Made available in DSpace on 2017-07-10T17:37:27Z (GMT). No. of bitstreams: 1 Luciana Iurkiv.pdf: 711564 bytes, checksum: 5c4d90728ef8f794863f124f3c97e9a7 (MD5) Previous issue date: 2009-02-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Induced resistance involves the activation of latent defense mechanisms in plants in response to the treatment with biotic or abiotic agents. The use of crude extracts aimed at inducing resistance mechanisms is an interesting alternative to chemical control, however, in this extracts can occurs besides de presence of inducers, the presence of suppressors. This work objectived a partial purification, through gel filtration chromatography (GFC) and by ammonium sulphate (AS) precipitation, of compounds in basidiocarps crude extracts from Pycnoporus sanguineus efficient to the control of Asian rust in soybean by induction of resistance. Crude extracts from P. sanguineus were submited to gel filtration chromatography (GFC), and five proteins and one carbohydrate peaks were obtained, with molecular weight ranging from 1,82 to 5,18 KDa. It was made the fractionated protein purification from 100 mL of crude extract from P. sanguineus. The fractions obtained were: 0-20%, 20-40%, 40-60%, 60-80% and 80-100% of ammonium sulphate. Incised soybean cotyledons were treated with fractions from GFC and AS precipitation, crude extract 20% (EB 20%), Saccharomyces cerevisiae (25 mg mL-1) and water. After incubation during 20 h, was made biochemical analyses to verify the phytoalexins content and peroxidases, polyphenoloxidases, β-1,3 glucanases and phenylalanine ammonia-lyase activities. The treatments from GFC, protein fractions III (3,44 KDa), IV (2,79 KDa) and V (1,82 KDa), and carbohydrate fraction, fractions 40-60%, 60-80% and 80-100%, obtained by ammonium sulphate precipitation, crude extract (CE) of basidiocarps at 20%, besides control treatments fungicide (tebuconazole, 0,5 g a.i. L-1) and water were used to evaluate the antimicrobial activity and induction of resistance in soybean against P. pacchirhizi in greenhouse. Soybean plants were treated and after three days were inoculated with the pathogen. Samples were collected 0, 1, 3, 6 and 9 days after the treatment for biochemical analyses and the severity was evaluated after 13 days. The data referring to evaluation of antimicrobial activity show that the fractions do not have inhibiting activity of germination of spores. As for severity, the treatments protein fraction III and CE 20% were efficient in the reduction of the number of injuries for cm2. For peroxidase the CE 20% presented tendency in reducing the enzymatic activity. Chitinases and polifenoloxidases did not presented statistical differences between the treatments. For β-1,3 glucanases there was local induction for the treatments with glicide fraction and protein fractions III and V, and fractions 40-60% and 60-80%, while CE 20% presented systemic induction for this enzyme. For phenylalanine ammonia-lyase the purified fractions of P. sanguineus reduced the enzymatic activity, except for CE 20%. It was possible to induce defense mechanisms in soybean against P. pachyrhizi for the application of partially purified fractions from P. sanguineus, indicating its possible use as an alternative method for controling this pathogen / A indução de resistência envolve a ativação de mecanismos de defesa latentes existentes nas plantas em resposta ao tratamento com agentes bióticos ou abióticos. A aplicação de extratos brutos visando à indução de mecanismos de resistência é uma alternativa interessante ao controle químico, entretanto, nestes extratos pode ocorrer além da presença de indutores, a presença de supressores. Este trabalho teve por objetivo a purificação parcial, por meio de cromatografia de filtração em gel (CFG) e precipitação com sulfato de amônio (SA), de compostos presentes em extrato bruto de Pycnoporus sanguineus, eficientes no controle de ferrugem asiática em soja por indução de resistência. Extrato bruto de P. sanguineus foi submetido à cromatografia de filtração em gel, sendo obtidos cinco picos protéicos e um pico glícido, com pesos moleculares variando de 1,82 a 5,18 KDa. Foi efetuada a precipitação fracionada das proteínas presentes em 100 mL de extrato bruto de basidiocarpos de P. sanguineus. As frações obtidas foram: 0-20%, 20-40%, 40-60%, 60-80% e 80-100% de SA. Cotilédones de soja incisados foram tratados com as frações provenientes da CFG e precipitação com SA, além dos tratamentos extrato bruto a 20% (EB 20%), Saccharomyces cerevisiae (25 mg mL-1) e água. Após incubação pelo período de 20 h efetuou-se análises bioquímicas dos cotilédones para verificar os teores de fitoalexinas, peroxidases, polifenoloxidases, β-1,3 glucanases e fenilalanina amônia-liase. Os tratamentos obtidos por CFG frações protéicas III (3,44 KDa), IV (2,79 KDa) e V (1,82 KDa), e glícida, além das frações 40-60, 60-80 e 80-100% obtidos por saturação com SA foram selecionados para avaliação de atividade antimicrobiana e de indução de resistência em soja contra P. pacchirhizi em casa de vegetação, utilizou-se também extrato bruto (EB) 20% de basidiocarpo de P. sanguineus e as testemunhas fungicida tebuconazole (0,5 g i.a. L-1) e