De nombreuses méthodes in vitro d’évolution d'enzymes existent mais elles sont souvent coûteuses en temps et en main d'œuvre. Une approche alternative, l’évolution dirigée in vivo, permet la sélection de variantes d'enzymes améliorées ou modifiées à moindre coût et peut être appliquée à des enzymes de structure inconnue. Des dispositifs automatisés de culture continue (GM3), ont été développés et construits au Genoscope. Le GM3 a été conçu pour maintenir indéfiniment des cultures bactériennes en suspension tout en les soumettant à des régimes de culture sélectifs de type turbidostat et chémostat. Le principal objectif du présent travail était l’optimisation par évolution dirigée in vivo d’enzymes de la famille des dioxygénases à α-cétoglutarate et fer-dépendantes (α-KAO). Les α-KAOs catalysent l'hydroxylation d’un atome de carbone non substitué et sont donc d’intéressants outils pour la fonctionnalisation de molécules en synthèse organique. La dynamique de fixation d’une mutation bénéfique dans une population en croissance en fonction du régime de culture et du format d’expression a été étudiée. Des souches d’Escherichia coli ont été génétiquement modifiées pour que leur croissance dépende strictement d’une activité α-KAO. Des populations de ces bactéries exprimant une α-KAO ont été soumises à des protocoles d’évolution dirigée in vivo pour sélectionner: dans un premier temps, des variants d’expression optimisée sur le substrat naturel de l’enzyme, dans un second temps, des variants utilisant des analogues de substrat non naturels. Deux variantes de la L-isoleucine dioxygénase (E65K, A62P) ont été obtenues. L’étude des paramètres cinétiques déterminera l’effet de ces mutations sur l’activité de l’enzyme. En collaboration avec l’équipe du Dr. Tolonen, la culture continue a été appliquée à l’évolution de la bactérie cellulolytique Clostridium phytofermentans. Cette bactérie anaérobie stricte qui dégrade la lignocellulose en éthanol et H2 présente un intérêt applicatif pour la valorisation de la biomasse. La dégradation de la lignocellulose libère des composés phénoliques, en particulier l’acide férulique. C. phytofermentans a été adapté à résister à des concentrations croissantes de ce composé inhibiteur. Des isolats résistant au ferulate ont été analysés et leur génomes séquencés (Cerisy et al., 2017). / Numerous methods of directed evolution in vitro have been developed. These methods are often expensive and time-consuming. An alternative approach, directed evolution in vivo, allows the selection of protein variants at lower cost and does not require prior knowledge of the protein’s structure and mechanism. An automated continuous culture device, the GM3, has been developed and built at the Genoscope. The GM3 technology enables the maintenance of steady-state microbial growth over an extended period of time.The main objective of the present study was the optimization by directed evolution in vivo of enzymes of the family of the Iron(II)/α-ketoacid-dependent oxygenases (α-KAOs). The α-KAOs catalyze the hydroxylation of non-activated C-H bonds and thus are interesting tools for the functionalization of organic molecules in synthetic chemistry.The dynamics of fixation of beneficial mutations in a growing cell population depending on the expression format and the culture regime has been explored.Strains of Escherichia coli have been genetically modified such that their growth strictly depended on a α-KAO activity. Populations of these bacteria expressing a α-KAO were subjected to protocols of directed evolution in continuous culture in order to select, in a first round, variants showing optimized expression on natural substrates of the enzyme, and in a second round, variants with activity towards non-natural analogs of the substrate. Two variants of L-isoleucine dioxygenase (E65K, A62P) were obtained. The determination of the kinetic parameters will reveal the influence of these mutations on the activity of the enzyme variants.In collaboration with the group of Dr. Tolonen, directed evolution in continuous culture was applied to the cellulolytic bacteria Clostridium phytofermentans. This anaerobic bacterium degrades lignocellulose into ethanol and H2 and can thus be applied for biomass valorization. The degradation of lignocellulose liberates phenolic compounds like ferulic acid known to be toxic to the bacteria. C. phytofermentans was adapted in the GM3 to resist to growing concentrations of this inhibitor. Resistant bacteria were analyzed and their genomes sequenced (Cerisy et al., 2017).

No description available.

