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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.
51

Conception, synthèse et étude biologique des nouveaux dérivés de sucre

Zhu, Chenjiang 04 November 2008 (has links) (PDF)
Les glucides sont omniprésents dans la nature et jouent un rôle clé dans divers processus de reconnaissance moléculaire. Nous avons réalisé la conception et la synthèse de quatre types de dérivés glucidiques qui se divisent en 4 chapitres. Le le` chapitre est consacré à la synthèse d'analogues C glycosidiques de l'arbutine, une hydroquinone glycosylée, inhibiteur de la tyrosinase utilisé pour le blanchissement de la peau. Le 2è' chapitre concerne la synthèse d'analogues C glycosidiques de la vitamine E avec comme objectif d'améliorer ses propriétés antioxydants. Le 3e' chapitre est consacré à la synthèse des glycosides photolabiles pour la réalisation de glycopuces. Via l'ouverture régiosélective du 4,6 0 (o nitro)benzylidene des methyl glycopyranosides, suivie d'une C 4 épimerization, plusieurs 6 0 (o nitro)benzyl glycopyranosides ont été préparés. Enfin, le chapitre 4 est consacré à la synthèse des C glycosyl amino acides comme inhibiteurs potentiels de PTP 1B, cible potentielle pour le traitement du diabète de type II. Les résultats biologiques montrent que ces molécules inhibent l'enzyme avec IC50 de l'ordre de micromolaire.
52

Ingénierie de glycoside hydrolases pour la glycosylation des protéines recombinantes

BLANCHARD, Sophie 07 December 2004 (has links) (PDF)
Le contrôle de la glycosylation des protéines recombinantes présente un enjeu considérable dans le développement de la production de protéines d'intérêt thérapeutique dans des systèmes d'expression hétérologues. La glycosylation joue en effet un rôle essentiel dans leurs propriétés, notamment pharmacocinétiques. Le remodelage de la N-glycosylation des protéines recombinantes a été envisagé via l'utilisation de la glycosynthase Cel7B E197A d'Humicola insolens. Cette enzyme doit tout d'abord être modifiée afin de pouvoir fixer un groupement N-acétylglucosaminyle dans le sous-site accepteur +1. Des études de modélisation moléculaire ont mis en évidence deux acides aminés qui pourraient empêcher le positionnement d'une unité glucosidique substituée en position 2. Différents mutants ont été préparés par mutagénèse dirigée afin d'étudier leur spécificité de substrat. Leurs activités glycosynthases ont été caractérisées, montrant l'influence des mutations introduites. Même si la spécificité de substrat désirée n'a pu être obtenue, une modification de la régiosélectivité de la glycosynthase a été mise en évidence vis-à-vis d'un substrat substitué en position 2 par un groupement de type azido.
53

Cytotoxic Compounds of Plant Origin – Biological and Chemical Diversity / Cytotoxiska föreningar från växter – biologisk och kemisk diversitet

Lindholm, Petra January 2005 (has links)
<p>The development of resistance by tumour cells to chemotherapeutic agents is a major problem in cancer treatments. One way to counter this is to find compounds with cytotoxic mechanisms other than those of drugs in clinical use today. The biological and chemical diversity encountered in Nature provide opportunities to discover completely new chemical classes of compounds. Some of these may represent previously unknown anticancer agents, and in some cases, novel, potentially relevant cytotoxic mechanisms. </p><p>The selection of plants for the cytotoxic investigation in this project was designed to cover large parts of the angiosperm system, providing a broad representation of species. Extracts of the plants were subjected to a polypeptide fractionation protocol, followed by bioassay-guided isolation, yielding series of fractions with increasing purity and cytotoxicity. The cytotoxicity assay included tumour cells from patients and a cell-line panel including ten different cell lines representing several types of resistant and non-resistant tumours. This screening strategy allowed fractions and compounds acting with novel mechanisms to be detected at an early stage. </p><p>The compounds isolated represent substantial chemical diversity and originate from diverse parts of the phylogenetic spectrum examined. They include the highly potent cytotoxic alkaloid, thiobinupharidine, the structure of which was determined by NMR techniques. Furthermore, two types of compound were shown to have previously unreported cytoxic activity: cyclotides (small macrocyclic polypeptides, in this case from violets) and polypeptides, possibly of thionine type, of loranthaceaeous mistletoes (collected in Panama). The well known cardiac glycosides from the foxglove, Digitalis, were identified as being responsible for the anti-tumour activity of this species.</p><p>In conclusion, the results obtained in this project show that selection based on phylogenetic information, together with a robust and reliable method to detect cytotoxicity, can be a useful approach for exploring the plant kingdom for cytotoxic substances.</p>
54

