Los semioquímicos son sustancias implicadas en la comunicación entre seres vivos. En el caso de los insectos, pueden ser herramientas de gran utilidad en la lucha contra plagas, como alternativas a la aplicación de insecticidas convencionales. Son sustancias de gran selectividad y muy activas a baja dosis. Los semioquímicos (feromonas y aleloquímicos) se emplean en el control de plagas tanto para la detección y seguimiento de poblaciones como, de forma directa, en las técnicas de confusión sexual, trampeo masivo y atracción y muerte, entre otras. En la presente tesis se describe la búsqueda y aplicación de semioquímicos para el control de diversas plagas de importancia agronómica, estudiando tres aspectos fundamentales. En primer lugar, el desarrollo y aplicación de la técnica de confusión sexual contra Aonidiella aurantii y Tuta absoluta. En el caso de A. aurantii, se trata de la primera aplicación eficaz de esta técnica contra una plaga de diaspinos y en el caso de T. absoluta, se establecen las condiciones para la aplicación de tratamientos eficaces de confusión sexual. En segundo lugar, se ha estudiado la existencia de valores óptimos de emisión para sistemas basados en la atracción de insectos, aplicado a las plagas Chilo suppressalis, Lobesia botrana, Bactrocera oleae y Ceratitis capitata. En el caso de los lepidópteros C. suppressalis y L. botrana y del tefrítido B. oleae se demostró que la atracción de machos se ve afectada por el nivel de emisión de feromona, de forma que velocidades de emisión por encima y por debajo del nivel óptimo obtenido tienen menor poder atrayente. Sin embargo, respecto a C. capitata, y su paraferomona trimedlure, su respuesta es de tipo asintótico, no se encontró un nivel óptimo y por tanto, mayores dosis de paraferomona atraían al mismo número de machos. La obtención de emisores con velocidades de emisión óptimas es un aspecto poco estudiado en la literatura científica que, sin embargo, es esencial para el desarrollo de los métodos de control basados en la atracción. Como tercera parte de esta tesis, se estudiaron detalladamente los compuestos emitidos por machos y hembras de C. capitata en diversos estadios, para intentar establecer compuestos y mezclas con posible poder atrayente. Para este trabajo se ha utilizado la técnica de microextracción en fase sólida, no empleada hasta el momento para este problema, y la metodología estadística del análisis de componentes principales. La disponibilidad de atrayentes eficaces para hembras de C. capitata supondría una gran ventaja para el control de esta plaga. / Vacas González, S. (2011). Uso de semioquímicos en el control de plagas. Estudios básicos y de aplicación [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/12499

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The main objective of this work was to evaluate the antioxidant and antibacterial activities of extracts of Brassica oleracea var. capitata, obtained by supercritical CO2 and ultrasound-assisted extractions, as well as to carry out the characterization of these extracts using gas chromatography coupled with mass detector. For supercritical CO2, five extractions were performed to investigate the influence of pressure (10-25 MPa) and temperature (20-60°C) on the extraction yield, chemical composition and antioxidant activity towards peroxyl, superoxide and hydroxyl radicals. The highest extraction yield was 0.47% at 60 °C and 25 MPa. In the characterization of the extracts was possible to identify compounds like sulforaphane and iberin nitrile. All extracts showed antioxidant activity for the three radicals, although the highest activity for all radicals was obtained using the extract obtained at 60 °C and 25 MPa (run 2). For the ultrasound-assisted extraction were evaluated the effects of solvent concentration and temperature. The extracts obtained in the optimized extraction condition, were subjected to different hydrolysis conditions before use in biological assays. It was evaluated the antioxidant activity against DPPH, superoxide and peroxyl radicals, besides the antibacterial activity against S. aureus and E. coli. Both crude and hydrolyzed extracts were characterized by gas chromatography coupled with mass detector. The best condition for extraction was 30 ° C and 60% (w /v) of ethanol. All extracts showed antioxidant activity towards DPPH, superoxide and peroxyl radicals, but the use of hydrolyzed extracts improved considerably the antioxidant activities. Antibacterial activity was detected only in extracts hydrolysates Brassica oleracea var. capitata. The main contributions of this work were that the use of supercritical CO2 extraction to obtain bioactive compounds from Brassica oleracea var. capitata showed a promising alternative to conventional methods of extraction, since it allowed the extraction of compounds of interest in science and industry. Besides, in this work was demonstrated that the hydrolysis of extracts can increase the antioxidant activity of plant extracts. / O objetivo principal deste trabalho foi avaliar a atividade antioxidante e antibacteriana de extratos de Brassica oleracea var. capitata, obtidos a partir de extração supercrítica e extração por ultrassom, além de caracterizá-los por cromatografia gasosa acoplada à detector de massas. Para os compostos bioativos apolares de Brassica oleracea var. capitata, utilizou-se extração com CO2 supercrítico e avaliou-se o potencial antioxidante dos extratos. Foram realizadas cinco extrações para investigar a influência da pressão (10 - 25 MPa) e temperatura (20 - 60°C) no rendimento da extração, na composição química e na atividade antioxidante frente os radicais peroxila, superóxido e hidroxila. Obteve-se o maior rendimento de extração 0,47% a 60 °C e 25 MPa. Na caracterização dos extratos foi possível a identificação de compostos como sulforafano e iberin nitrila. Todos os extratos apresentaram atividade antioxidante para os três radicais, porém a maior atividade para todos os radicais foi o extrato obtido a 60°C e 25 MPa. Para os compostos bioativos polares, otimizou-se a extração assistida por ultrassom. Os extratos obtidos nas melhores condições de extração foram submetidos a diferentes condições de hidrólise, antes da sua utilização nos ensaios biológicos. Avaliou-se a atividade antioxidante, frente ao radical DPPH, superóxido e peroxila, além da atividade antibacteriana, frente a S.aureus e E.coli. Tanto os extratos brutos quanto os hidrolisados, foram caracterizados por cromatografia gasosa acoplada à detector de massas. A melhor condição de extração foi a 30ºC e 60% (m/v) de etanol. Todos os extratos apresentaram atividade antioxidante frente aos radicais DPPH, superóxido e peroxila, mas o uso de extratos hidrolisados melhorou consideravelmente a atividade antioxidante. Em relação à atividade antibacteriana, apenas uma amostra, que foi submetida à condição de hidrólise alcalina apresentou ação frente a E.coli. Uma das principais contribuições deste trabalho foi que a utilização de extração com CO2 supercrítico, para obtenção de compostos bioativos de Brassica oleracea var. capitata, mostrou ser uma alternativa promissora em relação aos métodos convencionais de extração, pois permitiu a extração de compostos com interesse científico e industrial. Além disso, foi demonstrado que a hidrólise dos extratos pode aumentar consideravelmente a atividade antioxidante de extratos vegetais em relação aos extratos brutos.

