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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
11

Over Expression of the CMP-sialic Acid Transporter in Chinese Hamster Ovary Cells Leads to Increased Sialylation

Wong, Niki S.C., Yap, Miranda G.S., Wang, Daniel I.C. 01 1900 (has links)
Most glyco-engineering approaches used to improve quality of recombinant glycoproteins involve the manipulation of glycosyltransferase and/or glycosidase expression. We investigated whether the over expression of nucleotide sugar transporters, particularly the CMP-sialic acid transporter (CMP-SAT), would be a means to improve the sialylation process in CHO cells. We hypothesized that increasing the expression of the CMP-SAT in the cells would increase the transport of the CMP-sialic acid in the Golgi lumen, hence increasing the intra-lumenal CMP-sialic acid pool, and resulting in a possible increase in sialylation extent of proteins being produced. We report the construction of a CMP-SAT expression vector which was used for transfection into CHO-IFNγ, a CHO cell line producing human IFNγ. This resulted in approximately 2 to 5 times increase in total CMP-SAT expression in some of the positive clones as compared to untransfected CHO-IFNγ, as determined using real-time PCR analysis. This in turn concurred with a 9.6% to 16.3% percent increase in site sialylation. This engineering approach has thus been identified as a novel means of improving sialylation in recombinant glycoprotein therapeutics. This strategy can be utilized feasibly on its own, or in combination with existing sialylation improvement strategies. It is believed that such multi-prong approaches are required to effectively manipulate the complex sialylation process, so as to bring us closer to the goal of producing recombinant glycoproteins of high and consistent sialylation from mammalian cells. / Singapore-MIT Alliance (SMA)
12

Design and Synthesis of Sialic Acid Conjugates as Inhibitors of EKC-causing Adenoviruses

Johansson, Susanne January 2008 (has links)
The combat against viral diseases has been, and still is, a major challenge in the field of drug development. Viruses are intracellular parasites that use the host cell ma-chinery for their replication and release. Therefore it is difficult to target and destroy the viral particle without disturbing the essential functions of the host cell. This thesis describes studies towards antiviral agents targeting adenovirus type 37 (Ad37), which causes the severe ocular infection epidemic keratoconjunctivitis (EKC). Cell surface oligosaccharides serve as cellular receptors for many pathogens, including viruses and bacteria. For EKC-causing adenoviruses, cell surface oligo-saccharides with terminal sialic acid have recently been shown to be critical for their attachment to and infection of host cells. The work in this thesis support these re-sults and identifies the minimal binding epitope for viral recognition. As carbo-hydrate–protein interactions in general, the sialic acid–Ad37 interaction is very weak. Nature overcomes this problem and vastly improves the binding affinity by presenting the carbohydrates in a multivalent fashion. Adenoviruses interact with their cellular receptors via multiple fiber proteins, whereby it is likely that the ideal inhibitor of adenoviral infections should be multivalent. This thesis includes design and synthesis of multivalent sialic acid glycoconjugates that mimic the structure of the cellular receptor in order to inhibit adenoviral attachment to and infection of human corneal epithelial (HCE) cells. Synthetic routes to three different classes of sialic acid conjugates, i.e. derivatives of sialic acid, 3’-sialyllactose and N-acyl modified sialic acids, and their multivalent counterparts on human serum albumine (HSA) have been developed. Evaluation of these conjugates in cell binding and cell infectivity assays revealed that they are effective as inhibitors. Moreover the results verify the hypothesis of the multivalency effect and clearly shows that the power of inhibition is significantly increased with higher orders of valency. Potential inhibi-tors could easily be transferred to the eye using a salve or eye drops, and thereby they would escape the metabolic processes of the body, a major drawback of using carbohydrates as drugs. The results herein could therefore be useful in efforts to develop an antiviral drug for treatment of EKC.
13

Diagnostic and Prognostic Capacity of Serum Glycan Nodes in Different Types of Cancer

