Introduction : L'hémophilie est une maladie génétique de la coagulation due à un déficit en facteur VIII ou en facteur IX. Ces déficits sont responsables d'un déficit du complexe ténase intrinsèque (VIIIa-IXa). De plus, le complexe ténase extrinsèque (facteur tissulaire - VIIa) est physiologiquement rapidement inhibé par le TFPI lié au facteur Xa. Nous avons évalué la capacité d'une forme tronquée du facteur Xa (GDXa), dépourvue de domaine Gla à se lier au TFPI et à soulager l'inhibition physiologique du complexe ténase extrinsèque. Matériel et Méthodes : Dans une première partie, nous avons évalué la capacité du GDXa à restaurer la génération de thrombine de plasmas d'hémophiles A et B sévères sans et avec inhibiteurs. Nous avons également comparé les profils de génération de thrombine obtenus après addition du GDXa à ceux obtenus en présence d'anticorps neutralisants anti-TFPI ou anti-antithrombine. Enfin, nous avons comparé les cinétiques enzymatiques de neutralisation du facteur Xa et du GDXa par le TFPI et l'antithrombine. Dans une seconde partie, nous avons étudié in silico les interactions entre la chaîne lourde du facteur Xa et le TFPI pour détecter les zones d'interaction défavorables. Cette étude a identifié des acides aminés du facteur Xa qui pourraient être substitués pour optimiser l'interaction avec le TFPI. Les résultats in silico ont orienté nos choix de mutagenèse dirigée pour concevoir différents variants moléculaires du GDXa (R138F, R138G, R138I) où l'arginine 138 est substituée. Ces variants protéiques ont été produits de façon recombinante dans des cellules HEK293E. La capacité des différents variants à restaurer la génération de thrombine de plasmas d'hémophiles a été testée avec les surnageants de culture cellulaires correspondants. Résultats : Dans la première partie, nous avons montré que le GDXa est capable de restaurer la génération de thrombine de plasmas d'hémophiles A et B sans et avec inhibiteurs. Comparativement au facteur Xa, le GDXa montre une affinité moindre pour le TFPI tandis que les affinités du GDXa et du facteur Xa pour l'antithrombine sont identiques. Enfin, malgré une demi-vie courte, l'effet du GDXa sur la génération de thrombine est maintenu pendant au moins une heure. Dans la seconde partie, nous avons produit les différents variants R138F, R138G et R138I en cellules HEK293E et montré que les surnageants de culture cellulaire étaient capables de restaurer la génération de thrombine de plasmas d'hémophiles de façon plus efficace que le GDXa. Conclusion : Comme le GDXa est capable de restaurer la génération de thrombine de plasmas d'hémophiles, nos résultats suggèrent que le GDXa pourrait être une alternative efficace aux thérapeutiques hémostatiques court-circuitantes actuelles chez les hémophiles sans ou avec inhibiteurs. Les résultats obtenus renforcent l'hypothèse que l'activité pro-coagulante du GDXa serait liée à la formation d'un complexe GDXa-TFPI limitant la formation du complexe Xa-TFPI nécessaire à l'inhibition physiologique du complexe ténase extrinsèque. De plus, notre approche rationnelle basée sur une étude in silico visant à augmenter l'affinité du TFPI pour le GDXa a permis de produire différents variants moléculaires du GDXa dont l'activité procoagulante in vitro est augmentée par rapport au GDXa. / Background: Hemophilia is caused by deficiencies in coagulation factor VIII or IX, resulting in direct blockade of the intrinsic tenase complex and indirect blockade of the extrinsic tenase complex which is rapidly inhibited upon binding of factor Xa to tissue factor pathway inhibitor (TFPI). We evaluated the ability of Gla-domainless factor Xa (GDXa), a truncated form of factor Xa devoid of procoagulant properties, to bind to TFPI and to alleviate the physiological inhibition of the extrinsic tenase. Design and Methods: In the first part of this work, we evaluated the ability of GDXa to restore coagulation in plasmas from hemophilia A and B patients without and with inhibitors, using a thrombin generation assay triggered by a low concentration of tissue factor. We then compared its efficacy to generate thrombin to depletion of antithrombin or TFPI by specific antibodies. Finally, we compared the kinetics of neutralization of factor Xa and GDXa by antithrombin and TFPI. In the second part of this work, we realized an in silico study of the interactions between factor Xa heavy chain and TFPI. The aim was to detect unfavorable interactions and to identify amino-acid candidates for mutagenesis in order to increase affinity for TFPI. Taking into account the results of this in silico study, we produced by genic engienering different molecular variants of GDXa (R138F, R138G, R138I) where Arg138 was substituted by site directed mutagenesis. Proteins were produced in HEK293E cells. We tested dialyzed cell culture supernatants containing each variant to restore thrombin generation in plasmas from severe hemophilia patients. Results: In the first part of this work, we showed that GDXa was able to restore thrombin generation in plasma samples from hemophiliacs. This effect was observed for plasma from hemophilia A patients without or with inhibitors and for plasma from hemophilia B patients. GDXa had a lower affinity than factor Xa for TFPI whereas the affinities of both proteins for antithrombin were similar. Finally, despite a short half-life in plasma, the effect of GDXa on thrombin generation was sustained for at least one hour. In the second part of this work, we produced the different variants R138F, R138G et R138I in HEK293E cells and showed that cell culture supernatants were able to restore thrombin generation in a more efficient way than GDXa. Conclusions: As GDXa was able to restore thrombin generation in plasma from hemophilia patients, our results suggest that it may be an effective alternative to current treatments for hemophilia with or without inhibitors. Results sustained the hypothesis that GDXa coagulant activity is through TFPI binding and competition with factor Xa to bind TFPI resulting in limiting factor Xa-TFPI formation, which is essential for inhibition of extrinsic tenase complex. Furthermore, rational design of GDXa variants based on an in silico study lead to production of proteins whose coagulant activity is increased compared to GDXa."