Discovery of fiber-active enzymes in Populus wood

Aspeborg, Henrik January 2004 (has links)
Renewable fibers produced by forest trees provide excellentraw material of high economic value for industrialapplications. Despite this, the genes and corresponding enzymesinvolved in wood fiber biosynthesis in trees are poorlycharacterized. This thesis describes a functional genomicsapproach for the identification of carbohydrate-active enzymesinvolved in secondary cell wall (wood) formation in hybridaspen. First, a 3' target amplification method was developed toenable microarray-based gene expression analysis on minuteamounts of RNA. The amplification method was evaluated usingboth a smaller microarray containing 192 cDNA clones and alarger microarray containing 2995 cDNA clones that werehybridized with targets isolated from xylem and phloem.Moreover, a gene expression study of phloem differentiation wasperformed to show the usefulness of the amplificationmethod. A microarray containing 2995 cDNA clones representing aunigene set of a cambial region EST library was used to studygene expression during wood formation. Transcript populationsfrom thin tissue sections representing different stages ofxylem development were hybridized onto the microarrays. It wasdemonstrated that genes encoding lignin and cellulosebiosynthetic enzymes, as well as a number of genes withoutassigned function, were differentially expressed across thedevelopmental gradient. Microarrays were also used to track changes in geneexpression in the developing xylem of transgenic, GA-20 oxidaseoverexpressing hybrid aspens that had increased secondarygrowth. The study revealed that a number of genes encoding cellwall related enzymes were upregulated in the transgenic trees.Moreover, most genes with high transcript changes could beassigned a role in the early events of xylogenesis. Ten genes encoding putative cellulose synthases (CesAs) wereidentified in our ownPopulusESTdatabase. Full length cDNA sequences wereobtained for five of them. Expression analyses performed withreal-time PCR and microarrays in normal wood undergoingxylogenesis and in tension wood revealed xylem specificexpression of four putative CesA isoenzymes. Finally, an approach combining expressionprofiling,bioinformatics as well as EST and full length sequencing wasadopted to identify secondary cell wall related genes encodingcarbohydrate-active enzymes, such as glycosyltransferases andglycoside hydrolases. As expected, glycosyltransferasesinvolved in the carbohydrate biosynthesis dominated thecollection of the secondary cell wall related enzymes that wereidentified. Key words:Populus, xylogenesis, secondary cell wall,cellulose, hemicellulose, microarrays, transcript profiling,carbohydrate-active enzyme, glycosyltransferase, glycosidehydrolase
55

Cytotoxic Compounds of Plant Origin – Biological and Chemical Diversity / Cytotoxiska föreningar från växter – biologisk och kemisk diversitet

Lindholm, Petra January 2005 (has links)
The development of resistance by tumour cells to chemotherapeutic agents is a major problem in cancer treatments. One way to counter this is to find compounds with cytotoxic mechanisms other than those of drugs in clinical use today. The biological and chemical diversity encountered in Nature provide opportunities to discover completely new chemical classes of compounds. Some of these may represent previously unknown anticancer agents, and in some cases, novel, potentially relevant cytotoxic mechanisms. The selection of plants for the cytotoxic investigation in this project was designed to cover large parts of the angiosperm system, providing a broad representation of species. Extracts of the plants were subjected to a polypeptide fractionation protocol, followed by bioassay-guided isolation, yielding series of fractions with increasing purity and cytotoxicity. The cytotoxicity assay included tumour cells from patients and a cell-line panel including ten different cell lines representing several types of resistant and non-resistant tumours. This screening strategy allowed fractions and compounds acting with novel mechanisms to be detected at an early stage. The compounds isolated represent substantial chemical diversity and originate from diverse parts of the phylogenetic spectrum examined. They include the highly potent cytotoxic alkaloid, thiobinupharidine, the structure of which was determined by NMR techniques. Furthermore, two types of compound were shown to have previously unreported cytoxic activity: cyclotides (small macrocyclic polypeptides, in this case from violets) and polypeptides, possibly of thionine type, of loranthaceaeous mistletoes (collected in Panama). The well known cardiac glycosides from the foxglove, Digitalis, were identified as being responsible for the anti-tumour activity of this species. In conclusion, the results obtained in this project show that selection based on phylogenetic information, together with a robust and reliable method to detect cytotoxicity, can be a useful approach for exploring the plant kingdom for cytotoxic substances.
56

