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1
Smelt, Kathryn Helena. "Synthesis of L-fucose analogues." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362080.
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Yuan, Kun. "Effects of defucosylation on human breast cancer cells." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2010r/yuan.pdf.
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McCabe, N. R. "Training and fucose metabolism in chick brain." Thesis, Open University, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355645.
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Doknic, D. "SYNTHESIS OF FUCOSE-BASED LIGANDS FOR DC-SIGN." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/203241.
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DC-SIGN is a C-type lectin that is expressed in dendritic cells and that is involved in a number of infection processes such as HIV or Ebola. Within the scope of this work a library of monovalent, fucose-based ligands was synthesised to block DC-SIGN. The structures originate from three scaffolds containing 2-aminocyclohexane carboxylic acid in different configurations. The compounds were tested by means of SPR competition assays and were found to be similar to the natural ligand Lewis X in terms of affinity for DC-SIGN. On the one hand, the most potent ligand, and on the other hand, the most accessible ligand were selected for further functionalisation and polyvalent presentation by the attachment to dendrimers. The increased valency resulted in an improved affinity by up to one order of magnitude in comparison with the monomeric structure. Moreover, the functionalised monomers were used for the fabrication of glycan arrays and were screened with several commercially available lectins. Strongest binding could be detected with the lectin from the bacterial phytopathogen Ralstonia solanacearum.
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Sanz, Sender Silvia. "Synthesis and biological function of fucose in Plasmodium falciparum." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/587108.
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Malaria is a parasitic disease caused by Plasmodium parasites and it is transmitted by female Anopheles mosquito. P. falciparum has a complex life cycle that includes important stages in two different hosts: a mosquito and a human. The transmission between the human and the mosquito host also involves the transition between asexual and sexual forms of the parasites.
Glycobiology includes the study of carbohydrate metabolism and glycoconjugate (glycoprotein and glycolipid) structures. Protozoan parasites synthesize different glycoconjugates for protection and to respond to changes in the environment. Glycoconjugates coat the parasite surface with carbohydrates generally different from the host ones. They are crucial for parasite virulence and survival. Until very recently the only glycan structures described in P. falciparum were the GPI-anchors, however other glycan structures have been found in the past few years as the N-glycans or C-mannosylation. The glycome consists in the complete set of glycosylations that an organism or a cell produces at a certain time point, therefore the description of the parasite glycome may help to understand better the host- pathogen interactions in parasitic diseases. Sugar nucleotides are activated forms of monosaccharides that are the donors of glycosyltransferases to form glyconjugates. They can be synthesized by a de novo pathway that consists in the bioconversion of an existing sugar or sugar nucleotide; or by a salvage pathway that involves an activation and a further pyrophosphorylation. The identification and quantification of the sugar nucleotides present in malaria parasites may help to describe its glycosylation profile.
The first paper presented in the thesis describes the identification and quantification of the sugar nucleotides present in the parasite, among which we found: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. We also investigated the salvage pathways present in the parasite but we couldn’t elucidate the presence of a fucose salvage pathway. Plasmodium parasites conserve homolog genes for the de novo biosynthetic pathway of GDP-Fuc: GMD and FS. We were able to prove the in vitro activity of GMD and FS enzymes and to show that both enzymes are required for the synthesis of fucose. GMD and FS are expressed along the intraerythrocytic life cycle and both enzymes localize in the cytoplasm of the parasite, as well as other parasite enzymes related with carbohydrate metabolism. The expression of a putative O-fucosyltransferase (PoFUT2) present in the parasite genome, together with the uptake of GDP-Fuc by parasite extracts suggested the presence of a fucose containing glycan.
In the second paper, we characterized the enzymes responsible for the synthesis of GDP-Fuc, GMD and FS. We disrupted both genes in the parasite and analyzed the sugar nucleotide content present in the parasite. After GMD and FS disruption, GDP-Fuc was still detected in the parasite and no evidence of salvage mechanism was found. We described indirect evidence of a fucose containing glycan that was abrogated after the disruption of GMD.
The last work here presented (not yet published) tries to characterize the enzyme that is probably responsible for the transfer of fucose to the glycoconjugate. We disrupted, by double crossover recombination, PoFUT2 gene in the human and in the rodent malaria parasite. The disruption of PoFUT2 does not have any significant effect for the viability and growth of the parasite along the parasite cycle in the human and in the mosquito host.
These works open the door to new research lines to find an alternative pathway for obtaining fucose or GDP-Fuc. Obtaining evidence of the glycosylation state of PoFUT2 mutants and the characterization of other possible glycosylation reaction present in the parasite are other research topics to investigate.La malaria está causada por el parásito Plasmodium y se transmite mediante hembras del mosquito Anopheles. La glicobiología es el estudio de los procesos relacionados con los carbohidratos y las estructuras glicoconjugadas que forman. Los parásitos sintetizan glicoconjugados o proteínas de unión a glicanos, y muchas veces se encargan de mediar las interacciones huésped-patógeno. Los azúcares nucleótidos son formas activadas de monosacáridos que son usados por glicosiltransferasas para formar glicoconjugados. La identificación y cuantificación de estos azúcares nucleótidos en el parásito de la malaria puede contribuir a la definición de su perfil de glicosilación. El primer trabajo presentado en la tesis permitió identificar los azúcares nucleótidos presentes en el parásito, así como la posible presencia de un glicoconjugado que contenga fucosa, sintetizado a partir de la actividad O-fucosiltransferasa de una proteína homóloga anotada en el genoma del parásito, PoFUT2. El siguiente trabajo permitió caracterizar las enzimas implicadas en la síntesis de la GDP-fucosa, GMD y FS. Este artículo nos permitió mostrar evidencias indirectas de la presencia del glicococonjugado que contiene fucosa. A partir de la disrupción de los genes de biosíntesis descubrimos que el contenido de GDP-fucosa en parásitos mutantes no variaba con respecto a los parásitos salvajes. Sin embargo, la síntesis del gliconjugado sí que se reducía. El tercer trabajo (sin publicar) se centra en la caracterización de la enzima encargada de transferir la fucosa al glicoconjugado. La disrupción de PoFUT2 no parece tener ningún efecto en la viabilidad y crecimiento del parásito a lo largo del ciclo de éste en el humano y en el mosquito. Estos trabajos abren la puerta a nuevas investigaciones para descubrir una vía alternativa de obtención de GDP-Fucosa. La obtención de evidencias del estado de glicosilación de los mutantes de PoFUT2 y la caracterización de otras posibles glicosilaciones son otros temas a investigar.
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Bandini, Giulia. "Studies on fucosylation in Trypanosoma brucei." Thesis, University of Dundee, 2011. https://discovery.dundee.ac.uk/en/studentTheses/c74554c1-f4d3-4bb3-aa31-899fcf507e11.
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The biosynthesis of GDP-Fucose, the activated donor for fucose, has been recently shown to be essential in the parasite Trypanosoma brucei. Fucose is a common sugar modification on eukaryotic glycan structures, but it has not been well described in trypanosomatids. To elucidate the role of fucose in T. brucei we searched for putative fucosyltransferases in this parasite. A single putative T. brucei fucosyltransferase (TbFT) was identified and recombinantly expressed in Escherichia coli. The protein was active and structural characterization of its reaction product identified it as a GDP-Fuc: ß-D-galactose a-1,2-fucosyltransferase with preference for Galß1,3GlcNAc containing structures as glycan acceptors. A procyclic form conditional null mutant for TbFT was generated and this glycosyltransferase shown to be essential for parasite growth in vitro, with the mutant cells displaying a slightly abnormal morphology and an apparent reduction in the surface high molecular weight glycoconjugate complex. Here we also describe the various experimental approaches that were used to try to identify the fucosylated glycocojugates in T. brucei. Lastly, to better understand the biosynthesis of GDP-Mannose, the starting metabolite for the biosynthesis of GDP-Fuc, we biochemically characterized T. brucei phosphomannomutase (TbPMM). Here we show this enzyme could interconvert not only mannose-phosphates, but also glucose-phosphates.
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Muiry, Jennifer Anne Ross. "The bacterial transport systems for L-rhamnose and L-fucose." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315190.
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Yao, David C. "Roles of O-fucose Molecules in Notch Signaling and Hematopoiesis." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1311379342.
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Weidner, Stefan. "Enzymatische Synthese von GDP-[beta]-L-Fucose [GDP-beta-L-Fucose] ausgehend von D-Mannose Klonierung, Expression und Charakterisierung von Enzymen aus nicht-pathogenen Enterobacteriaceae /." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=972659331.