Submitted by Cristhiane Guerra (cristhiane.guerra@gmail.com) on 2017-02-03T15:51:13Z No. of bitstreams: 1 arquivototal.pdf: 2439266 bytes, checksum: 852d348e101a0679a14c24845b557b1e (MD5) / Made available in DSpace on 2017-02-03T15:51:13Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2439266 bytes, checksum: 852d348e101a0679a14c24845b557b1e (MD5) Previous issue date: 2015-07-31 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Phenolic compounds, among them the flavonoids, are holders of antimicrobial, antioxidant and anti-inflammatory effects, but not free from toxicity and/or adverse effects, that's why research in this area, whether they are in silico, in vitro and/or in vivo approach have been intensified to ensure the safe use of these molecules. Thus, the aim of this study was to investigate the probable pharmacological activities, toxicity and antibacterial and antioxidant effects of flavonoid flavone and its hydroxy derivatives: 3-hydroxyflavone, 5-hydroxyflavone and 6-hydroxyflavone, tracing a structure-activity relationship of such substances. It was investigated the pharmacological, pharmacokinetics and theoretical toxicological characteristics of flavonoids using in silico testing with the PASS online and Osiris softwares; the cytotoxicity on human erythrocytes of blood types A, B and O positive and negative Rh factor, running the models of hemolysis and Erythrocyte Osmotic Fragility (EOF); was analysed the antimicrobial activity in front of Gram-positive (B. subtilis CCT 0516, S. aureus ATCC 25619 and S. aureus ATCC 25925) and Gram-negative, including clinical importance (P. aeruginosa ATCC 8027, P. aeruginosa ATCC 23243, E. coli ATCC 2536, E. coli 101, E. coli 103, E. coli 104, E. coli 105 and E. coli 108); assessed the oxidant and antioxidant potential of these molecules in the presence of Reactive Oxygen Species (ROS - H2O2) and phenylhydrazinium (Ph) and, finally, the genotoxicity using the micronucleus test. The results obtained revealed numerous probable pharmacological activities to the flavonoids, as integrity agonists and membrane permeability inhibitors, anaphylatoxin receptor antagonists, inhibitors of kinase and peroxidase, antimutagenic potential and vase-protecting capacity; do not present significant theoretical toxicity risks and have good oral bioavailability. The 4 flavonoids have shown moderate hemolysis at concentrations of 500 and 1000 μg/mL, the example of 3-hydroxyflavone which induced 20.2 % and 53 % of hemolysis, respectively, in blood type A, Rh+; the flavonoids hydroxylated protected cells types A and O from osmotic stress. All flavonoids exhibited moderate antibacterial activity against Gram-positive strains and Gram-negative, being the flavone bactericide in the concentration of 200 μg/mL to the strains of P. aeruginosa ATCC 8027, S. aureus ATCC 25619 and E. coli 104, while other flavonoids have bacteriostatic action. It did not promote oxidation of erythrocyte and behaved as scavengers and antioxidants of H2O2 and phenylhydrazinium and finally the flavone did not show genotoxicity compared to cyclophosphamide, a proven genotoxic agent. It is concluded that the flavone, 3-hydroxyflavone, 5-hydroxyflavone and 6- hydroxyflavone have different pharmacological activities, good bioavailability and low theoreticals toxicity, reduced cytotoxicity, absence of genotoxicity as well as being moderate antibacterial and antioxidant, showing, with this study, the importance of the inclusion of computational chemistry techniques for targeting evaluation protocols of the biological effects of the molecules. / Compostos fenólicos, dentre eles os flavonoides, são detentores de efeitos antimicrobiano, antioxidante e anti-inflamatório, porém não isentos de toxicidade e/ou efeitos adversos, por isso pesquisas nesta área, sejam elas com uma abordagem in silico, in