Universiẗat, Diss., 2002--Göttingen.

TEIXEIRA, Claudener Souza. Determinação da estrutura tridimensional de uma lectina de sementes de Canavalia maritima (Aublet) complexada com o polifenol antioxidante resveratrol. 2015. 94 f. Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Vitor Campos (vitband@gmail.com) on 2016-09-01T23:33:14Z No. of bitstreams: 1 2015_tese_csteixeira.pdf: 4836976 bytes, checksum: abec6d8f925cfce27622f137363ea5f1 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-09-02T23:06:47Z (GMT) No. of bitstreams: 1 2015_tese_csteixeira.pdf: 4836976 bytes, checksum: abec6d8f925cfce27622f137363ea5f1 (MD5) / Made available in DSpace on 2016-09-02T23:06:47Z (GMT). No. of bitstreams: 1 2015_tese_csteixeira.pdf: 4836976 bytes, checksum: abec6d8f925cfce27622f137363ea5f1 (MD5) Previous issue date: 2015 / Resveratrol is a natural antioxidant polyphenol found especially in grape epicarp, walnuts, and pomegranates, which can inhibit the activation of proinflammatory mediators and cytokines at the early gene expression stage. It is well known that lectins are sugar-binding proteins that act as both pro- and anti-inflammatory molecules. Thus, the objective of this work was to verify the binding of a polyphenol compound with a lectin of Canavalia maritima (ConM) based on their ability to inhibit pro-inflammatory processes. To accomplish this, ConM was purified and crystallized, and resveratrol was soaked at 5 mM for 2 hours of incubation. The crystal belongs to the monoclinic space group C2, the final refinement resulted in an Rfactor of 16.0% and an Rfree of 25.5%. Resveratrol binds in the rigid β-sheet through H-bonds and hydrophobic interaction with amino acids that compose the fifth and sixth β-strands of the rigid β-sheet of ConM. The ConM and resveratrol inhibited DPPH oxidation, showing synergic activity with the most effective ratio of 2:3 and carbohydrate binding site is not directly related to antioxidant activity. It is the interaction between ConM and resveratrol that indicates the synergism of these two molecules in acting as free radicals scavengers and in reducing the inflammatory process through the inhibition of many pro-inflammatory events. / O resveratrol é um polifenol antioxidante natural encontrado especialmente na epicarpo da uva, em nozes e na romã o qual pode inibir a ativação de mediadores pró-inflamatório e citocinas na fase precoce de expressão do gene. Já é bem conhecido que as lectinas são proteínas de ligação de açúcares que atuam tanto como moléculas pró- e anti-inflamatórias. Assim, o objetivo do presente trabalho foi o de verificar a ligação de um composto de polifenol com uma lectina de Canavalia maritima (ConM) com base na sua capacidade para inibir processos pró-inflamatórios. Para alcançar este objetivo, a ConM foi purificada e cristalizada, uma solução de 5 mM de resveratrol 5 mM foi utilizada para soaking durante 2 horas de incubação. Os cristais obtidos pertencem ao grupo espacial monoclínico C2, o refinamento final resultou em um Rfactor de 16,0% e uma Rfree de 25,5%. Resveratrol se liga na rígida através de pontes de hidrogênio e interação hidrofóbica com aminoácidos que compõem a quinta e sexta fita-β da folha-β rígida da ConM. A ConM complexada com o resveratrol inibiu a oxidação do DPPH, mostrando atividade sinérgica entre a proteína e o ligante com a relação mais eficaz de 2:3. Foi verificado ainda que o sítio de ligação a carboidratos não está diretamente relacionado à atividade antioxidante. É a interação entre ConM e resveratrol, que indica o sinergismo destas duas moléculas em agir como agentes sequestrantes de radicais livres que podem estarem relacionados a capacidade de redução do processo inflamatório através da inibição de muitos mediadores pró-inflamatórios por lecitinas.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Tephrosia toxicaria Pers. (sin. Tephrosia sinapou (Bucâhoz) A. Chev), Fabaceae, à uma planta tropical ictiotÃxica, popularmente conhecida como âtimbÃâ e utilizada como defensivo agrÃcola natural. Stemodia maritima Linn. (Scrophulariaceae) à conhecida no Nordeste brasileiro como âmelosaâ e tambÃm encontrada nas ilhas do Caribe onde à usada para tratar vÃrios tipos de doenÃas. O estudo quÃmico das raÃzes de T. toxicaria permitiu o isolamento da flavanona obovatina que sofreu reaÃÃo de hidrogenaÃÃo para gerar um derivado hidrogenado TTR-1H. O estudo quÃmico das folhas de S. maritima forneceu dois diterpenos do tipo estemodanos, sendo eles a estemodina e estemodinol, alÃm da flavona jaceidina. O extrato etanÃlico das folhas de S. maritima apresentou atividade antioxidante comparÃvel à da vitamina C. A obovatina reduziu o infiltrado inflamatÃrio e a hiperalgesia facial em articulaÃÃo temporomandibular de ratos nas concentraÃÃes de 1 e 10 mg/Kg, exercendo atividades anti-inflamatÃria e analgÃsica em um modelo experimental de artrite induzida por zymosan. A estemodina, o