January 2018 (has links)
abstract: Glycans are monosaccharide-based heteropolymers that are found covalently attached to many different proteins and lipids and are ubiquitously displayed on the exterior surfaces of cells. Serum glycan composition and structure are well known to be altered in many different types of cancer. In fact, glycans represent a promising but only marginally accessed source of cancer markers. The approach used in this dissertation, which is referred to as “glycan node analysis”, is a molecularly bottom-up approach to plasma/serum (P/S) glycomics based on glycan linkage analysis that captures features such as α2-6 sialylation, β1-6 branching, and core fucosylation as single analytical signals. The diagnostic utility of this approach as applied to lung cancer patients across all stages as well as prostate, serous ovarian, and pancreatic cancer patients compared to certifiably healthy individuals, nominally healthy individuals and/or risk-matched controls is reported. Markers for terminal fucosylation, α2-6 sialylation, β1-4 branching, β1-6 branching and outer-arm fucosylation were most able to differentiate cases from controls. These markers behaved in a stage-dependent manner in lung cancer as well as other types of cancer. Using a Cox proportional hazards regression model, the ability of these markers to predict progression and survival in lung cancer patients was assessed. In addition, the potential mechanistic role of aberrant P/S glycans in cancer progression is discussed. Plasma samples from former bladder cancer patients with currently no evidence of disease (NED), non-muscle invasive bladder cancer (NMIBC), and muscle invasive bladder cancer (MIBC) along with certifiably healthy controls were analyzed. Markers for α2-6 sialylation, β1-4 branching, β1-6 branching, and outer-arm fucosylation were able to separate current and former (NED) cases from controls; but NED, NMIBC, and MIBC were not distinguished from one another. Markers for α2-6 sialylation and β1-6 branching were able to predict recurrence from the NED state using a Cox proportional hazards regression model adjusted for age, gender, and time from cancer. These two glycan features were found to be correlated to the concentration of C-reactive protein, a known prognostic marker for bladder cancer, further strengthening the link between inflammation and abnormal plasma protein glycosylation. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2018
14

Identification of the molecular origins of disease in a cohort of patients with suspected congenital disorders of glycosylation (CDG) / Identification de l'origine moléculaire d'une maladie dans un groupe de patients atteints de troubles congénitaux de la glycosylation

Sabry Zaki Tlep, Sahar 29 November 2016 (has links)
Contexte : Les désordres congénitaux de la glycosylation (CDGs) sont des maladies rares dues à des mutations dans des gènes codant pour des protéines de la biosynthèse des glycoconjugués. Les CDGs présentent avec des glycoprotéines sériques hypoglycosylées avec un spectre clinique large. Le diagnostic moléculaire des CDG est important dans le cadre du diagnostic prénatal et du développement de stratégies thérapeutiques. Objectif : Déterminer les mutations causales dans une cohorte de cas suspects de CDG. Deux cas ont fait l’objet d’explorations biochimiques afin de comprendre les conséquences des mutations et d’envisager des stratégies thérapeutiques. Sujets/méthodes : Des explorations biochimiques sur des fibroblastes cutanés d’une cohorte de patients présentant des signes cliniques suggérant un CDG et hypglycosylation des protéines sérique. Résultats et conclusions: Le premier patient présentait une maladie multisystémique sévère. Des mutations affectant le gène codant pour la dehydrodolichol diphosphate synthase (DHDDS) ont été trouvées. Une activité diminuée la DHDDS était accompagnée de la diminution du dolichol phosphate. Ce patient est le premier cas de DHDDS-CDG présentant une atteinte multi-viscérale. Dans une deuxième étude deux siblings présentaient une thrombopénie associée à des atteintes neurologiques. Une mutation bi-allélique dans le gène codant pour le transporteur golgien du CMP-acide sialique (SLC35A1) associé avec une hypoglycosylation des protéines sériques a été détectée. Des profils anormaux des glycosphingolipides ont été mis en évidence et supplémentation des cellules de patient par de l’acide sialique a augmenté la biosynthèse des gangliosides. / Background: Congenital disorders of glycosylation (CDGs) are rare inherited diseases caused by mutations in genes required for glycoconjugate biosynthesis. CDG clinical presentations range from monosystemic to multiorgan failure. Often these diseases are diagnosed biochemically by the presence of hypoglycosylated serum proteins. Molecular diagnosis of CDG is crucial for both antenatal diagnostics and development of treatment strategies. Aims: To determine the molecular origins of disease in suspected CDG patients. Two cases were chosen for more extended biochemical explorations in order to investigate the consequences of the mutations and possible treatment strategies. Subjects/Methods: Biochemical explorations of skin biopsy fibroblasts from a cohort of patients presenting with signs suggestive of CDG, and serum protein hypoglycosylation. Results and conclusions: In the first study, a patient presented with multisystemic disease suggesting CDG. Fibroblasts revealed both truncated dolichol-linked oligosaccharides and polymannose-type N-glycans. Mutations in the dehydrodolichol diphosphate synthase (DHDDS) gene were found as well as low DHDDS activity and dolichol phosphate levels. As previous cases of DHDDS-CDG present with retinitis pigmentosa only, we describe the first case of a CDG syndrome associated with mutations in DHDDS. In the second study, two siblings presented with thrombocytopenia and CNS signs. A biallelic mutation in the CMP-sialic acid transporter gene (SLC35A1) was associated with hyposialylated serum glycoproteins. Altered glycosphingolipid profiles were seen and sialic acid supplementation of patient cells increased the appearance of gangliosides
15