vitro e/ou in vivo têm se intensificado para que se garanta a segurança no uso dessas moléculas. Assim, o presente estudo se propôs a investigar as prováveis atividades farmacológicas, a toxicidade e os efeitos antibacteriano e antioxidante do flavonoide flavona e de seus derivados hidroxilados: 3-hidroxiflavona, 5-hidroxiflavona e 6-hidroxiflavona, traçando uma relação estrutura-atividade das referidas substâncias. Para tanto, investigou-se as características farmacológica, farmacocinética e toxicológica teóricas dos flavonoides utilizando ensaios in silico com os softwares PASS online e Osíris; a citotoxicidade sobre eritrócitos humanos dos tipos sanguíneos A, B e O e fator Rh positivo e negativo, executando-se os modelos de hemólise e Fragilidade Osmótica Eritrocitária (FOE); analisou-se a atividade antimicrobiana frente a bactérias Gram-positivas (B. subtilis CCT 0516, S. aureus ATCC 25619 e S. aureus ATCC 25925) Gram-negativas, inclusive de importância clínica (P. aeruginosa ATCC 8027, P. aeruginosa ATCC 23243, E. coli ATCC 2536, E. coli 101, E. coli 103, E. coli 104, E. coli 105 e E. coli 108); avaliou-se o potencial oxidante e antioxidante das referidas moléculas na presença de Espécies Reativas de Oxigênio (EROs - H2O2) e da fenilhidrazina (Ph) e, por último, a genotoxicidade por meio do teste do micronúcleo. Os resultados obtidos revelaram numerosas prováveis atividades farmacológicas para os flavonoides, como agonistas da integridade e inibidores da permeabilidade membranar, antagonistas do receptor de anafilatoxina, inibidores de quinase e peroxidase, potencial antimutagênico e capacidade vasoprotetora; não apresentam significativos riscos teóricos de toxicidade e detêm uma boa biodisponibilidade oral. Os 4 flavonoides demonstraram moderada hemólise nas concentrações de 500 e 1000 μg/mL, a exemplo da 3-hidroxiflavona que induziu 20,2 e 53 % de hemólise, respectivamente, no sangue tipo B,Rh-; os flavonoides hidroxilados protegeram os eritrócitos tipos A e O do estresse osmótico. Todos os flavonoides exibiram moderada atividade antibacteriana contra cepas Gram-positivas e Gram-negativas, sendo a flavona bactericida na concentração de 200 μg/mL para as linhagens de P. aeruginosa ATCC 8027, S. aureus ATCC 25619 e E. coli 104, enquanto que os demais flavonoides têm ação bacteriostática. As substâncias não promoveram oxidação dos eritrócitos e comportaram-se como sequestradores e antioxidantes de H2O2 e fenilhidrazina e, por fim, a flavona não apresentou genotoxicidade quando comparado com a ciclofosfamida, um comprovado agente genotóxico. Conclui-se que flavona, 3-hidroxiflavona, 5-hidroxiflavona e 6-hidroxiflavona possuem variadas atividades farmacológicas, boa biodisponibilidade e baixa toxicidade teóricas, reduzida citotoxicidade, ausência de genotoxicidade, além de serem moderadamente antibacterianos e antioxidantes, evidenciando-se, com este estudo, a importância da inserção de técnicas de química computacional para o direcionamento de protocolos de avaliação de efeitos biológicos de moléculas.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-06-20T11:50:06Z No. of bitstreams: 1 arquivototal.pdf: 10487656 bytes, checksum: 9f874336a1873756d7bc4d26e57ee76d (MD5) / Made available in DSpace on 2017-06-20T11:50:06Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 10487656 bytes, checksum: 9f874336a1873756d7bc4d26e57ee76d (MD5) Previous issue date: 2016-05-13 / This work describes the synthesis, characterization and antifungal potential of twelve mesoionic derivatives from the 1,3-thiazolium-5-thiolate system. The compounds, totally unprecedented, were divided in two classes: one class derived from the mesoionic 2-(4-chlorophenyl)-3-methyl-4-(4-isopropylphenyl)-1,3-thyazolium-5- thiolate with ten substituted acetamides and other class derived from the mesoionic 