Made available in DSpace on 2016-12-08T16:44:44Z (GMT). No. of bitstreams: 1 PGPV13MA109.pdf: 557511 bytes, checksum: d2cb3e9e50b7e2f27e43a1b3f1a189c1 (MD5) Previous issue date: 2013-02-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The family of Brassicaceae plays an important role in Brazilian horticulture, having the species of cabbage, broccoli, and cauliflower the higher at consumption by the population. The aim of this study was to evaluate the use of high dilutions preparation for production under organic system of cabbage, broccoli, and cauliflower. Experiments were conducted in the greenhouse and under fields conditions. For greenhouse, the experimental design was completely randomized. For field experiments the experiment design were randomized complete block design. Both designs were with four replications and nine treatments. For greenhouse, trays had of 64 plants from with 20 plant were taken as experiment plot. Each plot consisted of 20 plants for each specie and repetition. Ten plants distributed in double line was the plot in the field experiment. The treatments consisted of homeopathic preparations of Arnica montana, Silicea terra, Carbo vegetabilis, and Sulphur at 6CH and 30CH. For control water was used. The experiments were conducted separately for each species. In greenhouse, the treatments were applied every four days until transplanting (seven applications). Under field conditions, the treatments were applied every fifteen days until harvest (four applications). Shoot fresh mass, root fresh mass, shoot dry mass, root dry mass, plant height, root length, and stem diameter for seedlings were evaluated. For field, shape index, rate of the length of the heart and longitudinal diameter (C / D) for cabbage field crop were evaluated. Plant height and stem diameter for broccoli were evaluated. Fresh mass and dry mass of cabbage heads and broccoli inflorescences were also evaluated. The disease incidence of black-rot and alternaria and the occurrence of cabbage aphid and damage caused by diamondback moth were evaluated. The preparations of Sulphur at 6CH increased the shoot dry mass, root dry mass for seedlings and production of cabbage heads. The preparations of Sulphur at 6CH increased the plant height, root length, and stem diameter for broccoli. Silicea terra at 30CH increased the fresh mass and dry mass of broccoli inflorescences. Sulphur at 6CH reduced the aphid occurrence for cabbage and diamondback moth damage percentage for broccoli. Sulphur at 30CH increased the shoot dry mass, root dry mass, and height for cabbage seedlings. Sulphur at 30CH increased the shoot dry mass, root dry mass, and stem diameter. Aphid occurrence, alternaria; and black rot incidence were decreased when broccoli was treated by Sulphur at 30CH. Silicea terra at 30CH increased the shoot dry mass, root dry mass, height plant, stem diameter, and root length for broccoli seedlings. Silicea terra at 30CH also increased the shoot dry mass, root dry mass, and stem diameter of cauliflower seedlings. The preparations of Silicea terra at 30CH increased the production of cabbage heads and reduced the black rot incidence for broccoli. The high dilution preparations have influence on the yield and management of pests and diseases of cabbage, broccoli, and cauliflower growth under organic production system / A família Brassicaceae ocupa papel destacado na olericultura brasileira, sendo as espécies de repolho, brócolis e couve-flor de maior consumo pela população. O objetivo deste trabalho foi avaliar o uso de preparados em altas diluições na produção em sistemas orgânicos de repolho, brócolis e couve-flor. Os experimentos com repolho, brócolis e couve-flor foram realizados em casa de vegetação e a campo na Epagri e na comunidade de Pedras Brancas, Lages, SC. O delineamento foi inteiramente casualizado em casa de vegetação e de blocos ao acaso, a campo. Ambos os delineamentos foram com quatro repetições e nove tratamentos. Em casa de vegetação, as bandejas foram constituídas por 64 plantas por parcela para cada hortaliça. A parcela útil foi composta por 20 plantas centrais das 64 plantas da parcela. A campo, cada parcela de repolho e brócolis foi constituída por 10 plantas distribuídas em dupla linha. Os tratamentos utilizados, tanto para casa de vegetação como para campo, foram os preparados homeopáticos de Arnica montana, Silicea terra, Carbo vegetabilis e Sulphur na 6CH e 30CH; e água como testemunha. Os experimentos foram delineados em separado para cada uma das hortaliças e conduzidos em duplo cego. Em casa de vegetação, os tratamentos foram aplicados a cada quatro dias, sobre as mudas até seu transplante, totalizando sete aplicações. No campo, após o transplante das mudas, os tratamentos foram aplicados a cada quinze dias, sobre as plantas até sua colheita, totalizando quatro aplicações. Foram avaliadas, em mudas, características de massa fresca e seca da parte aérea e radicular, altura da planta, comprimento de raiz e diâmetro do caule. A campo, no momento da colheita, foram avaliadas as características de índice de formato, a relação do comprimento do coração e do diâmetro longitudinal (C/D) para repolho; para o brócolis, altura da planta e diâmetro do caule. Massa fresca e seca das cabeças em repolho e das inflorescências de brócolis também foram avaliadas. A incidência de alternariose e podridão-negra, bem como, a ocorrência do pulgão e dos danos da traça-das-crucíferas foram avaliadas em repolho e brócolis. O preparado de Sulphur na 6CH aumentou a massa seca da parte aérea e radicular em mudas e a produção de cabeças de repolho, bem como o incremento da massa fresca e seca das inflorescências em brócolis. O preparado de Sulphur na 6CH reduziu também a ocorrência do pulgão em plantas de repolho e brócolis e o percentual de danos da traça-das-crucíferas em brócolis.O preparado de Sulphur na 30CH aumentou a massa seca da parte aérea e radicular e altura da plântula em mudas de repolho, bem como o incremento da massa seca da parte aérea e radicular e diâmetro do caule em mudas de couve-flor. A ocorrência do pulgão, a incidência da alternariose e a podridão-negra foram reduzidas quando tratadas como o preparado de Sulphur na 30CH em plantas de brócolis. O preparado de Silicea terra na 30CH incrementou a massa seca da parte aérea e radicular, altura da plântula, comprimento de raiz e diâmetro do caule, em mudas de brócolis, bem como a massa seca da parte aérea e radicular e diâmetro do caule em mudas de couve-flor. O preparado de Silicea terra na 30CH aumentou a produção de cabeças de repolho e reduziu a incidência da podridão-negra em plantas de brócolis. Conclui- se que preparados em altas diluições influenciam na produção e no manejo de insetos-praga e doenças de plantas de repolho, brócolis e couve-flor, sob sistema orgânico

O presente trabalho investiga a aplicação do processo de extração com fluido supercrítico (EFS) para a obtenção de compostos fenólicos a partir de Butia capitata, coletado na Região Sul do Brasil. A planta, chamada popularmente de butiá, é uma espécie nativa da Argentina, Uruguai, Paraguai e Brasil. Os frutos do butiá são comestíveis e bastante apreciados pelas populações locais, que os consomem ao natural ou na forma de licores, geléias ou doces. Estudos recentes revelam que compostos fenólicos estão presentes nos frutos do butiá. Estes compostos merecem atenção por apresentarem atividade antioxidante, por prevenirem doenças cardiovasculares e reduzirem o risco de câncer. Atualmente existem diferentes formas para a obtenção de compostos fenólicos presentes em matrizes vegetais, porém muitas delas utilizam solventes orgânicos a altas temperaturas, o que deprecia o valor comercial dos extratos. Neste estudo, é utilizada uma tecnologia limpa, que tem como solvente o dióxido de carbono (CO2) em condições supercríticas. O CO2 é não-tóxico e não-inflamável, o que faz dele uma opção mais segura, quando comparado a outros solventes. Além disso, no estado supercrítico, o CO2 apresenta condições bastante favoráveis para a extração, como altas difusividades e massas específicas elevadas. Uma característica que deve ser ressaltada é a