JEREISSATI, Camila Barbosa Pinheiro. Análise proteômica de plastídeos do endosperma de sementes em desenvolvimento de pinhão manso (Jatropha curcas L.). 2015. 127 f. Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Vitor Campos (vitband@gmail.com) on 2016-09-01T22:55:24Z No. of bitstreams: 1 2015_teses_cbpjereissati.pdf: 1568499 bytes, checksum: 2f41598abfda8cf6bcab51817b1b742a (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-09-02T21:06:57Z (GMT) No. of bitstreams: 1 2015_teses_cbpjereissati.pdf: 1568499 bytes, checksum: 2f41598abfda8cf6bcab51817b1b742a (MD5) / Made available in DSpace on 2016-09-02T21:06:57Z (GMT). No. of bitstreams: 1 2015_teses_cbpjereissati.pdf: 1568499 bytes, checksum: 2f41598abfda8cf6bcab51817b1b742a (MD5) Previous issue date: 2015 / Jatropha curcas L. is a plant native to America and belongs to the Euphorbiaceae family. Currently it is gaining economical interest mainly because it is an oilseed crop with potential to produce biodiesel. However, presence of phorbol esters (a class of diterpenes) that are the major toxic constituents of the seeds, limits a better usage of the plant, by making the use of the residue, obtained after the oil extraction from the seeds, unfeasible for animal feed, due to its pro-carcinogenic activity and inflammatory action. Proteomic analysis of the plastids isolated from developing seeds of Jatropha is important because the synthesis of fatty acid as well as phorbol esters, the two most attractive compounds in the study of Jatropha curcas, occur in plastids. Proteomic analysis of this organelle is crucial to better understand and explore not only the biosynthetic pathway of these two compounds but other metabolic pathways , and addtionaly providing foundation for researchs that aimed to develope genotypes with more suitable characteristics for industrial applications. In this study, we performed a proteomic analysis of plastids isolated from the endosperm of developing Jatropha curcas seeds that were in the initial stage of deposition of protein and lipid reserves. Proteins extracted from the plastids were digested with trypsin, and the peptides were applied to an EASY-nano LC system coupled online to an ESI-LTQ-Orbitrap Velos mass spectrometer, and this led to the identification of 1103 proteins representing 804 protein groups, of which 923 were considered as true identifications, and this considerably expands the repertoire of J. curcas proteins identified so far. Of the identified proteins, only five are encoded in the plastid genome, and none of them are involved in photosynthesis, evidentiating the nonphotosynthetic nature of the isolated plastids. Homologues for 824 out of 923 identified proteins were present in three different plastids proteins databases i.e. PPDB, SUBA and PlProt, while homologues for 13 proteins were not found in any of these three databases but were marked as plastidial by at least one of the three prediction programs used (TargetP, ChloroP and PlantMPloc). Functional classification showed that proteins belonging to amino acids metabolism comprise the main functional class, followed by carbohydrate, energy, and lipid metabolisms. The small and large subunits of Rubisco were identified, and their presence in plastids is considered to be an adaptive feature counterbalancing for the loss of one-third of the carbon as CO2 as a result of the conversion of carbohydrate to oil through glycolysis. While several enzymes involved in the biosynthesis of several precursors of diterpenoids were identified, we were unable to identify any terpene synthase/cyclase, which suggests that the plastids isolated from the endosperm of developing seeds do not synthesize phorbol esters. In conclusion, this study provides insights into the major biosynthetic pathways and certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level. / O pinhão manso (Jatropha curcas L.) é uma planta nativa da América, pertencente à família Euphorbiaceae. Atualmente, ela desperta interesse econômico principalmente por se tratar de uma oleaginosa com potencial para a produção de biodiesel. Entretanto, a presença de ésteres de forbol (uma classe de diterpeno), que são os principais constituintes tóxicos das sementes, limita uma melhor utilização dessa planta, por inviabilizar o uso do resíduo de extração do óleo das sementes na alimentação animal, bem como, por apresentar atividade pró-carcinogênica e ação inflamatória. A análise proteômica de plastídeos, isolados de sementes em desenvolvimento de pinhão manso, é uma importante vertente de estudo, pois tanto a síntese de ácidos graxos como dos ésteres de forbol, os dois compostos mais atrativos no estudo de Jatropha curcas, ocorrem nos plastídeos. O estudo proteômico dessa organela torna-se crucial para melhor compreender e explorar não somente as vias biossintéticas desses dois compostos, como de outras vias metabólicas, além de proporcionar um conjunto de dados que pode ser utilizado em pesquisas voltadas para o desenvolvimento de genótipos com características mais adequadas para aplicações industriais. No presente trabalho, realizou-se uma análise proteômica de plastídeos isolados do endosperma de sementes em desenvolvimento do pinhão manso, que estavam nos estágios iniciais de deposição de lipídios e proteínas de reserva (25-30DAA), confirmados por meio de análises histológica e histoquímica. As proteínas extraídas dos plastídeos foram digeridas com tripsina e os peptídeos foram aplicados no sistema de nano-LC EASYII acoplado online ao espectrômetro de massa nano ESI LTQ-Orbitrap velos, o que resultou na identificação 1103 proteínas, representando 804 grupos de proteínas, dos quais 923 foram consideradas identificações verdadeiras. Isso expandiu consideravelmente o repertório de proteínas do pinhão manso até agora identificas. Dentre as proteínas identificadas, apenas 5 são codificadas pelo genoma plastidial, e nenhuma delas está envolvida na fotossíntese, o que evidencia a natureza não fotossintética dos plastídeos isolados. Homólogos de 824, dentre as 923 proteínas identificadas, estavam presentes nos bancos de dados PPDB, SUBA e PlProt, enquanto homólogos para 13 proteínas não foram encontrados em nenhum dos três bancos de dados plastidiais, mas foram detectados como plastidiais por pelo menos um dos três programas de predição de localização subcelular utilizados (TargetP, ChloroP, PlantMPloc). A classificação funcional mostrou que a maioria das proteínas identificadas pertencia ao metabolismo dos aminoácidos, seguido dos metabolismos dos carboidratos, energético e dos lipídios. As subunidades maiores e menores da Rubisco foram identificadas, e sua presença nos plastídeos foi considerada uma característica adaptativa para contrabalancear a perda de um terço do carbono na forma de CO2 como um resultado da conversão de carboidratos em óleo através da glicólise. Apesar de enzimas envolvidas na biossíntese de diversos precursores dos diterpenóides terem sido identificadas, não foi detectado nenhuma terpeno sintase/ciclase, o que sugere que os plastídeos isolados do endosperma de sementes em desenvolvimento não sintetizam ésteres de forbol, apesar de uma grande quantidade desse composto ser encontrada neste tecido. Como conclusão, o presente trabalho proporciona insights sobre as principais vias biossíntéticas, e sobre características peculiares dos plastideos isolados do endosperma de sementes em desenvolvimento.

