a therapeutic with considerable potential for treating lysosomal storage disorders and reducing viral infectious processes, was dose and time dependent so enabling the pharmokinetics of NB-DNJ to be observed. These studies also elucidated a transrenal method of clearance of glucosylated FOS and demonstrate that glucosylated FOS are excellent non-invasive in vivo biomarkers for a-glucosidase inhibition and protein misfolding in the ER.
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Rosenlöcher, Julia [Verfasser]. "Optimizing the glycosylation of recombinant proteins / Julia Rosenlöcher." Berlin : Freie Universität Berlin, 2016. http://d-nb.info/1112133437/34.
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Tey, Lai-Hock. "Studies of site-selective glycosylation of dihydrofolate reductase." Thesis, Cardiff University, 2008. http://orca.cf.ac.uk/55798/.
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Glycosylation is important for many molecular processes, although how glycosylation contributes to glycoprotein structure and function is not entirely clear. Other than that, the study of many of these events is complicated by the fact that natural glycoproteins normally occur as mixtures of glycoforms, therefore the isolation or synthesis of homogenous glycoproteins is an important task. Previous studies on deglycosylated proteins have shown that glycosylation reduces their catalytic activity and increase thermal stability. Therefore, we hypothesize that similar effects may be observed in the naturally unglycosylated dihydrofolate reductase from Escherichia coli (EcDHFR). Four different surface-exposed sites for the incorporation of a single cysteine residue were selected based on the protein crystal structure, which may or may not affect its dynamics. Homogeneous glycoproteins have been synthesized via chemoselective ligation of a glycosyl haloacetammide with the thiol of a cysteine, to produce site- selectively glycosylated forms of EcDHFR. Techniques such as mass spectrometry, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, ultraviolet (UV) visible spectroscopy and stopped-flow spectrophotometry have been used to identify and study the physical properties of different glycoforms of DHFR. Although there were some changes of the kinetic activity of the mutants of EcDHFR. the values were comparable to those of the wild-type protein. Interestingly, in four of the five cases studied. EcDHFRDM D87C, there was an increase in thermal stability upon site-selective glycosylation. The other mutants showed no effect. With the exception of the effect seen for the thermal stability of the D87C mutant, this is not in accordance with the original hypothesis. This suggest that the effect seen in the D87C mutant may be due to specific interactions of the carbohydrate moiety at certain points on the protein. An increase in resistance to thermal denaturation observed for proteins in sugar solutions may therefore also be due to binding of the sugars to specific sites on the protein. In conclusion, an effective method for the synthesis of homogeneous glycosylated and non-glycosylated proteins has been developed and applied to the site selective glycosylation of EcDHFR. The results also suggested that the kinetic properties of EcDHFR are not significantly affected by glycosylation. It may be the large effects in terms of protein stability which due to glycosylation only occur in naturally glycosylated proteins, and not in the naturally unglycosylated EcDHFR.
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Hamid, Umi Marshida Abd. "Glycosylation-based approaches for potential breast cancer biomarkers." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.540155.
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Anderson, Ross C. "Immunoassays for glycosylation of alpha-1-acid glycoprotein." Thesis, University of Sunderland, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337295.
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Fontinopoulou, Angeliki. "Investigating the glycosylation of recombinant glycoproteins with lectins." Thesis, University of Newcastle Upon Tyne, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432779.
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Jowett, Adrian K. "Glycosylation patterns during development of mouse molar teeth." Thesis, University of Manchester, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304035.
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