2- (4-chlorophenyl)-3-methyl-4-(4-methylphenyl)-1,3-thyazolium-5-thiolate also with ten substituted acetamides. This compounds synthesis, which presented satisfactory yields (85 to 95 %), occurred in four stages: first the intermediary N-methyl-Carylglicine was synthesized via Strecker reaction with p-substituted aldehydes, followed by its aroylation to obtain N-methyl-N-aroyl-C-arylglicine, the next step was its cyclodehydration with acetic anhydride, 1,3-dipolar cycloaddition and cyclorevertion induced by CS2 to obtain the mesoionic compounds from the 1,3- thiazolium-5-thyolate as free base. Lastly, the mesoionic compounds were converted into his respective salts by the treatment with ten substituted acetamides. All compounds were characterized by spectroscopic techniques IR, NMR 1H and NMR 13C, and their antifungal potential against five strains of Candida albicans were evaluated. Only four compounds exhibited efficient activity (MI-1-A3, MI-1-A4, MI- 1-A6 and MI-1-A7) with MIC between 256 – 512 μg mL-1. An in silico investigation of the mesoionic compounds, the substituted acetamides and the mesoionic derivatives was also made and the results showed that both mesoionic compounds and his derivatives are good candidates to be a drug while all substituted acetamides should present high toxicity. / Neste trabalho descreve-se a síntese, caracterização e potencial antifúngico de vinte derivados mesoiônicos do sistema 1,3-tiazólio-5-tiolato. Os compostos, inéditos, foram divididos em duas classes: uma classe de compostos derivados do mesoiônico 2-(4-clorofenil)-3-metil-4-(4-isopropilfenil)-1,3-tiazólio-5-tiolato com dez acetamidas substituídas e outra classe de compostos derivados do mesoiônico 2-(4-clorofenil)-3- metil-4-(4-metilfenil)-1,3-tiazólio-5-tiolato e também com dez acetamidas substituídas. A síntese desses compostos, que apresentou rendimentos satisfatórios (85 a 95 %), ocorreu em quatro etapas: primeiramente o intermediário N-metil-Carilglicina foi sintetizado via reação de Strecker com aldeídos p-substituídos, seguido de sua aroilação para obter o N-metil-N-aroil-C-arilglicina, seguida da ciclo desidratação com anidrido acético, cicloadição e cicloreversão 1,3-dipolar induzida por CS2 para obter os mesoiônicos do sistema 1,3-tiazólio-5-tiolato na forma de base livre. Por fim os mesoiônicos foram convertidos em seus respectivos sais através do tratamento com dez acetamidas substituídas. Todos os compostos, intermediários, mesoiônicos, acetamidas e derivados mesoiônicos, foram caracterizados por técnicas espectroscópicas de IV, RMN de 1H e 13C e avaliou-se o potencial antifúngico dos derivados mesoiônicos contra cinco cepas de Candida albicans. Dentre os vinte derivados mesoiônicos investigados, apenas quatro, MI-1-A3, MI-1-A4, MI-1-A6 e MI-1-A7, exibiram eficiente atividade com concentrações inibitórias mínimas entre 256 – 512 μg mL-1. Neste trabalho realizou-se ainda uma investigação in silico dos compostos mesoiônicos, das acetamidas substituídas e dos derivados mesoiônicos e os resultados indicaram que tanto os compostos mesoiônicos quanto seus derivados são bons candidatos a fármacos enquanto que as dez acetamidas substituídas investigadas devem apresentar elevada toxicidade.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-12-18T10:55:48Z No. of bitstreams: 1 Dissertação - Juliana Camila Lopes Cavaion - 2012.pdf: 2928983 bytes, checksum: 908363ad8b87e3a821dd54fe80dcb4ce (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-12-18T14:24:31Z (GMT) No. of bitstreams: 1 Dissertação - Juliana Camila Lopes Cavaion - 2012.pdf: 2928983 bytes, checksum: 908363ad8b87e3a821dd54fe80dcb4ce (MD5) / Made available in DSpace on 2014-12-18T14:24:32Z (GMT). No. of bitstreams: 1 Dissertação - Juliana Camila Lopes Cavaion - 2012.pdf: 2928983 bytes, checksum: 908363ad8b87e3a821dd54fe80dcb4ce (MD5) Previous issue date: 