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Staib, Lena [Verfasser], Thilo M. [Akademischer Betreuer] [Gutachter] Fuchs, and Hannelore [Gutachter] Daniel. "Investigation of propanediol and fucose degradation by Salmonella Typhimurium / Lena Staib ; Gutachter: Hannelore Daniel, Thilo M. Fuchs ; Betreuer: Thilo M. Fuchs." München : Universitätsbibliothek der TU München, 2016. http://d-nb.info/1121206808/34.
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Camirand, Anne. "Glycosyltransferases from pea membranes : glucose and fucose incorporation into cell wall polysaccharides." Thesis, McGill University, 1986. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75336.
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Synthesis from UDP-($ sp{14}$C) glucose of charged lipid-linked glucosyl compounds by pea membranes was short-lived, and of very limited magnitude compared to the synthesis of 1,4- and 1,3-linked B-glucans. Lipid-linked monophosphoryl glucose was the only charged lipid formed at initial stages, and had properties similar to that of dolichol-monophosphoryl glucose. It exhibited no turnover during pulse-chase experiments. Lipid-linked pyrophosphoryl-glucose or -oligosaccharides were not detected. Coumarin inhibited the synthesis of SDS-soluble products and glucans, but not of the lipid-P-glucose. Transfer of the label from endogeneous lipid-P-($ sp{14}$C) glucose or from dolichol-P-($ sp3$H) glucose into non-lipid products was minimal. It was concluded that the lipid-linked phosphoryl saccharide formed from UDP-glucose was not an obligate intermediate in the formation of B-glucans in pea membranes.Fucose-containing lipid-linked intermediates were not involved in the biosynthesis of xyloglucans. However, pea microsomal membranes catalysed the transfer of $ lbrack sp{14}{ rm C} rbrack$-fucose from GDP-$ lbrack sp{14}$C) fucose, with or without added unlabelled UDP-glucose, UDP-xylose or UDP-galactose, to an insoluble product with properties characteristic of xyloglucan. After digestion of the ethanol-insoluble pellet with Streptomyces griseus endocellulase, $ lbrack sp{14}$C) fucose residues occurred exclusively in a fragment identified as the xyloglucan nonasaccharide, Glc$ sb4$ Xyl$ sb3$ Gal Fuc. By comparison, in incubations with UDP-$ lbrack sp3$H) xylose, the maximum size of labeled oligosaccharide found following cellulase digestion of products was an octasaccharide. In the presence of both GDP-$ lbrack sp{14}$C) -fucose and UDP-$ lbrack sp3$H) xylose, a nonasaccharide containing both labels was produced. Fucose and xylose residues were transferred rapidly to acceptor molecules of MW up to 300,000. Such products did not elongate detectably over 60 min of incubation. We concluded that the nonasaccharide subunit of xyloglucan was generated in vitro by transfucosylation to preformed acceptor chains, and that its synthesis was dependent on exogenous GDP-fucose.
Microsomal membranes were separated by rate-zonal centrifugation on renografin gradients. Transfer to xyloglucan of labelled fucose and xylose from GDP- ($ sp{14}$C) fucose and UDP- ($ sp{14}$C) xylose occurred mainly in dictyosome-enriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with ($ sp3$H) fucose suggested that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles.
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Wisbrun, Natali Kristina [Verfasser]. "Klonierung und funktionelle Expression von Enzymen des L-Fucose-Stoffwechsels / Natali Kristina Wisbrun." Berlin : Freie Universität Berlin, 2007. http://d-nb.info/102241254X/34.
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Franzon, Vicki L. "Haemagglutinins of Vibrio cholerae : molecular characterization of the mannose-fucose resistant haemagglutinin (MFRHA) /." Title page, abstract and contents only, 1988. http://web4.library.adelaide.edu.au/theses/09PH/09phf837.pdf.
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Carchon, Gérald. "Mimes du L-fucose et de ses dérivés : eapplications à la synthèse d'analogues du ligand sialyl Lewis X et d'inhibiteurs des fucosyl transférases." Nancy 1, 1998. http://www.theses.fr/1998NAN10282.
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Processus vital, l'inflammation permet de juguler rapidement une infection. C’est la manifestation du système immunitaire qui se débarrasse de l'intrus avant qu'il ne se multiplie et n'envahisse l'organisme. Les principaux acteurs de cette ligne de défense sont les globules blancs. Pour accéder aux zones infectées, les leucocytes franchissent l'endothélium vasculaire grâce à des phénomènes d'adhérence. Les molécules, responsables de cette adhésion, situées à la surface des vaisseaux sanguins (sélectines) reconnaissent des motifs saccharidiques (sialyl Lewis X) arborés par des protéines localisées à la surface des globules blancs. Empêcher la fixation des leucocytes à l'endothélium pourrait être un bon moyen d'interrompre certaines pathologies complexes (inflammation aiguë, ischémie) ou certaines maladies auto-immunes (arthrite rhumatoïde, psoriasis, …), mais également la dissémination des métastases. Ceci conduit à utiliser le concept de mimes oligosaccharidiques pour concevoir à partir de la structure sialyl Lewis X des analogues solubles utilisable en thérapeutique. C’est dans ce contexte que se situe la première partie de notre travail dans laquelle nous avons essayé de concevoir un analogue du ligand sialyl Lewis X ou le fucose et le galactose seraient reliés par une suite de liaison C-glycosidique. Il en découle la mise au point d'une synthèse d'alpha-L-C-fucoside et aussi la nécessité de vérifier que ces structures adopteront bien la même conformation que leurs modèles naturels. L’inhibition des glycosyltransférases peut constituer une approche efficace du contrôle de la biosynthèse des oligosaccharides impliques dans certains processus biologiques. Les fucosyl-transférases sont responsables de la fucosylation des oligosaccharides comme le ligand sialyl Lewis X. Cette fucosylation utilise le guanosyl-diphosphofucose comme donneur glycosidique. Nous avons essayé d'apporter une solution à l'inhibition des fucosyl-transférases en préparant des analogues de ce substrat qui ne seraient plus des donneurs par remplacement de la liaison beta-O-fucosyl par une liaison C-glycosidique. De plus nous avons essayé de mimer le groupe phosphodiester par un enchainement non-charge afin d'augmenter les possibilités de pénétration cellulaire. Enfin nous avons utilisé un reste fructose pour mimer la partie fucose des inhibiteurs et préparer un analogue de l'état de transition de la réaction de fucosylation.
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Gunn, Francis James. "The overexpression, topology and purification of the L-fucose/proton symporter of Escherichia coli." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309370.
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Kossack, Wilhelm, Wycliffe Kiprop Kipnusu, Mateusz Dulski, Karolina Adrjanowicz, Olga Madejczyk, Ewa Kaminska, Emmanuel Urandu Mapesa, Martin Tress, Kamil Kaminski, and Friedrich Kremer. "The kinetics of mutarotation in L-fucose as monitored by dielectric and infrared spectroscopy." AIP Publishing, 2014. https://ul.qucosa.de/id/qucosa%3A21259.
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Fourier Transform Infrared Spectroscopy and Broadband Dielectric Spectroscopy are combined to trace kinetics of mutarotation in L-fucose. After quenching molten samples down to temperatures between T=313 K and 328 K, the concentrations of two anomeric species change according to a simple exponential time dependence, as seen by an increase in absorbance of specific IR-vibrations. In contrast, the dielectric spectra reveal a slowing down of the structural (α-) relaxation process according to a stretched exponential time dependence (stretching exponent of 1.5 ± 0.2). The rates of change in the IR absorption for α- and β-fucopyranose are (at T = 313 K) nearly one decade faster than that of the intermolecular interactions as measured by the shift of the α-relaxation. This reflects the fact that the α-relaxation monitors the equilibration at a mesoscopic length scale, resulting from fluctuations in the anomeric composition.
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Périon, Régis. "Nouvelles voies d'accès à l'acarbose et à des analogues hypoglycémiants - Synthèses et activités inhibitrices." Rennes 1, 2002. http://www.theses.fr/2002REN10121.
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Keeley, Tyler S. "Investigating the Roles of Fucosylation and Calcium Signaling in Melanoma Invasion." Scholar Commons, 2018. https://scholarcommons.usf.edu/etd/7535.
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Melanoma is the deadliest form of skin cancer. Prognosis for early stage melanoma patients is excellent, and surgery is often curative for these patients. However, once patients have presented with invasive disease, the average 5-year survival rate drops significantly from over 90% to between 10 and 15%. Several therapies have been developed to target a commonly mutated oncogene BRAF, or its downstream effectors. Unfortunately, while these treatments show robust initial response, most patients relapse within a year. Moreover, therapy-resistant tumors are often more invasive and metastatic. Therefore, it is important to investigate the molecular mechanisms underlying melanoma invasion and metastasis, and to prevent melanoma cell dissemination and metastatic progression. Invadopodia are proteolytic membrane protrusions used by metastatic cancer cells to degrade the extracellular matrix and to facilitate cancer cell invasion and metastasis. In my thesis research I have focused on protein fucosylation and store-operated calcium entry, two separate mechanisms involved in invadopodial regulation.