água. Visando avaliar o controle da doença ferrugem asiática e indução de enzimas relacionadas à defesa, montou-se ensaio em casa de vegetação onde plantas foram tratadas e após três dias inoculadas com o patógeno. Amostras foram coletadas 0, 1, 3, 6 e 9 dias após os tratamentos para análises bioquímicas e a severidade foi avaliada após 13 dias. Os dados referentes à avaliação de atividade antimicrobiana mostram que as frações não possuem atividade inibidora da geminação de esporos. Quanto à severidade, a fração III e o EB 20% foram eficientes na redução do número de lesões por cm2. Para peroxidases, o EB 20% apresentou tendência em reduzir a atividade enzimática. Quitinases e polifenoloxidases não apresentaram diferenças estatísticas entre os tratamentos. Para β-1,3 glucanases houve indução local pelas frações glícida e protéicas III e V, e pelas frações 40-60% e 60-80% em relação ao EB 20%, sendo que o mesmo apresentou indução sistêmica para essa enzima. Para fenilalaniana amônia-liase as frações purificadas de P. sanguineus apresentaram tendência em reduzir a atividade enzimática, com exceção para EB 20%. Foi possível induzir mecanismos de defesa em soja contra P. pachyrhizi pela aplicação de frações parcialmente purificadas de P. sanguineus, o que pode permitir o desenvolvimento de métodos alternativos para controle desse patógeno

Proteins that recognise the sugar surface structures on cells have an enormous potential to be used as tools in the characterisation of these structures. A group of proteins, called lectins, have been identified that can bind to carbohydrate complexes on the receptors of cells. The crude extract from Grevillea robusta seeds was found to contain lectin-like proteins that were different from most other lectins, as they would specifically target the receptors of white blood cells and not those found on red blood cells. Therefore, the lectin isolated from G.robusta could be used as a tool to identify the specific surface structures on white blood cells. The lectin was isolated using affinity chromatography where a complex (oligosaccharide) matrix was used. Agglutination, binding and sugar inhibition assays confirmed the isolated protein was a lectin. The lectin was found in low amounts (up to 5% of the total protein content) within the seeds of G.robusta. As a result of this low yield, the identification of the lectin by PAGE was difficult because the levels of protein were beyond the detection limit of the commercial staining reagents. The lectin was called the GR2 protein and was characterised as a monocot mannose binding lectin based on its sugar specificity for only mannose. A serine protease inhibitor was isolated from the seeds of G.robusta using two different chromatography methods, reverse phase HPLC (GR1.HPLC) and gel filtration chromatography (GR1.GF). Ion exchange chromatography was used to initially separate the proteins in the crude extract and the fraction containing the GR1 protein was further purified using reverse phase HPLC (GR1.HPLC). N-terminal sequencing results of the GR1.HPLC protein, showed evidence of proteolytic cleavage during the extraction process, which lead to the second purification method being established. Protease inhibitors were added to the buffers prior to being purified by gel filtration chromatography, which resulted in the GR1 protein being isolated from the crude extract without the presence of the contaminating protein. Mass spectroscopy identified the molecular weight of the GR1 protein to be 6669Da and the full amino acid sequence was derived by cDNA techniques. Sequence alignment studies of the GR1 protein showed significant similarities with the Bowman-Birk inhibitor. The positioning of the cysteine residues were conserved throughout the Bowman-Birk superfamily, however these residues were not conserved within the GR1 protein. Competitive inhibition assays on the GR1 protein revealed the protein could inhibit both trypsin and chymotrypsin at similar levels to that seen for the Bowman-Birk inhibitor. Therefore, the GR1 protein was characterised as a member of the Bowman-Birk superfamily of serine protease inhibitors. The three-dimensional structure of the GR1 protein was determined using two-dimensional NMR spectroscopy. Computer programs such as XEASY, DYANA and SYBYL® were used to tabulate the information taken from the 2D experiments, generate structures and minimise these structures respectively. The solution structure of the GR1 protein was found to contain a region of antiparallel β-sheet structure that corresponded to the trypsin binding site and the remainder of the protein consisted of loops and turns that were held together by disulfide bridges (the chymotrypsin-binding region). Structural similarities between the GR1 protein and the Bowman-Birk inhibitor existed only in the trypsin-binding site of the Bowman-Birk inhibitor. The GR1 protein is the first member of the Proteaceae family to be characterised as a Bowman-Birk inhibitor. This thesis outlines the isolation and biochemical characterisation of the two proteins found within Grevillea robusta and also describes the steps involved and results obtained in determining the three-dimensional structure of the GR1 protein.