Submitted by Katiane Souza (katyane.souza@gmail.com) on 2016-05-15T12:50:25Z No. of bitstreams: 1 arquivototal.pdf: 4121860 bytes, checksum: 395b66a7b8c8c0777cbe68012f0c3d4f (MD5) / Made available in DSpace on 2016-05-15T12:50:25Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 4121860 bytes, checksum: 395b66a7b8c8c0777cbe68012f0c3d4f (MD5) Previous issue date: 2008-09-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Among species that are being prospected for biodiesel production, the pinhão-manso (Jatropha curcas) has been outstanding as an oleaginous plant with essential qualities in biodiesel production. Perennial plant of the Euphorbiaceous family resistant to the adverse climate and soil conditions whose oil production's potential has been considered high. In the last years, a lot of researches had appeared around the crops for a better comprehension, once there's a lack of information regarding the agronomic plant requirements. By knowledge necessity for farming enlargement of that species, this work addressed to verify and to quantify the fertilizing effects in the edaphic and foliar region in pinhão-manso’s (Jatropha curcas) initial growth, as well as evaluate the Nitrogen and Potassium dosage effects in the initial plant growth through the foliar fertilization. The researches were accomplished in the period of April, 2006 to April, 2007, in the National Center of Research and Cotton - CNPA - EMBRAPA. Two experiments were implanted with randomized experimental design in blocks, in which the first one consisted of 17 treatments with three repeated sessions and four fertilizers application (bovine manure, biosolids, castor presscake and urea) each one with four different dosage (85, 170, 225 and 340 Kg/ha) via soil. The second experiment applied through foliar fertilization consisted by a factorial [(4 x 4) +1], with three repetitions, about seventeen treatments in four dosages types of Urea (6, 8, 10 and 12%) and Chloride of Potassium (2, 4, 6 and 8%). The following variables were estimated: stem height, diameter, foliar area, phytomass and vegetable analysis to the 15, 30, 45, 60, 75, 90, 105, 120, 135 DAE through soil. The same variables were estimated through foliar fertilization in the period of 65, 80, 95, 110, 125 and 140 DAE. When fertilized with biosolids and castor presscake treatments, the Pinhão Manso’s crops increased significantly, the plant growth promoting an additional of 40,90% to the height under the biosolid treatment and 40,60% to the stem diameter when fertilized with castor presscake, compared to the control. The pinhão-manso plants were benefited by fertilization, especially with the NPK-mineral, increasing the height of 96,14% when compared to the control, without the fertilizers usage. The crop through foliar/leaf fertilization had no significant differences among the practical treatments, obtaining a stem height addition with N4K4 dosage (amount) of 11,47% at the end of the experiment relatively to the control. The root length in the KCl/N45 dosage presented an increase of 172% in relation to witness when compared to the 24Kg/ha proportion. / Entre as espécies que estão sendo prospectadas para produção de biodiesel, o pinhãomanso (Jatropha curcas) têm se destacado como uma planta oleaginosa com as qualidades necessárias para a produção de biodiesel. Planta perene da família das Euforbiáceas, resistente às condições adversas de clima e solo cujo potencial para produção de óleo tem sido considerado elevado. Nos últimos anos, muitas pesquisas têm surgido entorno da cultura, com o propósito de conhecê-la melhor, pois pouco se sabe a respeito das exigências agronômicas da planta. Mediante a necessidade de conhecimento para ampliação do cultivo dessa espécie, o objetivo deste trabalho foi verificar e quantificar os efeitos das adubações no ambiente edáfico e foliar no crescimento inicial do pinhão manso (Jatropha curcas) e avaliar os efeitos das quantidades de Nitrogênio e Potássio no crescimento inicial da planta através da adubação foliar. Os experimentos foram realizados no período de abril de 2006 à abril de 2007, no Centro Nacional de Pesquisa e Algodão- CNPA- EMBRAPA. Implantouse dois experimentos com delineamento experimental em blocos casualizados, no qual o primeiro consistiu de 17 tratamentos com três repetições com aplicação de quatro fertilizantes (esterco bovino, biossólido, torta de mamona e uréia) cada um com quatro quantidades distintas (85, 170, 225 e 340 Kg/ha) via solo. O segundo experimento aplicado via adubação foliar consistiu em um fatorial [(4 x 4) +1], com três repetições, totalizando dezessete tratamentos em quatro tipos de quantidades de Uréia (6, 8, 10 e 12 %) e Cloreto de Potássio (2, 4, 6 e 8 %). Foram avaliadas as seguintes variáveis: altura caulinar, diâmetro, área foliar, fitomassa e análise vegetal aos 15, 30, 45, 60, 75, 90, 105, 120, 135 DAE via solo. As mesmas variáveis foram avaliadas via adubação foliar no período de 65, 80, 95, 110, 125 e 140 DAE. O pinhão manso nos primeiros 135 dias do seu ciclo, respondeu a adubação orgânica, com os produtos Biossólido e Torta de Mamona, na altura da planta e diâmetro caulinar de respectivamente 40,9% e 40,6% com relação ao controle, sem adubação orgânica. A cultura do pinhão manso foi beneficiada pela adubação, em especial com a NPK-mineral, proporcionando um incremento na altura de 96,14% quando comparados com a testemunha, sem o uso de fertilizantes. A cultura via adubação foliar não obteve diferenças significativas entre os tratamentos aplicados, obtendo um acréscimo na altura caulinar com a quantidade N4K4 ao final do experimento de 11,47% em relação à testemunha. O comprimento da raiz na dosagem de KCl/N45 apresentou um aumento de 172% em relação a testemunha quando avaliada na proporção de 24Kg/ha.