Post translational modifications of proteins are essential for their structure and function. Many cell surface proteins require modifications such as glycosylation for protein-protein interactions, cell adhesion, and signal transduction. Fucosylation is a form of glycosylation that adds L-fucose on glycan structures of proteins. There is evidence indicating that fucosylation plays an important but cancer-type and branching dependent role in cancer progression. Emerging evidence indicates that the fucose salvage pathway and protein fucosylation are altered during melanoma progression and metastasis. Here, we report that the fucose salvage pathway inhibits invadopodia formation and extracellular matrix degradation by promoting α(1,2) fucosylation of cell surface proteins. The activation of the fucose salvage pathway decreases invadopodia numbers and inhibits the proteolytic activity of invadopodia in WM793 melanoma cells. Inhibiting fucokinase, one of the critical enzymes in the fucose salvage pathway, in melanoma cells abrogates L-fucose-mediated inhibition of invadopodia, suggesting dependence on the fucose salvage pathway. The inhibition of invadopodia formation by L-Fucose treatment or fucokinase overexpression could be rescued by treatment with α(1,2), but not α(1,3/4) fucosidase, implicating an α(1,2) fucose linkage-dependent inhibitory effect. The ectopic expression of FUT1, an α(1,2) fucosyltransferase, is sufficient to inhibit invadopodia formation and ECM degradation. Our findings indicate that the fucose salvage pathway can inhibit invadopodia formation, and consequently, invasiveness in melanoma via α(1,2) fucosylation. Re-activation of this pathway in melanoma could be useful for preventing melanoma invasion and metastasis.
Calcium is a critical second messenger involved in a multitude of biological processes from cell proliferation to muscle contraction. In melanoma, previous studies have found that activation of the store operated calcium entry (SOCE) channel promotes tumor invasion and metastasis, in vitro and in xenograft models. The expression levels of STIM1, an essential component of the store operated calcium channels, has been found to increase with later stages of melanoma. In melanoma cell lines, the over expression of STIM1 enhances invadopodia number whereas STIM1 knockdown inhibits invadopodia formation. Similarly, gelatin degradation activity is enhanced with STIM1 overexpression and abrogated with STIM1 knockdown, implicating STIM1 as an important factor in the regulation of invadopodia formation and melanoma invasion. Though the studies published have shown a significant role of STIM1 in tumor progression, a robust transgenic animal model has not yet been established. Here, we developed a novel transgenic mouse model which, upon 4-hydroxytamoxifen (4OHT) treatment, induces the BRAFV600E mutation and PTEN, STIM1, and STIM2 deletions in melanocytes via an inducible Cre-lox system. Our investigation found that the loss of STIM1 exacerbates tumor growth and results in tumor formation significantly more quickly than STIM1 wild type mice. Whereas PCR analysis of 4OHT-treated skin showed deletion of STIM1 and PTEN, immunohistochemical staining of these genes in tumors did not convincingly demonstrate complete deletion. Therefore, it remains to be determined whether the effects we observed are due to STIM1 and STIM2 loss. These findings need to be corroborated in the future.
Our studies focus on two important mechanisms required for melanoma progression and metastasis. We found that α(1,2) fucosylation is able to inhibit invadopodia formation, and melanoma cell invasion. The reestablishment of α(1,2) fucosylation in melanoma could potentially be exploited to inhibit melanoma metastasis. Additionally, early evidence points to STIM1 having a tumor suppressive role in melanoma oncogenesis and tumor growth based on the transgenic mouse model. Although the phenotype is unexpected, further investigation of this model will likely provide important insight for the complicate roles of SOCE in melanoma initiation and progression.
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Joubert, Muriel. "Amino-analogues du L-fucose : synthèse par réaction d'hétéro-Diels-Alder asymétrique et inhibition de glycosidases." Mulhouse, 2000. http://www.theses.fr/2000MULH0619.
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Les α-(1,3)-fucosyltransférases sont des enzymes de transfert impliquées dans les processus inflammatoires. Lorsque la réponse inflammatoire s'avère néfaste pour l'organisme, les inhibiteurs de fucosyltransférases devraient permettre d'interférer avec ces processus inflammatoires et de le ralentir. Le mécanisme catalytique de ces enzymes passe par un état de transition similaire à celui de l'α-L-fucosidase, enzyme d'hydrolyse de polysaccharides fucosylés. En supposant la reconnaissance du fucose identique pour les deux classes d'enzymes, les inhibiteurs d'α-L-fucosidase pourront être des précurseurs d'inhibiteurs de fucosyltransférases. Notre travail présente la synthèse et à l'évaluation de l'activité d'amino-analogues du L-fucose, inhibiteurs puissants d'α-L-fucosidase. La synthèse envisagée pour préparer les aminosucres pipéridiniques utilise la réaction d'hétéro-Diels-Alder asymétrique entre un diène achiral et un diénophile nitrosé chiral. Nous avons synthétisé un dérivé α-chloronitrosé à partir du D-ribose, qui, après étude sur divers diènes, s'est révélé être un puissant inducteur chiral. Nous l'avons alors employé comme auxiliaire chiral dans la synthèse de la L-fuco-nojirimycine, analogue pipéridinique du L-fucose. Les aminosucres pyrrolidiniques ont été synthétisés au départ d'une réaction d'hétéro-Diels-Alder asymétrique entre un diène chiral, préparé à partir d'un ester pyroglutamique, et un diénophile acylnitrosé achiral. Pour obtenir la configuration du L-fucose, nous avons mis au point une nouvelle méthode efficace d'inversion du groupement méthyle en deux étapes. Nous avons ainsi synthétisé le 4-amino-4,5-didésoxy-L-lyxose, analogue pyrrolidinique du L-fucose. L'évaluation des propriétés inhibitrices de ces aminosucres ainsi que de leurs dérivés sulfitiques et 1-désoxylés, réalisée in vitro sur des glycosidases commerciales, a révélé de puissants inhibiteurs sélectifs d'α-L-fucosidase. Une étude de relation structure/activité a mis en évidence l'importance du groupement méthyle pour une inhibition efficace de cette enzyme.
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Murrey, Heather Elizabeth Dougherty Dennis A. Hsieh-Wilson Linda C. "Identification and characterization of the plasticity-relevant fucose-alpha(1-2)galactose glycoproteome from mouse brain /." Diss., Pasadena, Calif. : Caltech, 2009. http://resolver.caltech.edu/CaltechETD:etd-12182008-145714.
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Tuck, Laura. "Structural and synthetic biology study of bacterial microcompartments." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/33180.
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Bacterial microcompartments (BMCs) are proteinaceous metabolic compartments found in a wide range of bacteria, whose function it is to encapsulate pathways for the breakdown of various carbon sources, whilst retaining toxic and volatile intermediates formed from substrate breakdown. Examples of these metabolic processes are the 1,2- propanediol-breakdown pathway in Salmonella enterica (Pdu microcompartment), as well as the ethanolamine breakdown pathway in Clostridium difficile (Eut microcompartment). Some of the major challenges to exploiting BMCs as a tool in biotechnology are understanding how enzymes are targeted to microcompartments, as well as being able to engineer the protein shell of BMCs to make synthetic microcompartments that allow specific enzyme pathways to be targeted to their interior. Finally, the metabolic burden imposed by the production of large protein complexes requires a detailed knowledge of how the expression of these systems are controlled. This project explores the structure and biochemistry of an essential BMC pathway enzyme, the acylating propionaldehyde dehydrogenase. With crystal structures of the enzyme with the cofactors in the cofactor binding site and biochemical data presented to confirm the enzyme's substrate. The project also focuses on the creation of synthetic biology tools to enable BMC engineering with a modular library of BMC shell protein parts; forward engineered ribosome binding sites (RBS) fused to BMC aldehyde dehydrogenase localisation sequences. The parts for this library were taken from the BMC loci found in Clostridium phytofermentans and Salmonella enterica. Using a synthetic biology toolkit will allow the rapid prototyping of BMC constructs for use in metabolic engineering. The shell protein parts were used to generate a number of transcriptional units, to assess the effect of overexpression of individual BMC shell components on the morphology of BMCs and the effect these had on their host chassis. Different strength forward engineered RBS and localisation constructs have been designed to assess the possibility of controlling the levels of heterologous proteins targeted to the interior of microcompartment shell to allow metabolic engineering of encapsulated pathways. Along with looking at overexpression of a single shell protein, to assess viability of BMCs as scaffold-like structures, recombinant BMCs can be explored for their utility in bioengineering and their potential role in generating biofuels.