En el presente trabajo de tesis se evalúa la posible contribución de otros compuestos al desarrollo del olor sexual mediante la aplicación de técnicas de Head Space dinámico y cromatografía de filtración de gel. El estudio se efectuó en canales de cerdos enteros previamente clasificadas con concentraciónes bajas de escatol (<0,10 µg /g) y androstenona (0,50 µg/ g) , aunque presentaban , según las respuestas de un panel sensorial, olor sexual. La selección de muestras de grasa clasificadas sin olor sexual permitió la comparación estadística de los resultados obtenidos. El análisis de los compuestos volátiles mostró una mayor abundancia de aldehidos (hexanal, heptanal), ácidos grasos de cadena corta (hexanoico, heptanico nonanoico), 1,4-diclorobenceno y estireno en las muestras de grasa con defecto organoléptico, ue puede, debido a los atributos sensoriales de estas sustancias y sus bajos umbrales de detección, favorecer el desarrollo de aromas desagradables asociados al olor sexual por los miembros de un papel sensorial. El análisis de las fracciones obtenidas por filtración de gel mediante cromatografía de gases acoplada a la espectometría de masas permitió la identificación de la 4-fenil-3-buten-2-ona en las muestras clasificadas con olor sexual y concentraciones bajas de escatol y androstenona, que junto a los resultados obtenidos en el estudio sensorial, indican una influencia de este compuesto en el olor sexual. Por una parte la 4-fenil-3-buten-2-ona presentaría una acción sinérgica con la androstenona al potenciar su detección por parte de los panelistas a concentraciones inferiores de 0,01µg/g. Además, el olor a naftalina que presenta la 4-fenil-3-buten-2-ona puede confundir a los miembros del panel debido a que es uno de los descriptores sensoriales que se asocia con la presencia de escatol en las muestras de grasa evaluadas. Así mismo, la baja transferencia a la fase vapor del escatol y, especialmente, de la androstenona determinada mediante técnicas de Head Space estático, indica que la concentración de cada uno de los compuestos en la fase vapor no se corresponde con la concentración real en las muestras de grasa . Según estos resultados, las respuestas del panel test pueden estar influenciadas por la poca volatilidad de los compuestos implicados en el olor sexual y por la presencia en la fase vapor de otros compuestos aromáticos de mayor volatilidad.La validación de métodos de análisis para la determinación de indol-escatol y androstenona-androstenoles en grasa dorsal porcina mediante cromatografía líquida en fase normal y cromatografía de gases acoplada a la espectrometría de masas, respectivamente, fue otro de los objetivos planteados del presente estudio. La simplificación del tratamiento de las muestras de grasa fue uno de los aspectos analíticos que se tuvo en mayor consideración con la finalidad de ofrecer una mayor capacidad de muestras en un intervalo de tiempo corto.