extrato SMFE e obovatina foram submetidos a ensaios de atividade antibacteriana e demonstraram aÃÃo moderada frente a cepas de Staphylococcus aureus (12692), Staphylococcus aureus (358), Bacillus cereus (33018), Pseudomonas aeruginosa (15442) e Escherichia coli (27). As substÃncias isoladas foram identificadas por mÃtodos espectromÃtricos e espectroscÃpicos (IV, EM, RMN 1H e RMN 13C), incluindo RMN bidimensional (HMBC, HSQC, NOESY e COSY) e por comparaÃÃo com dados da literatura. / Inorganic matrices have been used as adsorbent mate rial for removal of chemical pollutants in aquatic systems, due to its good adsorption capacity for physical and chemical stabilities and this work was synthesized silica spheres functionalized with EDTA in order to remove metal ions in aqueous solution. The kinetics of adsorption of the ions under study (Cu2 + , Cd2 + and Zn2 +) was performed at pH 5.5 at a concentration of 60 mg L-1, where it was observed that equilibrium is reached very fast, about 10 minutes, indicating that limiting step of the adsorption process may be the chemisorption, as evidenced by t he best fitting model for the pseudo-second order. The adsorption isotherms were subjected to linear and nonlinear model for comparison, using the error function SSE and R2 as a reference. The study of simple and multielement ions was carried out at pH 5.5, concentrations ranging from 10-300 mg L-1, where the data were submitted to the models of Langmuir, Freundlich, Temkin and Redlich-Peterson. The results showed a better fit to the Langmuir model, which argues that the adsorption sites are homogeneous and monolayer adsorption. The maximum sorption capacities observed in studies of equilibrium for the system mono and multielement fo llow the order Cu2 +> Zn2 +> Cd2+. For comparison model method was used Akaike's where proved that the Langmuir model is the best. With respect to the study of adsorption column, the breakthrough curves indicate that saturation occurs in the same volume of 35 mL. The use of the eluent 0.1 M HCl, allowed to recover 100% of the adsorbed ions. The column was used for three cycles to verify their potential use, and it was proved that in the three-cycles can recover 100% of the metals, which means an optimum efficiency of the spheres of silica-APTS-EDTA.

Made available in DSpace on 2015-04-17T14:55:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In many parts of the world leguminous plant is one of the most widely known plants previously reported. There are many different types of plants in the family leguminous used as food, in this case, the genus Canavalia comprises a group of some 48 species. The term lectin (derived from the latin word legere, meaning, among other things, to select) represent a heterogeneous group of proteins ranging from size, structure, molecular organization and constitution of interaction points as well as performing several biological activities such as anti-inflammatory, antifungal and antibiotic. Until now, the lectins of plants are one of the most studied, although some lectins in animals are well-specified. This research aim to isolate, purify and define biological activities of a new lectin present in the seeds of the Canavalia maritime. The results indicate the presence of a new lectin with a preference to agglutinate native erythrocytes of rabbit. These lectins were both purified in column chromatography of Sephadex G-50 and chitin, molecular exclusion in HPLC system, respectively. The purity and molecular weight (approximate) of the new lectin (from 50 to 55 kDa) was detected by SDS polyacrylamide gel electrophoresis. The new lectin did not cause hemolysis in human erythrocytes type A, O and AB, it reflects the interaction level of the sugars. According to sugar inhibition assays, the new lectin (content 71,5μg) indicates it is specific for arabinose, fructose, maltose, saccharose and xylose. On the other hand, the new lectin did not prevent the growth of tested bacterial strains, however, it is able to promote the proliferation these microorganisms. It also presented an anti-inflammatory activity as peritonitis induced by carrageenan in mice reducing the migration of neutrophils in the peritoneum of mice and permeability of the blood vessels. It also produced antifungal activity on the growth of the fungus C. neoformans, on its growth assay, evidenced by CIM determination, in this case, the results demonstrates no growth in the presence of the new lectin. / As leguminosas estão entre as plantas mais conhecidas pelas pessoas de diversas partes do mundo. Nesta família, muitas são as plantas que usamos como alimento. O gênero Canavalia ao qual pertence o feijão da praia, dentro da família Leguminosae, é formado por um pequeno grupo de 48 espécies. O termo lectina (do latim legere , que significa escolher, selecionar), representa um grupo heterogêneo de proteínas que variam amplamente em