Discovery of fiber-active enzymes in Populus wood

Aspeborg, Henrik January 2004 (has links)
<p>Renewable fibers produced by forest trees provide excellentraw material of high economic value for industrialapplications. Despite this, the genes and corresponding enzymesinvolved in wood fiber biosynthesis in trees are poorlycharacterized. This thesis describes a functional genomicsapproach for the identification of carbohydrate-active enzymesinvolved in secondary cell wall (wood) formation in hybridaspen.</p><p>First, a 3' target amplification method was developed toenable microarray-based gene expression analysis on minuteamounts of RNA. The amplification method was evaluated usingboth a smaller microarray containing 192 cDNA clones and alarger microarray containing 2995 cDNA clones that werehybridized with targets isolated from xylem and phloem.Moreover, a gene expression study of phloem differentiation wasperformed to show the usefulness of the amplificationmethod.</p><p>A microarray containing 2995 cDNA clones representing aunigene set of a cambial region EST library was used to studygene expression during wood formation. Transcript populationsfrom thin tissue sections representing different stages ofxylem development were hybridized onto the microarrays. It wasdemonstrated that genes encoding lignin and cellulosebiosynthetic enzymes, as well as a number of genes withoutassigned function, were differentially expressed across thedevelopmental gradient.</p><p>Microarrays were also used to track changes in geneexpression in the developing xylem of transgenic, GA-20 oxidaseoverexpressing hybrid aspens that had increased secondarygrowth. The study revealed that a number of genes encoding cellwall related enzymes were upregulated in the transgenic trees.Moreover, most genes with high transcript changes could beassigned a role in the early events of xylogenesis.</p><p>Ten genes encoding putative cellulose synthases (CesAs) wereidentified in our own<i>Populus</i>ESTdatabase. Full length cDNA sequences wereobtained for five of them. Expression analyses performed withreal-time PCR and microarrays in normal wood undergoingxylogenesis and in tension wood revealed xylem specificexpression of four putative CesA isoenzymes.</p><p>Finally, an approach combining expressionprofiling,bioinformatics as well as EST and full length sequencing wasadopted to identify secondary cell wall related genes encodingcarbohydrate-active enzymes, such as glycosyltransferases andglycoside hydrolases. As expected, glycosyltransferasesinvolved in the carbohydrate biosynthesis dominated thecollection of the secondary cell wall related enzymes that wereidentified.</p><p><b>Key words:</b>Populus, xylogenesis, secondary cell wall,cellulose, hemicellulose, microarrays, transcript profiling,carbohydrate-active enzyme, glycosyltransferase, glycosidehydrolase</p>
57

