Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-01-29T13:33:40Z No. of bitstreams: 2 Dissertação - Paula Leticia de Melo Souza - 2015.pdf: 1524602 bytes, checksum: 34e108b82c97baa593fc6a90a1e38b4b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-02-01T11:43:57Z (GMT) No. of bitstreams: 2 Dissertação - Paula Leticia de Melo Souza - 2015.pdf: 1524602 bytes, checksum: 34e108b82c97baa593fc6a90a1e38b4b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-02-01T11:43:57Z (GMT). No. of bitstreams: 2 Dissertação - Paula Leticia de Melo Souza - 2015.pdf: 1524602 bytes, checksum: 34e108b82c97baa593fc6a90a1e38b4b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-02-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Biotransformations are powerful tools for optimization of active compounds, diversification and structural modifications, in addition to handling non-functionalized compounds unfeasible by chemical conventional methods. The versatility of microbial systems, in this context, and the numerous entitlements of filamentous fungi have leveraged the use of biotransformations with microorganisms. Glycosylations ate one of the most important and commom modification processes and the use of biocatalysts have set a favorable strategy to this type of reaction. Cunninghamella species, for its enzimatic arsenal, are widely applied, especially for enabling the production of novel drug and cell immobilization hás been evidenced as a tool to express its enzimatic activity.Considering the relevance of amino sugars and their enormous potential in various therapies, the aim of this study was to produce 5-(1-(3-fluorophenyl)-1H-pyrazol-4-yl)-1H-tetrazole (LQFM 021) derivatives, which contains tetrazole and pyrazole rings, by biotransformation with free and immobilized fungi cells of Cunninghamella echinulata ATCC 9244. Therefore, CLAE methodologies were developed for the analysis of biotransformation kinectics and characterization techniques employed (Nuclear Magnetic Resonance (NMR) and Mass Spectrometry with Ion cyclotron Resonance (FTICR-MS)) of the product. 96-hour incubations were proceeded with free cell Cunninghamella echinulata ATCC 9244, in the PDSM culture medium and immobilized cells in three separate experiments: biofilm incubated in PBS buffer, biofilm incubated in PDSM medium culture and reuse of biofilms, where one derivative was obtained. The structural characterization for the purified derivative obtained in sufficient quantities is a 1-(5-(1-(3-fluorophenyl)-1H-pyrazol-4-yl)-2H-tetrazol-2-yl)pentane-1,2,3,4,5-pentaol. / As biotransformações são ferramentas poderosas para otimização de compostos ativos, modificação e diversificação estrutural, além da manipulação de compostos não-funcionalizados inviável por métodos químicos tradicionais. A versatilidade dos sistemas microbiano, nesse contexto, e as inúmeras vantagens atribuídas aos fungos filamentosos alavancaram o uso das biotransformações com microorganismos. As glicosilações são um dos mais importantes e comuns processos de modificação e o uso de biocatalisadores tem configurado uma estratégia favorável a esse tipo de reação. Espécies de Cunninghamella, por seu arsenal enzimático, são extensamente aplicadas, principalmente por viabilizarem a produção de novos derivados de fármacos e a imobilização celular é evidenciada como uma estratégia para expressar essa atividade enzimática. Frente à relevância dos aminoaçúcares e seu enorme potencial terapêutico, o objetivo deste estudo foi produzir derivados do 5-(1-(3-fluorofenil)-1H-pirazol-4-il)-1H-tetrazol (LQFM 021), que contém anéis tetrazólico e pirazólico, a partir da biotransformação por células fúngicas livres e imobilizadas de Cunninghamella echinulata ATCC 9244. Para tanto, foram desenvolvidas metodologias para análise da cinética de biotransformação do LQFM 021, bem como empregadas técnicas de caracterização (Ressonância Magnética Nuclear (RMN) e Espectrometria de Massas com Ressonância Ciclotrônica de Íons (FTICR-MS) do produto obtido. Foram realizadas incubações de 96 horas com células livres de Cunninghamella echinulata ATCC 9244, em meio de cultura PDSM e com células imobilizadas em três experimentos distintos: biofilme incubado em tampão PBS, biofilme incubado em meio de cultura PDSM e reutilização dos biofilmes, em que foi obtido um derivado do LQFM 021.A proposta estrutural para o derivado obtido, purificado em quantidades suficientes, é de um 1-(5-(1-(3-fluorofenil)-1H-pirazol-4-il)-2H-tetrazol-2-il)pentano-1,2,3,4,5-pentaol.

of Ph.D. thesis Molecular mechanisms of regulation of trafficking and function of different subtypes of NMDA receptors in hippocampal neurons Mgr. Kristýna Skřenková N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors that play a key role in the mammalian central nervous system. Under physiological conditions, these receptors are important for excitatory synaptic transmission and memory formation. However, under pathological conditions, their abnormal regulation or activation may lead to many neurological and psychiatric disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy or schizophrenia. Previous studies have shown that the number and type of NMDA receptors on the cell surface are regulated at multiple levels, including their synthesis, folding, internalization or degradation. During the trafficking of NMDA receptors to the cell surface membrane, both the agonist binding and receptor activation are examined. Moreover, NMDA receptors undergo many posttranslational modifications such as palmitoylation, phosphorylation or N-glycosylation. In this thesis, we studied the molecular mechanisms that may affect the trafficking and functional properties of NMDA receptors in mammalian cells and rat hippocampal neurons. Specifically, we studied i)...