possibilidade de utilizar baixas temperaturas na extração, comparativamente aos processos que utilizam solventes líquidos, uma vez que os compostos fenólicos são termolábeis. Nos experimentos, foi utilizada apenas a polpa desidratada. O planejamento experimental foi elaborado avaliando a influência de três variáveis de processo em três níveis: pressão (150, 200 e 250 bar), temperatura (40, 50 e 60°C) e teor de cossolvente (1, 3 e 5% de etanol). Os extratos obtidos foram analisados em cromatografia líquida de alta eficiência (CLAE), sendo os rendimentos expressos em mg do composto identificado por CLAE /100g de polpa de butiá desidratada (PBD), e pelo método Folin-Ciocalteau com os rendimentos em compostos fenólicos totais (CFT) expressos em mg de rutina equivalente (RE)/100g PBD. Foram identificados cinco compostos majoritários nos extratos nas análises em CLAE. As maiores concentrações foram encontradas nas condições de 250 bar, 60°C e 5% de etanol. Os compostos identificados foram o ácido clorogênico (0,46mg/100g PBD), o ácido ferúlico (1,45mg/100g PBD), a rutina (3,47mg/100g PBD), a quercetina (0,90mg/100g PBD) e o canferol (1,93mg/100g PBD). Em termos de CFT, a melhor condição de extração foi de 200 bar, 50°C e 5% de etanol, resultando em 23,09mg RE/100g PBD. Os extratos também foram avaliados quanto ao poder de redução do radical DPPH•, pelo método IC50. O método fornece a quantidade necessária de extrato pra varrer 50% do radical. Como esperado, o extrato obtido na corrida experimental de maior rendimento em CFT apresentou maior atividade antioxidante (17,4 mg/mL), seguido da corrida experimental de maior rendimento em compostos fenólicos isolados (56,1 mg/mL) e, com menos atividade, o extrato obtido por solvente orgânico (80,0 mg/mL). / In this work it is investigated the application of supercritical fluid extraction to obtain phenolic compounds from Butia capitata, collected in southern Brazil. This plant, known popularly as butiá, is a native species in Argentina, Uruguay, Paraguay and Brazil. The edible fruits are popular and eaten raw or as liqueurs, jellies or jams. In recent studies, it is shown that phenolic compounds are present in the fruits of butiá. These compounds deserve attention because they have antioxidant activity, preventing cardiovascular disease and reducing the risk of cancer. There are different processes to obtain phenolic compounds from plant matrices, but many of them make use of organic solvents at high temperatures, which depreciates the extract value. In this study, it is used a clean technology, with carbon dioxide (CO2) as solvent in supercritical conditions. The CO2 is nontoxic and nonflammable that makes it a safer option than other solvents. Furthermore, in supercritical conditions the CO2 has favorable conditions for extraction, as high diffusivities and densities. A feature that should be emphasized is the possibility of using low temperatures in the extract, when compared to processes using liquid solvents, since the phenolic compounds are decomposed by high temperature. In the experiments only the dried pulp was used. The experimental design was developed to assess the influence of three process variables at three levels: pressure (150, 200 e 250 bar), temperature (40, 50 e 60°C) and cosolvent content (1, 3 e 5% of ethanol). The extracts were analyzed by high performance liquid chromatography (HPLC), in which the yields were expressed in terms of mg of the identified phenolic compounds by HPLC/100g of dried pulp and by the Folin-Ciocalteau method, with yields, as total phenolic compounds, expressed in mg of rutin equivalent/100g of dried pulp. It were identified five majority phenolic compounds by HPLC. Its highest concentrations were found in the conditions of 250 bar, 60°C and 5% of cosolvent. The identified compounds were chlorogenic acid (0,46mg/100g of dried pulp), ferulic acid (1,45mg/100g of dried pulp), rutin (3,47mg/100g of dried pulp), quercetin (0,90mg/100g of dried pulp) and kaempferol (1,93mg/100g of dried pulp). In terms of total phenolic compounds, the best conditions were 200 bar, 50°C and 5% of cosolvent, which resulted in 23,09mg of rutin equivalent/100g of dried pulp. Also, the antioxidant activity of the extracts was measured by scavenging of the DPPH• radical, by the IC50 method. The method provides the necessary amount of extract to scavenging 50% of the radial. As expected, the extract obtained in experimental run with the highest yield in CFT showed a higher antioxidant activity (17.4 mg / mL), followed by the experimental run with the highest yield in phenolic compounds isolated (56.1 mg / mL), and, with less activity, the extract obtained by organic solvent (80.0 mg / mL).

Σα αρνητικά και θετικά κατά Gram βακτήρια E. coli και S. αureus αντίστοιχα αναγνωρίζονται και δεσμεύονται στην επιφάνεια των αιμοκυττάρων της C. capitata. Η πρόσδεση των βακτηρίων ενεργοποιεί τόσο τις β1 ιντεγκρίνες, όσο και σηματοδοτικά μονοπάτια που περιλαμβάνουν τα μόρια μεταγωγής σήματος Ras, Raf, MEK, ERK, FAK, Src, και GRB2. Οι παραπάνω ενεργοποιήσεις, σε συνδυασμό με τη συμμετοχή του κυτταροσκελετού της ακτίνης και της τουμπουλίνης, καταλήγουν στην επαγωγή της έκκρισης μορίων απαραίτητων για την κυτταροφαγία των βακτηρίων που είναι και το τελικό αποτέλεσμα των παραπάνω διαδικασιών. Σα συνθετικά πολυμερή σφαιρίδια, αλλά και πιθανόν και άλλοι αβιοτικοί παράγοντες, παρότι δεν έχουν καμία προηγούμενη εξελικτική σχέση με τα αιμοκύτταρα, ως σύγχρονο προϊόν της ανθρώπινης γνώσης, αναγνωρίζονται και δεσμεύονται στην επιφάνεια των αιμοκυττάρων από άγνωστους μέχρις στιγμής υποδοχείς. Η κυτταροφαγία τους προωθείται μέσω ενεργοποίησης σηματοδοτικών μονοπατιών που περιλαμβάνουν την ενεργοποίηση των μορίων FAK, Src και MAP κινασών καθώς και με τη συμμετοχή του κυτταροσκελετού της ακτίνης και της τουμπουλίνης. Ο LPS αναγνωρίζεται και δεσμεύεται στην επιφάνεια των αιμοκυττάρων, ενεργοποιεί άγνωστους μέχρις στιγμής υποδοχείς και διαμέσου σηματοδοτικών μονοπατιών που περιλαμβάνουν τις Ras, ενεργοποιεί τις MAP κινάσες και το σύστημα της έκκρισης. Αν και ενεργοποιεί και τις τρεις MAP κινάσες, μόνο η ERK και η p38 απαιτούνται τόσο στη διαδικασία της έκκρισης, όσο και στη διαδικασία της ενδοκυττάρωσής του. Η FAK, αν και ενεργοποιείται από τον LPS, δεν εμπλέκεται στην διαδικασία της ενδοκυττάρωσής του. Σα παραπάνω δείχνουν ότι τα αιμοκύτταρα έχουν αναπτύξει διακριτούς μηχανισμούς για την κυτταροφαγία των παθογόνων, των μικρομορίων και των αβιοτικών παραγόντων, γεγονός που δείχνει την ικανότητα εξέλιξης των εντόμων έτσι ώστε να καλύπτουν τις ανάγκες της επιβίωσή τους. / Gram negative and Gram positive bacteria, E. coli and S. αureus respectively, are recognized and they are bound on the C. capitata hemocyte surface. After binding, they activate β1 integrins and intracellular signalling pathways, involving the kinases Ras, Raf, MEK, ERK, FAK, Src, and GRB2. This signal transduction, with the participation of the cytoskeleton of actin and tuboulin filaments, leads to a regulated secretion, that is a prerequisite for phagocytosis. Latex beads and probably other abiotic factors, despite having no previous evolutionary relation to the hemocytes, being a new product of human knowledge, are recognized and they are bound on the hemocyte surface, by hitherto unknown receptors. They activate intracellular signalling pathways that involves FAK, Src and MAP kinases and they promote, with the participation of actin and tuboulin cytoskeleton, their phagocytosis. LPS is recognized and bound on the hemocyte surface and activates so far unknown receptors and through unknown intracellular signalling pathways involving Ras, activates the MAP kinases and the regulated secretion. Although it activates all three MAPKs, only the ΕRΚ and p38 are required not only for the secretion, but also for its internalization. Although FAK is activated by LPS, it does not get involved in the process of its internalization. All of the above mentioned results indicate that the hemocytes have developed distinct mechanisms for phagocytosis of pathogens, micromolecules and abiotic factors, a fact that underlines insects evolutionary adaptations, so that they can survive.