Elucidation of the Catalytic Mechanism of Golgi alpha-mannosidase II

Shah, Niket 26 February 2009 (has links)
The central dogma of molecular biology outlines the process of information transfer from a DNA sequence, to a protein chain. Beyond the step of protein synthesis, there are a variety of post-translational modifications that can take place, one of which is addition of carbohydrate chains to nascent proteins, known as glycosylation. The N-linked glycosylation pathway is responsible for the covalent attachment of multifunctional carbohydrate chains on asparagine residues of nascent proteins at Asn-X-Ser/Thr consensus sequences. These carbohydrate chains are thought to aid in cell signaling, immune recognition, and other processes. Golgi alpha-mannosidase II (GMII) is the enzyme in the N-glycosylation pathway that is responsible for cleaving two mannose linkages in the oligosaccharide GnMan5Gn2 (where Gn is N-acetylglucosamine and Man is mannose), thereby producing GnMan3Gn2 , which is the committed step in complex N-glycan synthesis. It has been speculated that GMII is an excellent therapeutic target for cancer treatment, as the unusual distribution of carbohydrates on the surface of tumour cells has been characterized in many cancers. In addition, swainsonine-—a strong, yet nonspecific inhibitor of GMII—-has been shown to block metastasis and improve the clinical outcome of patients with certain cancers, including those of the colon, breast and skin. This thesis examines Golgi alpha-mannosidase II from Drosophila melanogaster (dGMII) as a model for all GMII enzymes. First, a 1.80 Angstrom resolution crystal structure of a weak inhibitor, kifunensine, binding to dGMII provides mechanistic insights into the substrate distortion in the GMII reaction. It is hypothesized that the GMII reaction proceeds via a 1 Sinterintermedi-ate. Second, a 1.40 Angstrom resolution structure of a mutant dGMII bound to its natural substrate, GnMan5Gn, identifies key substrate binding and catalytic residues, as well as expanding the definition of the GMII active site to include two distant sugar−binding subsites. Finally, the results are taken together, with knowledge of other related enzymes to synthesize a plausible itinerary for the GMII reaction.
58

Elucidation of the Catalytic Mechanism of Golgi alpha-mannosidase II

Shah, Niket 26 February 2009 (has links)
The central dogma of molecular biology outlines the process of information transfer from a DNA sequence, to a protein chain. Beyond the step of protein synthesis, there are a variety of post-translational modifications that can take place, one of which is addition of carbohydrate chains to nascent proteins, known as glycosylation. The N-linked glycosylation pathway is responsible for the covalent attachment of multifunctional carbohydrate chains on asparagine residues of nascent proteins at Asn-X-Ser/Thr consensus sequences. These carbohydrate chains are thought to aid in cell signaling, immune recognition, and other processes. Golgi alpha-mannosidase II (GMII) is the enzyme in the N-glycosylation pathway that is responsible for cleaving two mannose linkages in the oligosaccharide GnMan5Gn2 (where Gn is N-acetylglucosamine and Man is mannose), thereby producing GnMan3Gn2 , which is the committed step in complex N-glycan synthesis. It has been speculated that GMII is an excellent therapeutic target for cancer treatment, as the unusual distribution of carbohydrates on the surface of tumour cells has been characterized in many cancers. In addition, swainsonine-—a strong, yet nonspecific inhibitor of GMII—-has been shown to block metastasis and improve the clinical outcome of patients with certain cancers, including those of the colon, breast and skin. This thesis examines Golgi alpha-mannosidase II from Drosophila melanogaster (dGMII) as a model for all GMII enzymes. First, a 1.80 Angstrom resolution crystal structure of a weak inhibitor, kifunensine, binding to dGMII provides mechanistic insights into the substrate distortion in the GMII reaction. It is hypothesized that the GMII reaction proceeds via a 1 Sinterintermedi-ate. Second, a 1.40 Angstrom resolution structure of a mutant dGMII bound to its natural substrate, GnMan5Gn, identifies key substrate binding and catalytic residues, as well as expanding the definition of the GMII active site to include two distant sugar−binding subsites. Finally, the results are taken together, with knowledge of other related enzymes to synthesize a plausible itinerary for the GMII reaction.
59

Estudos funcionais e estruturais de uma endoglucanase de Phanerochaete chrysosporium da família 45 das hidrolases de glicosídeos / Structural and functional studies of an endoglucanase from Phanerochaete chrysorporium belonging to the glycoside hydrolase family 45