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Gallagher, Julie Marie. "The synthesis of 1-acetamido-2,6-anhydro-1,7-deoxy-L-glycero-L-galactitol (N-[β-L-fucopyranosylomethyl]-acetamide) and related derivatives." Scholarly Commons, 1989. https://scholarlycommons.pacific.edu/uop_etds/2182.
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One important goal of this thesis is the hydrogenation of the glycosyl cyanide, which has never been mentioned by any of the groups who have done work in this area, except for B. Coxon and G. Fletcher, who in 1964, reduced a tetra-O-acetyl-β-D-galactopyranosyl cyanide with lithium aluminum hydride. We were hoping to obtain, by reduction with hydrogen on Pd/C, an aminomethyl C-glycoside. It is believed that these aminomethyl C-glycosides are of potential biological importance especially in the area of AIDS and HIV therapy.
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VASCONCELOS, Juliana Lúcia de Albuquerque. "Avaliação do valor diagnóstico e prognóstico do carboidrato L-fucose e das fucosiltransferases 3 e 6 em tumores prostáticos humano." Universidade Federal de Pernambuco, 2012. https://repositorio.ufpe.br/handle/123456789/18485.
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Câncer é um conjunto de alterações celulares, que leva a uma divisão celular sem controle, podendo invadir tecidos adjacentes através da circulação sanguínea e do sistema linfático. O Câncer de Próstata (CP) é o segundo tumor mais comum entre a população masculina, e é considerado o câncer da terceira idade. A carcinogênese é um mecanismo complexo no qual ocorre mudanças na expressão de proteínas e glicoconjugados. Glicosilação é mediada por glicosiltransferases, enzimas que tem função de inserir resíduos de carboidratos específicos, e é um dos mais importantes processos biológicos pós-tradução de modificações na estrutura final e função de lipídios e proteínas. As fucosiltransferases (FUT) participam da transferência de resíduos de L-fucose, um sacarídeo associado ao câncer e a processos inflamatórios, da GDP-L-fucose. Neste estudo objetivou-se avaliar a expressão dos genes FUT3 e FUT6 através da Imunohistoquímica em Adenocarcinoma Prostático e Hiperplasia Prostática Benigna correlacionando com o padrão de expressão de L-fucose empregando a histoquímica com as lectinas UEA-I (Ulex europaeus) e LTA (Lotus tetragonolobus). As enzimas FUT3 e FUT6 apresentaram-se com uma alta expressão tanto no Adenocarcinoma Prostático como na Hiperplasia Benigna Prostática, principalmente a FUT 6. Os resultados da histoquímica com lectinas mostraram uma baixa distribuição/accessibilidade de L-fucose. Sugere-se que, as enzimas FUT3 e FUT6 possam representar potenciais biomarcadores para avaliar alterações benignas e malignas prostáticas refletindo uma variação no perfil de L-fucose nestes tumores que podem estar associados às suas características biológicas.
Cancer is a set of cellular changes, leading to uncontrolled cell division that may invade surrounding tissues via bloodstream and lymphatic system. Prostate Cancer (PC) is the second most common tumor in men and is considered the cancer of the elderly. Carcinogenesis is a complex mechanism in which changes occur in the expression of proteins and glycoconjugates where glycosylation plays key roles since modulates the carbohydrate moieties in glycoconjugates being one of the most important biological processes of posttranslational modifications in the final structure and function of lipids and proteins. Fucosyltransferases (FUTs) are enzymes that catalyze the transfer of the L-fucose residues, a saccharide which has been linked to cancer and inflammation features, from GDP-Fuc. This study the objective to evaluate the expression of genes FUT 3 and FUT 6 by immunohistochemistry in Prostatic Adenocarcinoma and Benign Prostatic Hyperplasia and to correlates with the expression pattern of L-fucose using lectin histochemistry with UEA-I (Ulex europaeus) and LTA (Lotus tetragonolobus). FUT3 and FUT6 showed a high expression in both prostatic tissues, especially FUT6. The results of lectin histochemistry showed a low distribution/accessibility of L-fucose residues. It is suggested that FUT3 and FUT6 may represent potential biomarkers to evaluate benign and malignant alterations in prostate reflecting a variation in the profile of L-fucose residues in these tumors which can be associated to their biological features.
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Fitchette, Anne-Catherine. "Immunolocalisation de la xylosylation et le la fucosylation des glycannes complexes dans l'appareil de Golgi des cellules de sycomore (Acer pseudoplatanus L. )." Rouen, 1993. http://www.theses.fr/1993ROUES003.
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Les glycanes complexes portés par les glycoprotéines végétales diffèrent de ceux rencontrés chez les mammifères par l'absence d'acide sialique et par la présence d'un résidu fucose α1,3 lié au GlcNAc de la partie réductrice de l'unité chitobiose et d'un xylose lié en β1,2 au β-mannose. La séquence des évènements de maturation des glycanes n'est pas aussi bien connue dans la cellule végétale que pour la cellule animale. Dans cette étude, nous nous sommes principalement intéressés aux xylosyl- et fucosyl-transférases spécifiques aux plantes et intervenant dans la glycosylation tardive. Nous avons préparé des anticorps dirigés contre les glycanes végétaux contenant des résidus xylose ou fucose. Par immunodétection à l'aide de ces anticorps, nous avons visualisé la distribution subcellulaire des glycoprotéines portant ces glycanes complexes dans les cellules de sycomore. De plus, cette approche immunocytochimique a permis une localisation indirecte des xylosyl- et fucosyl-transférases en détectant les glycanes produits par ces enzymes dans les empilements golgiens des cellules de sycomore. Nos résultats indiquent que les glycanes complexes N-liés aux glycoprotéines vacuolaires ou pariétales sont xylosylés principalement dans le Golgi médian, alors que leur fucosylation est un évènement tardif intervenant dans le Golgi trans
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Barker, Andrew. "Haemagglutinins of vibrio cholerae 01 : studies on the organisation of the genes encoding the mannose-fucose-resistant haemagglutinin (MFRHA) /." Title page, contents and abstract only, 1994. http://web4.library.adelaide.edu.au/theses/09PH/09phb244.pdf.
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Lau, Tak Bun Stephen. "Mechanistic investigation of 3, 5-epimerases / reductases involved in the biosynthesis of GDP-L-Fucose and GDP-L-Galactose." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/14851.
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GDP-fucose synthase (GFS), also known as GDP-4-keto-6-deoxy-mannose 3, 5- epimerase / 4-reductase (GMER), is the key enzyme in the GDP-L-fucose biosynthetic pathway. The pathway begins with a dehydratase that converts GDP-D-mannose into 6-deoxy-4-keto- GDP-D-mannose. Then GFS performs two consecutive epirnerizations, and a final NADPH dependent reduction to generate GDP-L-fucose. The detailed mechanism of GFS has remained a mystery due to limited mechanistic studies as well as the lack of an x-ray crystal structure with any bound GDP-sugar. This thesis investigates the mechanism of the reaction as well as the roles of the active site residues in GFS. The results support a sequential ordered epimerization mechanism where the stereocenter at C-3”is inverted first, followed by the inversion of
stereochemistry at C-5”. This identifies GDP-6-deoxy-D-altrose as a reaction intermediate for the first time. The findings of this thesis also leads to the assignment of the catalytic residue Cys 109,
as the base which deprotonates both epimerization sites, and His179 as the acid which reprotonates the corresponding enol intermediates. GDP-mannose 3, 5-epimerase (GME), is the key enzyme that catalyzes the first committed step of vitamin C biosynthesis in plants. It oxidizes the C-4” position of GDP mannose, then sequentially inverts the stereochemistry at C-5” and C-3”, and finally reduces the C-4” carbonyl to produce GDP-L-galactose. The proposed sequential order of epimerization is based on the observation that GDP-L-gulose (the C-5” epimeric product) is produced whereas GDP-D-altrose (the C-3” epimeric product) is not. Structural studies on GME suggest that Cys145 acts as the catalytic base and Lys 217 acts as the catalytic acid in both epimerization steps. However, very limited mechanistic studies on GME have been performed. This thesis provides mechanistic evidence that supports the proposed sequential epimerization order as well as the assignment of roles for the catalytic residues. It also suggests a model whereby protonated Cys145 is unable to exchange its proton with solvent during the lifetime of the enol(late) intermediate.
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Goupille, Caroline. "Commutation fucose/acide sialique au niveau d'un variant du CD44 : conséquences sur le comportement de cellules de carcinome colique." Nantes, 1997. http://www.theses.fr/1997NANT04VS.