tamanho, estrutura, organização molecular, bem como na constituição de seus sítios de interação, desempenhando atividades biológicas diversas como antiinflamatórias, antifúngicas e antibióticas. As lectinas de plantas são as mais estudadas até então, embora algumas presentes em animais e microorganismos já tenham sido bem caracterizadas. O objetivo geral deste trabalho foi isolar, purificar e determinar atividades biológicas de uma nova lectina presente em sementes da leguminosa Canavalia maritima. Os resultados indicaram a presença de uma nova lectina com preferência em aglutinar eritrócitos nativos de coelho, purificada através de cromatografias de afinidade em Sephadex G-50 e quitina, e de exclusão molecular em sistema HPLC, respectivamente. Por meio de eletroforese em gel de poliacrilamida na presença de SDS, foi constatada a pureza e o peso molecular aproximado da nova lectina de 50 a 55 kDa. A nova lectina não promoveu hemólise em eritrócitos humanos dos tipos A, O e AB, em graus variáveis, o que reflete a sua especificidade de interação com diferentes açúcares. Com uma composição de 71,5 μg de carboidratos, a nova lectina demonstrou, pelo teste de inibição por açúcares, especificidade para os açúcares arabinose, frutose, maltose, sacarose e xilose. Por outro lado, a nova lectina, não inibiu o crescimento das linhagens bacterianas testadas, entretanto, observou-se que ela é capaz de promover a proliferação destes microorganismos. Apresentou atividade antiinflamatória, no modelo de peritonite induzido por carragenina em camundongos, reduzindo a migração dos neutrófilos no peritônio de camundongos e a permeabilidade dos vasos sanguíneos. Também produziu atividade antifúngica sobre o crescimento dos fungos C. neoformans, nos ensaios de crescimento das cepas, evidenciado pela determinação da CIM, onde os resultados demonstraram que não houve crescimento na presença da nova lectina.

Made available in DSpace on 2015-04-01T14:16:01Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1333665 bytes, checksum: 2f915f0ace8bbb6bb5fbbb794e7c40d5 (MD5) Previous issue date: 2013-06-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In many parts of the world leguminous plant is one of the most widely known plants previously reported. There are many different types of plants in the family leguminous used as food, in this case, the genus Canavalia comprises a group of some 48 species. The term lectin (derived from the latin word legere, meaning, among other things, to select) represent a heterogeneous group of proteins ranging from size, structure, molecular organization and constitution of interaction points as well as performing several biological activities such as anti-inflammatory, antifungal and antibiotic. Until now, the lectins of plants are one of the most studied, although some lectins in animals are well-specified. This research aim to isolate, purify and define biological activities of a new lectin present in the seeds of the Canavalia maritime. The results indicate the presence of a new lectin with a preference to agglutinate native erythrocytes of rabbit. These lectins were both purified in column chromatography of Sephadex G-50 and chitin, molecular exclusion in HPLC system, respectively. The purity and molecular weight (approximate) of the new lectin (from 50 to 55 kDa) was detected by SDS polyacrylamide gel electrophoresis. The new lectin did not cause hemolysis in human erythrocytes type A, O and AB, it reflects the interaction level of the sugars. According to sugar inhibition assays, the new lectin (content 71,5μg) indicates it is specific for arabinose, fructose, maltose, saccharose and xylose. On the other hand, the new lectin did not prevent the growth of tested bacterial strains, however, it is able to promote the proliferation these microorganisms. It also presented an anti-inflammatory activity as peritonitis induced by carrageenan in mice reducing the migration of neutrophils in the peritoneum of mice and permeability of the blood vessels. It also produced antifungal activity on the growth of the fungus C. neoformans, on its growth assay, evidenced by CIM determination, in this case, the results demonstrates no growth in the presence of the new lectin. / As leguminosas estão entre as plantas mais conhecidas pelas pessoas de diversas partes do mundo. Nesta família, muitas são as plantas que usamos como alimento. O gênero Canavalia ao qual pertence o feijão da praia, dentro da família Leguminosae, é formado por um pequeno grupo de 48 espécies. O termo lectina (do latim legere , que significa escolher, selecionar), representa um grupo heterogêneo de proteínas que variam amplamente em tamanho, estrutura, organização molecular, bem como na constituição de seus sítios de interação, desempenhando atividades biológicas diversas como antiinflamatórias, antifúngicas e antibióticas. As lectinas de plantas são as mais estudadas até