Made available in DSpace on 2015-03-26T13:51:54Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2031314 bytes, checksum: b64e48cf16628db68c74198ac88fc30e (MD5) Previous issue date: 2011-02-21 / The physic nut (Jatropha curcas L.) is an important source of raw material for biodiesel production. This species has high ecological adaptability and can grow in a wide variety of environmental conditions, presenting good development in both dry tropical regions and humid equatorial zones. In the regions where physic nut is being cultivated a large number of spores of Arbuscular Mycorrhizal Fungi (AMF) has been found and the plants have beneficial relationships in association with these fungi. The objective of this work was to survey the AMF species in association with different accesses of physic nut, as well as to compare the diversity of these fungi when physic nut is subjected to different edaphoclimatic conditions, using classical methods of identification for these microorganisms, complemented with molecular techniques. In this context, a total of 19 AMF species were identified in the rhizosphere of physic nut, besides individuals identified only at the genus level. Differences in the occurrence of AMF species among the areas of Viçosa, Canaã and Nova Porteirinha, as well among the different accesses cultivated in this last area were observed. In all areas, the numbers of AMF spores ranged from 89 to 728 per 100 cm3 of soil and the mycorrhizal colonization percentage ranged from 50,67 to 72 %. The species of the genus Glomus were the most abundant. Individuals of physic nut of the same origin, grown in remote regions but with similar edaphoclimatic conditions, present similar AMF species. However, the different accesses showed a variation in AMF population in the rhizospheric soil from the same area. The sum of morphological and molecular methods showed a more complete set of information on the diversity of AMF in the rizosphere of physic nut. These results represent the first reports related to the composition of AMF species in physic nut plantations in different geographical areas. / A cultura do pinhão-manso (Jatropha curcas L.) é considerada uma importante fonte de matéria-prima para produção do biodiesel. Esta espécie possui uma alta adaptabilidade ecológica, permitindo seu crescimento em uma grande variedade de condições ambientais, podendo ter um bom desenvolvimento tanto em regiões tropicais secas quanto em zonas equatoriais úmidas. Regiões onde a cultura do pinhão-manso está sendo implantada apresentam um elevado número de esporos de fungos micorrízicos arbusculares (FMAs) e as plantas apresentam relações benéficas nas associações com estes fungos. O objetivo deste trabalho foi realizar um levantamento das espécies de FMAs em associação com diferentes acessos de pinhão-manso, bem como comparar a diversidade desses fungos quando esta cultura é submetida a diferentes condições edafoclimáticas, utilizando métodos clássicos de identificação para estes micro-organismos, complementados com avaliações moleculares. Neste contexto, um total de 19 espécies de FMAs foi identificado na rizosfera das plantas de pinhão-manso, além dos indivíduos apresentados somente em nível de gênero. Houve diferenças na ocorrência destas espécies entre as áreas de Viçosa, Canaã e Nova Porteirinha, bem como entre os diferentes acessos cultivados nesta última área. Em todas as áreas, o número de esporos de FMAs variou de 89 a 728 por 100 cm3 de solo e a porcentagem de colonização micorrízica variou entre 50,67 a 72 %. As espécies do gênero Glomus foram as mais abundantes. Indivíduos de pinhão-manso de mesma procedência, plantadas em regiões distantes, mas, com condições edafoclimáticas semelhantes, possuem populações de FMAs similares. Os acessos diferentes apresentaram uma variação na população de FMAs no solo rizosférico em uma mesma área. A soma das metodologias morfológicas e moleculares apresentou informações mais completas a respeito da diversidade de FMAs presentes na rizosfera de pinhão-manso. Estes resultados representam os primeiros relatos sobre a composição das espécies de FMAs em povoamentos de pinhão-manso em diferentes áreas geográficas de estudo.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Um nÃmero crescente de peptÃdeos isolados a partir dos mais diversos tecidos vegetais tem destacado essas molÃculas como alvo potencial de estudo. Estes peptÃdeos apresentam uma grande diversidade funcional e, por isso, vem sendo descritos frequentemente na literatura cientÃfica dos Ãltimos anos. No presente trabalho sÃo relatadas a prospecÃÃo molecular e caracterizaÃÃo funcional de âshort-peptidesâ vegetais. Inicialmente, o lÃtex de Calotropis procera foi particionado, gerando cinco fraÃÃes: hexÃnica (49,4%), diclorometano (5,2%), acetato de etila (2,0%), butanÃlica (2,1%) e aquosa (41,1%) (CapÃtulo 1). O perfil fitoquÃmico e as anÃlises espectroscÃpicas mostraram que a fraÃÃo diclorometano foi quimicamente a mais heterogÃnea. As fraÃÃes diclorometano e acetato de etila apresentaram toxicidade in vitro contra linhagens de cÃlulas tumorais (DL50 variando de 0,05 a 3,9 Âg/mL) e, in vivo, contra o crustÃceo Artemia salina (DL50 variando de 10,9 a 65,7 Âg/mL). Estas mesmas fraÃÃes apresentaram atividade antiinflamatÃria no modelo de peritonite induzida por carragenana em ratos, associadas a uma inibiÃÃo da migraÃÃo de neutrÃfilos de 67% (diclorometano) e 56% (acetato de etila). A reaÃÃo positiva com tolidina e ninidrina sugere a presenÃa de ciclopeptÃdeos na fraÃÃo bioativa acetato de etila. No CapÃtulo 2, vinte e uma sequÃncias primÃrias de peptÃdeos, oriundas da fraÃÃo 1 de sementes de Jatropha curcas, foram deduzidas por meio de sequenciamento de novo e confirmadas atravÃs de buscas contra um banco de dados genÃmico de Jatropha. A comparaÃÃo do padrÃo de fragmentaÃÃo por MS/MS entre o Ãon duplamente carregado de m/z de 671,79, presente nas fraÃÃes 1 e PII-C, e um peptÃdeo sintÃtico, anÃlogo Ãquele, mostrou que o uso de passos analÃticos adicionais para a identificaÃÃo e caracterizaÃÃo destes peptÃdeos nÃo seria necessÃrio. A ferramenta de busca BlastP mostrou que a sequÃncia APTLSGGSVPRDAD apresenta homologia e està inserida no domÃnio conservado das proteÃnas de embriogÃnese tardia (LEA_6). No CapÃtulo 3, os dois peptÃdeos sintÃticos anÃlogos, linear (1342,44 g/mol) e cÃclico (1324,44 g/mol), obtidos por sÃntese quÃmica em fase sÃlida, mostraram diferenÃas que caracterizam seus arranjos, quando comparados pelo padrÃo de fragmentaÃÃo por MS/MS e mobilidade iÃnica. O estado de carga e o tempo de retenÃÃo foram similares, do ponto de vista fÃsico-quÃmico. Os estudos de dicroÃsmo circular mostraram a presenÃa de um estado desordenado para os peptÃdeos. Os ensaios biolÃgicos com os peptÃdeos nÃo revelam sinais aparentes de atividade funcional, porÃm nÃo se pode descartar a possibilidade de que modelos biolÃgicos adicionais sejam testados. Os resultados advindos deste estudo geram novas perspectivas e servem de base para a realizaÃÃo de trabalhos subsequentes que tratem do isolamento e caracterizaÃÃo de peptÃdeos nas espÃcies Calotropis procera e Jatropha curcas. / An increasing number of peptides isolated from a range of tissue plants have highlighted these molecules as a potential study target. Those peptides demonstrate a large functional variety and, therefore, have been frequently described in the scientific literature of the last years. The present work reports a molecular prospecting and functional characterization of plant "short-peptides". Initially, the Calotropis procera latex was partitioned into five fractions: hexane (49.4%), dichloromethane (5.2%), ethyl acetate (2.0%), butanol (2.1%) and aqueous (41.1%) (Chapter 1). The phytochemical profile and the spectroscopic analysis showed that the dichloromethane fraction was chemically the most heterogeneous. The dichloromethane and ethyl acetate fractions showed in vitro cytotoxicity against tumor cell lines (LD50 ranging from 0.05 to 3.9 mg/mL) and in vivo citotoxity against the crustacean Artemia salina (LD50 ranging from 10.9 to 65.7 mg/mL). These same fractions showed anti-inflammatory activity in carrageenan-induced peritonitis model in rats, associated to an inhibition of neutrophil migration for the dichloromethane (67%) and ethyl acetate (56%) fractions. A positive reaction with tolidine and ninhydrin suggests the presence of cyclopeptides in the bioactive ethyl acetate fraction. In Chapter 2, twenty primary sequences of peptides derived from the 1 fraction of Jatropha curcas seeds were deduced by de novo sequencing and confirmed by searches against genomic Jatropha database. The comparison of MS/MS fragmentation patterns between the doubly charged ion (671.79 m/z) found in fractions 1 and PII-C and its analogue synthetic peptide has shown that the use of additional analytical steps for the identification and characterization of these peptides would be not necessary. The BlastP search tool showed that the sequence APTLSGGSVPRDAD is homologous to and it is inserted into the conserved domain of the late embryogenesis protein (LEA_6). In Chapter 3, the two analogues synthetic peptides, linear (1342.44 g/mol) and cyclic (1324.44 g/mol), obtained by solid phase synthesis, showed differences that characterize their arrangement when compared to the pattern fragmentation by MS/MS and ion mobility. The net charge and the retention time had similar physicochemical parameters in both peptides. Dichroism circular studies showed the presence of a disordered state to both peptides. The biological assays of peptides showed no apparent signs of functional activity, however this fact do not exclude the possibility to test additional biological models. The results arising from this study generate new prospects and provide a basis for further work aiming the isolation and characterization of peptides in C. procera and J. curcas species.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / In this work were studied diverse physiological mechanisms, as the osmotic adjustment, photosynthesis, including gas exchange and chlorophyll fluorescence parameters, as well the oxidative responses in physic nuts submitted to different abiotic stresses as: salinity, drought and high temperature. The first experiment aimed to study the effects of increase of the NaCl concentrations (0, 25, 50, 75 and 100 mM) in the ions accumulate (Na+, Cl- and K+) and some growth variables, as well evaluate the water state and the principals solutes (organic and inorganic) involved on the osmotic adjustment of physic nuts plants under this stressful condition. Physic nuts plants showed sensibility to salt stress, presenting a reduction by 50% in dry matter from by 47 mM of NaCl concentration during 15-d. This sensibility should be due the leaf Na+ and Cl- high accumulation, associated a strong reduction in the K+ concentration, induced by high Na+ content. On the other hand, physic nuts plants were able osmotic adjust to salinity due a severe decrease on the osmotic potential and increase of leaf water state, principally in the higher NaCl levels. Of the solutes studied, was observed that salt ions (Na+ and Cl-) contributed with the most of the osmotic adjustment, while that the K+ contribution was decreased strongly by NaCl. The glycinebetaine compared to proline was more important to the osmotic adjustment of physic nuts leaves, as in the absence as in presence of different NaCl levels in the nutrient solution. The second experiment evaluated the resistance of photosynthetic apparatus of physic nuts plants submitted to different time of exposure (7-d and 14-d of treatment and 3-d of recovery) to salt stress (100 mM of NaCl).The changes caused on photochemistry activity and leaf gas exchange were evaluate by Na+ and Cl- accumulation and decrease of K+/Na+ ratio in the leaves. After 7-d of treatment was observed a major action of osmotic effects. However, after 14-d of treatment, the ionic effects caused by Na+ and Cl- excessive