Pathogenicity of IgG-Fc desialylation and its association with Th17 cells in an animal model of systemic lupus erythematosus / 全身性エリテマトーデスの動物モデルにおけるIgG-Fc脱シアル化の病原性とTh17細胞との関連

Nishida, Yuri 23 January 2024 (has links)
京都大学 / 新制・課程博士 / 博士(医学) / 甲第24994号 / 医博第5028号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 上野 英樹, 教授 椛島 健治, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
16

Effect of Sialylation of Histophilus somni Lipooligosaccharide on Virulence and Resistance to Host Defenses

Balyan, Rajiv 19 September 2007 (has links)
Incorporation of N-acetyl neuraminic acid (NANA), or sialic acid, onto lipooligosaccharide (LOS) enhances the virulence of several bacterial species. In the present study, we assessed the effect of sialylation of Histophilus somni LOS on complement-mediated killing, binding of complement factor H (which converts C3b to inactive C3b (iC3b) and inhibit the alternative complement pathway) to the bacteria, complement activation by the LOS, and phagocytosis and killing of the bacteria by bovine polymorphonuclear leukocytes (PMN). Killing of H. somni by alternative complement pathway was measured by incubation of sialylated or non-sialylated H. somni with antibody-free precolostral calf serum (PCS) followed by viable plate count. A complement dose-dependent response to killing of non-sialylated H. somni by PCS was observed. However, sialylated H. somni were significantly (P = 0.001) more resistant to killing at any of the concentrations of PCS used. Sialylated H. somni LOS activated (P = 0.025) and consumed (P = 0.001) less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized sheep red blood cells or rabbit red blood cells, and by western blotting of C3 activation products. Sialylated H. somni bound more factor H than non-sialylated bacteria (determined by enzyme-linked immunosorbent assay) (P = 0.004), supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni inhibited both PMN phagocytosis of 3H-thymidine-labelled bacteria (P = 0.004) and intracellular killing of the bacteria (P = 0.0001), compared to non-sialylated bacteria. Therefore, sialylation of the LOS results in enhanced binding of complement factor H to the bacteria, resulting in diminished complement activation, resistance to complement-mediated lysis, and PMN phagocytosis and killing. / Master of Science
17

Investigation of Haemophilus somnus Virulence Factors: Lipooligosaccharide Sialylation and Inhibition of Superoxide Anion Production

Howard, Michael D. 20 April 2005 (has links)
Virulent strains of the bovine opportunistic pathogen Haemophilus somnus (Histophilus somni) cause multi-systemic diseases in cattle. One of the reported virulence factors that H. somnus may use to persist in the host is resistance to intracellular killing. It is reported in this dissertation that H. somnus significantly (P <0.001) inhibited production of superoxide anion (O2-) by bovine mammary and alveolar macrophages as well as by polymorphonuclear leukocytes. Inhibition of O2- production was time- and dose-dependent and did not occur after incubation with Escherichia coli, H. influenzae, or Brucella abortus. Non-viable H. somnus, purified lipooligosaccharide (LOS), or cell-free supernatant from mid-log phase cultures did not inhibit O2- production, indicating that O2- inhibition required contact with live H. somnus. Commensal isolates of H. somnus were less capable or incapable of inhibiting macrophage O2- production compared to isolates tested from disease sites. H. somnus shares conserved epitopes in its LOS with Neisseria gonorrhoeae, N. meningitidis, and H. influenzae, and can also undergo structural phase variation of these LOS epitopes. Sialylation of the terminal galactose of H. somnus LOS is another reported virulence mechanism. Current sequencing of the genomes of H. somnus strains 2336 (pathogenic) and 129Pt (commensal) has enabled in silico identification of three open reading frames (ORFs) involved in sialylation. The ORFs-1 (hsst-I) and -2 (hsst-II) had BLASTx homology to sialyltransferases, while ORF-3 (neuAhs) had BLASTx homology to CMP-sialic acid synthetases. These ORFs were amplified by PCR and cloned into the expression vector pCWOri+. Thin layer chromatography of the hsst-I gene product showed this sialyltransferase exhibited preference for sialylation of terminal N-acetyllactosamine (LacNAc, beta-Gal-[1,4]-beta-GlcNAc-R). However, Hsst-II preferentially sialylated lacto-N-biose (LNB, beta-Gal-[1,3]-beta-GlcNAc-R). In this study, phase variation of the terminal linkage in isolate 738 from a 3 linked galactose (LNB) to a 4 linked galactose (LacNac) was demonstrated. Such variation of a glycose linkage appears to be a novel mechanism of LOS phase variation. Furthermore, the ability of sialylated strain 738 LOS vs de-sialylated strain 738 LOS to induce Toll-like receptor 4 signaling was decreased by 28%, as determined by ELISA for Macrophage Inflammatory Protein-2. Therefore, sialylated LOS may aid H. somnus to avoid host innate immunity. / Ph. D.
18