2012-06-04 / Filamentous fungi have cytochrome P450 enzyme system similar to mammals whose application allows catalyze phase I oxidative reactions of metabolism. Obtaining functionalized derivatives by these microorganisms was the main purpose of this dissertation. Studies in silico to predict metabolism in the programs MetaPrint2D and SMARTCyp indicated S-oxidation, aromatic hydroxylation and dealkylation reactions, as the most likely S-oxidation in thiomorpholine ring of 2-[4-(1,4-thiazinan-4-ylsulfonyl)phenylcarbamoyl]benzoic acid (LASSBio-596). The filamentous fungi Absidia blakesleana ATCC 26617, Aspergillus candidus ATCC 1009, Beauveria bassiana ATCC 7159, Cunninghamella echinulata ATCC 9244, Cunninghamella echinulata ATCC 9245, Cunninghamella elegans ATCC 36112, Fusarium roseum ATCC 14717 and Streptomyces vendulae ATCC 8664 were used for biotransformation of LASSBio-596. In the reaction conditions applied, substrate dissolved in methanol with concentration of 0.25 mg/mL added to liquid culture medium PDSM and incubated at 27 °C with 200 rpm for 96 hours, the fungus Beauveria bassiana ATCC 7159 performed S-oxidation in the thiomorpholine ring resulting in sulfone derivative of LASSBio-596, called LaBioCon 214, yield of 5.84 %, and it was found the hydrolysis of amide reaction obtaining the compound 4-(thiomorpholinosulfonyl)aniline, LaBioCon 202, yield of 13.8 %. It was noted the solvent used affects the biotransformation process. The monitoring of the reactions and purification of the derivatives obtained were performed by high performance liquid chromatography with ultraviolet detector at 267 nm and preparative chromatography with column Microsorb 100-5 C18 (250 x 10 mm, 5 μm). Subsequently, sulfone derivative was obtained, called LaBioCon 223, by synthesis. Structural characterization was made by classical methods such as 1H NMR and mass spectrometry. / Os fungos filamentosos possuem o sistema enzimático citocromo P450 semelhante ao de mamíferos, cuja aplicação permite catalisar reações oxidativas de fase I do metabolismo. A obtenção de derivados funcionalizados por estes microrganismos foi o objetivo principal deste trabalho. Estudos in silico de previsão do metabolismo nos programas MetaPrint2D e SMARTCyp indicaram reações de S-oxidação, hidroxilação aromática e desalquilação, sendo mais provável a S-oxidação no anel tiomorfolina do ácido 2-[4-(1,4-tiazinan-4-ilsulfonil)fenilcarbamoil]benzóico (LASSBio-596). Os fungos filamentosos Absidia blakesleana ATCC 26617, Aspergillus candidus ATCC 1009, Beauveria bassiana ATCC 7159, Cunninghamella echinulata ATCC 9244, Cunninghamella echinulata ATCC 9245, Cunninghamella elegans ATCC 36112, Fusarium roseum ATCC 14717 e Streptomyces vendulae ATCC 8664 foram empregados para biotransformação do LASSBio-596. Nas condições reacionais aplicadas, substrato solubilizado em metanol com concentração de 0,25 mg/mL adicionado ao meio de cultura líquido PDSM e incubado a 27 °C com 200 rpm por 96 horas, a Beauveria bassiana ATCC 7159 promoveu a S-oxidação no anel tiomorfolina resultando no derivado sulfona do LASSBio-596, denominado LaBioCon 214, com rendimento de 5,84 %, e constatou-se a reação de hidrólise da amida obtendo o composto 4-(tiomorfolina-sulfonil)anilina, LaBioCon 202, com rendimento de 13,8 %. Verificou-se que o solvente utilizado interfere no processo de biotransformação. O monitoramento das reações e a purificação dos derivados obtidos foram realizados por cromatografia líquida de alta eficiência com detector ultravioleta em 267 nm e cromatografia preparativa com coluna Microsorb 100-5 C18 (250 x 10 mm, 5 μm). Posteriormente, foi obtido o derivado sulfona, LaBioCon 223, por via sintética. A caracterização estrutural foi realizada por métodos clássicos como a espectroscopia de ressonância magnética nuclear de hidrogênio e espectrometria de massas.