Made available in DSpace on 2017-07-10T17:40:55Z (GMT). No. of bitstreams: 1 Edilaine_D_V_GoncalvesTrevisoli.pdf: 2707364 bytes, checksum: 7b7a664f9727d5909c470f377ee155e8 (MD5) Previous issue date: 2016-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The induction of resistance in plants to pathogens is an alternative method of disease control, wich involves activation of plant resistance mechanisms such as induction of phytoalexins. The elicitors molecules are able to induce and activate those responses, and therefore, techniques have sought to isolate and characterize fractions with elicitor character. The study aimed to purify, through ion exchange chromatography and gel filtration chromatography, eliciting molecules from pathogenic nematodes, and test them in phaseolin induction in beans hypocotyls beans and gliceolin in soybean cotyledons. The buffer solution Tris HCl 0.05 M (pH 6.8) was used as control and the acibenzolar-S-methyl (50 mg a.i. L-1) and Saccharomyces cerevisiae (20 mg mL-1) were used as induction standard treatments. Ion exchange chromatography (IEC) and gel filtration chromatography (GC) were performed to separate fractions with eliciting power from 500 female nematodes of Meloidogyne incognita and Meloidogyne javanica. For purification of elicitors from Meloidogyne javanica, through IEC, six glycidic fractions and six glycoproteins were obtained. These were purified on GC, obtaining sixty-three fractions. They have been classified according to their nature, as twenty-six glycidic and thirty-seven glycoprotein with molecular weights ranging from 29.19 to 2989.25 kDa. Regarding the elicitors purification of Meloidogyne incognita through IEC, nine glycidic and five glycoprotein fractions were obtained. From these fractions, a total of fifty-eight fractions was obtained through GC, twenty-five glycidic and thirty-three glycoprotein with molecular weights ranging from 37.42 to 200.32 kDa. From the fractions purified from Meloidogyne javanica eight had inducing potential of phaseolin. For gliceolin fifteen fractions showed inducing effect. Regarding the fractions purified from Meloidogyne incognita, no fraction has inductive potential of phaseolin superior to the standard treatment. However, twenty-two fractions suppressed phytoalexin inducing activity. For gliceolin ten fractions induced the same, whereas, twenty-three fractions suppressed the induction of gliceolin. Chromatography was efficient in the purification of elicitors compounds. Compounds with suppressing characteristics of gliceolin and phaseolin were checked in bioassays. For those fractions obtained through IEC, and then submitted to GC that did not induce phytoalexin, it is suggested that molecules need to act together to have elicitor effect and thus induce defense response in the plant / A indução de resistência em plantas contra patógenos é um método de controle alternativo de doenças, e que envolve a ativação dos mecanismos de resistência da planta, como a indução de fitoalexinas. As moléculas eliciadoras possuem a capacidade de induzir e ativar tais repostas, e assim sendo, técnicas têm buscado isolar e caracterizar frações com caráter eliciador. O trabalho teve por objetivo purificar, por cromatografia de troca iônica cromatografia de filtração em gel, moléculas eliciadoras a partir de nematoides fitopatogênicos, e testá-las na indução de faseolina em hipocótilos de feijoeiro e gliceolina em cotilédones de soja. O tampão Tris HCl 0,05 M (pH 6,8) foi utilizado como tratamento controle e o acibenzolar-S-metil (50 mg i.a. L-1) e o Saccharomyces cerevisiae (20 mg mL-1) foram utilizados como tratamento padrão de indução. Cromatografia de troca iônica (CTI) e cromatografia de filtração em gel (CFG) foram realizadas para separar frações com poder eliciador a partir de quinhentas fêmeas de nematoides de Meloidogyne incognita e Meloidogyne javanica. Para a purificação de eliciadores a patir de Meloidogyne javanica, por CTI, foram obtidos seis frações glicídicas e seis glicoproteicas. Estas, por sua vez, foram purificadas em CFG, sendo obtidos no total sessenta e três frações. As mesmas foram classificadas de acordo com sua natureza, sendo vinte e seis glicídicas e trinta e sete glicoproteicas, com massas moleculares variando de 29,19 a 2.989,25 kDa. Em relação a purificação de eliciadores de Meloidogyne incognita por CTI, foram obtidos nove frações glicídicas e cinco glicoproteicas. A partir destas, foram obtidos por CFG um total de cinquenta e oito frações, sendo vinte e cinco glicídicas e trinta e três glicoproteicas, com massas moleculares variando de 37,42 a 200,32 kDa. Das frações purificadas a partir de Meloidogyne javanica oito apresentaram potencial indutor de faseolina. Para gliceolina quinze frações mostraram efeito indutor. Em relação as frações purificadas a partir de Meloidogyne incognita, nenhuma fração apresentou potencial indutor de faseolina superior ao tratamento padrão. Entretanto, vinte e duas frações suprimiram a atividade de indução de fitoalexina. Para gliceolina dez frações induziram a mesma, enquanto que, vinte e três frações suprimiram a indução da gliceolina. A cromatografia foi eficiente na purificação de compostos eliciadores. Compostos com características supressoras de gliceolina e faseolina foram verificadas nos bioensaios. Para aquelas frações obtidas por CTI e posteriormente submetidas a CFG que não induziram fitoalexina, sugere-se que as moléculas necessitam atuar juntas para haver efeito eliciador e assim induzir a resposta de defesa no vegetal