Final publication is available at http://dx.doi.org/10.1016/j.bbrc.2015.12.114 CreativeCommonsAttribution Non-Commercial No Derivatives License の記載が必要 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19924号 / 医博第4144号 / 新制||医||1017(附属図書館) / 33010 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 野田 亮, 教授 小川 修 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L’analyse protéomique par spectrométrie de masse s’est imposée comme une méthode incontournable pour la caractérisation des protéines. Grâce aux progrès de l’instrumentation et de la bioinformatique, l’interprétation automatisée des spectres MS/MS permet aujourd’hui d’identifier des milliers de protéines dans un type cellulaire. Cependant, cette méthodologie s’applique encore difficilement aux organismes dont les génomes n’ont pas été séquencés, et donc pour lesquels il n’existe pas de banques de séquences peptidiques de référence. Notre travail a porté sur le développement et l’application d’une méthodologie d’interprétation des données MS/MS pour les organismes à génomes non séquencés. Cette méthodologie est basée sur le séquençage de novo suivi de recherche MS-BLAST. Ainsi nous avons pu : Identifier les différents partenaires de complexes protéiques tels que les protéines des complexes TgGAP50, TgAlba, TgSORTLR impliqués dans la motilité, la virulence ou le trafic intracellulaire des protéines du parasite Toxoplasma gondii, Identifier et caractériser des variants d’hémoglobine humaine, Identifier les protéines différentiellement exprimées lors des interactions vigne et champignons à génomes non séquencés dans la maladie de l’esca, Caractériser finement la N-glycosylation de l’invertase vacuolaire du raisin. Nous avons pu réaliser nos études sur des échantillons d’origines très différentes : homme, plantes, champignons, parasites et nous avons apporté des éléments de réponses moléculaires aux questions biologiques. / The proteomic analysis by mass spectrometry is now an essential method for the characterization of proteins. Thanks to advances in instrumentation and bioinformatics, automated interpretation of MS/MS spectra can now identify thousands of proteins in a cell type. However, this methodology remains poorly applied to the organisms that genomes are not sequenced and therefore where there is no database of reference for peptides sequences. Our work has focused on the development and application of a methodology for the interpretation of MS/MS data for the organisms that genomes are not sequenced. This methodology is based on the de novo sequencing followed by MS-BLAST search. Thus we have: Identify different partners of protein complexes such as proteins TgGAP50, TgAlba and TgSORTLR complex, involved in motility, virulence or intracellular protein trafficking of Toxoplasma gondii, Identify and characterize human hemoglobin variants, Identify the proteins differentially expressed during interaction of vines and fungi that genomes are not sequenced in esca disease, Finely characterize the N-glycosylation of the grape vacuolar invertase. We have achieved our studies on samples of very different origins: human, plants, fungi, parasites, and we provided evidence of molecular responses to biological questions.

Anion exchanger 1 (AE1) is a 95 kDa glycoprotein that facilitates Cl-/HCO3- exchange across the erythrocyte plasma membrane. Seven conserved tryptophan (Trp) residues are in the AE1 membrane domain; at the membrane interface (Trp648, Trp662, and Trp723), in transmembrane segment (TM) 4 (Trp492 and Trp496), and in hydrophilic loops (Trp831, and Trp848). All 7 Trp residues were individually mutated into alanine (Ala) and phenylalanine (Phe) and transiently expressed in human embryonic kidney (HEK)-293 cells. The 7 Trp residues could be grouped into three classes according to the impact of the mutations on the functional expression of AE1: class 1, normal expression, class 2, expression decreased, and class 3, expression decreased by Ala substitution. These results indicate that Trp residues play differential roles in AE1 expression depending on their location in the protein and suggest that Trp mutants with a low expression are misfolded and retained in the ER.

Anion exchanger 1 (AE1) is a 95 kDa glycoprotein that facilitates Cl-/HCO3- exchange across the erythrocyte plasma membrane. Seven conserved tryptophan (Trp) residues are in the AE1 membrane domain; at the membrane interface (Trp648, Trp662, and Trp723), in transmembrane segment (TM) 4 (Trp492 and Trp496), and in hydrophilic loops (Trp831, and Trp848). All 7 Trp residues were individually mutated into alanine (Ala) and phenylalanine (Phe) and transiently expressed in human embryonic kidney (HEK)-293 cells. The 7 Trp residues could be grouped into three classes according to the impact of the mutations on the functional expression of AE1: class 1, normal expression, class 2, expression decreased, and class 3, expression decreased by Ala substitution. These results indicate that Trp residues play differential roles in AE1 expression depending on their location in the protein and suggest that Trp mutants with a low expression are misfolded and retained in the ER.