então, embora algumas presentes em animais e microorganismos já tenham sido bem caracterizadas. O objetivo geral deste trabalho foi isolar, purificar e determinar atividades biológicas de uma nova lectina presente em sementes da leguminosa Canavalia maritima. Os resultados indicaram a presença de uma nova lectina com preferência em aglutinar eritrócitos nativos de coelho, purificada através de cromatografias de afinidade em Sephadex G-50 e quitina, e de exclusão molecular em sistema HPLC, respectivamente. Por meio de eletroforese em gel de poliacrilamida na presença de SDS, foi constatada a pureza e o peso molecular aproximado da nova lectina de 50 a 55 kDa. A nova lectina não promoveu hemólise em eritrócitos humanos dos tipos A, O e AB, em graus variáveis, o que reflete a sua especificidade de interação com diferentes açúcares. Com uma composição de 71,5 μg de carboidratos, a nova lectina demonstrou, pelo teste de inibição por açúcares, especificidade para os açúcares arabinose, frutose, maltose, sacarose e xilose. Por outro lado, a nova lectina, não inibiu o crescimento das linhagens bacterianas testadas, entretanto, observou-se que ela é capaz de promover a proliferação destes microorganismos. Apresentou atividade antiinflamatória, no modelo de peritonite induzido por carragenina em camundongos, reduzindo a migração dos neutrófilos no peritônio de camundongos e a permeabilidade dos vasos sanguíneos. Também produziu atividade antifúngica sobre o crescimento dos fungos C. neoformans, nos ensaios de crescimento das cepas, evidenciado pela determinação da CIM, onde os resultados demonstraram que não houve crescimento na presença da nova lectina.

Made available in DSpace on 2015-04-01T14:16:05Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2925990 bytes, checksum: c09f8e05395a813c660d9c2568ea71c5 (MD5) Previous issue date: 2014-08-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Legume lectins are historically recognized as carbohydrate-binding proteins, and this property is roughly linked to the majority of their biological activities, such as pro and antiinflamatory processes, apoptosis and mitogenic mechanisms. However, several studies have demonstrated the capability of these proteins to bind diverse other compounds, such as phytohormones and hydrophobic molecules, although the biological relevance of these interactions remains elusive. Many biological reactions involve proteins interactions with nucleic acids and the protein interaction with DNA plays a key role in the cellular regulation and all vital functions. In the present work, it is reported the crystal structure of ConM, a lectin isolated from Canavalia maritima seeds, in complex with Interleukine 1β primer. A cubic crystal, belonging to the F23 space group, was obtained by the diffusion vapor method and submitted to X-ray diffraction. In the structure, an electron density map corresponding to four nucleotides from the interleukin-1β primer was found in the interface of ConM non-canonical dimer. The nucleotides stabilization involves H-bonds and electrostatic forces with the amino acids His127, Ser110, Lys114, Ser190, Asp192, Thr194, His51 and Ser190. Based on these crystallographic results and in a molecular docking simulation, it is hypothesized that the lectin may function as a transcription factor, which could help us to understand some of their biological activities. / Lectinas de leguminosas são historicamente reconhecidas como proteínas de ligação a carboidratos e essa propriedade é ligada à maioria das suas atividades biológicas, tais como pró e antiinflamatórias, apoptose e mecanismos mitogênicos. No entanto, vários estudos demonstraram a capacidade destas proteínas de se ligarem a diversos outros compostos, tais como fitohormônios e moléculas hidrofóbicas, embora a relevância biológica destas interações permaneça indefinida. Muitas reações biológicas envolvem a interação de proteínas com ácidos nucleicos e a interação da proteína com o DNA desempenha um papel chave em toda a regulação celular e das funções vitais. No presente trabalho relatamos a estrutura cristalina da ConM, uma lectina isolada de sementes de Canavalia maritima, em complexo com o primer da Interleucina 1β. Um cristal cúbico, pertencente ao grupo espacial F23, foi obtido através do método da difusão de vapor e submetido à difração de raios X. Na estrutura, o mapa de densidade eletrônica correspondente a quatro nucleotídeos do primer da interleucina-1β foi encontrado na interface do dímero não-canônico. A estabilização dos nucleotídeos envolve pontes de hidrogênio e forças eletrostáticas com os aminoácidos His127, Ser110, Lys114, Ser190, Asp192, Thr194, His51 e Ser190. Com base nestes resultados cristalográficos e em uma simulação de docking molecular, é hipotetizado que a lectina pode funcionar como um fator de transcrição, o que poderia nos ajudar a entender algumas de suas atividades biológicas.