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Stahl, Martin. "Colonization of the Intestinal Mucus Layer by Campylobacter jejuni." Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/22861.
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Campylobacter jejuni is a major cause of bacterial gastroenteritis in the developed world; however, many aspects of its biology remain poorly understood, including its colonization of the mucus layer lining the gastrointestinal tract. In this study, we utilized microarray transposon tracking to compile a list of 195 genes essential for the growth of C. jejuni in vitro under microaerophilic conditions. Then we characterized C. jejuni growing in an extracted intestinal mucus medium. We found that C. jejuni will grow efficiently in a medium comprised of either chick and piglet intestinal mucus, and that these media have a dramatic impact on its transcriptome. Within the genes identified as differentially expressed during growth in a mucus medium, we identified a single operon, (cj0481-cj0490), which we have subsequently characterized as being responsible for both the uptake and metabolism of L-fucose. This represents the first observation of carbohydrate metabolism by the otherwise asaccharolytic C. jejuni. We further found that the inability to utilize L-fucose puts C. jejuni at a competitive disadvantage when colonizing the piglet intestine, but not the chick cecum. Finally, we examined C. jejuni’s ability to utilize mucins as a carbon source while growing within the mucus layer. We found that despite mucins being a major source of L-fucose and amino acids within the intestine, C. jejuni has a minimal ability to degrade and utilize mucins on its own. However, close proximity to mucolytic bacteria within the microbiota of the intestine, allows for increased C. jejuni growth. Together, this paints the picture of an organism that is well adapted to survival within the mucus lining of the intestine and establishing itself as part of the intestinal microbiota.
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Hemming, Richard John. "The presence of fucose- and galactose-containing glycoproteins in the cell nucleus as shown by radioautographic and lectin binding studies /." Thesis, McGill University, 1986. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66045.
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Hüllen, Andreas Jürgen [Verfasser], and Britta [Akademischer Betreuer] Brügger. "GFUS-CDG Identifizierung eines neuen und behandelbaren Defekts in der GDP-L-Fucose Synthase / Andreas Jürgen Hüllen ; Betreuer: Britta Brügger." Heidelberg : Universitätsbibliothek Heidelberg, 2021. http://nbn-resolving.de/urn:nbn:de:bsz:16-heidok-303559.
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Olivier, Stéphane. "Développement de la plateforme cellulaire EB66 dérivée de cellules souches embryonnaires de canard pour la production industrielle d’anticorps thérapeutiques à activité ADCC améliorée." Nantes, 2010. http://www.theses.fr/2010NANT33VS.
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Le marché des anticorps monoclonaux (Acm) présente la croissance la plus forte du secteur pharmaceutique. Mais le coût prohibitif des traitements impose le développement de molécules plus actives et moins onéreuses. Une des stratégies repose sur l’amélioration de l’activité ADCC (antibody-dependent cell cytotoxicity) permise par le contrôle de la fucosylation des Acms. Les cellules EB66, dérivées de cellules souches embryonnaires de canard, ont des atouts règlementaires et industriels uniques: elles sont génétiquement stables, immortelles et sont capables de croître à de fortes densités en milieu sans sérum. De plus, les espèces aviaires produisant naturellement des anticorps peu fucosylés, la cellule EB66 pourrait constituer une plateforme précieuse pour la production d’Acm à activité ADCC renforcée. Ce travail a consisté à établir et à optimiser les technologies requises à une production industrielle d’Acm thérapeutiques dans la cellule EB66. Un procédé de sélection de clones producteurs a été développé. L’optimisation d’un vecteur d’expression spécifique associée au développement du procédé de production ont permis d’atteindre un rendement supérieur à 1 g/L. Ces Acm ont présenté une glycosylation comparables à ceux produits en cellules de hamster CHO à l’exception d’un contenu en fucose naturellement réduit. Ce faible taux de fucose a été associé à une activité ADCC améliorée. Egalement, la capacité de fucosylation des clones producteurs semble être corrélée avec le niveau d’expression de l’alpha 1,6-fucosyltransférase. La cellule EB66 possède ainsi le potentiel pour devenir une plateforme de production industrielle d’Acm thérapeutique à forte activité cytotoxiqueMonoclonal antibodies (mAbs) represent the fastest growing class of pharmaceuticals. However, prohibitive therapeutic costs call to develop cheaper and more active molecules. To this end, one of the promising strategies is to enhance mAbs efficacy through an improved antibody dependent cell cytotoxicity (ADCC) correlated with mAbs fucosylation level. EB66 cell line, a duck embryonic stem cell-derived substrate, displays unique regulatory and industrial features: they are genetically stable, immortal, and reach high cell densities in serum-free medium. The fact that avian species have been described to naturally produce low-fucosylated antibodies prompted the investigation on the use of the duck EB66 cells for the production of mAbs with reduced fucose content and enhanced ADCC activity. The aim of this work was to establish and optimize technologies dedicated to the development of the EB66 cellular platform for the production of therapeutic mAbs. A selection procedure has been developed to isolate producer clones. A yield titer higher to 1 g/l has been reached thanks to the optimization of a specific expression vector associated to the development of the production process. The mAbs produced on EB66 cells display a glycosylation profile comparable with mAbs produced on the Chinese hamster ovary cells, with a naturally reduced fucose content resulting in a strongly enhanced ADCC activity. Furthermore, we observed a correlation between mAbs fucosylation and expression level of the alpha 1,6-fucosyltransferase within producer clones. The EB66 cells have therefore the potential to evolve as a novel cellular platform for the production of high potency therapeutic antibodies
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Martinez, Michael. "Simulation ab initio de modules d'intérêt biologique : modes de vibrations pour la spectroscopie infrarouge." Paris 6, 2006. http://www.theses.fr/2006PA066620.
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Bardonnet, Pierre-Louis. "Formulation et caractérisation de liposomes porteurs de glycolipides synthétiques : application au ciblage d'Helicobacter pylori." Phd thesis, Université Claude Bernard - Lyon I, 2007. http://tel.archives-ouvertes.fr/tel-00382031.
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Ce travail traite de la formulation et de la caractérisation de liposomes porteurs de glycolipides synthétiques, en vue du ciblage d'une bactérie : Helicobacter pylori. Après avoir passé en revue les différents systèmes à temps de résidence gastrique prolongé, il décrit la synthèse et l'utilisation de néoglycolipides de type "ancre-espaceur-sucre ", constitué respectivement du cholestérol, du tétraéthylène glycol et enfin du fucose (ou N-acétylglucosamine). Ont été étudiées dans ce travail l'organisation supramoléculaire des néoglycolipides seuls en fonction de leur état d'hydratation, les altérations de la bicouche liposomale suite à l'incorporation du néoglycolipide, l'accessibilité des sucres à la surface des liposomes, la variation du pH intraliposomal en fonction de pH externes acides, et enfin, l'interaction de quatre formulations de liposomes contenant ou non les néoglycolipides avec 2 souches d'H. pylori
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Bardonnet, Pierre-Louis. "Formulation et caractérisation de liposomes porteurs de glycolipides synthétiques : application au ciblage d'Helicobacter pylori." Phd thesis, Lyon 1, 2007. https://theses.hal.science/docs/00/38/20/31/PDF/Bardonnet_Pierre_Louis.pdf.
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Ce travail traite de la formulation et de la caractérisation de liposomes porteurs de glycolipides synthétiques, en vue du ciblage d’une bactérie : Helicobacter pylori. Après avoir passé en revue les différents systèmes à temps de résidence gastrique prolongé, il décrit la synthèse et l’utilisation de néoglycolipides de type "ancre-espaceur-sucre ", constitué respectivement du cholestérol, du tétra-éthylène glycol et enfin du fucose (ou N-acétylglucosamine). Ont été étudiées dans ce travail l’organisation supramoléculaire des néoglycolipides seuls en fonction de leur état d’hydratation, les altérations de la bicouche liposomale suite à l’incorporation du néoglycolipide, l’accessibilité des sucres à la surface des liposomes, la variation du pH intraliposomal en fonction de pH externes acides, et enfin, l’interaction de quatre formulations de liposomes contenant ou non les néoglycolipides avec 2 souches d’H. PyloriThis thesis is about the formulation and characterisation of synthetic glycolipids-loaded liposomes in order to target a bacterium: Helicobacter pylori. Gastroretentive systems are first reviewed. Secondly, the synthesis and use of the system “anchor-spacer-sugar”, i. E. Cholesterol, tetraethylene glycol and fucose (or N-acetylglucosamine) respectively, are described. During this work, we studied the neoglycolipids supramolecular organization in function of their hydration rate, the alteration of the liposomale bilayer following the neoglycolipid incorporation, the accessibility of the sugar moieties at the liposomes surface, the intraliposomal pH variation in function of acidic external pH, and finally, the interaction between four liposomal formulations bearing or not neoglycolipids with two strains of H. Pylori
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Apanga, Ludovic. "Mise au point de procédés de préparation d'oligosaccharides contenant du fucose à partir d'exopolysaccharides issus de souches mutées de bactéries du sol." Amiens, 2008. http://www.theses.fr/2008AMIE0116.