Marina Paglione Ramia 07 December 2015 (has links)
A importância do estudo das celulases não se limita à aquisição de conhecimento científico, mas também ao grande potencial biotecnológico que elas representam. Isso se deve ao fato da celulose ser a molécula mais abundante presente na natureza e prover uma vasta gama de produtos e processos sustentáveis. Muitas famílias de celulases já foram bem caracterizadas, enquanto outras permanecem ainda desconhecidas. Dentre estas últimas, a família 45 das hidrolases de glicosídeos é a família de celulases fúngicas menos caracterizada tanto estruturalmente quanto funcionalmente. Recentemente foi proposta a divisão dessa família em três subfamílias e, até agora, apenas membros da subfamília A tiveram enzimas estruturalmente elucidadas. Nesse trabalho reportamos a estrutura cristalográfica da proteína recombinante endoglucanase de Phanerochaete chrysosporium (PcCel45A), a primeira das hidrolases de glicosídeos da subfamília C, e seu complexo com celobiose a 1,4 &Aring; e 1,7 &Aring; de resolução, respectivamente. A PcCel45A é uma enzima de domínio único, com uma estrutura em &beta;-barril e seu empacotamento geral remete ao formato de âncora. O sítio ativo da enzima forma um longo sulco na superfície da estrutura, sendo que o seu centro catalítico é diferente das outras enzimas publicadas dessa família e o aspartato catalítico, que atua como aceptor de próton na reação de inversão, (Asp10) não é conservado. Adicionalmente, a estrutura cristalográfica dessa enzima apresenta mais similaridades com as &beta;-expansinas (proteínas de plantas) e transglicosilases líticas (proteínas que clivam o peptidoglicano de bactérias) do que com as outras representantes da família 45, o que a torna ainda mais singular. Para entendermos melhor seu funcionamento foram realizadas mutações sítio-dirigidas nos principais resíduos do sítio ativo. O Asp121, conhecido por participar da reação de inversão das outras enzimas da família como doador de próton, mostrou-se essencial para a atividade da enzima, enquanto que outros resíduos conservados como a Tyr25, o Trp161 e o Asp92 afetaram, mas não aniquilaram a atividade da enzima, apresentando aproximadamente 20%, 50% e 10% da atividade da enzima nativa, respectivamente. / The importance of the study of the cellulases is not limited to generating significant scientific knowledge, since these enzymes represents an enormous potential in biotechnology. This is partly because cellulose is the most abundant molecule in nature and provides a wide range of products and sustainable process. Many cellulases families have been well characterized, while others still remain unknown. Among them, the glycoside hydrolase family 45 is the least well characterized both structurally and functionally, between fungal cellulases. It was recently proposed the subdivision of this family into three subfamilies, with structural information available only for subfamily A. In this work, we report the chrystallographic structure of the recombinant endoglucanase from Phanerochaete chrysosporium (PcCel45A), the first GH45 subfamily C and its complex with cellobiose at 1.4 &Aring; and 1.7 &Aring; respectively. The PcCel45A is a single domain enzyme, which has a &beta;-barrel structure with the overall shape resembling an anchor. The active site of the enzyme has a long cleft on the surface, being remarkably different from those members of subfamily A, and the catalytic aspartate responsible for acting as proton acceptor (Asp10) is not present. Additionally, the chrystallographic structure of this enzyme has shown more similarity with &beta; -expansins (plant proteins) and lytic transglycosylase (proteins that cleave the peptidoglycan of bacteria) than others representants of family 45, which makes it more singular. For a better understanding of its function, we perform pontual mutations in the main residues from active site. The Asp121, known for acting as proton acceptor in the inversion reaction of others enzymes, proved to be essential for the enzyme activity, while others conserved residues as Tyr25, Trp161 and Asp92 affected but not annihilated the enzyme activity, leaving approximately 20%, 50% and 10% of the native enzyme activity.
60

Estudos funcionais e estruturais de enzimas frutosiltransferases das famílias 32 e 68 de hidrolases de glicosídeos / Hidrolases de glicosídeos 32 e 68, Frutosiltransferases, Frutooligossacarídeos Functional and Structural studies of the fructosyltransferases enzymes from the families 32 and 68 of glycoside hydrolases