Les travaux présentés dans cette thèse ont porté sur la specificité des réponses cellulaires de l’halophyte obligatoire Cakile maritima au choc salin et la régulation des événements précoces impliqués dans la mort cellulaire programmée et la survie en condition de salinité. Dans une première étape, nous avons montré que cette plante est aussi tolérante aux chocs salins répétés qu’au stress salin progressif. Cependant, on a observé de zones de mort cellulaires sur les feuilles âgées soumises à un choc salin sévère (400 mM NaCl). Pour mieux cerner la cascade d’événements impliqués dans ce processus de mort cellulaire, nous avons poursuivi nos expériences sur des suspensions cellulaires de C. maritima, dont nous avons-nous même optimisé les conditions d’obtention, et des suspensions cellulaires d’Arabidopsis thaliana (glycophyte modèle). Chez les deux espèces, nous avons observé une mort cellulaire programmée qui dépend de la durée et l’intensité du traitement salin appliqué, et qui met en jeu les mêmes événements cellulaires notamment la dépolarisation de la membrane plasmique due à l’entrée de Na+ par les NSCCs, un dysfonctionnement mitochondrial, une production d’anions superoxydes et une activation de protéines de type caspase. La tolérance de C. maritima au stress salin serait potentiellement due à une forte accumulation d’ascorbate qui permettrait à cette halophyte de mieux réduire les dommages générés par le stress oxydatif. C. maritima s’est aussi distinguée par une meilleure capacité de contrôler l’accumulation cytoplasmique de Na+, conduisant à la survie de ses cellules en condition de salinité. Cette étude sur la mort cellulaire induite par le NaCl chez les cellules en culture de C. maritima nous a aussi permis de mettre en évidence deux types de comportement dans cette population de cellules en culture : l’un lié à une dépolarisation soutenue en réponse au NaCl conduisant probablement à la mort de ces cellules, l’autre lié à une dépolarisation transitoire indiquant que l’influx de Na+ au travers des NSCC était régulé permettant probablement aux cellules présentant ce comportement de survivre en évitant l'accumulation excessive de Na+ dans le cytosol. Dans la dernière partie de ce travail, nous avons mis en évidence la capacité de C. maritima d’exclure Na+ via le système SOS. Ce résultat suggère l’existence d’une deuxième voie de signalisation induite parallèlement à celle conduisant à la mort cellulaire. Cette voie, impliquant une production rapide d’oxygène singulet, pourrait permettre un influx de Ca2+ dans le cytoplasme activant la protéine SOS3 et en cascade SOS2 et SOS1 et les H+-ATPases de la membrane plasmique permettant un efflux du Na+ via SOS1 hors des cellules. / AbstractThis work aimed at understanding the specificity of cellular responses of the obligate halophyte Cakile maritima to salt shock and regulation of early events involved in programmed cell death and survival under salinity conditions. In a first step, we have shown that this plant is tolerant upon both repetitive salt shocks and gradual salt application. However, we have observed a cell death zones on older leaves subjected to a severe shock saline (400 mM NaCl). To better understand the cascade of events involved in the cell death process, we continued our experiments on suspension culture of C. maritima, which we have optimized ourselves the conditions for establishment and suspension culture of Arabidopsis thaliana (glycophyte model). In both species, salinity induced programmed cell death that depends on the duration and the intensity of the applied salt treatment. Also, the same cellular events, including depolarization of the plasma membrane due to the Na+ influx by NSCCs, mitochondrial dysfunction, production of superoxide anions and activation of caspase-like proteins, occurs early in response to salt stress. C. maritima tolerance to salt stress is potentially due to a strong accumulation of ascorbate that would allow this halophyte to better reduce damage generated by oxidative stress. C. maritima is also distinguished by a better ability to control the cytoplasmic accumulation of Na+, leading to the survival of its cells under salinity conditions. This study on cell death induced by NaCl in cell culture of C. maritima also allowed us to identify two types of behavior in this population of cells in culture: one related to a sustained depolarization in response to NaCl probably leading to death of these cells, the other linked to a transient depolarization indicating that the Na+ influx through the NSCC was probably regulated allowing cells exhibiting this behavior to survive by avoiding excessive accumulation of Na+ in the cytosol. In the last part of this work, we have demonstrated the ability of C. maritima to exclude Na+ via the SOS system. This result suggests the existence of a second signaling pathway induced in parallel to that leading to cell death. This pathway, involving a rapid production of singlet oxygen, could allow a Ca2+ influx in the cytoplasm that acts as an elicitor for activation of SOS3 protein and SOS2-SOS1 cascade and H+- ATPases of the plasma membrane allowing Na+ efflux via SOS1 out of cells.