Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-08-31T19:17:42Z No. of bitstreams: 1 2016 - Francine Fran?a Monken.pdf: 2589456 bytes, checksum: 06c9e0d64d97d48b268dfd24bee047a6 (MD5) / Made available in DSpace on 2017-08-31T19:17:42Z (GMT). No. of bitstreams: 1 2016 - Francine Fran?a Monken.pdf: 2589456 bytes, checksum: 06c9e0d64d97d48b268dfd24bee047a6 (MD5) Previous issue date: 2016-09-30 / CAPES / This study aimed determinate the chemical composition of raw secretions expelled by two species of Aparasphenodon genus from compounds isolation, purification and structural identification. For this, individuals of A. brunoi (n=5) e A. arapapa (n=3) were collected in Ilha da Marambaia, municipality of Mangaratiba (RJ), and in municipality of Ilh?us (BA), respectively. The secretions were obtained by smooth electric stimulus and were translucent and little viscous, with approximated yield of 4 mg of sample/individual. For each species, was realized chromatography of thin layer, the revelator reagent ninhydrin indicated presence of amine groups after violet color appearing. Samples than were submitted with the same retention time (0.88 and 1.44 min) in both analysis, indicating presence of similar compounds in both species. In same way, samples purification realized by semi preparative liquid chromatography of high efficiency presented the same fractions number for both species. The structural determination was made by LC-MS/MS only for A. brunoi and reveled high collagen incidence and other uncharacterized proteins. Biological assays were realized in concentrations of 1?g.mL-1 and 10?g.mL-1 for both species. The assay of cellular viability did not present survival reduction of yeast, such as dosing of lipid peroxidation that did not show reaction with malonaldehyde in all tested cases. In other hand, the assay of mitochondrial damage reveled toxicity in concentration of 10 ?g.mL-1, reducing for discreet form the cellular viability in both samples. We conclude that both species have proteins as the main compound and, in a cellular level, are not harmful in 1 ?g.mL-1, but can promote damage in 10 ?g.mL-1. Besides that, as reveled profiles to both extracts, both chemical and biological assays were very close, the existence of a pattern in chemical composition of cutaneous secretions in amphibians of the same genus can be reinforced, even that species be in distinct environmental conditions. / O presente estudo buscou determinar a composi??o qu?mica das secre??es brutas expelidas por duas esp?cies de Aparasphenodon, a partir do isolamento, purifica??o e identifica??o estrutural destes compostos. Para tal, indiv?duos de A.brunoi (n=5) e A.arapapa (n=3) foram coletados na Ilha da Marambaia, Mangaratiba (RJ) e no munic?pio de Ilh?us (BA), respectivamente. As secre??es foram obtidas por est?mulo el?trico suave e mostraram-se translucidas e pouco viscosas, com rendimento aproximado de 4 mg de amostra/indiv?duo. Para cada esp?cie, foi realizada cromatografia em camada delgada; o reagente revelador ninidrina indicou presen?a de grupos am?nicos ap?s aparecimento da cor violeta. As amostras foram ent?o submetidas ? cromatografia liquida de alta efici?ncia anal?tica, sendo detectados dois picos majorit?rios com mesmo tempo de reten??o (0,88 e 1,44 min) em ambas as an?lises, indicando assim a presen?a de compostos semelhantes nas duas esp?cies. Da mesma forma, a purifica??o das amostras realizada por cromatografia liquida de alta efici?ncia semi-preparativa apresentou o mesmo n?mero de fra??es para ambas as esp?cies. A determina??o estrutural foi determinada por LC-MS/MS apenas para A. brunoi e revelou alta incid?ncia de col?geno e outras prote?nas n?o caracterizadas. Os ensaios biol?gicos foram realizados nas concentra??es de 1 ?g.mL-1 e 10 ?g.mL-1 para ambas as esp?cies. O ensaio de viabilidade celular n?o apresentou redu??o da sobreviv?ncia das leveduras, assim como a dosagem de peroxida??o lip?dica n?o mostrou rea??o com malonalde?do, em todos os casos testados. Por outro lado, o ensaio de dano mitocondrial revelou toxicidade na concentra??o de 10 ?g.mL-1, reduzindo de forma discreta a viabilidade celular, em ambas as amostras. Conclu?mos ent?o que as duas esp?cies possuem prote?nas como seu principal componente e, a n?vel celular, n?o s?o nocivas em 1 ?g.mL-1, mas podem promover danos quando em 10 ?g.mL-1. Al?m disso, como os perfis revelados para ambos os extratos, tanto nos ensaios qu?micos como nos biol?gicos, foram muito pr?ximos, a exist?ncia de um padr?o na composi??o qu?mica das secre??es cut?neas em anf?bios de um mesmo g?nero pode ser refor?ada, ainda que as esp?cies se encontrem em condi??es ambientais distintas.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-06-02T19:54:07Z No. of bitstreams: 1 2015 - Soraia John da Silva.pdf: 1762972 bytes, checksum: aae3e43996736241367a1924339662c0 (MD5) / Made available in DSpace on 2017-06-02T19:54:07Z (GMT). No. of bitstreams: 1 2015 - Soraia John da Silva.pdf: 1762972 bytes, checksum: aae3e43996736241367a1924339662c0 (MD5) Previous issue date: 2015-08-20 / CAPES / The glyphosate, herbicide used in different countries, inhibits the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), blocking the synthesis of aromatic amino acids. With this, it still affects the activity of phenylalanine ammonia lyase (PAL) and the concentration of metabolites in plants. Its activity was first reported as a metal chelator. Therefore, to make use of this herbicide, may be formation of complexes with metals from soil, occurring change in the effectiveness of the product and loss of minerals. Thus, to avoid such problems,this study aimed to compare the effectiveness of three forms of glyphosate: purified, previously complexed with metals, and a commercial form (Roundup WG?). The effects of glyphosate complexes with copper, cobalt and nickel with different stoichiometries and different pH values on the in vitro activity of EPSPs were evaluated. Selected complex for the first experiment in vivo were Cu421 and Co821 (first number for pH and the latter two to stoichiometry glyphosate: metal). These complexes were used in the in vivo experiment I, as well as distilled water (control), purified Roundup and glyphosate on transgenic soybean and B. decumbens. In the following experiments (II and III), mineral oil was used in all treatments to reduce leaching of the compounds. The complex selected for continuation of this study was Cu421 due to its emphasis on the results obtained in the first experiment in vivo. When comparing the effect of the compounds in Falker Chlorophyll Index (ICF) of B. decumbens compared to control, there was fall of this index when using Roundup, Cu421 and purified glyphosate (the latter only in the second experiment). Later it was analyzed fresh mass accumulation and collected fresh material for determination of enzyme activities and N-NO3- levels. The complex Cu421 caused a more significant fresh mass reduction and still allowed the increase in the content of soluble nitrate in Brachiaria decumbens, probably due to the presence of nitrate in this complex. In the case of transgenic soybean there was no significant change in ICF. The use of Roundup and the complex did not change significantly the fresh mass accumul ation in this species. The application of the purified glyphosate, however, caused increase in mass accumulation and all treatments altered soybeans in nitrate content. In experiments with mineral oil in B. decumbens, the Cu421 complex inhibited significantly the EPSPs activity, as well as purified Roundup and glyphosate (the latter only in the experiment III). No inhibition occurred in transgenic soybean. All treatments in vivo stimulated PAL activity only on weed, which suggests that the increase in this activity is a consequence of the inhibition of EPSPs and other deleterious effects of compounds on susceptible plants. This study showed that especially Cu421 complex might be used effectively as a herbicide since it inhibits EPSPs, activates the PA,L reduces fresh mass accumulation and influences photosynthesis in the tested weed. Besides, the transgenic soybean showed little sensitive to this compound, which further increases the prospect of using the same as herbicide. / O glifosato, herbicida usado mundialmente, atua inibindo a enzima 5-enolpiruvil-chiquimato- 3-fosfato-sintase (EPSPs), bloqueando a s?ntese de amino?cidos arom?ticos. Com isso, ainda influencia a atividade de fenilalanina am?nia liase (PAL) e a concentra??o de metab?litos nas plantas. A sua primeira a??o reportada foi como agente quelante de metais. Portanto, ao fazer uso deste herbicida, pode haver forma??o de complexos com metais do solo, ocorrendo altera??o da efic?cia do produto e defici?ncia mineral. Assim, de modo a evitar tais problemas, este trabalho teve como objetivo, comparar a efici?ncia de tr?s formas de glifosato: purificado, previamente complexado com metais, e uma das suas formas comerciais (Roundup WG?). Os efeitos de complexos de glifosato com cobre, cobalto e n?quel, sintetizados sob diferentes estequiometrias e valores de pH, foram avaliados sobre a atividade in vitro de EPSPs. Os complexos selecionados para o primeiro experimento in vivo foram: Cu421 e Co821 (primeiro n?mero referente ao pH e os dois ?ltimos ? estequiometria glifosato:metal). Estes complexos foram usados no experimento in vivo I, assim como ?gua destilada (controle), Roundup e glifosato purificado, sobre soja transg?nica e Brachiaria decumbens. Nos experimentos seguintes utilizou-se ?leo mineral em todos os tratamentos para reduzir a lixivia??o dos compostos. O complexo selecionado para continua??o deste estudo foi o Cu421 devido a seu destaque nos resultados obtidos no primeiro experimento in vivo. Ao comparar o efeito dos compostos no ?ndice de Clorofila Falker (ICF) de B. decumbens em rela??o ao controle, observou-se queda deste ?ndice ao usar Roundup, Cu421 e glifosato purificado (este ?ltimo, somente no segundo experimento). Posteriormente foi analisado ac?mulo de massa fresca e coletado material fresco para determina??o das atividades enzim?ticas e dos teores N-NO3-. O complexo Cu421 foi o que provocou maior redu??o da massa fresca e ainda possibilitou o aumento no teor sol?vel de nitrato em B. decumbens, provavelmente devido ? presen?a de nitrato neste complexo. No caso da soja transg?nica n?o houve altera??o significativa de ICF. O uso de Roundup e dos complexos n?o alterou de forma significativa o ac?mulo de massa fresca nesta esp?cie. A aplica??o do glifosato purificado, entretanto, causou incremento no ac?mulo de massa e todos os tratamentos alteraram o teor de nitrato em soja. Nos experimentos com ?leo mineral em B. decumbens, o complexo Cu421 inibiu de forma significativa a atividade de EPSPs, assim como Roundup e glifosato purificado (este ?ltimo, somente no experimento III). Em soja transg?nica n?o houve inibi??o. Todos os tratamentos in vivo estimularam a atividade de PAL somente na gram?nea, permitindo sugerir que o aumento nesta atividade seja consequ?ncia da inibi??o de EPSPs e de outros efeitos delet?rios dos compostos sobre plantas suscept?veis. O presente estudo mostrou que principalmente o complexo Cu421 pode vir a ser utilizado de forma eficaz como herbicida, uma vez que inibe a EPSPs, ativa a PAL, reduz ac?mulo de massa fresca e influencia a fotoss?ntese na planta daninha testada. E, em contrapartida, a soja transg?nica mostrou-se pouco sens?vel a este composto, o que aumenta ainda mais a perspectiva do uso do mesmo como herbicida.