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Le L-fucose et les oligosaccharides contenant du L-fucose présentent des propriétés biologiques intéressantes notamment dans la prévention des métastases, dans la réaction d’inflammation, dans le traitement des arthrites rhumatoïdes, dans la vaccination (antigènes) et dans le domaine cosmétique contre la déshydration de la peau. Une production du L-fucose tout comme des oligosaccharides contenant du L-fucose en vue de leur utilisation dans des thérapies, a poussé des nombreux laboratoires à un screening des organismes synthétisant des polysaccharides contenant du L-fucose. Notre travail, s’est dans un premier temps sur la production et la caractérisation des polysaccharides des souches Sinorhizobium M5N1 cPRk290 notée N et Enterobacter notée Sc. Dans un second temps nous avons produit des oligosaccharides contenant du L-fucose dérivés des polysaccharides. Cette production d’oligosaccharides a nécessité l’utilisation de plusieurs méthodes (hydrolyse acide, hydrolyse thermique, production dans le milieu de culture et dégradation enzymatique). La méthode enzymatique a permis d’obtenir des oligosaccharides de manière reproductibleL-fucose and oligosaccharides containing of L-fucose present interesting biological properties notably in the prevention of the metastases, in the reaction of inflammation, in the treatment of rheumatoid arthritics, in the vaccination (antigens) and in the cosmetic domain against the dehydration of the skin. A production of L-fucose as of oligosaccharides containing of L-fucose with the aim of their use in therapies, pushed numerous laboratories to a screening of organisms synthetizing polysaccharides containing of L-fucose. Our work, concerned first of all the production of the polysaccharides by Sinorhizobium M5N1 cPRK290 noted N and Enterobacter noted Sc strains, later these polysaccharides were characterized. And secondly we produced oligosaccharides containing of L-fucose from polysaccharides. This production of oligosaccharides required the use of several methods (acid hydrolysis, thermal hydrolysis, in the medium of culture and enzymatic degradation). The enzymatic method allows to obtain oligosaccharides in a reproducible way
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Cândido, Teresinha Cristina [UNESP]. "Comparação entre os métodos de ELISA - Antígeno total e ELISA - Ligante de Fucose e Manose em cães sintomáticos e oligossintomáticos para leishmaniose visceral." Universidade Estadual Paulista (UNESP), 2007. http://hdl.handle.net/11449/92184.
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A Leishmaniose visceral canina (LVC), conhecida como calazar, é uma antropozoonose endêmica no Brasil, causada pela Leishmania L. chagasi. O teste sorológico de ELISA tem sido empregado na rotina de inquéritos epidemiológicos e no auxílio diagnóstico clínico de cães suspeitos. O método de ensaio imunoenzimático em fase sólida (ELISA) usando o antígeno total de Leishmania L. chagasi (ELISA-AgT), assim como, o ELISA - Ligante de Fucose e Manose (ELISA-FML), tem demonstrado boa sensibilidade e especificidade para detectar a doença em cães assintomáticos e oligossintomáticos. O presente trabalho teve como objetivo comparar dois antígenos pelo método de ELISA no sorodiagnóstico de cães naturalmente infectados pela LVC, positivos no exame parasitológico, e agrupados em: grupo sintomático e, grupo oligossintomáticos, tendo como grupo controle cães de área não endêmica. Nos animais oligossintomáticos, a sensibilidade observada para ELISA-AgT foi de 86,7%, já para o método ELISA-FML apresentou valor de 90%. A especificidade foi de 100% no método de ELISA-AgT e 96,7 % para o ELISA-FML. Nos animais sintomáticos, a sensibilidade e especificidade para o ELISA-AgT foram de 90% e 93,3%, respectivamente; já o método de ELISA-FML apresentou sensibilidade e especificidade de 86,7% e 96,7%. No teste ELISA-AgT o valor preditivo positivo foi de 93,1% nos sintomáticos e 100% nos oligossintomáticos, enquanto que nos animais submetidos ao teste ELISA-FML, foi observado 96,3% para os sintomáticos e 96,4% para os oligossintomáticos. O índice Kappa foi utilizado para medir o grau de concordância real entre os dois métodos imunoenzimáticos empregados e o exame parasitológico direto, mostrando boa concordância entre os métodos realizados.
Visceral canine leishmaniasis or calazar is Brazilian an endemic antropozoonosis caused by Leishmania L. chagasi. ELISA sorologycal test has been used in routine of epidemiological studies and in diagnosis of clinical suspect dogs. The immunoenzymatic assay in solid phase (ELISA) using Leishmania L. chagasi total antigens AgT-ELISA and Fucose Manose ligant-ELISA (FML-ELISA) has been showed good sensibility and specificity for detection disease in asymptomatic and oligosymptomatic dogs. The present paper has the goal of compare the efficiency of two antigens by ELISA methods in dogs naturally affect by leishmaniasis with positive parasitological exam and grouped by symptoms. Group composed by symptomatic dogs, and Group by oligosymptomatic dogs. Control group was constituted of dogs from Leishmania free areas. Dogs of Group oligosymptomatics presented 86,7% of sensibility in AgT-ELISA and 90% at FML-ELISA. The specificity was 100% at AgT-ELISA and 96.7% for FML-ELISA. At Group symptomatics the sensibility and specificity for AgTELISA and FML-ELISA were respectively 90% and 93.3% and, the FMLELISA showed 86.7% and 96.7% corresponding to sensibility and specificity. On ELISA-AgT the positive predictive valor was 93.1% at symptomatic and 100% at oligosymptomatic, moreover in the dogs tested by FML-ELISA the valor was 96.3% for the symptomatic and 96.4% for oligosymptomatics. The Kappa was used and showed a good concordance between both methods tested. Our results had shown that in oligosymptomatics animals the FML-ELISA presented greater sensitivity, while that, in the symptomatic animals, the AgT-ELISA showed more sensible in the detention of positive.
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Cândido, Teresinha Cristina. "Comparação entre os métodos de ELISA - Antígeno total e ELISA - Ligante de Fucose e Manose em cães sintomáticos e oligossintomáticos para leishmaniose visceral /." Araçatuba : [s.n.], 2007. http://hdl.handle.net/11449/92184.
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Resumo: A Leishmaniose visceral canina (LVC), conhecida como calazar, é uma antropozoonose endêmica no Brasil, causada pela Leishmania L. chagasi. O teste sorológico de ELISA tem sido empregado na rotina de inquéritos epidemiológicos e no auxílio diagnóstico clínico de cães suspeitos. O método de ensaio imunoenzimático em fase sólida (ELISA) usando o antígeno total de Leishmania L. chagasi (ELISA-AgT), assim como, o ELISA - Ligante de Fucose e Manose (ELISA-FML), tem demonstrado boa sensibilidade e especificidade para detectar a doença em cães assintomáticos e oligossintomáticos. O presente trabalho teve como objetivo comparar dois antígenos pelo método de ELISA no sorodiagnóstico de cães naturalmente infectados pela LVC, positivos no exame parasitológico, e agrupados em: grupo sintomático e, grupo oligossintomáticos, tendo como grupo controle cães de área não endêmica. Nos animais oligossintomáticos, a sensibilidade observada para ELISA-AgT foi de 86,7%, já para o método ELISA-FML apresentou valor de 90%. A especificidade foi de 100% no método de ELISA-AgT e 96,7 % para o ELISA-FML. Nos animais sintomáticos, a sensibilidade e especificidade para o ELISA-AgT foram de 90% e 93,3%, respectivamente; já o método de ELISA-FML apresentou sensibilidade e especificidade de 86,7% e 96,7%. No teste ELISA-AgT o valor preditivo positivo foi de 93,1% nos sintomáticos e 100% nos oligossintomáticos, enquanto que nos animais submetidos ao teste ELISA-FML, foi observado 96,3% para os sintomáticos e 96,4% para os oligossintomáticos. O índice Kappa foi utilizado para medir o grau de concordância real entre os dois métodos imunoenzimáticos empregados e o exame parasitológico direto, mostrando boa concordância entre os métodos realizados.Abstract: Visceral canine leishmaniasis or calazar is Brazilian an endemic antropozoonosis caused by Leishmania L. chagasi. ELISA sorologycal test has been used in routine of epidemiological studies and in diagnosis of clinical suspect dogs. The immunoenzymatic assay in solid phase (ELISA) using Leishmania L. chagasi total antigens AgT-ELISA and Fucose Manose ligant-ELISA (FML-ELISA) has been showed good sensibility and specificity for detection disease in asymptomatic and oligosymptomatic dogs. The present paper has the goal of compare the efficiency of two antigens by ELISA methods in dogs naturally affect by leishmaniasis with positive parasitological exam and grouped by symptoms. Group composed by symptomatic dogs, and Group by oligosymptomatic dogs. Control group was constituted of dogs from Leishmania free areas. Dogs of Group oligosymptomatics presented 86,7% of sensibility in AgT-ELISA and 90% at FML-ELISA. The specificity was 100% at AgT-ELISA and 96.7% for FML-ELISA. At Group symptomatics the sensibility and specificity for AgTELISA and FML-ELISA were respectively 90% and 93.3% and, the FMLELISA showed 86.7% and 96.7% corresponding to sensibility and specificity. On ELISA-AgT the positive predictive valor was 93.1% at symptomatic and 100% at oligosymptomatic, moreover in the dogs tested by FML-ELISA the valor was 96.3% for the symptomatic and 96.4% for oligosymptomatics. The Kappa was used and showed a good concordance between both methods tested. Our results had shown that in oligosymptomatics animals the FML-ELISA presented greater sensitivity, while that, in the symptomatic animals, the AgT-ELISA showed more sensible in the detention of positive.