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O resveratrol à um polifenol antioxidante natural encontrado especialmente na epicarpo da uva, em nozes e na romà o qual pode inibir a ativaÃÃo de mediadores prÃ-inflamatÃrio e citocinas na fase precoce de expressÃo do gene. Jà à bem conhecido que as lectinas sÃo proteÃnas de ligaÃÃo de aÃÃcares que atuam tanto como molÃculas prÃ- e anti-inflamatÃrias. Assim, o objetivo do presente trabalho foi o de verificar a ligaÃÃo de um composto de polifenol com uma lectina de Canavalia maritima (ConM) com base na sua capacidade para inibir processos prÃ-inflamatÃrios. Para alcanÃar este objetivo, a ConM foi purificada e cristalizada, uma soluÃÃo de 5 mM de resveratrol 5 mM foi utilizada para soaking durante 2 horas de incubaÃÃo. Os cristais obtidos pertencem ao grupo espacial monoclÃnico C2, o refinamento final resultou em um Rfactor de 16,0% e uma Rfree de 25,5%. Resveratrol se liga na rÃgida atravÃs de pontes de hidrogÃnio e interaÃÃo hidrofÃbica com aminoÃcidos que compÃem a quinta e sexta fita-β da folha-β rÃgida da ConM. A ConM complexada com o resveratrol inibiu a oxidaÃÃo do DPPH, mostrando atividade sinÃrgica entre a proteÃna e o ligante com a relaÃÃo mais eficaz de 2:3. Foi verificado ainda que o sÃtio de ligaÃÃo a carboidratos nÃo està diretamente relacionado à atividade antioxidante. à a interaÃÃo entre ConM e resveratrol, que indica o sinergismo destas duas molÃculas em agir como agentes sequestrantes de radicais livres que podem estarem relacionados a capacidade de reduÃÃo do processo inflamatÃrio atravÃs da inibiÃÃo de muitos mediadores prÃ-inflamatÃrios por lecitinas. / Resveratrol is a natural antioxidant polyphenol found especially in grape epicarp, walnuts, and pomegranates, which can inhibit the activation of proinflammatory mediators and cytokines at the early gene expression stage. It is well known that lectins are sugar-binding proteins that act as both pro- and anti-inflammatory molecules. Thus, the objective of this work was to verify the binding of a polyphenol compound with a lectin of Canavalia maritima (ConM) based on their ability to inhibit pro-inflammatory processes. To accomplish this, ConM was purified and crystallized, and resveratrol was soaked at 5 mM for 2 hours of incubation. The crystal belongs to the monoclinic space group C2, the final refinement resulted in an Rfactor of 16.0% and an Rfree of 25.5%. Resveratrol binds in the rigid β-sheet through H-bonds and hydrophobic interaction with amino acids that compose the fifth and sixth β-strands of the rigid β-sheet of ConM. The ConM and resveratrol inhibited DPPH oxidation, showing synergic activity with the most effective ratio of 2:3 and carbohydrate binding site is not directly related to antioxidant activity. It is the interaction between ConM and resveratrol that indicates the synergism of these two molecules in acting as free radicals scavengers and in reducing the inflammatory process through the inhibition of many pro-inflammatory events.

Chan, Kwok Ho. / Thesis submitted in: November 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 124-129). / Abstracts in English and Chinese. / Statement --- p.I / Acknowledgements --- p.II / Abstract --- p.III / 摘要 --- p.IV / Table of Contents --- p.V / Abbreviations --- p.XIII / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- GTPase in general --- p.1 / Chapter 1.2 --- G proteins and GTP switch --- p.2 / Chapter 1.3 --- Structural similarities in GTPase --- p.3 / Chapter 1.4 --- G proteins in bacteria --- p.3 / Chapter 1.5 --- Background information of the protein family EngB --- p.4 / Chapter 1.6 --- Basic information of EngB in Thermotoga maritima --- p.5 / Chapter 1.7 --- Objectives of this work --- p.6 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Chemical reagents --- p.8 / Chapter 2.1.2 --- Buffers / Chapter 2.1.2.1 --- Preparation of buffers --- p.10 / Chapter 2.1.2.2 --- Buffers for common use --- p.11 / Chapter 2.1.3 --- Expression strains and plasmids --- p.14 / Chapter 2.1.4 --- Primer list --- p.14 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Preparation of competent cells --- p.15 / Chapter 2.2.2 --- Cloning / Chapter 2.2.2.1 --- Cloning of target genes by PCR --- p.15 / Chapter 2.2.2.2 --- Agrose gel electrophoresis --- p.17 / Chapter 2.2.2.3 --- Extraction and purification of DNA from agarose gel --- p.17 / Chapter 2.2.2.4 --- Restriction digestion of DNA --- p.18 / Chapter 2.2.2.5 --- Ligation of digested insert and expression vector --- p.18 / Chapter 2.2.2.6 --- Transformation and plating out transformants for miniprep --- p.19 / Chapter 2.2.2.7 --- Verification of insert by PCR --- p.20 / Chapter 2.2.2.8 --- Mini-preparation of plasmid DNA --- p.21 / Chapter 2.2.2.9 --- Confirmation of miniprep product by restriction enzyme digestion..… --- p.22 / Chapter 2.2.2.10 --- Sequencing of the plasmid DNA --- p.23 / Chapter 2.2.3 --- Expression of the recombinant MBP-TM EngB protein and SBP-CBP EC EngB / Chapter 2.2.3.1 --- Transformation for protein expression --- p.23 / Chapter 2.2.3.2 --- Preparation of starter culture --- p.24 / Chapter 2.2.3.3 --- Expression of recombinant protein --- p.24 / Chapter 2.2.3.4 --- Cell harvesting --- p.24 / Chapter 2.2.3.5 --- Releasing the cell content --- p.25 / Chapter 2.2.3.6 --- Check for protein expression by SDS-PAGE --- p.25 / Chapter 2.2.4 --- Purification of TM EngB / Chapter 2.2.4.1 --- SP ion-exchange chromatography --- p.27 / Chapter 2.2.4.2 --- Thrombin digestion to remove MBP tag --- p.28 / Chapter 2.2.4.3 --- Heparin affinity chromatography --- p.29 / Chapter 2.2.4.4 --- Gel filtration chromatography --- p.29 / Chapter 2.2.5 --- Purification of SBP-CBP EC EngB / Chapter 2.2.5.1 --- SP ion-exchange chromatography --- p.30 / Chapter 2.2.5.2 --- Gel filtration chromatography --- p.31 / Chapter 2.2.6 --- Protein concentration quantitation --- p.32 / Chapter 2.2.7 --- Crystallography of TM EngB / Chapter 2.2.7.1 --- Crystallization preparation --- p.32 / Chapter 2.2.7.2 --- Crystallization screening by sitting drop method --- p.32 / Chapter 2.2.7.3 --- Optimization of crystallization conditions --- p.33 / Chapter 2.2.7.4 --- X-ray diffraction --- p.33 / Chapter 2.2.8 --- Thermodynamics studies of proteins / Chapter 2.2.8.1 --- Preparation of protein sample --- p.34 / Chapter 2.2.8.2 --- Guanidine-induced denaturation experiment --- p.34 / Chapter 2.2.8.3 --- Thermal-induced denaturation experiment --- p.35 / Chapter 2.2.9 --- Binding assay to study affinity for ligands --- p.36 / Chapter 2.2.9.1 --- Using GDP analogue mant-GDP to detect formation of enzyme-ligand complex (TM EngB-mant-GDP) --- p.36 / Chapter 2.2.9.2 --- Basic information of Fluorescence spectroscopy --- p.36 / Chapter 2.2.9.3 --- Determination of λem and λex --- p.37 / Chapter 2.2.9.4 --- Studying ligand affinity by titration with ligand analogue --- p.37 / Chapter 2.2.10 --- Pull down experiment to study interacting partner of E. coli EngB --- p.38 / Chapter 2.2.10.1 --- Preparing protein extracts from E. coli --- p.38 / Chapter 2.2.10.2 --- Preparing streptavidin resin --- p.39 / Chapter 2.2.10.3 --- Binding of dual-tagged E. coli EngB to streptavidin resin --- p.39 / Chapter 2.2.10.4 --- Purifying protein using the prepared streptavidin resin --- p.40 / Chapter 2.2.10.5 --- Preparing calmodulin resin --- p.41 / Chapter 2.2.10.6 --- Binding of dual-tagged E.coli EngB to calmodulin resin --- p.41 / Chapter 2.2.10.7 --- Analysis of dual-tag affinity purified protein --- p.42 / Chapter 2.2.11 --- Silver staining of acrylamide gel / Chapter 2.2.11.1 --- Staining reagents --- p.42 / Chapter 2.2.11.2 --- Staining procedures --- p.43 / Chapter Chapter 3 --- Structure determination of T. maritima EngB by X-ray crystallography / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Generation of TM EngB expression construct --- p.45 / Chapter 3.3 --- Expression and purification of TM EngB --- p.46 / Chapter 3.4 --- TM EngB was crystallized with freshly purified TM EngB --- p.47 / Chapter 3.5 --- Data processing of diffraction data and structure refinement of TM EngB …… --- p.48 / Chapter 3.6 --- Apo-form TM EngB was obtained by unfolding and refolding --- p.49 / Chapter 3.7 --- Crystallization of apo-form TM EngB --- p.50 / Chapter 3.8 --- Data processing of diffraction data and structure refinement of apo-form TM EngB --- p.51 / Chapter 3.9 --- Producing EngB-GDP complex crystal from apo-from EngB --- p.52 / Chapter 3.10 --- TM EngB is a monomer in solution --- p.54 / Chapter 3.11 --- Summary of chapter three --- p.55 / Tables and figures of chapter three --- p.57 / Chapter Chapter 4 --- Structural