Made available in DSpace on 2014-08-20T13:32:57Z (GMT). No. of bitstreams: 1 dissertacao_lara_dias.pdf: 2572863 bytes, checksum: 41207696775098ff734edf871864bf12 (MD5) Previous issue date: 2011-02-14 / The current challenges in plant breeding are to maximize the productivity of major crop species and to create means for exploring novel crop environments. One of these environments is the lowland hydromorphic soils that are proper for the irrigated rice crop. Adapting other crops to this environment could reduce the incidence of diseases, pests and weeds, therefore benefiting from a crop rotation system. When a plant is exposed to abiotic stresses, it has to cope with environmental changes through physiological and anatomic changes that need quick gene expression responses, i.e., changes in active/silenced status as well as in the rates of transcription. Cis-acting regulatory elements have straight relationship with transcription factors (TF) in complex signaling networks. This TF binding sites (cis-elements) are the functional DNA elements that influence temporal and spatial transcriptional activity. We investigated possible patterns of sequences that can be inferred about the mechanisms that plants use to develop under flooding stress. This search for possible homologies between the various cis-elements would lead us to performed interactive analyses about how plants use their molecular mechanisms responding to abiotic stresses. Online databases were searched, looking for genes previously described in literature which are expressed in response to flooding in Oryza sativa, Arabidopsis thaliana and their homologous in Glycine max and Zea mays. The 1.0 Kb upstream portion of each gene was extracted and analyzed in silico. Besides, all the promoters of these four species were subjected to a tool for searching for novel signals, intending to find new motif patterns. Our in silico analysis shows that from 259 cis elements found in PLACE for all promoters of Arabidopsis and rice, 12 of them are common to both species, and are distinguished by having high frequency. Using the MEME program two consensus motifs could be found among the species Oryza sativa and Zea mays. These could represent new cis elements patterns, because they had relatively high occurrences in the gene promoters and they are related to conserved sequences in monocots. The analysis here presented shows important points for future studies related to the waterlogging stress and unmasking molecular tolerance mechanisms to this typical stress. From the data generated, it will be possible to direct experiments on genetic transformation with target genes and/or cis elements in order to attribute some characteristic in plants, such as those found in rice, so they can develop in an environment with O2 deprivation. / Os atuais desafios no melhoramento de plantas são maximizar a produtividade das principais espécies cultivadas e criar meios para explorar uma variedade de ambientes de cultivo. Um desses ambientes são os solos hidromórficos de planícies alagadas. A adaptação de outras culturas a este ambiente poderia reduzir a incidência de doenças, pragas e plantas daninhas, se beneficiando de um sistema de rotação de culturas. Quando uma planta é exposta a estresses abióticos ela tem que lidar com as alterações ambientais através de mudanças fisiológicas e anatômicas, as quais necessitam de rápidas mudanças na expressão gênica, ou seja, no seu estado inicial, bem como nas taxas de transcrição. Elementos regulatórios de ação cis têm relação direta com fatores de transcrição (FT) em complexas redes de sinalização. Estes sítios de ligação de FTs são elementos funcionais de DNA que influenciam a atividade transcricional de forma temporal e espacial. Neste trabalho, foram investigados possíveis padrões de seqüências que se possam inferir sobre os mecanismos que as plantas utilizam para se desenvolver na condição do alagamento. Essa busca por possíveis homologias entre os vários elementos cis permite realizar análises interativas sobre como as plantas utilizam seus mecanismos moleculares para responder a estresses abióticos. Bancos de dados online foram utilizados, na busca de genes previamente descritos em literatura e que são expressos em resposta ao alagamento em Oryza sativa, Arabidopsis thaliana e seus homólogos em Glycine max e Zea mays. A porção de 1,0 Kb a montante de cada gene foi extraída e analisada in silico. Além disso, todos os promotores das quatro espécies foram submetidos a busca por uma variedade de sinais, com a intenção de encontrar novos padrões de motivos de DNA. Esta análise mostrou que, dos 259 elementos cis encontrados em promotores de Arabidopsis e arroz, 12 deles são comuns a ambas as espécies e se distinguem do restante por terem alta freqüência. Utilizando o programa MEME dois motivos consenso foram encontrados entre as espécies Oryza sativa e Zea mays. Estes podem representar novos padrões de elementos cis, pois apresentaram ocorrências relativamente elevada nos promotores de genes e são relacionados com seqüências conservadas em monocotiledôneas. A análise aqui apresentada mostra pontos importantes para futuros estudos relacionados ao estresse por alagamento e auxilia na descoberta de mecanismos moleculares de tolerância ao alagamento nas plantas. A partir dos dados gerados, será possível direcionar experimentos de transformação genética a fim de atribuir alguma característica às plantas, tais como aqueles encontrados no arroz, para que possam se desenvolver em um ambiente com privação de O2.