Dendritic cells (DCs) are promising mediators of anti-tumour immune responses. Unfortunately, a major hindrance to the development of highly effective DC vaccines is their short lifespan. Tumour antigen presentation may also not be optimal. We hypothesize that the introduction of exogenous survival factors (SFs) would prolong DC longevity and that modulation of TAA glycosylation will improve antigen presentation. To this end, we have constructed bicistronic lentivectors (LVs) encoding the xeno Tumour-Associated-Antigen (TAA), rHER-2/neu, and one of five candidate SFs. We demonstrated that our LVs can effectively protect transduced DCs from apoptosis when subjected to apoptosis-inducing conditions. TAA glycosylation has been proposed to obstruct the processing and presentation of peptides on MHC molecules. To address this second issue, we have engineered a LV that encodes a partially deglycosylated rHER-2/neu. Overall, we have generated the tools to alter intrinsic DC properties, which we believe will be integral to improving DC vaccine efficacy.

Dendritic cells (DCs) are promising mediators of anti-tumour immune responses. Unfortunately, a major hindrance to the development of highly effective DC vaccines is their short lifespan. Tumour antigen presentation may also not be optimal. We hypothesize that the introduction of exogenous survival factors (SFs) would prolong DC longevity and that modulation of TAA glycosylation will improve antigen presentation. To this end, we have constructed bicistronic lentivectors (LVs) encoding the xeno Tumour-Associated-Antigen (TAA), rHER-2/neu, and one of five candidate SFs. We demonstrated that our LVs can effectively protect transduced DCs from apoptosis when subjected to apoptosis-inducing conditions. TAA glycosylation has been proposed to obstruct the processing and presentation of peptides on MHC molecules. To address this second issue, we have engineered a LV that encodes a partially deglycosylated rHER-2/neu. Overall, we have generated the tools to alter intrinsic DC properties, which we believe will be integral to improving DC vaccine efficacy.

CONTEXTE: Les " Type I Congenital disorders of glycosylation " (CDG-I) comportent des déficits de biosynthèse de l'oligosaccharide lié au dolichol (DLO) qui est nécessaire pour la N-glycosylation des protéines. Ces déficits induisent : 1) une hypoglycosylation des protéines qui serait à l'origine de la pathologie ; et 2) une accumulation de DLO tronqués à partir desquels, par un mécanisme encore inconnu, des structures oligosaccharidiques libres phosphorylées (OSP) sont générées dans le cytosol. Afin de comprendre le rôle de ce processus dans le CDG, il était donc nécessaire de caractériser l'activité qui est à l'origine des OSP.RESULTATS: J'ai caractérisé biochimiquement une DLO diphosphatase (DLODP) qui génère des OSP et du dolichol phosphate à partir de DLO. L'activité DLODP co-fractionne avec un marqueur de l'appareil de Golgi (AG) mais pas avec les enzymes réticulaires qui utilisent le dolichol phosphate. Cette localisation inattendue de DLODP m'a conduit à étudier la génération des OSP dans les cellules en utilisant la bréfeldine A (BFA) qui fusionne l'AG avec le RE. La BFA ne modifie pas les taux de DLO tronqués ni ceux des OSP cytoplasmiques dans un modèle cellulaire de CDG-I. Cependant, dans ces cellules et dans les cellules témoins, la BFA induit une forte augmentation des OSP dans le système endomembranaire à partir de DLO non-tronqués.CONCLUSION: L'identification de différents pools d'OSP, topologiquement distincts et pouvant être modulés de façon indépendante, révèle la multiplicité des mécanismes pour la génération d'OSP et suggère que la DLODP Golgienne n'est pas forcément l'enzyme responsable de la génération des OSP dans le contexte de CDG-I. / BACKGROUND: Type I congenital disorders of glycosylation (CDG-I) are caused by genetic defects in the biosynthetic pathway for the dolichol-linked oligosaccharide (DLO) that is required for protein N-glycosylation. These mutations result in the accumulation of truncated DLO and protein hypoglycosylation. Although protein hypoglycosylation is thought to be the main pathogenic factor in CDG-I, the role of truncated DLO intermediates in cellular homeostasis is not clear. Truncated DLO intermediates are known to give rise to cytoplasmic oligosaccharyl phosphates (OSP) by an uncharacterized mechanism. To understand this DLO editing process biochemical and molecular characterization of the activity that generate OSP is needed.RESULTS: I biochemically characterized a DLO diphosphatase (DLODP) that generates OSP and dolichol phosphate from DLO. Subcellular fractionation of mouse liver homogenates demonstrated a microsomal activity that co-distributes with a Golgi apparatus (GA) marker but not with endoplasmic reticulum (ER)-situated dolichol phosphate utilizing enzymes. This unexpected localization of DLODP prompted me to study OSP generation in cells using brefeldin A (BFA), which fuses the GA with the ER. BFA did not affect the levels of truncated DLO or cytoplasmic OSP, present in a cellular model of CDG-I. However, in these, and control cells, BFA caused striking increases of OSP within the endomembrane system. CONCLUSION: the identification of topologically distinct, independently modulated, OSP pools indicates multiple mechanisms for OSP generation and suggest that the GA-situated DLODP may not be the enzyme responsible for OSP generation in CDG-I.

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