accumulation and by K+/Na+ ratio strong reduction in the leaves, caused permanent damages on the photosynthesis of physic nuts plants due as the stomatal limitations as non-stomatal ones. The third experiment aimed to study the comparative effects between the salt stress (50 mM of NaCl) and water stress (induced for PEG 6000), both with osmotic potential of â0.22 MPa on the photosynthesis, water relations and growth of physic nuts plants. The water stress effects induced for PEG in the leaf growth, electrolyte leakage and leaf gas exchange were more deleterious than by NaCl ones. In the both stresses was observed decrease in the leaf CO2 assimilation due the stomatal and non-stomatal limitations. However, the chlorophyll fluorescence parameters did not affect. The fourth experiment aimed to evaluate the relative contribution of organic and inorganic solutes on the osmotic adjustment of leaves and roots physic nuts plants in different water restriction levels. Of the solutes studied, the K+ and soluble sugar were the most involved in the osmotic adjustment as in the leaves as in roots. Others solutes as, Na+, Cl-, total amino acids and glycibetaine, also presented a effective role in the reduction of osmotic potential in both organs. On the other hand, the leaf proline content, although has increased significantly, was not sufficient to promote an effective participation of this amino acid in the osmotic adjustment of physic nuts plants. The same experiment aimed to observe the isolated and combined effects of water stress and high temperature on the photosynthesis and evaluate the oxidative defenses system in physic nuts plants. The photosynthetic apparatus was more sensitive to water stress than heat ones, been that the combination of them caused deleterious effects yet large in this complex. Additionally, the oxidative damages also were more marked in the combined stress. In general, the data shown that physic nuts plants, although present ability to adjust osmotically to salinity and drought, have their photosynthetic apparatus very affected in this stressful conditions. Even as, the defense system against oxidative damages appears has not been efficient in plants exposure at the drought and heat isolated and combined stresses / In this work were studied diverse physiological mechanisms, as the osmotic adjustment, photosynthesis, including gas exchange and chlorophyll fluorescence parameters, as well the oxidative responses in physic nuts submitted to different abiotic stresses as: salinity, drought and high temperature. The first experiment aimed to study the effects of increase of the NaCl concentrations (0, 25, 50, 75 and 100 mM) in the ions accumulate (Na+, Cl- and K+) and some growth variables, as well evaluate the water state and the principals solutes (organic and inorganic) involved on the osmotic adjustment of physic nuts plants under this stressful condition. Physic nuts plants showed sensibility to salt stress, presenting a reduction by 50% in dry matter from by 47 mM of NaCl concentration during 15-d. This sensibility should be due the leaf Na+ and Cl- high accumulation, associated a strong reduction in the K+concentration, induced by high Na+ content. On the other hand, physic nuts plants were able osmotic adjust to salinity due a severe decrease on the osmotic potential and increase of leaf water state, principally in the higher NaCl levels. Of the solutes studied, was observed that salt ions (Na+ and Cl-) contributed with the most of the osmotic adjustment, while that the K+contribution was decreased strongly by NaCl. The glycinebetaine compared to proline was more important to the osmotic adjustment of physic nuts leaves, as in the absence as in presence of different NaCl levels in the nutrient solution. The second experiment evaluated the resistance of photosynthetic apparatus of physic nuts plants submitted to different time of exposure (7-d and 14-d of treatment and 3-d of recovery) to salt stress (100 mM of NaCl).The changes caused on photochemistry activity and leaf gas exchange were evaluate by Na+ and Cl- accumulation and decrease of K+/Na+ ratio in the leaves. After 7-d of treatment was observed a major action of osmotic effects. However, after 14-d of treatment, the ionic effects caused by Na+ and Cl- excessive accumulation and by K+/Na+ ratio strong reduction in the leaves, caused permanent damages on the photosynthesis of physic nuts plants due as the stomatal limitations as non-stomatal ones. The third experiment aimed to study the comparative effects between the salt stress (50 mM of NaCl) and water stress (induced for xv PEG 6000), both with osmotic potential of â0.22 MPa on the photosynthesis, water relations and growth of physic nuts plants. T e water stress effects induced for PEG in the leaf growth, electrolyte leakage and leaf gas exchange were more deleterious than by NaCl ones. In the both stresses was observed decrease in the leaf CO2 assimilation due the stomatal and nonstomatal limitations. However, the chlorophyll fluorescence parameters did not affect. The fourth experiment aimed to evaluate the relative contribution of organic and inorganic solutes on the osmotic adjustment of leaves and roots physic nuts plants in different water restriction levels. Of the solutes studied, the K+ and soluble sugar were the most involved in the osmotic adjustment as in the leaves as in roots. Others solutes as, Na+, Cl-, total amino acids and glycibetaine, also presented a effective role in the reduction of osmotic potential in both organs. On the other hand, the leaf proline content, although has increased significantly, was not sufficient to promote an effective participation of this amino acid in the osmotic adjustment of physic nuts plants. The same experiment aimed to observe the isolated and combined effects of water stress and high temperature on the photosynthesis and evaluate the oxidative defenses system in physic nuts plants. The photosynthetic apparatus was more sensitive to water stress than heat ones, been that the combination of them caused deleterious effects yet large in this complex. Additionally, the oxidative damages also were more marked in the combined stress. In general, the data shown that physic nuts plants, although present ability to adjust osmotically to salinity and drought, have their photosynthetic apparatus very affected in this stressful conditions. Even as, the defense system against oxidative damages appears has not been efficient in plants exposure at the drought and heat isolated and combined stresses

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-10-04T11:12:58Z No. of bitstreams: 2 Dissertação - Ricardo Alexandre Florentino Barbosa - 2016.pdf: 1068079 bytes, checksum: b33551290eb02664f6c8ccb4ea75fb0b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-10-04T11:14:02Z (GMT) No. of bitstreams: 2 Dissertação - Ricardo Alexandre Florentino Barbosa - 2016.pdf: 1068079 bytes, checksum: b33551290eb02664f6c8ccb4ea75fb0b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-10-04T11:14:02Z (GMT). No. of bitstreams: 2 Dissertação - Ricardo Alexandre Florentino Barbosa - 2016.pdf: 1068079 bytes, checksum: b33551290eb02664f6c8ccb4ea75fb0b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-07-22 / Jatropha is an oilseed with high potential for biodiesel and biofuel production, but there are few studies addressing its water needs and nutritional demands. Therefore, the objective of this work was to evaluate the effect of potassium fertilization and irrigation on the yield and composition of the oil of jatropha seeds. The treatments consisted of four doses of potassium fertilization: 40 kg ha-1 of K2O, 80 kg ha-1 of K2O, 120 kg ha -1 of K2O, 120 kg ha-1 of K2O, 120 kg ha-1 of K2O, 120 Kg ha-1 of K2O and 160 kg ha-1 of K2O, and four irrigation slides: 0% of evaporation of the Class A tank (ECA); 40% of the RCT, 80% of the RCT; And 120% of RCT. The experimental design was in randomized blocks with subdivided plots with 4 replicates. For extraction of the oil of seeds was used the chemical method via Soxhlet and to obtain the composition of oil was used the gas chromatography. The treatments of fertilization and irrigation promoted an increase in the yield and quality of the oil of jatropha seeds. Plants fertilized with 120 kg ha-1 of K2O and irrigated with the irrigation blade of 80% of ECA obtain the best results. / O pinhão manso é uma oleaginosa com alto potencial para produção de biodiesel e biolubrificantes, porém existem poucos trabalhos abordando suas necessidades hídricas e demandas nutricionais. Portanto, o objetivo deste trabalho foi avaliar o efeito da adubação potássica e da irrigação sobre o rendimento e composição do óleo de sementes de pinhão manso. O experimento foi desenvolvido na área experimental da Escola de Agronomia da Universidade Federal de Goiás, Goiânia, Goiás. Os tratamentos consistiram de quatro doses de adubação potássica, sendo: 40 kg ha-1 de K2O, 80 kg ha-1 de K2O, 120 kg ha-1 de K2O e 160 kg ha-1 de K2O, e quatro lâminas de irrigação: 0% da evaporação do tanque Classe A (ECA); 40% da ECA, 80% da ECA; e 120% da ECA. O delineamento experimental foi em blocos casualizados com parcelas subdivididas com 4 repetições. Para extração do óleo de sementes foi utilizado os método químico via Soxhlet e para obtenção da composição de óleo utilizou-se a cromatografia gasosa. Os tratamentos de adubação e irrigação promoveram aumento no rendimento e qualidade do óleo de sementes de pinhão manso. Plantas adubadas com 120 kg ha-1 de K2O e irrigadas com a lâmina de irrigação de 80% da ECA obtém os melhores resultados.

Jatropha curcas L., também conhecida no Brasil como pinhão-manso, é uma oleaginosa que atraiu a atenção do mundo para bioenergia dado a qualidade e o alto conteúdo de óleo na semente. Contudo, a espécie necessita de programas de melhoramento genético para fixar características de interesse em bancos de germoplasma. Este trabalho objetivou identificar e analisar a diversidade alélica, metabólica e físico-química relacionada à síntese de óleo e ésteres de forbol em uma amostra de 22 genótipos do BAG do Instituto Agronômico de Campinas para seleção de genótipos elite. Os genes MFP2, KASIII e 3-N-D, envolvidos na biossíntese de óleo e de taxol, foram selecionados a partir de Gomes et al. (2010). Os resultados mostraram que há baixa diversidade alélica para os genes KAS III e MFP2, por outro lado, a expressão desses mesmos genes e a composição de ácidos graxos foi bastante variável entre os genótipos e ao longo do desenvolvimento da semente de J. curcas indicando uma provável regulação diferenciada da via de óleo. O estudo realizado permite combinar a diversidade alélica, expressão dos genes, conteúdo metabólico e poder calorífico para a seleção de genótipos e identificação de parentais direcionando cruzamentos no quadro do programa de melhoramento do IAC. / Jatropha curcas L., also know in Brazil as physic nut, is an oilseed that call world´s attention to bioenergy due to quality and high oilseed content. However, this species needs of breeding programs so that interest traits may to be fixed in germplasm banks. This study aimed to identify and analyze allelic, metabolic and physicochemical diversity related to oil and phorbol esters synthesis in a 22 genotypes sample from Instituto Agronômico de Campinas-BAG for elite genotypes selection. The MFP2, KASIII and 3-ND genes involved in oil and taxol biosynthesis were selected from Gomes et al. (2010). The results showed that there is low allelic diversity for KAS III and MFP2 genes, on the other hand, the expression of these same genes and fatty acid composition was quite variable between genotypes through J. curcas seed development indicating a putative differential regulation in oil pathway. This study allows combining allelic diversity, gene expression, metabolic content and calorific values for parental genotypes identification and selection driving crosses within IAC breeding program.

O pinhão-manso (Jatropha curcas L) é uma espécie da família da Euforbiácea que tem se destacado por ser uma planta perene, rústica e com elevada produção de óleo. A avaliação dos efeitos da poda e da irrigação no crescimento e produção do pinhão-manso é relevante para o manejo de um cultivo com essa espécie. Este estudo teve como objetivo principal avaliar os efeitos de diferentes tipos de poda e manejos hídricos no cultivo do pinhão-manso em Piracicaba, SP. O experimento foi conduzido na área experimental do Departamento de Engenharia de Biossistemas (ESALQ/USP), com plantas de quatro anos de idade dispostas em espaçamento 3 x 4 m, totalizando 833 plantas ha-1. Os tratamentos foram arranjados de forma aleatória em blocos compostos de quatro repetições, sendo que os fatores foram constituídos de dois manejos hídricos (irrigado e sem irrigação) e três tipos de poda, sendo P1 (sem poda), P2 (1,5 m de altura x 1,5 m de diâmetro de copa) e P3 (poda a 2 m de altura e 1,5 m de diâmetro da copa). A irrigação teve influência no diâmetro da copa, nas taxas de crescimento absoluto em altura e diâmetro, na taxa de crescimento relativo em diâmetro e nas variáveis produtivas. O fator poda apresentou diferenças em todas as variáveis de crescimento, sendo a poda drástica (P2) a que exibiu as maiores taxas médias de crescimento. As plantas irrigadas apresentaram os maiores rendimentos por planta e por hectare. A análise de ressonância magnética nuclear de hidrogênio (RMN de 1H) revelou que a irrigação e a poda não influenciaram o teor de óleo nas sementes de pinhão-manso. Em relação ao perfil graxo, a irrigação teve influência em plantas submetidas a poda drástica (P2), de modo que a ausência de irrigação favoreceu o maior acúmulo de ácido oleico no óleo. Pela análise energética, observou-se que os fertilizantes foram os insumos com maior participação na demanda energética (42,57 GJ ha-1) para os dois sistemas estudados, seguido dos combustíveis (32,96 GJ ha-1). A área irrigada consumiu 100,4 GJ ha-1, sendo 16% devido ao acionamento do pivô central. A energia bruta dos frutos de pinhão-manso foi de 69,82 e 45,31 GJ ha-1 para o sistema irrigado e o sem irrigação, respectivamente. O balanço energético (BE) para as duas áreas foi negativo e a lucratividade energética (EROI) foi de 0,63 para a área irrigada e 0,49 para a área sem irrigação, evidenciando a ineficiência e a não sustentabilidade do cultivo de pinhão-manso nas condições deste estudo para geração de energia. / Jatropha (Jatropha curcas L) is a species from Euforbiácea family that has been widely studied because of some particular characteristics such as being a perennial plant, its rusticity and the high oil production. The evaluation of pruning and irrigation on growth development and yield of jatropha is very important for the culture management. The objective of this study was to evaluate the effects on jatropha under different types of pruning and water management in Piracicaba, SP. The experiment was conducted in the experimental area of the Department of Biosystems Engineering (ESALQ/USP) during the fourth year crop growing and with 3 x 4 m planting spacing, totalizing 833 plants ha-1. The treatments were randomly arranged in blocks with two water management (irrigation and rainfed) and three pruning types (P1, no pruning; P2, with 1.5 m height x 1.5 m canopy diameter and; P3, with 2.0 height x 1.5 m canopy diameter). In each block, there were three pruning types with 4 blocks in each water management. Irrigation have influenced the canopy diameter values, absolute growth rates for height and canopy diameter, relative growth rate for canopy diameter and yield parameters., pruning management have influenced in all growth parameters, with drastic pruning (P2) representing the highest average growth rates. The irrigation have showed the highest yield per plant and per hectare. The analysis of Nuclear Magnetic Resonance of hydrogen (1H NMR) have shown that the irrigation and pruning have not influenced the oil content on jatropha seeds. Regarding the fatty profile, the irrigation management have influenced only the P2, since that no irrigation have influenced to largest accumulation of oleic acid in the oil. In energy analysis, it was noted that fertilizers were the inputs with the largest participation in energy input (42.57 GJ ha-1) for both water management, followed by fuels (32.96 GJ ha-1). The irrigated area consumed 100.4 GJ ha-1 energy, representing 16% due to the central pivot usage. The gross energy of jatropha fruits was 69.82 and 45.31 GJ ha-1 for irrigated and rainfed system, respectively. The energy balance for the two areas was negative and the energy return over investment, with 0.63 and 0.49 for irrigated and rainfed, respectively, have showed the inefficiency and unsustainability of jatropha cultivation to generate energy.

Made available in DSpace on 2017-07-24T19:37:55Z (GMT). No. of bitstreams: 1 Renato Ferraz Penteado.pdf: 5510615 bytes, checksum: a1e326883a6f1e7ad0decd6835add201 (MD5) Previous issue date: 2016-08-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Knowledge of protein structures is of huge importance, since this information allows to understand the mechanisms through which proteins carry out their biological functions. Lipases constitute an enzymatic family capable to perform synthesis or hydrolysis of ester bonds of triacyl glycerols (TAGs) with long chain fatty acids.These enzymes are the theme of many investigations given their potential to be used in a wide variety of apllications involving chemicals with the ester functional group,e.g., in organic synthesis. On the other hand, structural knowledge of some enzymes is important for the development of new therapeutic drugs or even to contribute for the understanding of structural evolutionary features, like those belonging to metabolic pathways. In this work were accomplished the homology modeling of the lipase from Jatropha curcas and the structure determination of the triosephosphate isomerase from Naegleria gruberi from three X ray diffraction data sets. Among three experimental structures obtained, two belong to C2 space group, with different unit cells, and one to P4122 space group. Initial phases were obtained by molecular replacement procedure using the Phaser program and all structures were refined interactively with Coot and Phenix programs. In one structure it was possible to model three molecules of the precipitant agent Jeffamine present in the crystallization solution and one molecule of Tris buffer (placed at the active site). Structural comparisons were performed among the refined and validated model and some of its homologues, taking into account the differences observed in the structural-based alignment among them and characteristics noticed during the refinement procedure. Circular dichroism experiments have shown that thermal denaturation is irreversible to triosephosphate isomerase of Naegleria gruberi. / O conhecimento da estrutura de proteínas é de grande importância, uma vez que esta informação permite o entendimento dos mecanismos pelos quais elas desempenham suas funções biológicas. Lipases constituem uma família enzimática capaz de realizar a síntese ou hidrólise de ligações éster de substratos triacilgliceróis (TAGs) contendo ácidos graxos de cadeia longa. São alvo de muitos estudos dadas suas potencialidades em um grande número de aplicações envolvendo o grupo funcional éster, por exemplo, em química orgânica síntética. Já o conhecimento estrutural de algumas enzimas é importante para o desenvolvimento de novas drogas terapêuticas ou mesmo contribuir para o entendimento de aspectos evolutivos estruturais, como daquelas pertencentes a vias metabólicas. Neste trabalho foram realizadas a modelagem por homologia da estrutura lipase da planta Jatropha curcas e a determinação experimental da estrutura da triose fosfato isomerase do microrganismo Naegleria gruberi a partir de três conjuntos de imagens de difração de Raios X. Das três estruturas experimentais obtidas, duas pertencem ao grupo de espaço C2, com células unitárias diferentes, e uma ao grupo de espaço P4122. As fases iniciais foram obtidas com o procedimento de substituição molecular utilizando o programa PHASER e todas as estruturas foram refinadas iterativamente com o auxílio dos programas COOT e PHENIX. Em uma das estruturas foi possível modelar três moléculas do agente precipitante Jeffamine® presente na condição de cristalização e uma molécula do tampão Tris (no sítio ativo do monômero B). Comparações estruturais foram realizadas entre o modelo refinado e validado e algumas das proteínas homólogas, tendo em vista diferenças observadas no alinhamento baseado em estrutura entre elas e características notadas durante o procedimento de refinamento. Experimentos de dicroísmo circular mostraram que a desnaturação térmica é irreversível para esta proteína.