Made available in DSpace on 2014-08-20T13:42:10Z (GMT). No. of bitstreams: 1 Dissertacao_Joceani_Dal_Cero.pdf: 1522635 bytes, checksum: 6a5a31f6f6667ef5f524442ba0bb1e72 (MD5) Previous issue date: 2010-02-26 / Regulatory molecules, such as transcription factors, have been thoroughly investigated, especially in hormone-mediated responses that involve gene expression modulation. Frequently, the main determinant of gene expression is its transcriptional rate. Thus, molecular mechanisms underlying transcription regulation have become an important topic in genetic studies of ethylene signaling. The present work aimed to investigate the ERF (Ethylene Response Factor) family employing bioinformatic tools, integrating publicly available datasets from the model species Arabidopsis thaliana and phylogenetic analyses to help elucidating the biological roles of the family in apple. The preliminary survey of the ERF sequences in Malus has provided basic information to be incorporated in further studies of the functional role of ERFs in this perennial species. Expression analyses of MdERF1 and MdERF in apple fruits suggest that other factors, besides ethylene, are involved in their transcriptional regulation in Malus. The second chapter reports the investigation of the transcriptional profiling of those ERF genes in response to pathogen attack, using a biological assay of in vitro propagated plants inoculated with the fungus Venturia inaequalis (apple scab disease). The study has provided evidences of the involvement of MdERF1 in eliciting the plant response; whereas, MdERF2 does not appear to be participate in the pathogenesis. / Moléculas que participam dos processos regulatórios, como os fatores de transcrição, têm recebido atenção especial, pois uma das principais ações dos estímulos hormonais é a modulação da expressão gênica. Como a taxa de transcrição de um gene é o maior determinante da sua expressão, os mecanismos moleculares pelos quais a transcrição gênica é regulada têm se tornado um dos tópicos principais de estudos em genética molecular envolvendo o hormônio etileno. O objetivo deste trabalho foi realizar análises de bioinformática para a família ERF (Ethylene Response Factors), integrar bases de dados existentes na internet no modelo Arabidopsis, bem como análise filogenética que permitam avaliar os papéis dos diferentes membros da família. Este levantamento preliminar das seqüências ERF em Malus forneceu informação básica para estudos posteriores mais aprofundados, com relação aos mecanismos moleculares da família nesta importante cultura perene. A análise da expressão de MdERF1 e MdERF2 em frutos de maçã indica que outros fatores além do etileno estão envolvidos na regulação da transcrição dos ERF em Malus. O segundo capítulo refere-se à resposta dos ERF frente ao ataque de patógenos. Para isso, foram infectadas plantas de macieira provenientes de cutivo in vitro com o fungo Venturia inaequalis (sarna da maçã). As evidências desses estudos sugerem o envolvimento do gene MdERF1 no processo de patogênese, enquanto que o gene MdERF2 parece não estar envolvido no processo.

La réactivation aberrante du gène TWIST1 embryonnaire a été identifiée dans le laboratoire comme un mécanisme d'inactivation récurrente des voies dépendantes de p53 et Rb dans de nombreuses tumeurs. En diminuant la sénescence et l’induction de l'apoptose, TWIST1 coopère avec des protéines oncogéniques dans la transformation cellulaire in vitro et intervient dans l'initiation et la progression tumorale in vivo. Comme TWIST1 est très faiblement exprimée dans la plupart des cellules adultes différenciées, elle constitue une cible attrayante pour des thérapeutiques futures. Récemment, l’héterodimère TWIST1/E2A (E12 ou E47, deux produits d'épissage alternatif du gène TCF3) joue un rôle pro-métastatique dans le cancer de la prostate (Gajula et al., 2015), alors que ce complexe est la forme oncogénique de TWIST1 dans des cellules épithéliales mammaires humaines (Jacqueroud et al., 2016). L'hétérodimérisation par l'intermédiaire des domaines HLH est une condition sine qua non pour la formation de domaine de liaison d'ADN (Murre et Massari 2000). Une approche in silico basé que la modélisation par homologie des complexes de TWIST1 met en évidence le rôle déterminant des boucles inter-hélicales dans le maintien de complexes de TWIST1 à l’ADN en étudiant plusieurs insertions de TWIST1 observées chez les patients atteints de syndrome Saethre-Chotzen (Bouard et al., 2014). Ensuite des approches in silico et in vitro nous ont permis de comprendre les mécanismes moléculaires sous-jacents à la reconnaissance des séquences E-box des promoteurs de gènes cibles par l'héterodimère TE et leur stabilisation sur l'ADN (bouard et al., 2016). Nous avons décrit trois états différents du complexe TWIST1/E12 lié à l'ADN à des séquences E-box fonctionnels et modifiés en fonction de l'affinité de reconnaissance des E-box par TWIST1 / E12 (Bouard et al, 2016). Et enfin, cette approche in silico nous a permis de montrer l’impact de la phosphorylation de TWIST1 sur la dimérisation et sur la liaison à l’ADN des complexes de TWIST1. Cette dernière étude met en exergue l’importance des régulations post-traductionnelles dans l’étude de l’activité de la protéine TWIST1 et dans la recherche d’inhibiteurs / Aberrant reactivation embryonic TWIST1 gene has been identified in the laboratory as a recurrent inactivation mechanism dependent pathways of p53 and Rb in many tumors. Decreasing senescence and the induction of apoptosis, TWIST1 cooperates with oncogenic proteins in cell transformation in vitro and is involved in tumor initiation and progression in vivo. As TWIST1 is very weakly expressed in most adult differentiated cells, it is an attractive target for future therapies. Recently, the heterodimer TWIST1 / E2A (E12 or E47, two alternative splice product of the TCF3 gene) plays a pro-metastatic role in prostate cancer (Gajula et al., 2015), while the complex is the oncogenic form of TWIST1 in human mammary epithelial cells (Jacqueroud et al., 2016). The heterodimerization through HLH areas is a prerequisite for DNA binding domain of training (Massari and Murre 2000). An in silico approach that based homology modeling of complex TWIST1 highlights the key role of inter-hélicales loops in the DNA TWIST1 complex retention student TWIST1 several insertions observed in patients Saethre syndrome -Chotzen (Bouard et al., 2014). Then approaches in silico and in vitro have enabled us to understand the molecular mechanisms underlying the recognition of E-box sequences of the promoters of target genes by TE heterodimer and stabilize DNA (Bouard et al. 2016). We have described three different states of the complex TWIST1 / E12 bound to DNA to functional E-box sequences and modified according to the affinity recognition by E-box TWIST1 / E12 (Bouard et al, 2016). Finally, this approach in silico allowed us to show the impact of TWIST1 phosphorylation on dimerization and binding to DNA TWIST1 complexes. This latest study highlights the importance of post-translational regulation in the study of the activity of the protein and TWIST1 in the search for inhibitors