Modification des capacités de glycosylation des cellules d'insectes

Marchal, Ingrid 21 September 2001 (has links) (PDF)
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.
19

Immunomodulatory properties of IgG glycosylation and the anti-inflammatory mechanism of intravenous immunoglobulin

Yu, Xiaojie January 2013 (has links)
The IgG Fc domain mediates a range of antibody effector functions, including antibody dependent cell-mediated cytotoxicity (ADCC), complement activation, phagocytosis, and the recently emerged general anti-inflammatory effect of immunoglobulin therapy (IVIg). The conserved N-glycan attached to Fc N297 maintains the Fc structural integrity for the effector functions, while its glycoform is known to modulate the affinity for the Fc γ-receptors (FcγRs), complement, and the C-type lectin DC-SIGN. IgG Fc exhibits protein-directed glycosylation characterized by a series of biantennary complex type glycoforms, with a small population of sialylated species. The sialylated Fc has been proposed to bind DC-SIGN and initiate an anti-inflammatory signalling pathway. The restricted Fc glycan processing is partially attributed to the hydrophobic interaction between Fc glycan and the hydrophobic Fc protein backbone. Mutations within the hydrophobic Fc protein-glycan interface dramatically increases Fc glycan processing, while concomitantly decreases Fc affinity for the FcγRs. However, it is unclear whether this disrupted Fc-FcγR interaction was due to the increased terminal glycan processing, or the perturbed Fc protein-glycan interface. Here, the integrity of the Fc protein-glycan interface was demonstrated to be important in maintaining the productive Fc-FcγR interaction independently of glycoform. This glycoform-independent effect was exploited to generate novel inhibitory Fc variants. In addition, the interaction between sialylated IgG and the putative IVIg receptor DC-SIGN was re-evaluated. Analysis shows that IVIg binds DC-SIGN in a glycan-independent, Fab-mediated manner. Furthermore, the effect of IVIg sialylation on human antigen presenting cells was examined; evidence presented here indicate that IVIg deglycosylation, not desialylation, has an anti-inflammatory effect on human dendritic cells (DCs). These data suggest the need for a general re-evaluation of the current mechanistic model of anti-inflammatory IVIg.
20