Με στόχο την απομόνωση του γονιδίου hsp83 της μεσογειακής μύγας, πραγματοποιήθηκε διαλογή μιας cDNA βιβλιοθήκης από προνύμφες 3ου σταδίου, με ανιχνευτή το δεύτερο εξώνιο του hsp83 ομόλογου γονιδίου της Drosophila auraria. Από τη διαλογή αυτή προέκυψαν αρκετοί αλληλεπικαλυπτόμενοι κλώνοι, ο μεγαλύτερος των οποίων (CM-1) είχε μέγεθος 2.593 kb και περιελάμβανε ένα ανοιχτό αναγνωστικό πλαίσιο 715 αμινοξέων, από τη μετάφραση του οποίου προκύπτει ένα πολυπεπτίδιο προβλεπόμενου μοριακού βάρους 81,74 kDa. Η προβλεπόμενη αμινοξική αλληλουχία έδειξε πολύ μεγάλη ταυτότητα με όλα τα μέλη της οικογένειας HSP90 και ειδικότερα με τις HSP83 ομόλογες πρωτεΐνες της Drosophila. Επιπλέον, η προβλεπόμενη αμινοξική αλληλουχία περιείχε όλες τις συντηρημένες περιοχές των μελών της οικογένειας των HSP90 και στο καρβοξυτελικό της άκρο, έφερε το πενταπεπτίδιο MEEVD, το οποίο είναι χαρακτηριστικό όλων των κυτταροπλασματικών ισομορφών αυτής της οικογένειας. Με βάση αυτά τα αποτελέσματα ο κλώνος CM-1, ονομάστηκε Cchsp83. Ο κλώνος αυτός, εκτός από ολόκληρη την κωδική περιοχή του γονιδίου, περιείχε μέρος της 5’ μη μεταφραζόμενης περιοχής και ολόκληρη την 3’ μη μεταφραζόμενη περιοχή του hsp83 γονιδίου της μεσογειακής μύγας. Ανάλυση κατά Southern σε γονιδιωματικό DNA με διάφορα ένζυμα περιορισμού και κατάλληλους cDNA ανιχνευτές, έδειξε ότι το Cchsp83 γονίδιο υπάρχει σε ένα μόνο αντίγραφο στο γονιδίωμα της μεσογειακής μύγας. Ανάλυση “Northern” έδειξε την ύπαρξη ενός μεταγράφου με μέγεθος 2,7 kb περίπου. Το Cchsp83 γονίδιο, χαρτογραφήθηκε με in situ υβριδοποίηση σε μία θερμοεπαγόμενη χρωμοσωματική διόγκωση των πολυταινικών χρωμοσωμάτων των σιελογόνων αδένων του εντόμου (6R:94C). Η μελέτη του προτύπου έκφρασης του γονιδίου Cchsp83 έγινε σε επίπεδο RNA με RT-PCR σε ολικό RNA και σε επίπεδο πρωτεΐνης με ανάλυση Western σε ολικά πρωτεϊνικά εκχυλίσματα. Αντισώματα για την CcHSP83 αναπτύχθηκαν μετά από έκφραση ενός σημασμένου με 6 His τμήματος του Cchsp83 cDNA (490-690aa) σε βακτήρια E. coli. Η αντίδραση του Cchsp83 γονιδίου στην αύξηση της θερμοκρασίας είναι πολύ γρήγορη και ευαίσθητη. Μετάγραφα του γονιδίου ανιχνεύονται μετά από 5 λεπτά θερμικού στρες και σε θερμοκρασίες αρκετά χαμηλές (300C). Η επαγωγή του γονιδίου γίνεται μέγιστη μετά από 30-60 λεπτά θερμικού στρες στους 37-390C. Η επαναφορά της έκφρασης του γονιδίου στα φυσιολογικά επίπεδα μετά από θερμικό στρες είναι αργή, αφού για να γίνει αυτό απαιτούνται 4 ώρες ανάκαμψης στους 250C, μετά από μόλις 30 λεπτά θερμικού στρες στους 380C. Αναπτυξιακή μελέτη του προτύπου έκφρασης του Cchsp83 έδειξε ότι το γονίδιο εκφράζεται σε όλη τη διάρκεια της ανάπτυξης της μεσογειακής μύγας. Στους 250C τόσο τα επίπεδα του RNA, όσο και τα επίπεδα της πρωτεΐνης, είναι υψηλά στα εμβρυικά στάδια, χαμηλά στα προνυμφικά και μέτρια στα νυμφικά και ενήλικα στάδια. Μετά από θερμικό στρες στους 380C, τα επίπεδα των μεταγράφων αυξάνονται αρκετά, ιδιαίτερα στα στάδια που σε φυσιολογικές συνθήκες είναι χαμηλά. Όσον αφορά στα επίπεδα της πρωτεΐνης, τα αποτελέσματα ήταν παρόμοια μετά από θερμικό στρες στους 350C, αλλά όχι στους 37-390C, όπου δεν παρατηρήθηκε καθόλου επαγωγή. Το γεγονός αυτό υποδηλώνει ότι σε υψηλές θερμοκρασίες καταστέλλεται η ωρίμανση ή και η μετάφραση του Cchsp83 RNA. Με στόχο την απομόνωση των ρυθμιστικών περιοχών του γονιδίου Cchsp83 της μεσογειακής μύγας, πραγματοποιήθηκε διαλογή μιας χρωμοσωματικής λEMBL-4A βιβλιοθήκης, με ανιχνευτή το δεύτερο εξώνιο του hsp83 ομόλογου γονιδίου της Drosophila auraria. Από τη διαλογή αυτή απομονώθηκαν δύο κλώνοι, ένας από τους οποίους περιελάμβανε μέρος της κωδικής περιοχής, την 5’ μη μεταφραζόμενη περιοχή και 3,5 kb της 5’ ανοδικής περιοχής του γονιδίου Cchsp83. Σύγκριση της γονιδιωματικής αλληλουχίας με τη cDNA αλληλουχία, αποκάλυψε την ύπαρξη ενός μικρού εσωνίου 275 bp ανάμεσα στην 5’ μη μεταφραζόμενη περιοχή και στην αρχή της κωδικής περιοχής του γονιδίου. Βιοπληροφορική ανάλυση και πειράματα 5’ RACE, υποδηλώνουν ότι το σημείο έναρξης της μεταγραφής του γονιδίου βρίσκεται 144 bp ανοδικά του 5’ άκρου του εσωνίου και 23 bp καθοδικά ενός τυπικού στοιχείου TATA (ΤΑΤΑΑΑΤΑ). Δύο πιθανά στοιχεία απόκρισης στη θερμοκρασία (HSEs) εντοπίστηκαν στην εγγύς 5’ περιοχή του γονιδίου, 35 και 330 bp ανοδικά του στοιχείου TATA. Επιπλέον, βρέθηκαν 4 ακόμα πιο απομακρυσμένα HSEs, 1.595, 2.861, 2.880 και 2.890 bp, ανοδικά του σημείου έναρξης της μεταγραφής, καθώς και ένα HSE μέσα στο εσώνιο. Λειτουργική ανάλυση της εγγύς 5’ ανοδικής περιοχής του γονιδίου Cchsp83 πραγματοποιήθηκε με τη μέθοδο του γενετικού μετασχηματισμού. Τρία αλληλεπικαλυπτόμενα τμήματα του υποκινητή του γονιδίου Cchsp83, μήκους 519 bp (-380/+139, PL), 230 bp (-86/+144, PM) και 193 bp (-55/+139, PS) τοποθετήθηκαν μπροστά από το γονίδιο αναφοράς lacZ και εισήχθησαν στο γονιδίωμα της μεσογειακής μύγας με το σύστημα μετασχηματισμού Minos. Η έκφραση του γονιδίου αναφοράς, ελέγχθηκε σε όλες τις διαγονιδιακές σειρές που προέκυψαν με την ποσοτική ενζυματική μέθοδο της β-γαλακτοζιδάσης. Οι PM-lacZ και PS-lacZ σειρές δεν έδειξαν ανιχνεύσιμα επίπεδα έκφρασης του lacZ γονιδίου τόσο σε φυσιολογικές συνθήκες, όσο και σε συνθήκες θερμικού στρες. Αντιθέτως, οι περισσότερες από τις PL-lacZ σειρές έδειξαν σημαντικά επίπεδα συστατικής έκφρασης. Το αναπτυξιακό πρότυπο έκφρασης του γονιδίου αναφοράς μελετήθηκε σε μία PL-lacZ σειρά και βρέθηκε παρόμοιο με εκείνο του ενδογενούς γονιδίου, υποδηλώνοντας ότι η -380/+139 περιοχή του υποκινητή περιλαμβάνει όλα τα ρυθμιστικά στοιχεία που απαιτούνται για την ορθή χρονική έκφραση του Cchsp83 γονιδίου σε φυσιολογικές συνθήκες. Αν και η περιοχή αυτή του υποκινητή περιλαμβάνει δύο δυνητικά στοιχεία απόκρισης στη θερμοκρασία καθώς και την 5’ UTR, εντούτοις δεν είχε την ικανότητα να οδηγήσει σε θερμοεπαγόμενη έκφραση το γονίδιο αναφοράς. Τα αποτελέσματά μας υποδεικνύουν ότι η περιοχή -380/+139 του Cchsp83 γονιδίου μπορεί να χρησιμοποιηθεί για την ανάπτυξη διαγονιδιακών συστημάτων σήμανσης της μεσογειακής μύγας, τα οποία αναμένεται να συμβάλλουν στη διατήρηση και ανίχνευση κατάλληλων στελεχών που χρησιμοποιούνται σε προγράμματα βιολογικού ελέγχου του επιβλαβούς αυτού εντόμου. / By using the second exon of the D. auraria hsp83 gene as a probe, a number of overlapping cDNA clones were isolated from a Ceratitis capitata (medfly) cDNA library. The longest cDNA had a size of 2,593 bp and contained an open reading frame coding for a putative polypeptide of 715 amino acids with a predicted molecular weight of 81.74 kDa. In addition, it contained a part of the 5’-untranslated region and the complete 3’-untranslated region of a medfly hsp83 homolog, named Cchsp83. The predicted amino acid sequence showed a high degree of identity to all known sequences of the HSP90 family and was more closely related to the Drosophila HSP83 homologs. The putative medfly HSP83 contained all the conserved sequences of the members of the HSP90 family and was ended, at the C-terminal, with the pentapeptide MEEVD that characterizes all the cytosolic members of this family. Genomic Southern blot analysis, with several restriction enzymes and appropriate cDNA probes, indicated that the Cchsp83 gene exists as a single copy in the medfly genome. Northern blot hybridization revealed a single transcript of approximately 2.7 kb. In situ hybridization analysis showed that the Cchsp83 gene maps at the 94C region of the 6th chromosome (6R:94C), which corresponds to one of the major heat shock puffs of the medfly salivary gland polytene chromosomes. Evaluation of the Cchsp83 gene transcript and protein levels was performed by RT-PCR and western analysis, using total RNA and protein from synchronized animals. Anti-HSP83 antibodies were prepared against a histidine tag purified chimeric polypeptide from E. coli cells, transformed with a part of the coding region (aa 490-690) of the Cchsp83 cDNA. The response of the Cchsp83 gene to heat was fast and sensitive. Heat-induced transcript levels could be detected within 5 min at temperatures as low as 300C. Maximum transcript levels were obtained after 30-90 min treatments at 35-390C. Following recovery at 250C, after a 30 min heat shock, the accumulated transcripts remained at high levels for approximately 3h and declined to the non-induced levels 1h later. Developmental studies showed that the Cchsp83 gene is expressed constitutively throughout medfly development. At 250C, both transcript and protein levels were high in embryonic stages, low in larval stages and moderate in pupal and adult stages. Following heat shock at 380C, the transcript levels increased approximately 3- to 5-fold, depended on the developmental stage. Similar results were obtained for the protein levels after a heat shock at 350C, but not at 380C, suggesting that the Cchsp83 mRNAs are not translated efficiently at high temperatures. Screening of a genomic λEMBL-4A library from 24-h-old medfly embryos with the second exon of the D. auraria hsp83 gene, resulted in the isolation of a genomic clone containing part of the coding region, the untranslated leader region (5’ UTR) and 3.5 kb from the 5’ flanking region of the gene. Comparison of the genomic and cDNA nucleotide sequences revealed the presence of a small intron of 275 bp between the 5’ untranslated and coding regions of the gene. Thus the Cchsp83 gene is organized into two exons separated by a small intron. Computational analysis and 5’ RACE experiments, suggested that the putative transcription initiation site of the gene is located 144 bp upstream of the 5’ splicing site of the intron and 23 bp downstream of a typical TATA box (TATAΑΑΤΑ). Two putative heat shock elements (HSEs) were identified in the proximal 5’ flanking region of the gene, 35 and 330 bp, upstream of the TATA box. In addition to them, four distal HSEs, 1,595, 2,861, 2,880 and 2,890 bp, upstream of the putative transcription initiation site and one HSE inside the intron were identified. Functional analysis of the proximal 5’ flanking region of the Cchsp83 gene was performed by germline transformation. Three overlapping promoter fragments PL (-380/+139), PM (-86/+144) and PS (-55/+139), were fused to the lacZ reporter gene and the resulting constructs were introduced into the medfly genome via Minos-element mediated germline transformation. The expression of the reporter gene, in at least 8 homozygous transformed lines for each construct, was evaluated by quantitative β-galactosidase assays. The PM-lacZ and PS-lacZ lines did not show detectable levels of lacZ expression at neither normal or heat shock conditions. On the other hand, most of the PL-lacZ lines showed significant levels of constitutive lacZ expression. Developmental expression studies in one of these lines showed that the reporter gene exhibited similar developmental expression pattern to the endogenous one, suggesting that the PL promoter region includes all the necessary regulatory elements for driving correct temporal expression of the Cchsp83 at normal conditions. Although this promoter region contained the two proximal HSEs and the 5’ UTR, it was unable to drive heat-induced expression of the reporter gene suggesting that additional upstream and/or downstream sequences are necessary for the heat-induced expression of the Cchsp83 gene. Our data indicate that the PL promoter region of the Cchsp83 gene can be used as a driver for the development of robust transgenic marker systems in medfly. Such systems are important for detecting, maintaining and recognizing medfly strains that are used today in population control programs of this agricultural pest.