Orientador: Maria Cecília Rui Luvizotto
Coorientador: Valéria Marçal Felix de Lima
Banca: Marcia Dalastra Laurenti
Banca: Gisele Fabrino Machado
Mestre
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D, Nolfi. "Studio morfologico e funzionale dell’apparato di Golgi in relazione ad una struttura LTL-positiva nelle cellule di carcinoma prostatico DU145." Doctoral thesis, Università di Siena, 2020. http://hdl.handle.net/11365/1095701.
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The Golgi apparatus is a complex structure present in all eukaryotic cells. This organelle, which was first observed in 1989, still represents a fascinating enigma for much of its structural and functional peculiarity. Generally, the Golgi apparatus is known as the heart of the secretory pathway and glycosylation, which is one of the major post-traductional modifications.
Most of the reactions of the glycosylation pathway occur in the Golgi apparatus, where glycosyltransferases and glycosidases modify glucidic chains by adding or removing monosaccharides. All the steps follow a precise sequential order from the cis-Golgi to the trans-Golgi network, depending on the exclusive presence of peculiar enzymes in each Golgi compartment.
The sequential order of all these reactions is guaranteed by the morphological stability of the Golgi apparatus, which appears as many staked cisternae fused together forming a unique ribbon structure in vertebrates’ cells. The integrity of the Golgi ribbon is the result of a perfect harmonization between Golgi matrix proteins and the cytoskeleton.
Defect in glycosylation has been observed in many pathologies, such as in neurodegenerative disease and malignant transformation of cancer cells. Particularly, the Golgi apparatus of cancer cells loses its typical ribbon shape and splits in smaller vesicles scattered in the cytosol.
This thesis focuses on the defects of glycosylation in cancer cells, with a particular regard to fucosylation, since we observed a novel Golgi-derived α 1,2 fucosylated tubular structure exclusive of high proliferative cells. This structure, which was highlighted thanks to the α 1,2 fucose binding Lotus tetragonolobus lectin (LTL), seems to be responsible for a peculiar uptake system that lets many molecules, including LTL itself, enter the cell.
To better understand 1) the relation between the morphological scattering of the cisternae and the functionality of the Golgi apparatus and 2) the link between the Golgi disarrangement and the origin of the LTL-positive tubular system, we analysed cells from the human prostatic tumor cell line DU145 by confocal microscopy using the lectins LTL and AAA (Aleuria aurantia Agglutinin), both specific for fucose, and different antibodies able to mark the organelle.
Microscopic observations were parallelly performed with SDS-PAGE on DU145 extracts to analyze the localization of the Golgi proteins and glycans in the nuclear, cytoplasmic, membrane, and cytoskeleton fractions obtained using the Qproteome Cell Compartment Kit.
Furthermore, in order to evaluate the relationship between the Golgi apparatus/tubular system and the actin cytoskeleton, DU145 were left to grow in presence of Cytochalasin D, a fungal toxin capable to depolymerize the microfilaments.
Finally, we performed an uptake experiment to test the functionality of the tubular system, both in the presence and absence of cytochalasin.
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COLLIOU, VIRGINIE. "Synthese de c-disaccharides contenant le d-galactose et le l-fucose : application a la n-acetyl-c-lactosamine et au lewis a mixte." Paris 6, 1997. http://www.theses.fr/1997PA066049.
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La synthese d'analogues carbones de disaccharide, en particulier de c-disaccharides, molecules ou l'oxygene interglycosidique a ete remplace par un groupement methylene, suscite un interet croissant. Dans cette optique, la strategie de l'agrafe moleculaire a ete etendue a la synthese de c-disaccharides complexes. Elle met en jeu l'addition d'un radical anomere sur une double liaison presente dans la molecule. Une etude basee sur les derives d'acides 3-desoxy-ulosoniques, nous a permis de mettre en evidence le caractere inerte des radicaux tertiaires anomeres obtenus. Malgre l'elaboration de l'agrafe a la fonction ester, nous n'avons pas pu obtenir de c-disaccharides de cette famille. La n-acetyl-c-lactosamine a ete synthetisee suivant deux strategies. La voie directe met en jeu une unite accepteur de radical possedant deja la fonction acetamido. Dans la voie indirecte, le produit de cyclisation en serie neutre est fonctionnalise par une double inversion permettant d'introduire le groupement acetamido. La n-acetyl-c-lactosamine obtenue est glycosylee par voie enzymatique afin d'obtenir un lewis x mixte. Dans une troisieme partie, nous avons effectues la synthese du trisaccharide c,o-lewis a a partir d'un c-gal-beta-(1-3)glcnac, glycosyle par la suite. Apres introduction de la fonction acetamido sur le c-galactoside par la strategie mise au point dans le chapitre precedent, la fucosylation du c-disaccharide obtenu nous a conduit au lewis a mixte desire. Parallelement, nous avons synthetise un c-fuc-alpha-(1-4)glcnac, motif qui constitue le lewis a.
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Mreyen, Marcus. "Glycosylation in Dictyostelium discoideum Characterisation and location of glycans on the spore coat protein SP96 in wild type and fucose mutants of the cellular slime mold /." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=962671746.
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Duarte, Monica Machado. "Distribuição de glicoproteinas no ligamento periodontal e no periodonto relacionado ao esmalte do incisivo de camundongo, em diferentes condições funcionais : estudo radioautografico pela incorporação de [3H]-Fucose." [s.n.], 2004. http://repositorio.unicamp.br/jspui/handle/REPOSIP/289385.