Made available in DSpace on 2015-07-22T20:50:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-25 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Células iNKT restritas a CD1d são protetoras contra o desenvolvimento de melanoma murino, B16F10-Nex2, subcutaneamente em animais singênicos. Essa proteção pode ser deduzida pelo desenvolvimento acelerado do tumor em animais geneticamente deficientes na expressão de CD1d (CD1d-KO), com sobrevida significantemente menor do que a observada com animais WT submetidos ao mesmo desafio de células tumorais. CD1d é uma família de glicoproteínas relacionadas a MHC classe I, envolvidas na apresentação, principalmente em células dendríticas, de antígenos lipídicos para células iNKT. No presente trabalho focalizamos a identificação do lipídeo endógeno expresso em células de melanoma capaz de induzir uma resposta de vigilância imune baseada em células iNKT. Verificamos também a possibilidade de proteger animais contra o desenvolvimento tumoral utilizando células dendríticas primadas com o lipídeo endógeno. A extração dos componentes lipídicos de melanoma foi realizada a partir de tumores crescidos in vivo, evitando-se assim artefatos do cultivo celular in vitro. Foram testados três diferentes protocolos de extração (A, B e C), obtendo-se 14 frações diferentes que foram testadas na ativação do hibridoma DN32.D3, uma linhagem de células NKT murinas imortalizadas. A fração F3 do protocolo A (F3A) ativou o hibridoma DN32.D3 medido pela produção de IL-2. Para uma eficiente apresentação dos lipídeos dessa fração utilizamos com sucesso células dendríticas de medula óssea (BMDC) nos ensaios in vitro e in vivo, para apresentação de F3A e glicolipídeos ativadores de células NKT, agalactosilceramida (a-GalCer) e isoglobotrihexosilceramida (iGb3). Na tentativa de isolar o composto estimulatório presente em F3A de melanoma, essa fração foi analisada por HPTLC revelada com diversos reagentes específicos para resíduos de ácido siálico, açúcares neutros, fosfato e lipídeos totais. A fração também foi submetida a separações em colunas de sílica, ensaio de “immunostaining” quimioluminescente com lectina de Bandeiraea simplicifolia e análises em espectrometria de massa, onde foram identificados gangliosídeos como GM3 bem como glicoesfingolipídeos neutros como iGb3, Gb3, iGb4 e Gb4 por ESI-LIT-MS (electrosptray ionization-linear íon trap-mass spectrometry). Quando iGb3 foi incubado com BMDC e testado com células DN32D3 essas foram ativadas produzindo IL-2. GM3 consistentemente era um inibidor dessa ativação de células iNKT. Ensaios de citotoxicidade foram então realizados e verificamos que células de NKT estimuladas por BMDC incubadas com a-GalCer ou iGb3 circundavam as células tumorais B16F10-Nex2 visualizadas em microscopia de fluorescência e, em ensaio in vitro, as células NKT promoviam lise de até 40% das células tumorais B16F10-Nex2. Realizamos então ensaios in vivo, onde camundongos foram inoculados endovenosamente com células do melanoma murino e tratados ou não com células dendríticas primadas com a-GalCer ou iGb3. Ao excisarmos os pulmões dos animais, notamos que os grupos tratados com lipídeos ativadores de células NKT tinha cerca de 4 vezes menos nódulos pulmonares que o grupo não tratado. Nossos resultados mostram que o melanoma murino B16F10-Nex2 possui a molécula iGb3 e sua precursora iGb4, capaz de ativar células NKT conferindo a essas capacidade citotóxica in vitro contra o melanoma. Esse lipídeo (iGb3) também mostrou um efeito protetor contra metástases pulmonares oriundas do melanoma murino quando apresentado por células dendríticas utilizadas em protocolo de terapia celular. Esse é o primeiro trabalho mostrando que efetivamente um glicolipídeo endógeno ligante de CD1d é capaz de ativar células NKT com efeito protetor antitumoral, através de terapia celular com células dendríticas. Palavras chave: Melanoma B16F10-Nex2, células iNKT, glicoesfingolipídeos iGb3 e iGb4 GM3, células dendríticas, imuno vigilância. / CD1d-restricted iNKT cells are protective against the murine melanoma B16F10- Nex2 growing subcutaneously in syngeneic animals. This is inferred from the fast tumor growth in animals genetically deficient in CD1d (CD1d-KO), which showed a survival time significantly shorter than that of WT animals equally challenged with tumor cells. CD1d belongs to a family of glycoproteins resembling MHC class I, involved in the presentation, chiefly in dendritic cells, of lipidic antigens to iNKT cells. In the present work we focus on the identification of an endogenous lipid component expressed in melanoma cells able to induce an immunosurveillance response based on iNKT. We also investigated the possibility of animal protection against tumor development by using dendritic cells primed with the endogenous lipid. The extraction of membrane lipid components was carried out from in vivo grown tumors, thus avoiding artifacts from the in vitro grown cultures. Three different extraction protocols were tested (A, B, C), and 14 different fractions were obtained and tested for the activation of hybridoma DN32.D3, a cell line of immortalized murine NKT cells. Fraction F3 of protocol A (F3A) activated hybridoma DN32.D3 to produce IL-2. For an efficient presentation of lipids from this fraction we successfully used bone marrow dendritic cells (BMDC) on in vitro and in vivo assays. F3A and NKT-cell activating glycolipids, agalactosylceramide (a-GalCer) and isoglobohexosylceramide (iGb3), were tested. In the attempt to isolate the stimulatory component present in the melanoma F3A fraction, HPTLC was used and revealed with several specific reagents for sialic acid residues, neutral sugars, phosphate and total lipids. The fraction was also separated in silica columns, immunostained with Bandeiraea simplicifolia BS-1 lectin and analyzed by mass spectrometry. Ganglioside GM3 and neutral glycosphingolipids iGb3, Gb3, iGb4 and Gb4 were identified by ESI-LIT-MS (electrospray ionization- linear ion trap- mass spectrometry). By incubation of iGb3 with BMDC and these with DN32.D3 cells, the latter were activated to produce IL-2. GM3 consistently inhibited the activation of iNKT cells. Cytotoxicity assays were then carried out, in which we found that NKT cells stimulated by BMDC, primed with a-GalCer or iGb3, encircled the B16F10-Nex2 tumor cells as visualized by fluorescent microscopy. NKT cells promoted lysis of up to 40% of tumor cells. In vivo tests showed that mice injected endovenously with murine melanoma cells and treated with dendritic cells primed with a-GalCer or iGb3, had lungs with 4-fold fewer nodules than an equally challenged but untreated group. The present results show that the murine melanoma B16F10-Nex2 expresses iGb3 and its precursor iGb4, being able to activate NKT cells and conferring them a cytotoxic activity in vitro against melanoma. Such lipid (iGb3) was also protective in vivo reducing the melanoma pulmonary metastases when presented by dendritic cells used in cellular therapy protocol. This is the fist work showing effectively that an endogenous CD1d-restricted glycolipid able to activate iNKT cells display a protective antitumor effect, using cellular therapy with dendritic cells. / TEDE / BV UNIFESP: Teses e dissertações