Production d'IgG sialylées en CHO et impact sur leurs fonctions effectrices

Raymond, Céline 10 1900 (has links)
La sialylation des N-glycanes du fragment Fc des immunogobulines G (IgG) est une modification peu fréquente des IgG humaines. Pourtant, elle est l’objet de beaucoup d’attention depuis que deux articles fondateurs ont été publiés, qui montrent l’un que la sialylation des IgG diminue leur capacité à déclencher la cytotoxicité cellulaire dépendant de l’anticorps (ADCC), et l’autre que les IgG sialylées en α2,6 seraient la fraction efficace des IgG intraveineuses (IgIV) anti-inflammatoires. Les anticorps monoclonaux thérapeutiques, qui sont le plus souvent des IgG recombinantes produites en culture de cellules de mammifère, connaissent depuis la fin des années 90 un succès et une croissance phénoménaux sur le marché pharmaceutique. La maîtrise de la N-glycosylation du Fc des IgG est une clé de l’efficacité des anticorps monoclonaux. Si les IgG sialylées sont des molécules peu fréquentes in vivo, elles sont très rares en culture cellulaire. Dans cette étude, nous avons développé une méthode de production d’IgG avec une sialylation de type humain en cellules CHO. Nous avons travaillé principalement sur la mise au point d’une stratégie de production d’IgG sialylées par co-expression transitoire d’une IgG1 avec la β1,4-galactosyltransférase I (β4GTI) et la β-galactoside-α2,6-sialyltransférase I (ST6GalI). Nous avons montré que cette méthode permettait d’enrichir l’IgG1 en glycane fucosylé di-galactosylé mono-α2,6-sialylé G2FS(6)1, qui est le glycane sialylé présent sur les IgG humaines. Nous avons ensuite adapté cette méthode à la production d’IgG présentant des profils de glycosylation riches en acides sialiques, riches en galactose terminal, et/ou appauvris en fucosylation. L’analyse des profils de glycosylation obtenus par la co-expression de diverses combinaisons enzymatiques avec l’IgG1 native ou une version mutante de l’IgG1 (F243A), a permis de discuter des influences respectives de la sous-galactosylation des IgG1 en CHO et des contraintes structurales du Fc dans la limitation de la sialylation des IgG en CHO. Nous avons ensuite utilisé les IgG1 produites avec différents profils de glycosylation afin d’évaluer l’impact de la sialylation α2,6 sur l’interaction de l’IgG avec le récepteur FcγRIIIa, principal récepteur impliqué dans la réponse ADCC. Nous avons montré que la sialylation α2,6 augmentait la stabilité du complexe formé par l’IgG avec le FcγRIIIa, mais que ce bénéfice n’était pas directement traduit par une augmentation de l’efficacité ADCC de l’anticorps. Enfin, nous avons débuté le développement d’une plateforme d’expression stable d’IgG sialylées compatible avec une production à l’échelle industrielle. Nous avons obtenu une lignée capable de produire des IgG enrichies en G2FS(6)1 à hauteur de 400 mg/L. Cette étude a contribué à une meilleure compréhension de l’impact de la sialylation sur les fonctions effectrices des IgG, et a permis d’augmenter la maîtrise des techniques de modulation du profil de glycosylation des IgG en culture cellulaire. / Only a fraction of the N-glycans present on the Fc fragment of the human IgGs is sialylated. However, a new interest for sialylation has risen since two major articles were published, one showing that sialylation reduces the capacity of the antibody to trigger antibody-dependent cell cytotoxicity (ADCC), whereas the other showed that the IgGs carrying α2,6-sialic acids on their Fc N-glycans were responsible for the anti-inflammatory activity of intravenous immunoglobulins (IVIGs) injected at high doses. Therapeutic monoclonal antibodies (mAbs) are in majority recombinant IgGs produced in mammalian cell culture. Since the end of the nineties, mAbs have become a major class of pharmaceutical products, and their success is still growing. The control of Fc N-glycosylation is a key parameter for the improvement of the therapeutic efficacy of mAbs. Sialylated IgGs are found only as traces in the classic CHO cell culture processes. In this study, we developed a method for the production of IgGs with a human-like sialylation in CHO cells. We focused on a production strategy relying on the transient co-expression of an IgG1 with the β1,4-galactosyltransferase I (β4GTI) and the β-galactoside-α2,6-sialyltransferase I (ST6GalI). We showed that this method allowed the enrichment of the IgG1 glycoprofile in the fucosylated di-galactosylated mono-α2,6-sialylated glycane G2FS(6)1, which is the main sialylated glycan found in human IgGs. We then adapted this method to the production of highly galactosylated or highly sialylated IgGs with and without core-fucosylation. The analysis of the glycosylation profiles obtained using the various enzyme combinations co-expressed with the native IgG1 or the mutant IgG1 F243A allowed us to discuss the influence of the under-galactosylation found in IgGs produced in CHO cells versus the Fc structural constraints on the limitation of IgG sialylation in CHO cells. We used the IgG1 glycovariants produced with our method to assess the impact of Fc α2,6-sialylation on the interaction of the IgG with the receptor FcγRIIIa, which is the main receptor mediating the ADCC response. We showed that the presence of α2,6-sialylation in the Fc increased the stability of the IgG-FcγRIIIa complex. This benefit however did not translate into an improved ADCC capacity. Finally, we initiated the development of a stable expression platform for the production of sialylated IgGs at yields relevant for the industry. We obtained a cell line capable of producing IgGs enriched in G2FS(6)1 at 400 mg/L. This may eventually represent a novel approach to manufacture a recombinant IVIG surrogate. With this work, we contributed to a better understanding of the impact of sialylation on the effector functions of IgGs. We also improved our understanding of the techniques allowing for the modification and control of the glycosylation profile of IgGs in cell culture.

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