Mariana Zuliani Theodoro de Lima 22 October 2015 (has links)
A busca por substâncias benéficas à saúde humana tem impulsionado o desenvolvimento de pesquisas visando o estudo de enzimas e seus produtos através da otimização de bioprocessos. Um dos principais componentes utilizados como base para a indústria de alimentos funcionais são carboidratos denominados frutooligossacarídeos (FOS) derivados da sacarose. Estes são sintetizados por enzimas denominadas frutosiltransferases que podem ser encontradas em plantas, bactérias e fungos. Os FOS têm atraído grande interesse da indústria, devido às suas características fisiológicas e biomoduladoras. Por serem polissacarídeos prebióticos não-digeríveis, têm a capacidade de estimular seletivamente o crescimento de bifidobactérias e lactobacilos, auxiliando na prevenção da cárie dentária e câncer de cólon em humanos. Podem também contribuir na diminuição do colesterol total e triglicerídeos no sangue, promover a reabsorção de cálcio e magnésio e serem utilizados em dietas com restrições alimentares, por serem açúcares de baixo valor calórico e elevado valor nutricional. Tendo em vista a existência de diferentes formas de FOS sintetizados por diferentes mecanismos, o presente trabalho buscou realizar a caracterização estrutural e funcional de um conjunto de 13 frutosiltransferases de bactérias e fungos. Os genes alvo foram clonados e as enzimas expressas e purificadas. Ensaios estruturais e de atividade enzimática, incluindo de hidrólise e polimerização da sacarose, foram conduzidos para a melhor compreensão das bases moleculares envolvidas no reconhecimento do substrato. Oito enzimas, duas &beta;-frutofuranosidases de B. adolescentis, três sucrose-6-phosphate hydrolase de B. licheniformis e L. gasseri e uma invertase de A.niger foram cristalizadas e as enzimas de B. adolescentis e de L. gasseri tiveram suas estruturas resolvidas e seus sítios catalíticos mapeados. Estas apresentam em sua estrutura uma região &beta;-propeller, local identificado como sítio catalítico, conectada à um módulo &beta;-sanduíche. Ambas as enzimas apresentaram atividade hidrolítica da sacarose e a enzima de L. gasseri apresentou a formação dos FOS nistose e 1-cestose com concentrações de 1 M de sacarose bem como em tempos de 8 e 12 horas de incubação. Estes estudos, somados às análises das outras enzimas, permitirão o melhoramento na produção em larga escala, além da otimização e o controle destes processos de obtenção de FOS. / The search for beneficial substances to human health has driven the development of researches on the study of enzymes and their products through bioprocess optimization. One of the main components used as a basis for functional food industry are carbohydrates called fructooligosaccharides (FOS) derived from sucrose. These are synthesized by enzymes called fructosyltransferases which can be found in plants, bacteria and fungi. The FOS has attracted great interest of the industry due to its physiological and biomodulator properties. Because it is non-digestible polysaccharides prebiotics, have the ability to selectively stimulate the growth of bifidobacteria and lactobacilli, assisting in the prevention of tooth decay and colon cancer in humans. They can also contribute to decrease total cholesterol and triglycerides in the blood, to promote the absorption of calcium and magnesium and can be used in diets with dietary restrictions, because they are low-calorie sugars presenting high nutritional value. Considering there are different forms of FOS synthesized by different mechanisms, the present work attempts to make structural and functional characterization of a set of 13 fructosyltransferases of bacteria and fungi. The target genes were cloned and the enzymes were expressed and purified. Structural testing of X-ray crystallography and enzymatic activity, including sucrose hydrolysis and polymerization were carried out for a better understanding of the molecular basis involved in substrate recognition. Eight enzymes, two &beta;-frutofuranosidases B. adolescentis, three sucrose-6-phosphate hydrolase of B. licheniformis and L. gasseri and A. niger invertase, were crystallized and the enzymes from B. adolescentis and from L. gasseri had their structures determined and their catalytic site mapped. These are similar to each other and present in their structure one &beta;-propeller region which was identified as catalytic site, connected to one &beta;-sandwich module. Both enzymes showed hydrolytic activity of the sucrose and L. gasseri showed the formation of FOS, 1-kestose and nystose with 1 M sucrose concentrations and times of 8 and 12 hours of incubation. These studies together with the analysis of other enzymes will enable the improvement in large-scale production, besides the optimization and control of these processes for the production of FOS.

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