L'élastine est la protéine matricielle responsable de l'élasticité des tissus retrouvée dans des tissus soumis à de fortes contraintes mécaniques tels que les poumons, les artères ou la peau. La dégradation de cette protéine lors de processus physiopathologiques produit des peptides bologiquement actifs nommés peptides d'élastine portant le motif GXXPG essentiel à leur activité. Ces peptides régulent diverses fonctions biologiques telles que le chimiotactisme, la synthèse de protéases, la prolifération. Tous ces effets dépendent de la fixation des peptides d'élastine au complexe récepteur de l'élastine. Ce complexe est composé de trois sous-unités : une protéine périphérique de 67 kDa, l'Elastin Binding Protein (EBP), et deux protéines associées à la membrane, la Protective Protein/Cathepsin A (PP/CA) et la Neuraminidase-1 (Neu-1) de 55 et 61 kDa respectivement. L'activité sialidase de Neu-1 est responsable de l'activation de ERK 1/2 après fixation des peptides d'élastine au complexe récepteur de l'élastine.Dans cette étude, nous démontrons que l'EBP et les radeaux lipidiques sont colocalisés à la membrane plasmique. Nous montrons, de plus, que la déstructuration de ces microdomaines aussi bien que leur déplétion en glycolipides bloque la signalisation du récepteur. L'utilisation d'un anticorps monoclonal bloquant dirigé contre le GM3 montre qu'il est essentiel à la signalisation. Après traitement par les peptides d'élastine, le contenu cellulaire en GM3 diminue alors que celui en lactosylcéramide augmente suggérant une conversion du GM3 en lactosylcéramide. L'utilisation de lactose ou de siRNA Neu-1 bloque cette conversion ce qui tend à démontrer que le complexe récepteur de l'élastine est impliqué dans ce mécanisme. Une analyse par cytométrie en flux confirme cette production de lactosylcéramide induite par les peptides d'élastine.L'analyse par spectrométrie de masse mettrait en évidence deux lactosylcéramides (C23:0 et C24:1) potentiellement bioactifs dont la synthèse chimique a été entreprise. La purification des radeaux lipidiques par ultracentrifugation différentielle en gradient de saccharose ainsi que leur identification par Dot-blot couplé à la fluorescence montre un changement de densité de ces microdomaines après stimulation par les peptides d'élastine.L'évaluation biologique in vitro de ces lactosylcéramides montre qu'ils miment les effets des peptides d'élastine sur l'activation de ERK 1/2, la prolifération et la synthèse de MMP-1. Enfin, l'évaluation ex vivo des lactosylcéramides démontre une réduction de la zone de tissu cardiaque nécrosé suggérant un rôle cardioprotecteur de ces molécules. Ce travail propose un mécanisme original de transduction du signal à la membrane plasmique et nous laisse envisager le complexe récepteur de l'élastine, les peptides d'élastine et le lactosylcéramide comme de nouveaux agents thérapeutiques potentiels. / Elastin is the matrix protein responsible for the elasticity of tissues where resilience is required such as lung, arteries or skin. Elastin degradation during physiopathological processes produces biologically active peptides named elastin peptides bearing the GXXPG pattern essential for their activity. These peptides regulate various biological functions such as chemotaxis, proteases synthesis and proliferation. These effects are dependent of elastin peptide binding to the elastin receptor complex (ERC). This complex is composed of three subunits: a peripheral protein of 67 kDa called elastin binding protein (EBP) and two membrane-associated proteins, protective protein/cathepsin A (PP/CA) and neuraminidase-1 (Neu-1) of 55 and 61 kDa, respectively. The sialidase activity of Neu-1 is responsible for ERK 1/2 pathway activation following binding of elastin peptide on the elastin receptor complex.In this study, we demonstrate that EBP and lipid rafts colocalize at the plasma membrane. We also show that the disruption of these microdomains and their depletion in glycolipids block the receptor signaling. The use of a monoclonal anti-GM3 blocking antibody shows that this glycosphingolipid is essential for signaling. Following elastin peptide treatment, cellular GM3 level decreases while the lactosylceramide one increases consistently with a GM3/LacCer conversion. The use of lactose or Neu-1 siRNA blocks this process suggesting that the elastin receptor complex is involved in this mechanism. Flow cytometry analysis confirms this elastin peptide-driven LacCer generation.Mass spectrometry analysis of elastin peptide-stimulated cell membrane extracts identified two potentially bioactive lactosylceramides (C23:0 and C24:1) and their synthesis has been realized. Lipid rafts purification by differencial ultracentrifugation in sucrose gradient shows a variation of the microdomains density as well as their identification by fluorescence linked-Dot-blot following elastin peptide stimulation.In vitro biological evaluation of these lactosylceramides shows that they mimic the elastin peptide effects on ERK 1/2 activation, proliferation and MMP-1 synthesis. Finally, ex vivo lactosylceramides evaluation demonstrates a decrease of cardiac tissue necrosis area suggesting that these molecules could be cardioprotective agents. This work proposes an original mechanism of signal transduction at the plasma membrane and let us foresees the elastin receptor complex, elastin peptides and lactosylceramide as new potential therapeutical targets.