The development of materials with tailored functionalities and with continuously shrinking linear dimensions towards (and below) the nanoscale is not only going to revolutionize state of the art fabrication technologies, but also the computational methodologies used to model the materials properties. Specifically, atomistic methodologies are becoming increasingly relevant in the field of materials science as a fundamental tool in gaining understanding on as well as for pre-designing (in silico material design) the behavior of nanoscale materials in response to external stimuli. The major long-term goal of atomistic modelling is to obtain structure-function relationships at the nanoscale, i.e. to correlate a definite response of a given physical system with its specific atomic conformation and ultimately, with its chemical composition and electronic structure. This has clearly its pendant in the development of bottom-up fabrication technologies, which also require a detailed control and fine tuning of physical and chemical properties at sub-nanometer and nanometer length scales. The current work provides an overview of different applications of atomistic approaches to the study of nanoscale materials. We illustrate how the use of first-principle based electronic structure methodologies, quantum mechanical based molecular dynamics, and appropriate methods to model the electrical and thermal response of nanoscale materials, provides a solid starting point to shed light on the way such systems can be manipulated to control their electrical, mechanical, or thermal behavior. Thus, some typical topics addressed here include the interplay between mechanical and electronic degrees of freedom in carbon based nanoscale materials with potential relevance for designing nanoscale switches, thermoelectric properties at the single-molecule level and their control via specific chemical functionalization, and electrical and spin-dependent properties in biomaterials. We will further show how phenomenological models can be efficiently applied to get a first insight in the behavior of complex nanoscale systems, for which first principle electronic structure calculations become computationally expensive. This will become especially clear in the case of biomolecular systems and organic semiconductors.

Modern methods of processing genomic data yield good results, however they are often redeemed by time consummation. This is mainly the reason why this thesis deals with numerical methods of bacterial genome processing which could be a suitable alternative for current methods, both in terms of quality and computational demands. This thesis presents the current methods used for processing of genomes in silico and proposes suitable numerical presentations for the whole genome analysis. Some of these methods are programmed and used for processing further in this diploma thesis. In it, various mathematical and statistical methods are being tested, ones that could lead to successful species delineation of bacteria by numerical presentations of their genomes.

The development of materials with tailored functionalities and with continuously shrinking linear dimensions towards (and below) the nanoscale is not only going to revolutionize state of the art fabrication technologies, but also the computational methodologies used to model the materials properties. Specifically, atomistic methodologies are becoming increasingly relevant in the field of materials science as a fundamental tool in gaining understanding on as well as for pre-designing (in silico material design) the behavior of nanoscale materials in response to external stimuli. The major long-term goal of atomistic modelling is to obtain structure-function relationships at the nanoscale, i.e. to correlate a definite response of a given physical system with its specific atomic conformation and ultimately, with its chemical composition and electronic structure. This has clearly its pendant in the development of bottom-up fabrication technologies, which also require a detailed control and fine tuning of physical and chemical properties at sub-nanometer and nanometer length scales. The current work provides an overview of different applications of atomistic approaches to the study of nanoscale materials. We illustrate how the use of first-principle based electronic structure methodologies, quantum mechanical based molecular dynamics, and appropriate methods to model the electrical and thermal response of nanoscale materials, provides a solid starting point to shed light on the way such systems can be manipulated to control their electrical, mechanical, or thermal behavior. Thus, some typical topics addressed here include the interplay between mechanical and electronic degrees of freedom in carbon based nanoscale materials with potential relevance for designing nanoscale switches, thermoelectric properties at the single-molecule level and their control via specific chemical functionalization, and electrical and spin-dependent properties in biomaterials. We will further show how phenomenological models can be efficiently applied to get a first insight in the behavior of complex nanoscale systems, for which first principle electronic structure calculations become computationally expensive. This will become especially clear in the case of biomolecular systems and organic semiconductors.