O presente trabalho investiga a aplicação do processo de extração com fluido supercrítico (EFS) para a obtenção de compostos fenólicos a partir de Butia capitata, coletado na Região Sul do Brasil. A planta, chamada popularmente de butiá, é uma espécie nativa da Argentina, Uruguai, Paraguai e Brasil. Os frutos do butiá são comestíveis e bastante apreciados pelas populações locais, que os consomem ao natural ou na forma de licores, geléias ou doces. Estudos recentes revelam que compostos fenólicos estão presentes nos frutos do butiá. Estes compostos merecem atenção por apresentarem atividade antioxidante, por prevenirem doenças cardiovasculares e reduzirem o risco de câncer. Atualmente existem diferentes formas para a obtenção de compostos fenólicos presentes em matrizes vegetais, porém muitas delas utilizam solventes orgânicos a altas temperaturas, o que deprecia o valor comercial dos extratos. Neste estudo, é utilizada uma tecnologia limpa, que tem como solvente o dióxido de carbono (CO2) em condições supercríticas. O CO2 é não-tóxico e não-inflamável, o que faz dele uma opção mais segura, quando comparado a outros solventes. Além disso, no estado supercrítico, o CO2 apresenta condições bastante favoráveis para a extração, como altas difusividades e massas específicas elevadas. Uma característica que deve ser ressaltada é a possibilidade de utilizar baixas temperaturas na extração, comparativamente aos processos que utilizam solventes líquidos, uma vez que os compostos fenólicos são termolábeis. Nos experimentos, foi utilizada apenas a polpa desidratada. O planejamento experimental foi elaborado avaliando a influência de três variáveis de processo em três níveis: pressão (150, 200 e 250 bar), temperatura (40, 50 e 60°C) e teor de cossolvente (1, 3 e 5% de etanol). Os extratos obtidos foram analisados em cromatografia líquida de alta eficiência (CLAE), sendo os rendimentos expressos em mg do composto identificado por CLAE /100g de polpa de butiá desidratada (PBD), e pelo método Folin-Ciocalteau com os rendimentos em compostos fenólicos totais (CFT) expressos em mg de rutina equivalente (RE)/100g PBD. Foram identificados cinco compostos majoritários nos extratos nas análises em CLAE. As maiores concentrações foram encontradas nas condições de 250 bar, 60°C e 5% de etanol. Os compostos identificados foram o ácido clorogênico (0,46mg/100g PBD), o ácido ferúlico (1,45mg/100g PBD), a rutina (3,47mg/100g PBD), a quercetina (0,90mg/100g PBD) e o canferol (1,93mg/100g PBD). Em termos de CFT, a melhor condição de extração foi de 200 bar, 50°C e 5% de etanol, resultando em 23,09mg RE/100g PBD. Os extratos também foram avaliados quanto ao poder de redução do radical DPPH•, pelo método IC50. O método fornece a quantidade necessária de extrato pra varrer 50% do radical. Como esperado, o extrato obtido na corrida experimental de maior rendimento em CFT apresentou maior atividade antioxidante (17,4 mg/mL), seguido da corrida experimental de maior rendimento em compostos fenólicos isolados (56,1 mg/mL) e, com menos atividade, o extrato obtido por solvente orgânico (80,0 mg/mL). / In this work it is investigated the application of supercritical fluid extraction to obtain phenolic compounds from Butia capitata, collected in southern Brazil. This plant, known popularly as butiá, is a native species in Argentina, Uruguay, Paraguay and Brazil. The edible fruits are popular and eaten raw or as liqueurs, jellies or jams. In recent studies, it is shown that phenolic compounds are present in the fruits of butiá. These compounds deserve attention because they have antioxidant activity, preventing cardiovascular disease and reducing the risk of cancer. There are different processes to obtain phenolic compounds from plant matrices, but many of them make use of organic solvents at high temperatures, which depreciates the extract value. In this study, it is used a clean technology, with carbon dioxide (CO2) as solvent in supercritical conditions. The CO2 is nontoxic and nonflammable that makes it a safer option than other solvents. Furthermore, in supercritical conditions the CO2 has favorable conditions for extraction, as high diffusivities and densities. A feature that should be emphasized is the possibility of using low temperatures in the extract, when compared to processes using liquid solvents, since the phenolic compounds are decomposed by high temperature. In the experiments only the dried pulp was used. The experimental design was developed to assess the influence of three process variables at three levels: pressure (150, 200 e 250 bar), temperature (40, 50 e 60°C) and cosolvent content (1, 3 e 5% of ethanol). The extracts were analyzed by high performance liquid chromatography (HPLC), in which the yields were expressed in terms of mg of the identified phenolic compounds by HPLC/100g of dried pulp and by the Folin-Ciocalteau method, with yields, as total phenolic compounds, expressed in mg of rutin equivalent/100g of dried pulp. It were identified five majority phenolic compounds by HPLC. Its highest concentrations were found in the conditions of 250 bar, 60°C and 5% of cosolvent. The identified compounds were chlorogenic acid (0,46mg/100g of dried pulp), ferulic acid (1,45mg/100g of dried pulp), rutin (3,47mg/100g of dried pulp), quercetin (0,90mg/100g of dried pulp) and kaempferol (1,93mg/100g of dried pulp). In terms of total phenolic compounds, the best conditions were 200 bar, 50°C and 5% of cosolvent, which resulted in 23,09mg of rutin equivalent/100g of dried pulp. Also, the antioxidant activity of the extracts was measured by scavenging of the DPPH• radical, by the IC50 method. The method provides the necessary amount of extract to scavenging 50% of the radial. As expected, the extract obtained in experimental run with the highest yield in CFT showed a higher antioxidant activity (17.4 mg / mL), followed by the experimental run with the highest yield in phenolic compounds isolated (56.1 mg / mL), and, with less activity, the extract obtained by organic solvent (80.0 mg / mL).

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2017-02-15T10:50:25Z No. of bitstreams: 1 texto completo.pdf: 1856190 bytes, checksum: 405040c338d9d0453f53e6b127a24ccd (MD5) / Made available in DSpace on 2017-02-15T10:50:25Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1856190 bytes, checksum: 405040c338d9d0453f53e6b127a24ccd (MD5) Previous issue date: 2016-03-29 / O Brasil está entre os maiores produtores de frutas do mundo, ocupando a terceira posição no ranking, com produção anual de 40 milhões de toneladas. O Vale de São Francisco manteve em 2014 o patamar de 85% das exportações brasileiras de manga. Entre as frutas, a manga apresenta produção nacional ascendente, com quase 1,2 milhão de toneladas em 2014. Entre os obstáculos, a maior participação do setor no mercado internacional está nos aspectos fitossanitários, principalmente as moscas- das-frutas (Diptera: Tephritidae). O objetivo deste trabalho foi analisar a flutuação populacional de moscas-das-frutas em pomares comerciais de mangueira, bem como estudar a influência dos fatores abióticos na flutuação populacional. A partir dos resultados, discute-se a viabilidade de ampliação do número de propriedades aptas a exportar mangas in natura para países que exigem que os frutos sejam oriundos de Áreas de Baixa Prevalência de Pragas (ABPP). O estudo foi desenvolvido a partir de dados coletados de 2009 a 2014 em 44 pomares comerciais de manga localizados nos Projetos de Irrigação Senador Nilo Coelho, Maria Tereza e Bebedouro, em Petrolina, PE. A espécie exótica Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) foi detectada em todos os pomares estudados, e os dados sugerem que ela tenha deslocado as espécies nativas do gênero Anastrepha (Diptera: Tephritidae). A população apresentou níveis elevados em todas as áreas monitoradas, principalmente de outubro a janeiro, em todos os períodos estudados. A temperatura e a umidade relativa influenciaram fortemente a captura de insetos adultos, em que a temperatura apresentou correlação positiva e a umidade, correlação negativa. A população de C. capitata esteve em níveis acima do admitido para implantação de ABPP, de forma que o aumento de exportações de manga para mercados mais exigentes dependerá grandemente da execução de um programa para supressão da população. / Brazil is a major fruit grower worldwide, occupying the third position and harvesting 40 x 106 ton. In 2014, Sao Francisco Valley responded for 85% of mango exports of the country. Mango production is going through a period of increase in production and approximately 1.2 million tons were harvested in 2014. Phytosanitary measures are major constraints to export, mainly those established to avoid the entry of fruit flies (Diptera: Tephritidae). This work aimed to analyse the population fluctuation of fruit flies in commercial mango orchards, as well as study the influence of abiotic factors in population density. The feasibility of increasing the number of properties certified according to international standards of areas of low pest prevalence (ALPP) is discussed, as this is a measure established by several international markets. Data gathered from 2009 to 2014 for 44 commercial orchards in the districts Senador Nilo Coelho, Maria Tereza, and Bebedouro, in Petrolina, PE, were used. The exotic species Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) was detected in all orchards and data suggest that it may have displaced the native species of the genus Anastrepha (Diptera: Tephritidae). Population density was high in all considered areas, mainly from October to January. Temperature and relative humidity had significant impact on population density, the former showing a positive correlation and the later, a negative one. C. capitata population was higher than the threshold implementation in standards for LPPA so that the increase of mango exports for more restrictive countries will largely depend on the conduction of a suppression program. / Não foi localizado o currículo lattes do autor.

O presente trabalho investiga a aplicação do processo de extração com fluido supercrítico (EFS) para a obtenção de compostos fenólicos a partir de Butia capitata, coletado na Região Sul do Brasil. A planta, chamada popularmente de butiá, é uma espécie nativa da Argentina, Uruguai, Paraguai e Brasil. Os frutos do butiá são comestíveis e bastante apreciados pelas populações locais, que os consomem ao natural ou na forma de licores, geléias ou doces. Estudos recentes revelam que compostos fenólicos estão presentes nos frutos do butiá. Estes compostos merecem atenção por apresentarem atividade antioxidante, por prevenirem doenças cardiovasculares e reduzirem o risco de câncer. Atualmente existem diferentes formas para a obtenção de compostos fenólicos presentes em matrizes vegetais, porém muitas delas utilizam solventes orgânicos a altas temperaturas, o que deprecia o valor comercial dos extratos. Neste estudo, é utilizada uma tecnologia limpa, que tem como solvente o dióxido de carbono (CO2) em condições supercríticas. O CO2 é não-tóxico e não-inflamável, o que faz dele uma opção mais segura, quando comparado a outros solventes. Além disso, no estado supercrítico, o CO2 apresenta condições bastante favoráveis para a extração, como altas difusividades e massas específicas elevadas. Uma característica que deve ser ressaltada é a possibilidade de utilizar baixas temperaturas na extração, comparativamente aos processos que utilizam solventes líquidos, uma vez que os compostos fenólicos são termolábeis. Nos experimentos, foi utilizada apenas a polpa desidratada. O planejamento experimental foi elaborado avaliando a influência de três variáveis de processo em três níveis: pressão (150, 200 e 250 bar), temperatura (40, 50 e 60°C) e teor de cossolvente (1, 3 e 5% de etanol). Os extratos obtidos foram analisados em cromatografia líquida de alta eficiência (CLAE), sendo os rendimentos expressos em mg do composto identificado por CLAE /100g de polpa de butiá desidratada (PBD), e pelo método Folin-Ciocalteau com os rendimentos em compostos fenólicos totais (CFT) expressos em mg de rutina equivalente (RE)/100g PBD. Foram identificados cinco compostos majoritários nos extratos nas análises em CLAE. As maiores concentrações foram encontradas nas condições de 250 bar, 60°C e 5% de etanol. Os compostos identificados foram o ácido clorogênico (0,46mg/100g PBD), o ácido ferúlico (1,45mg/100g PBD), a rutina (3,47mg/100g PBD), a quercetina (0,90mg/100g PBD) e o canferol (1,93mg/100g PBD). Em termos de CFT, a melhor condição de extração foi de 200 bar, 50°C e 5% de etanol, resultando em 23,09mg RE/100g PBD. Os extratos também foram avaliados quanto ao poder de redução do radical DPPH•, pelo método IC50. O método fornece a quantidade necessária de extrato pra varrer 50% do radical. Como esperado, o extrato obtido na corrida experimental de maior rendimento em CFT apresentou maior atividade antioxidante (17,4 mg/mL), seguido da corrida experimental de maior rendimento em compostos fenólicos isolados (56,1 mg/mL) e, com menos atividade, o extrato obtido por solvente orgânico (80,0 mg/mL). / In this work it is investigated the application of supercritical fluid extraction to obtain phenolic compounds from Butia capitata, collected in southern Brazil. This plant, known popularly as butiá, is a native species in Argentina, Uruguay, Paraguay and Brazil. The edible fruits are popular and eaten raw or as liqueurs, jellies or jams. In recent studies, it is shown that phenolic compounds are present in the fruits of butiá. These compounds deserve attention because they have antioxidant activity, preventing cardiovascular disease and reducing the risk of cancer. There are different processes to obtain phenolic compounds from plant matrices, but many of them make use of organic solvents at high temperatures, which depreciates the extract value. In this study, it is used a clean technology, with carbon dioxide (CO2) as solvent in supercritical conditions. The CO2 is nontoxic and nonflammable that makes it a safer option than other solvents. Furthermore, in supercritical conditions the CO2 has favorable conditions for extraction, as high diffusivities and densities. A feature that should be emphasized is the possibility of using low temperatures in the extract, when compared to processes using liquid solvents, since the phenolic compounds are decomposed by high temperature. In the experiments only the dried pulp was used. The experimental design was developed to assess the influence of three process variables at three levels: pressure (150, 200 e 250 bar), temperature (40, 50 e 60°C) and cosolvent content (1, 3 e 5% of ethanol). The extracts were analyzed by high performance liquid chromatography (HPLC), in which the yields were expressed in terms of mg of the identified phenolic compounds by HPLC/100g of dried pulp and by the Folin-Ciocalteau method, with yields, as total phenolic compounds, expressed in mg of rutin equivalent/100g of dried pulp. It were identified five majority phenolic compounds by HPLC. Its highest concentrations were found in the conditions of 250 bar, 60°C and 5% of cosolvent. The identified compounds were chlorogenic acid (0,46mg/100g of dried pulp), ferulic acid (1,45mg/100g of dried pulp), rutin (3,47mg/100g of dried pulp), quercetin (0,90mg/100g of dried pulp) and kaempferol (1,93mg/100g of dried pulp). In terms of total phenolic compounds, the best conditions were 200 bar, 50°C and 5% of cosolvent, which resulted in 23,09mg of rutin equivalent/100g of dried pulp. Also, the antioxidant activity of the extracts was measured by scavenging of the DPPH• radical, by the IC50 method. The method provides the necessary amount of extract to scavenging 50% of the radial. As expected, the extract obtained in experimental run with the highest yield in CFT showed a higher antioxidant activity (17.4 mg / mL), followed by the experimental run with the highest yield in phenolic compounds isolated (56.1 mg / mL), and, with less activity, the extract obtained by organic solvent (80.0 mg / mL).

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The Mediterranean fruit fly (medfly), Ceratitis capitata, is a highly invasive species throughout the world and considered as one of the most successful agricultural and economical pests. The increase of global trade in fruit and human travel combined with the biology of the medfly has allowed the species to spread from its proposed Afrotropical origin, to a number of locations throughout the world. In the Western Cape various control strategies have been implemented to control medfly populations, including insecticides and more environmentally-friendly techniques such as the Sterile Insect Technique (SIT). In order to be effective, however, an SIT program requires some knowledge of the population structure and the movement of individuals between pest-occupied sites. The identification of sites from which re-invasion is most likely to occur and knowledge regarding the primary routes through which pests are likely to re-establish are critical to ensure successful SIT programmes. To provide this important information to SIT and area-wide pest control programs in South Africa, sampling at two different spatial scales (regional- and fine-scale) in South Africa was undertaken. Regional scale sampling was done at 13 locations in the Western Cape and fine scale sampling was done at 13 locations within the Ceres-valley. All individuals were genotyped at 11 polymorphic microsatellite markers and selected individuals from the regional scale were sequenced for the mitochondrial gene COI. Our results show that populations at regional- and fine-scale in the Western Cape are characterized by high levels of genetic diversity (HEregional = 0.805; HEfine = 0.803). Little or weak population differentiation was detected at the regional- and fine-scales, suggesting overall high levels of gene flow among sampling locations. These findings were supported by coalescent based methods indicating sufficient levels of gene flow to prevent population differentiation between neighbouring (200m) and distant (350km) populations. However, natural dispersal in C. capitata has been shown to rarely exceed 10 km. As such, high levels of gene flow between distant populations are more likely the result of humanmediated dispersal, linked to the movement of fresh produce within South Africa. This high level of gene flow has important implications for pest management practices, as my results suggests that area-wide pest management should be undertaken at a regional scale, rather than on a farm or valley scale. My results are placed within a management framework, and I argue for more stringent control when fruit are transported within South Africa. Of particular interest for future studies is the investigation of gene flow at broader spatial scales (i.e. the whole of South Africa) and a comparison of the genetic diversity, population differentiation and gene flow patterns of C. capitata with that of Ceratitis rosa will be important to establish a successful pest management strategy in South Africa. / AFRIKAANSE OPSOMMING: Die Mediterreense vrugtevlieg (medvlieg), Ceratitis capitata, is ‘n indringerspesie wêreldwyd en word beskou as een van die mees suksesvolle ekonomiese en landbou peste. Die medvlieg het ‘n Afrotropiese oorsprong, maar die toename in wêreldwye handel en reis, gekombineer met die biologie van die medvlieg het gelei tot die verspreiding van die spesie na ‘n groot aantal bestemmings regoor die wêreld. Die Wes-Kaap provinsie van Suid-Afrika implementeer tans verskeie strategieë om medvlieg bevolkings te beheer. Hierdie strategieë sluit in die gebruik van plaagdoders sowel as meer omgewingsvriendelike tegnieke soos die Steriele Insektegniek (SIT). Om ‘n effektiewe SIT program te implementeer vereis basiese kennis ten opsigte van die genetiese struktuur van die bevolking sowel as van die beweging van individue tussen verskillende pesvoorkomsgebiede. Die identifisering van areas van waar herkolonisering mees waarskynlik kan plaasvind en kennis in verband met die primêre roetes waardeur pes spesies hervestig, is van kritiese belang om ‘n suksesvolle SIT program te verseker. Medvlieg individue is op twee verskillende ruimtelike skale (streeks- en plaaslike-skaal) versamel om die nodige inligting aan SIT en area-wye pes beheer programme in Suid-Afrika te verskaf. Streeks-skaal individue is by 13 lokaliteite regoor die Wes-Kaap versamel en plaaslike-skaal individue by 13 lokaliteite in die Ceres-vallei. Alle versamelde individue is vir 11 polimorfiese mikrosatelliet merkers gegenotipeer en DNS volgordebepaling van geselekteerde individue vanuit die streek-skaal is gedoen vir die mitochondriale geen COI. My resultate toon dat bevolkings op beide skale gekarakteriseer word deur hoë vlakke van genetiese diversiteit (HEstreeks = 0.805; HEplaaslik = 0.803) en geen of swak bevolkings differensiasie. Hierdie resultate dui daarop dat daar hoë vlakke van geenvloei tussen bevolkings is. Hierdie bevindinge word verder ondersteun deur metodes gebaseer op die statistiese eienskappe van die genealogiese verhouding tussen allele onder sekere mutasie en demografiese modelle, wat voldoende vlakke van geenvloei aandui tussen nabye (200m) sowel as verafgeleë (350km) bevolkings om bevolkings differensiasie te verhoed. Natuurlike beweging in C. capitata is egter selde meer as 10 km, sodanig kan die hoë vlakke van geenvloei toegeskryf word aan die verspreiding van individue met menslike hulp, spesifiek in die vervoer van varsprodukte in Suid-Afrika. Hierdie hoë vlak van geenvloei het verreikende implikasies vir pes beheer praktyke, omdat my resultate voorstel dat area-wye pes beheer onderneem moet word op ‘n streeks-skaal eerder as op ‘n plaas-tot-plaas of vallei wye area. Ek plaas my resultate in ‘n bestuursraamwerk, waarin ek streng beheer van vrugtevervoer in Suid-Afrika beklemtoon. Verdere navorsing moet fokus op die ondersoek van geenvloei op ‘n landswye skaal (hele Suid-Afrika) in C. capitata, sowel as die vergelyking van die genetiese diversiteit, bevolkings differensiasie en geenvloei patrone van C. capitata met die van Ceratitis rosa om ‘n suksesvolle pes beheer strategie vir Suid-Afrika te formuleer.