Made available in DSpace on 2016-04-28T14:58:31Z (GMT). No. of bitstreams: 1 Nathalia da Silva Braga .pdf: 530379 bytes, checksum: b05d80dc10a6d1d8763bdea93cfc086c (MD5) Previous issue date: 2010-02-22 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The National Program for Production and Use of Biodiesel of the federal government has encouraged through public policy the production of raw materials to meet the demand for biodiesel. The Jatropha curcas has shown potential to represent an important supplier of raw materials. However, there is little information about the culture, which highlights the need for caution on the part of producers. This study aims to adjust the methodology for accelerated aging and electrical conductivity to assess the effect of Jatropha seeds and the germination and seed vigor of primed Jatropha curcas under different water potentials of the substrate, and establishment of seedlings in the greenhouse. For definition of methodologies for testing vigour (first experiment), the experimental design was completely randomized design with four replications. Five lots were tested for electrical conductivity at 12, 24, 36 and 48 hours and the accelerated aging test at 41 ?C for 48 and 72 hours, with or without the saturated NaCL. To evaluate the priming technique (second experiment) the experimental design was the triple factorial (5 lots x 2 treatments x 4 conditioning potentials of germinating) with four replicates for germination tests. And, for tests of vigour was used the double factorial (5 x 2 lots of conditioning treatments). The seeds of the five lots were submitted or not to priming at 20 ? C for 144 hours, with subsequent drying at 32 ?C. For each lot, under substrate water potential of zero, -0.2, -0.4, -0.6 MPa, was tested for germination and the percentage of normal seedlings in the first count. Also, other vigor tests were installed (seedling length, seedling emergence in sand). For the evaluation of seedling emergence under greenhouse conditions, the height, stem diameter and leaf number were evaluated at 10, 20, 30 and 40 days after sowing. It can be observed that the seeds with levels near 50% of water present primary root emission after 192 hours of soaking. The Pre-hydration promotes the synchronization of the germination process and mainly favors the seed germination vigor. In greenhouse conditions, pre-hydration of seeds favors the survival of plants and these have increased the number of leaves at 20 and 30 days after the installation. The accelerated aging and electrical conductivity undertaken jointly in the routine, allow the classification of seed lots of Jatropha. / O Programa Nacional de Produ??o e Uso do Biodiesel, do governo federal, vem estimulando por meio de pol?ticas p?blicas a produ??o de mat?ria prima para suprir a demanda por biodiesel. O pinh?o manso (Jatropha curcas L.) tem apresentado potencial podendo representar importante fornecedora de mat?ria prima. Contudo, ainda existem menos informa??es a respeito da cultura, o que evidencia a necessidade de cautela por parte dos produtores Este trabalho tem por objetivo ajustar metodologias para os testes de envelhecimento acelerado e de condutividade el?trica para avalia??o do vigor de sementes de pinh?o manso, bem como avaliar a germina??o e o vigor de sementes condicionadas de pinh?o manso sob diferentes potenciais h?dricos do substrato, e o estabelecimento destas mudas em casa de vegeta??o. Para defini??o de metodologias para testes de vigor (primeiro experimento), o delineamento experimental adotado foi inteiramente casualizado, com quatro repeti??es. Cinco lotes foram submetidos ao teste de condutividade el?trica durante 12, 24, 36 e 48 horas e ao teste de envelhecimento acelerado a 41?C durante 48 e 72 horas, usando ou n?o a solu??o saturada de NaCl. Para avalia??o da t?cnica de condicionamento (segundo experimento), o delineamento experimental utilizado foi o fatorial triplo (5 lotes x 2 tratamentos de condicionamento x 4 potenciais h?dricos de substrato de germina??o), com quatro repeti??es para os testes de germina??o. E, para os testes de vigor foi utilizado o fatorial duplo (5 lotes x 2 tratamentos de condicionamento). As sementes dos cinco lotes foram submetidas ou n?o ao condicionamento a 20?C durante 144 horas, com posterior secagem a 32 ?C. Por lote, sob potenciais h?dricos do substrato de zero, -0,2; -0,4; -0,6 MPa, foi avaliada a germina??o e a porcentagem de pl?ntulas normais na primeira contagem. Tamb?m, foram instalados outros testes de vigor (comprimento de pl?ntulas, emerg?ncia de pl?ntula em areia, ?ndice de velocidade de emerg?ncia de pl?ntulas e avalia??o de mudas). Para a avalia??o da emerg?ncia de pl?ntulas em casa de vegeta??o, foi avaliada a altura, o di?metro de caule e o n?mero de folhas, aos 10, 20, 30 e 40 dias ap?s a semeadura. Pode-se observar que as sementes com teores pr?ximos a 50% de ?gua apresentam emiss?o de raiz primaria ap?s 192 horas de embebi??o. A pr?-hidrata??o promove a sincroniza??o do processo de germina??o e favorece principalmente o vigor das sementes. Em condi??es de casa de vegeta??o, a pr?-hidrata??o das sementes favorece a sobreviv?ncia das plantas e estas apresentam aumento do n?mero de folhas aos 20 e 30 dias ap?s a instala??o. Os testes de envelhecimento acelerado e de condutividade el?trica, realizados em conjunto na rotina, possibilitam a classifica??o de lotes de sementes de pinh?o manso.