details of TM EngB / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Overall fold of TM EngB --- p.67 / Chapter 4.3 --- Mode of nucleotide binding of TM EngB --- p.68 / Chapter 4.4 --- Structural differences in switch I region between chain A and chain B in crystal structure of TM EngB/GDP complex --- p.70 / Chapter 4.5 --- Structural difference between TM EngB/GDP complex and apo TM EngB --- p.73 / Chapter 4.6 --- Summary of chapter four --- p.73 / Tables and figures of chapter four --- p.76 / Chapter Chapter 5 --- Purified TM EngB is Active for binding guanine nucleotide but inactive for GTPase hydrolysis activity / Chapter 5.1 --- Introduction --- p.88 / Chapter 5.2 --- Studying ligand affinity by competitive binding experiment --- p.88 / Chapter 5.3 --- GDP binds to TMEngB with higher affinity than GTPyS --- p.91 / Chapter 5.4 --- TM EngB showed very low intrinsic GTPase activity --- p.92 / Chapter 5.5 --- Discussion --- p.93 / Tables and figures of chapter five --- p.95 / Chapter Chapter 6 --- Thermostability of EngB of T. maritima / Chapter 6.1 --- Introduction --- p.98 / Chapter 6.2 --- Guanidine hydrochloride - induced unfolding --- p.98 / Chapter 6.3 --- Thermal-induced unfolding --- p.99 / Chapter 6.4 --- Structural comparison of thermophilic and mesophilic EngB --- p.100 / Chapter 6.5 --- Discussion --- p.102 / Tables and figures of chapter six --- p.105 / Chapter Chapter 7 --- Construction of a dual-tag affinity pull-down system for finding interacting partner of EngB / Chapter 7.1 --- Introduction --- p.112 / Chapter 7.2 --- Preparation of dual-tagged E.coli EngB / Chapter 7.2.1 --- Cloning of SBP-CBP-EC EngB expression construct --- p.113 / Chapter 7.2.2 --- Expression and purification of SBP-CBP-EC EngB --- p.114 / Chapter 7.3 --- Pull down using dual tagged E.coli EngB as bait to isolate potential interacting partners of EngB --- p.114 / Chapter 7.4 --- Discussion --- p.115 / Tables and figures of chapter seven --- p.117 / Chapter Chapter 8 --- Conclusion --- p.122 / References --- p.124

The purple sandpipers (Calidris maritima) are the most common waders in the high arctic archipelago of Svalbard, Norway. There they have to cope with a very short summer season and high metabolic costs of migrating far north and breeding in an arctic environment. The food on land is usually scarce, whereas there are rich feeding grounds in the littoral zone, such as in the intertidal zone of river flats. These feeding grounds are though only available to the purple sandpipers during low tide and as long as the estuary is not covered by sea ice. One of these intertidal flats was used as the fieldwork area in this study. To study when the birds are coming to this intertidal flat for feeding, a count study was performed during the entire stay of the purple sandpipers in Svalbard in summer 2010. Point counts were performed at low tide during 118 different days. Additionally, point counts were performed at twenty days during the six hours of the entire low tide period, to study when during the tidal cycle most sandpipers were feeding at the estuary. Most sandpipers were counted at the intertidal flat at the beginning of June with the highest number, 921 individuals, on 8th June. When the tundra was free of snow and the birds could start breeding, numbers where rapidly declining with very few sandpipers left in the estuary in July and the first part of August. From the end of August numbers were increasing again with a second but lower peak in the end of September and beginning of October. By the end of October all sandpipers had left the estuary. The study on the appearance of purple sandpipers at the estuary at the different periods of low tide showed that there were significantly more sandpipers between low tide and half an hour later than at the rest of the low tide period. This might be due to better access to their prey at that time. This knowledge could be used in future studies aiming at recording the maximum numbers. The result of the phenologic study could be included in a long term monitoring to see if the numbers and the timing of purple sandpipers are stable in this area or not: Are the peak numbers differing significantly? Is the timing of the arrival, the stay on the tundra and the timing of leaving the archipelago in the fall changing? Long-term monitoring would be especially interesting in the view of possible influences of the climate change on the purple sandpipers. Rising sea level as a result of the climate change would change the morphology of the estuaries and thereby influence the food resources available for sandpipers.