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Orientador: Jose MerzelDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-03T22:57:31Z (GMT). No. of bitstreams: 1 Duarte_MonicaMachado_M.pdf: 1989817 bytes, checksum: 5a5900a55bbb2d95070f23c019f2fc91 (MD5) Previous issue date: 2004
Resumo: Para analisar se o aumento da quantidade de proteínas não colágenas no ligamento periodontal de incisivos de ratos desimpedidos (hipofuncionais) está relacionado com o aumento da velocidade de erupção que ocorre nestes dentes, utilizamos a [3H]-fucose, precursor específico para glicoproteínas, e, através da radioautografia, observamos sua incorporação nos tecidos periodontais de incisivos de camundongos. Camundongos fêmeas foram divididos em três grupos com 4 animais cada. Em dois grupos o incisivo inferior esquerdo foi cortado à altura da papila gengival,tornando-o hipofuncional enquanto o contralateral direito tornou-se hiperfuncional. No terceiro grupo os incisivos foram mantidos intactos e portanto normofuncionais. A [3H]-fucose foi injetada na veia caudal no seguinte esquema: 1) no primeiro grupo, 1 dia após a instalação da alteração funcional; 2) no 2o grupo 7 dias após a desoclusão do incisivo esquerdo (assim mantida com cortes a cada 48 horas) e 3) no grupo normofuncional no mesmo tempo que no grupo 2. Os camundongos foram perfundidos com fixador de Karnovsky, em grupos de dois, 8 e 96 horas após a injeção do precursor radioativo. As hemimandíbulas foram removidas, descalcificadas, divididas em segmentos transversais e incluídas em araldite. Cortes de 1mm das regiões relacionadas à crista alveolar, primeiro molar e região odontogênica, foram cobertos com emulsão nuclear Ilford K-5 e expostos durante 14 dias. As radiografias foram analisadas quantitativamente utilizando um sistema de análise de imagens, determinando-se a densidade de área de grãos de prata nos tecidos periodontais. O exame microscópico das radioautografias mostrou que todos os tecidos dentais e periodontais do incisivo de camundongo apresentaram incorporação de [3H]-fucose, em graus variados de intensidade. Nos tecidos periodontais houve um aumento da biossíntese de glicoproteínas fucosiladas nos dentes desimpedidos por 7 dias, comparado aos normofuncionais, hiperfuncionais e hipofuncionais de 1 dia, particularmente no periodonto relacionado ao esmalte e no ligamento periodontal relacionado ao osso alveolar. Estes resultados sugerem que o aumento da quantidade de glicoproteínas fucosiladas (entre as quais fibronectina, integrinas, receptor de EGF) nos tecidos periodontais, não está diretamente relacionado a erupção acelerada que se estabelece nos dentes hipofuncionais no 10 dia de desoclusão. Como este aumento foi mais expressivo no tecido conjuntivo perivascular dos tecidos periodontais e ocorreu também na polpa, é possível que parte desta maior quantidade seja devida ao aumento dos receptores de EGF
Abstract: The periodontal ligament of unimpeded rat incisors show an increase in the amount of non-collagens proteins. To analyse if such increase is related to the higher eruption rate shown by those hipofunctional teeth we visualized through radiautography the uptake of 3H-fucose by periodontal tissues. Female mice were divided in three groups of four animals each. In two of these groups the left mandibular incisor was cut at the level of the gingival papila rendering the tooth hipofunctional while the contralateral incisor became hiperfunctional. In the third group the incisors were kept normofunctional. [3H]-fucose was injected throug the caudal vein: 1) in the first group, 1 day after the shortening of the left incisor; 2) in the second group, 7 days after the left incisor became unimpeded (and so maintained by repeating the shortening every 48h); and 3) in the normofunctional group at same time of the second group. Two animals of each group were killed by intracardiac perfusion of Karnovsky¿s fixative at 8 and 96h after injection. The hemimandibules were removed, decalcified, divided in transversal segments and embedded in araldite. 1mm-thick sections of the regions related to the alveolar crest, first lower molar and odontogenic organ were covered with Ilford K-5 nuclear emultion and exposed for 14 days. The area density of silver grains, over periodontal tissues was determined using an image analyzing system. In different degrees of intensity all dental and periodontal tissues were labeled by 3H-fucose. At 8h after the injection the silver grain density was markedly higher in the periodontal ligament related to the alveolar bone and in the enamel related periodontium of incisors kept unimpeded for 7 days as compared with all other groups. These results suggest that the increase of fucosileted glycoproteins (among them fibronectin, integrins and EGF-receptor) in the periodontal tissues of unimpeded incisors is not related to the accelerated rate of the eruption which begins 1 day after their crown is shortened. The uptake of 3H-fucose was conspicuously increased in the perivascular regions of the periodontal tissues and also in the pulp of 7 days hipofunctional incisors, indicating that the higher content of glycoproteins in these tissues may be related to the increase of EGF-receptors
Mestrado
Histologia e Embriologia
Mestre em Biologia Buco-Dental
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Yu, Zhen. "Substrate-Selective Copper Catalysts as Catalytic Metallodrugs: from G-Quadruplex Targeting Small-Molecular Nucleases to Artificial Glycosidases." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1500316480959497.
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Faust, Thomas. "Synthese und Charakterisierung von Uridin-5'-diphospho-alpha-D-fucose sowie Nachweis, partielle Reinigung und Charakterisierung einer UDP-fucose:Digitoxigenin 3-O-Beta-D-Fucosyltransferase aus Digitalis lanato in lanata EHRH /." [S.l. : s.n.], 1994. http://www.gbv.de/dms/bs/toc/17134751X.pdf.
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Jansing, Julia Verfasser], Rainer [Akademischer Betreuer] [Fischer, and Ralph [Akademischer Betreuer] Panstruga. "CRISPR/Cas9-mediated knockout of six plant-specific glycosyltransferase genes in Nicotiana benthamiana for the production of α-1,3-fucose- and β-1,2-xylose-free recombinant proteins / Julia Jansing ; Rainer Fischer, Ralph Panstruga." Aachen : Universitätsbibliothek der RWTH Aachen, 2018. http://d-nb.info/1186069554/34.
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Jansing, Julia [Verfasser], Rainer [Akademischer Betreuer] Fischer, and Ralph [Akademischer Betreuer] Panstruga. "CRISPR/Cas9-mediated knockout of six plant-specific glycosyltransferase genes in Nicotiana benthamiana for the production of α-1,3-fucose- and β-1,2-xylose-free recombinant proteins / Julia Jansing ; Rainer Fischer, Ralph Panstruga." Aachen : Universitätsbibliothek der RWTH Aachen, 2018. http://d-nb.info/1186069554/34.
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Basak, Prakriti. "Synthesis of conjugates of L-fucose and ortho-carborane as potential agents for boron neutron capture therapy and Synthesis of 2,3-dideoxy-2,3-methanoribofuranoside glycosyl donors and a study of their use in stereocontrolled glycosylation reactions." Connect to this title online, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1041010809.
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Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xiii, 279 p.: ill. (some col.). Includes abstract and vita. Advisor: Todd L. Lowary, Dept. of Chemistry. Includes bibliographical references (p. 150-154).
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Basak, Prakitri. "Synthesis of conjugates of L-fucose and ortho-carborane as potential agents for boron neutron capture therapy and synthesis of 2,3-dideoxy-2,3-methanoribofuranoside glycosyl donors and a study of their use in stereocontrolled glycosylation reactions." Columbus, OH : Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1041010809.
Full textAbstract:
Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xiii, 279 p.: ill. (some col.). Includes abstract and vita. Advisor: Todd L. Lowary, Dept. of Chemistry. Includes bibliographical references (p. 150-154).
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Taupin, Vanessa. "La différenciation sporale chez les microsporidies : imagerie 3D et isolement des stades de développement, analyse de l'expression différentielle de protéines structurales et première identification des glycanes." Phd thesis, Clermont-Ferrand 2, 2006. https://theses.hal.science/docs/00/70/27/83/PDF/2006CLF21679.pdf.
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La microsporidie, Encephalitozoon cuniculi, parasite intracellulaire, est un pathogène opportuniste. Des reconstructions tridimensionnelles à partir de coupes sériées ont permis de visualiser les différents stades cellulaires au cours de la sporogenèse. L'immunolocalisation de protéines pariétales couplée à l'hybridation in situ des ARNm correspondants ont révélé leur expression différentielle durant le développement intracellulaire. L'étude sur la glycosylation des protéines a permis de démontrer l'absence de N-glycosylation et l'existence d'une voie de O-mannosylation. Semblables à celles des champignons, les chaînes sont linéaires d'une longueur maximale de 8 mannoses liés en alpha1,2 et les mannoprotéines sont localisées dans le capuchon polaire. Des protéines fucosylées sont présentes dans la paroi sporale. La mise au point d'un protocole de séparation des stades sporogoniques en gradient de densité, offre des perspectives d'analyses biochimiques comparatives
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Taupin, Vanessa. "La différenciation sporale chez les microsporidies : imagerie 3D et isolement des stades de développement, analyse de l'expression différentielle de protéines structurales et première identification des glycanes." Phd thesis, Université Blaise Pascal - Clermont-Ferrand II, 2006. http://tel.archives-ouvertes.fr/tel-00702783.
Full textAbstract:
La microsporidie, Encephalitozoon cuniculi, parasite intracellulaire, est un pathogène opportuniste. Des reconstructions tridimensionnelles à partir de coupes sériées ont permis de visualiser les différents stades cellulaires au cours de la sporogenèse. L'immunolocalisation de protéines pariétales couplée à l'hybridation in situ des ARNm correspondants ont révélé leur expression différentielle durant le développement intracellulaire. L'étude sur la glycosylation des protéines a permis de démontrer l'absence de N-glycosylation et l'existence d'une voie de O-mannosylation. Semblables à celles des champignons, les chaînes sont linéaires d'une longueur maximale de 8 mannoses liés en alpha1,2 et les mannoprotéines sont localisées dans le capuchon polaire. Des protéines fucosylées sont présentes dans la paroi sporale. La mise au point d'un protocole de séparation des stades sporogoniques en gradient de densité, offre des perspectives d'analyses biochimiques comparatives
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Ralle, Georg. "Günter Bruno Fuchs und seine literarischen Vorläufer Quirinus Kuhlmann, Peter Hille und Paul Scheerbart /." Hannover-Laatzen : Wehrhahn, 2007. http://deposit.d-nb.de/cgi-bin/dokserv?id=2935577&prov=M&dok_var=1&dok_ext=htm.
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