圖形化模型編輯器是模型編輯器開發長久以來一直努力的方向。Eclipse.org 已提供兩個功能強大的圖形化編輯器設計框架：GEF 圖形化編輯器設計框架，和將 EMF 與 GEF 結合並簡化其設計流程的 GMF 圖形化模型編輯器設計框架，來協助開發者發展圖形化模型編輯器。 / 使用設計框架時所面臨的高學習門檻是開發者選用設計框架上的一大障礙。為降低開發者在 GMF 使用上的學習門檻，我們希望能提供一個以模型開發為導向的圖形編輯器設計專屬模型語言，讓程式設計師能夠以其慣用的程式設計角度來描述其圖形化模型編輯器的組成架構。 / 本篇研究主要是在探討 GMF 圖形化模型編輯器開發流程的簡化。在研究中希望藉由提供 GMF 圖形化模型編輯器設計框架的 GM3 專屬模型語言，讓開發者能夠透過 MDA 的角度來開發圖形化模型編輯器的架構，藉以縮短 GMF 的開發流程。實驗中使用 JavaCC 文法剖析器產生工具來開發 GM3專屬模型語言與 GMF 各項模型定義間的模型轉換。 / The availability of a visual graphical editor for a target domain is the prerequisite of visual graphical modeling, which has been adopted by classical software development for decades and is especially emphasized in today's model-driven engineering. However, compared with traditional textual editors, developing a visual graphical editor from scratch is not an easy work. As a result, there were frameworks developed such as GEF and GMF aimed to simplify the construction of graphical editors. Even so, however, it is still though hard for an average programmer to construct a visual graphical editor by using these frameworks without a long time of learning. / Our result is a modeling langauge of graphcial editors called GM3, serving as a bridge betwen developers of graphical editors and the GMF framework. With GM3, the developer can specify the model of his editing domain, how each model element should be presented by which kind of graphical elements in the editor, and how the tool pallete should be filled with various kind of creation and manipulation tools for model elements. After the GM3 specification of an editor is produced, the GM3 transformation engine developed by us using the JavaCC parser generator can be used to generate all files required of the GMF framework and, finally, a subsequent application of the standard GMF code generation procedure can produce a complete graphical editor on Eclipse platform.

No description available.

Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways.

Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways.