Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-04-26T13:48:20Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_cassyo_de_araujo_rufino.pdf: 1782663 bytes, checksum: 48211778344c5f757ff01c4c9282d9b8 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-05-02T18:29:29Z (GMT) No. of bitstreams: 2 tese_cassyo_de_araujo_rufino.pdf: 1782663 bytes, checksum: 48211778344c5f757ff01c4c9282d9b8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-05-02T18:31:26Z (GMT) No. of bitstreams: 2 tese_cassyo_de_araujo_rufino.pdf: 1782663 bytes, checksum: 48211778344c5f757ff01c4c9282d9b8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-02T18:31:51Z (GMT). No. of bitstreams: 2 tese_cassyo_de_araujo_rufino.pdf: 1782663 bytes, checksum: 48211778344c5f757ff01c4c9282d9b8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-03-05 / Sem bolsa / A utilização de produtos naturais conjuntamente com nanopartículas de prata tem atraído interesse de pesquisadores, tanto do ponto de vista tecnológico quanto cientifico devido à ação desses produtos no controle de doenças e pragas. O objetivo desse trabalho foi testar produtos naturais (associados ou não a nanoprata) com ação antimicrobiana sobre Bipolaris oryzae, in vitro, e no tratamento de sementes de arroz. Para o estudo dos compostos contendo quitina e própolis adicionadas opcionalmente nanoprata foram utilizados sementes de arroz na cultivar IRGA 424 inoculadas com um isolado de Bipolaris oryzae. Os compostos utilizados são naturais, têm propriedades antiadesivas e atividade antimicrobiana contra microrganismos, possuem propriedades biocompatíveis e não fitotóxicas aos vegetais em geral. O processo de sínteses dos compostos contendo própolis, quitina e nanoprata, foi realizado por meio da aplicação de sonicação para a dispersão e mistura entre as substâncias. Foram testados os produtos no desenvolvimento e crescimento do fungo Bipolaris oryzae em placas de petri e em sementes de arroz infectadas. Os tratamentos foram: quitina, própolis, prata, quitina+própolis, quitina+própolis+nanoprata, quitina+prata, própolis+nanoprata e testemunha (T - sementes sem tratamentos), totalizando neste estudo 10 tratamentos. Logo a eficiência dos produtos e suas soluções foram testados por meio do blotter test (teste de sanidade de sementes), porcentagem de inibição e diâmetro de crescimento do fungo, germinação e comprimento de plântulas das sementes tratadas com os produtos. Os resultados demonstram que os oligômeros de quitosana e os compósitos de quitosana com extrato de própolis e nanopartículas de prata controlam o crescimento do patógeno. A importância deste achado reside no fato de que é um passo em direção ao objetivo de diminuir o uso de fungicidas químicos. A partir de testes de germinação poderá concluir-se que os melhores resultados foi o tratamento com quitosano/prata compósito NPs, com uma taxa de germinação de 91%. Os novos materiais poliméricos são considerados como muito promissores para o controle de Bipolaris oryzae no arroz, tornando-se assim alternativas ecológicas aos fungicidas químicos. / The use of natural products in conjunction with silver nanoparticles has attracted interest of many researches, from both scientific and technological point of view due to their action for disease and pests control. The objective was to test natural products (with or without nanosilver) with antimicrobial action against Bipolaris oryzae, in vitro, and in the treatment of rice seeds. To study the compounds containing chitin and propolis optionally added nanosilver rice seeds were used in IRGA inoculated with an isolated 424 Bipolaris oryzae. The compounds used are natural, have anti-adhesive properties and antimicrobial activity against microorganisms, have biocompatible properties and not phytotoxic to plants in general. The process of synthesis of compounds containing propolis, chitin and nanosilver was performed by applying the sonication for the dispersion and mixing of the substances. Products were tested on the development and growth of Bipolaris oryzae fungus in petri dishes and infected rice seeds. The treatments were: chitin, propolis, silver, chitin + propolis, chitin + Propolis + nanosilver, chitin + silver, propolis + nanosilver and control (T - treatments seeded), totaling 10 treatments in this study. The results were conclusive in showing that chitosan oligomers and the composites of chitosan with propolis extract and silver nanoparticles led to a remarkable reduction in growth of the pathogen. The importance of this finding lies in the fact that it is a step towards the goal of decreasing the use of chemical fungicides in plant pathology. From germination tests it could be concluded that the best results was the treatment with chitosan/silver NPs composite, with a germination rate of 91%. The novel polymeric materials can thus be deemed as very promising for the control of the Bipolaris oryzae of rice and pave the way towards eco-friendly alternatives to chemical fungicides.

Submitted by Jaqueline Silva (jtas29@gmail.com) on 2014-09-15T20:06:22Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação Karinna Bannach Reis.pdf: 3955087 bytes, checksum: fbfd1023491588a9a5495cd281d72a41 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-09-15T20:06:45Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação Karinna Bannach Reis.pdf: 3955087 bytes, checksum: fbfd1023491588a9a5495cd281d72a41 (MD5) / Made available in DSpace on 2014-09-15T20:06:45Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação Karinna Bannach Reis.pdf: 3955087 bytes, checksum: fbfd1023491588a9a5495cd281d72a41 (MD5) Previous issue date: 2013-12-18 / Ruta graveolens has been successfully applied for many human diseases treatment and promises a well succeed alternative for plant diseases control because it has also phytoalexins in its composition. The aim of this study was to standardise the R. graveolens liquid extract and evaluate its potential for controlling rice (Oryza sativa) leaf blast (Magnaporthe oryzae). The drug has been characterized, the liquid extract obtained and the methodology for quantifying the standards components, furanocoumarins psoralen and bergapten, validated by high performance liquide chromatography. The components of essential oil obtained from standardized liquid extract were characterized by gas chromatography-mass spectrometry. In a completely randomized design, conducted in artificial hydrophobic surface with three replications and eleven treatments composed of M. oryzae conidial suspension (105 con.ml-1) mixed with R. graveolens standardized extract (0.01 to 0.10 g.mL-1), or furocoumarins psoralen (0.18 to 1.82 μg. mL-1), or bergapten (0.29 to 2.91 μg. mL-1), or water (control). It was evaluated the inhibition of conidial germination and appressorium formation and the median lethal dose (LD50). A second assay, in a completely randomized design in three replications was conducted in greenhouse conditions. It was composed of 21 days old rice plants, which were sprayed with a mixture containing M. oryzae conidial suspension (3x105 con.ml-1) and R. graveolens extract, without dilution, or furocoumarins psoralen (18.26 μg. mL-1), or bergapten (29.14 μg. mL-1), or water (control). Nine days after inoculation, leaf blast severity was scored with a diagrammatic scale, the data were statistically analyzed and means compared. The standardized plant extract inhibited M. oryzae conidial germination (LD50=0.237mg) and appressorium formation (LD50=0.121 mg) up to 100% and reduced 80.84% of leaf blast. By fluorescence microscopy it was possible to observe that standardized plant extract did not damage M. oryzae cell wall and plasma membrane, indicating another type of interaction to inhibit conidia development. Isolated standards furanocoumarins psoralen and bergapten did not inhibit conidial germination and appressorium formation and reduce leaf blast severity proportionally, suggesting that synergistic interactions between extract and essential oil components were responsible for the success of R. graveolens in suppressing rice disease, making it an alternative to compose rice blast management. / Ruta graveolens é utilizada com sucesso no tratamento de diversas patologias humanas e possui potencial para o controle alternativo de fitopatógenos, pois também apresenta fitoalexinas em sua composição. O objetivo deste estudo foi padronizar o extrato vegetal líquido de R. graveolens e avaliar seu potencial para o controle da brusone foliar. O material vegetal foi caracterizado, o extrato líquido obtido e a metodologia para a quantificação dos padrões psoraleno e bergapteno foi validada por cromatografia líquida de alta eficiência. Os componentes do óleo essencial obtidos a partir do extrato líquido padronizado foram caracterizados por cromatografia gasosa acoplada à espectrometria de massas. No ensaio conduzido em superfície hidrofóbica artificial, com três repetições e onze tratamentos, 10 μL da suspensão de conídios de Magnaporthe oryzae (105 con/mL) foram misturadas com o extrato vegetal padronizado (0,01 a 0,10 g/mL), ou as furanocumarinas psoraleno (0,18 a 1,82 μg/mL) e bergapteno (0,29 a 2,91 μg/mL), ou água (controle). Avaliou-se a inibição da germinação conidial e da formação dos apressórios determinando-se a dose letal capaz de inibir 50% dos indivíduos (DL50). No experimento conduzido em casa de vegetação e com três repetições, plantas de arroz com 21 dias após plantio foram pulverizadas com uma suspensão de conídios de M. oryzae (3x105 com/mL) misturada com extrato vegetal padronizado sem diluição, ou psoraleno (18,26 μg/mL), ou bergapteno (29,14 μg/mL), ou água (controle). Após nove dias da inoculação, avaliou-se a severidade de brusone foliar com uma escala de 10 graus, os dados foram analisados estatisticamente e as médias comparadas. O extrato vegetal padronizado inibiu a formação de tubo germinativo (DL50= 0,237 mg) e a formação de apressório (DL50= 0,121 mg) de M. oryzae em até 100%, e reduziu a severidade de brusone nas folhas em 80,84%. Através de microscopia de fluorescência, não foi observada ação do extrato padronizado em membrana plasmática e parede celular de M. oryzae, o que indica outro tipo de interação para inibir o desenvolvimento do conídio. Os padrões psoraleno e bergapteno não inibiram proporcionalmente a germinação e a formação de apressório, como também não reduziram a severidade de brusone nas folhas em sua forma isolada, o que sugere que interações sinérgicas entre os diversos componentes do extrato e do óleo essencial de R. graveolens foram responsáveis pelo sucesso do extrato padronizado em suprimir a brusone foliar, tornando-o uma alternativa para compor o manejo de brusone em arroz.

Rhizopus oryzae is a filamentous fungi which produces lactic acid and ethanol in fermentations. R. oryzae has numerous advantages for use industrial production of L-(+)-lactic acid but the yield of lactic acid produced on the basis of carbon consumed is low. Metabolic flux analysis of R. oryzae has shown that most of the pyruvate produced at the end of the glycolysis is channelled to ethanol, acetyl-CoA and oxaloacetate production. This study aimed to answer some questions addressed on the regulation of pyruvate branch point in R. oryzae and for this purpose biochemical characterisation of the enzymes acting at this branch point and cloning the genes coding for these enzymes have been done. Pyruvate decarboxylase was purified and characterised for the first time from R. oryzae. The purified enzyme has a Hill coefficient of 1.84 and the Km of the enzyme is 8.6 mM for pyruvate at pH 6.5. The enzyme is inhibited at pyruvate concentrations higher than 30 mM. The optimum pH for enzyme activity shows a broad range from 5.7 and 7.2. The monomer molecular weight was estimated as 59&plusmn / 2 kDa by SDS-PAGE analysis. Pyruvate decarboxylase (pdcA and pdcB) and lactate dehydrogenase (ldhA and ldhB) genes of R. oryzae have been cloned by PCR-cloning approach and the filamentous fungi Aspergillus niger was transformed with these genes. The A. niger transformed with either of the ldh genes of R. oryzae showed enhanced production of lactic acid compared to wild type. Citric acid production was also increased in these transformants while no gluconate production was observed Cloning of hexokinase gene from R. oryzae using degenerate primers was studied by the use of GenomeWalker kit (Clontech). The results of this study were evaluated by using some bioinformatics tools depending on the unassembled clone sequences of R. oryzae genome.

Plusieurs insectes se développant dans des milieux nutritionnellement déficients vivent en symbiose durable avec des bactéries intracellulaires (endosymbiotes) qui complémentent leur alimentation et améliorent leur pouvoir adaptatif. Alors que ces associations ont été largement étudiées sur les plans physiologiques et évolutifs, peu de travaux se sont consacrés à l’étude des mécanismes impliqués dans la tolérance et le contrôle des endosymbiotes par l’hôte. L’objectif de cette thèse est d’étudier, chez les charançons des céréales du genre Sitophilus, les particularités moléculaires et immunitaires du bactériome, un organe que l’insecte développe pour héberger les symbiotes et les isoler de sa réponse immunitaire systémique. Le bactériome du charançon exprime une réponse immunitaire modulée : des études transcriptomiques ont montré que les effecteurs de l’immunité sont peu exprimés dans cet organe, à l’exception d’un gène codant un peptide antimicrobien, la coléoptéricine A. Cette dernière interagit avec les endosymbiotes et participe à leur confinement intracellulaire. Dans une première partie, j’ai montré avec une approche d’interférence à l’ARN que l’expression du gène colA serait contrôlée par un système original qui impliquerait les gènes relish et tollip. Cette régulation « interne » au bactériome semble assurer le maintien des endosymbiotes et l’homéostasie de l’organe. Afin de comprendre comment le bactériome répond à une infection par les bactéries exogènes, j’ai suivi par RT-qPCR l’expression de gènes effecteurs de l’immunité dans le bactériome après injection systémique de bactéries à Gram positif ou négatif. Ceci a mis en évidence une réponse « externe », induite en cas d’infection, et qui aurait un rôle de protection des endosymbiotes contre les bactéries exogènes. Enfin, je me suis consacré à l’étude des changements de régulation accompagnant le passage du stade larvaire au stade adulte, marqué par une symbiose très dynamique. Le nombre d’endosymbiotes augmente fortement pendant les premiers jours de vie imaginale, puis diminue jusqu’à leur élimination complète par recyclage autophagique. Une analyse RNAseq a permis d’identifier les voies de signalisation dont l’activité accompagne cette dynamique. Une approche de RT-qPCR a également montré que l’immunité du bactériome est maintenue à un faible niveau d’activation pendant tout le processus de recyclage. Ce travail montre qu’au cours de leur évolution, les insectes ont sélectionné plusieurs stratégies pour assurer le maintien et l’ajustement de leur charge endosymbiotique en fonction de leurs besoins physiologiques : une signalisation immunitaire assurerait le confinement intracellulaire des endosymbiotes, et un ensemble de processus cellulaires incluant l’apoptose et l’autophagie semble être en associé aux voies métaboliques pour assurer le contrôle de la dynamique bactérienne et garantir le compromis bénéfice/coût de la symbiose. / Many insect species living on nutritionally unbalanced media depend on intracellular mutualistic bacteria, called obligatory endosymbionts, for their development and reproduction. Endosymbionts are housed in specialized host cells called bacteriocytes, that group together to form the bacteriome organ. Although such associations have been widely investigated on a physiological and evolutionary point of view, little is known about the mechanisms involved in the tolerance and the control of endosymbionts by the host. This work aims at deciphering the molecular and immune specificities of the bacteriome using the model system Sitophilus oryzae, the cereal weevil, and its obligate endosymbiont Sodalis pierantonius. The weevil bacteriome expresses a modulated immune response: transcriptomic studies showed that immune effector genes were lowly expressed despite the massive bacterial presence, with the exception of colA, a gene encoding for Coleoptericin A, an antimicrobial peptide. Coleoptericin A interacts with endosymbionts and participates in their intracellular seclusion. In a first chapter, I used RNA interference to demonstrate that colA gene expression may be controlled by an original system involving the genes relish and tollip. This “internal” regulation for endosymbiont control seems to maintain bacteriome homeostasis. In a second chapter, in order to understand how the bacteriome responds to an infection by exogenous bacteria, I followed up by RT-qPCR the expression of immune effector genes in the bacteriome after injection of Gram positive and Gram negative bacteria. This highlighted an “external” immune response, inducible upon infections, which may enable endosymbiont protection against exogenous intruders. In a third and last chapter, I focused on the regulation changes that accompany the switch from the larval stage to the imaginal stage, the latter being characterized by a very dynamic symbiosis. Endosymbiont load drastically increases during the first days of imaginal life, then rapidly decreases until complete elimination of the bacteria by autophagic recycling. RNAseq analysis allowed the identification of signaling pathways linked to this dynamic. A complementary RT-qPCR approach also showed that bacteriome immunity was laid low during the whole recycling process. This work shows that several strategies have been selected during host-symbiont coevolution to ensure the maintenance of the endosymbionts and the adjustment of their population depending on the insects physiological needs: immunity allows the intracellular seclusion in the bacteriocytes, and cell processes including autophagy and apoptosis are associated to metabolic pathways to control the endosymbiotic dynamics and secure the cost and benefit trade-off of symbiosis.

The present thesis deals with the production of microbial enzymes and their subsequent stabilization through encapsulation. The theoretical part focuses on microbial enzymes, especially extracellular hydrolases, their producers and characteristics. Within the theory is also discussed the possibility of the application of enzymes in the field of pharmacy and medicine. Experimental work was focused on the actual production of microbial enzymes and methods for their to stabilization. The production of proteolytic and lipolytic enzymes in dependence on time and the used culture substrate were followed. The highest enzyme production was observed in Aspergillus oryzae when cultured on wheat bran at the third day of cultivation. In the experimental part was further carried out the identification, isolation and purification of enzymes. A substantial part of the experiment was to stabilize produced microbial enzymes by encapsulation. Enzymes were entrapped into alginate particles with encapsulation efficiency in the range of 55-70 %. The highest efficiency exhibited encapsulated enzymes from Aspergillus oryzae. Subsequently, long-term stability of the encapsulated enzyme in two environments (in water and gel) was followed during six weeks incomparison with free enzyme. During storage of free enzyme a significant decrease in enzyme activities occured, especially between the fourth and sixth week of storage. On the contrary, in encapsulated increased enzyme activities were observed. Empty particles exhibited higher stability during storage in the gel than in water. In this thesis potential use of enzymes in the pharmaceutical industry as agents promoting digestion was tested too. According to the results, particles with encapsulated microbial enzymes could be considered as suitable for some pharmaceutical applications.

Bread waste represents a significant portion of global food waste, necessitating innovative approaches for its valorization. This research project explores the utilization of bread waste through fermentation with Aspergillus oryzae to produce fungal film which could be used for textile applications. While previous studies have examined various applications of food waste, this project specifically targets the textile industry, aiming to mitigate pollution associated with conventional textile manufacturing. The objective of this project was to investigate the feasibility of creating fungal films derived from bread waste. Additionally, to analyze the material's properties through assessments of tensile strength, microscopic analysis, and the identification of an appropriate methodology for this investigation. The biomass suspension was prepared using an ultrafine grinder, and a kitchen blender was subsequently employed to minimize the presence of remaining solids from the grinding process. Additionally, a range of strategies for film casting and wet laying were implemented and evaluated. Wet laying involved combining fungal biomass with tannin to mimic the characteristics of leather. On the other hand, casting utilized pre-treated biomass suspension to assess the formation and quality of the films. As the research progressed and different tannins were used to treat the biomass, a methodology was developed, and glycerol was introduced as a plasticizer. Furthermore, nanocellulose was later incorporated exclusively for the casting of the films to serve as a binder. The films that were produced gave interesting results are observed in casted sheets containing nanocellulose and glycerol-infused biomass (3% BM + 2% Cellulose + 0.13 g Glycerol), exhibiting exceptional tensile strength (35.1 ± 3.42 MPa) and elongation (16.7 ± 5.98%). Wet laid biomass sheets treated with Tara and glycerol display tensile strength (19.9 ± 3.55 MPa) and elongation (6.66 ± 3.02%). These findings signify the potential for developing fungal films from bread waste, necessitating further research to refine methodologies. Overall, this research project paves the way for future advancements in fungal films derived from bread waste. By investigating the use of Aspergillus oryzae and employing wet laying and casting techniques, the project establishes a foundation for sustainable textile production. The successful utilization of bread waste not only addresses the issue of food waste but also contributes to reducing pollution in the textile industry.

Algunas plagas causan daños importantes en productos y granos almacenados, lo cual conlleva consecuentemente a pérdidas de producción y calidad en estos productos.Las principales plagas del arroz almacenado en España son, Sitophilus oryzae L. (Coleoptera Curculionidae), Rhyzopertha dominica Fabricius (Coleoptera: Bostrichidae), y Cryptolestes pusillus Schönherr (Coleoptera: Cucujidae). Las dos primeras son plagas primarias que atacan directamente el grano y resultan bastante destructivas debido a que sus larvas se alimentan y desarrollan dentro de él. C.pusillus es, sin embargo, una plaga secundaria que se beneficia de granos que ya están dañados y rotos.Actualmente, el uso de fumigantes e insecticidas de síntesis sigue siendo el principal método de lucha para controlar las plagas de almacén, si bien recientemente (dadas las continuas restricciones al uso de agroquímicos) existe un gran interés en la utilización de otras alternativas tales como el control biológico, el almacenamiento a bajas temperaturas, o los tratamientos con calor entre otros.Igualmente muchos productos obtenidos principalmente de plantas y que derivan del metabolismo secundario de las mismas ofrecen una fuente de bioinsecticidas que podrían representar una alternativa ecológica frente a los insecticidas de síntesis ya que su uso masivo e indiscriminado ha ocasionado problemas tales como la aparición de resistencias en determinadas especies de insectos frente a diferentes materias activas, desequilibrios ecológicos y problemas medioambientales sin olvidar el riesgo que entrañan para la salud humana.Con este trabajo se ha pretendido estudiar la actividad plaguicida de los aceites esenciales extraídos de tres plantas: Coriandrum sativum L. (Umbelliferae), Carum carvii L. (Umbelliferae) y Ocimum basilicum L. (Labiatae) y su posterior fraccionamiento para identificar dentro de estos aceites los compuestos químicos responsables de esta actividad insecticida sobre tres plagas de almacén de arroz (S. oryzae, R.dominica y C.pusillus).Del estudio de estos monoterpenoides, linalol, S-carvona y estragol resultaron tener una alta actividad insecticida sobre estas plagas. Sin embargo el E-anetol fue más selectivo para R.dominica y C.pusillus, así como el limoneno, γ-terpineno, geraniol y eucaliptol sólo resultaron activos frente a C.pusillus.Algunos monoterpenoides podrían actuar de sinergistas potenciando la actividad de otros, como podría ser el caso del alcanfor, acetato de geranilo y E-anetol con linalol en R.dominica y C.pusillus, o el caso del metoxicinamaldehido, p-anisaldehido y linalol que pueden tener efecto sinergista sobre el estragol.Este trabajo también abarcó el estudio de un posible modo de acción de estos monoterpenoides, la inhibición de la acetilcolinesterasa, para alcanzar un mayor entendimiento del comportamiento de estas sustancias en el interior del insecto.Así se pudo observar como la mayoría de monoterpenoides estudiados inhibían en cierta medida esta enzima, siendo fenchona, S-carvona y linalol los monoterpenoides que mayor inhibición originaron.Del mismo modo se observó como fenchona, γ-terpineno, geraniol y linalol inhibían competitivamente la acetilcolinesterasa, mientras que S-carvona, estragol y alcanfor producían una inhibición mixta para esta enzima.Sin embargo no se observó inhibición de la acetilcolinesterasa por parte del E-anetol a las concentraciones de monoterpenoides ensayadas.Para completar este trabajo se examinó de igual forma la capacidad que tenían estos bioinsecticidas de generar resistencia en estas tres plagas así como el mecanismo de resistencia implicado en el desarrollo de este proceso. Para ello se fueron seleccionando las poblaciones de insectos mediante la aplicación de los diferentes monoterpenoides a dosis crecientes durante 7 generaciones. De este modo se pudo calcular el factor de resistencia en cada una de las plagas y para cada uno de los monoterpenoides comparando las concentraciones letales 50 de las poblaciones seleccionadas con las poblaciones iniciales (sensibles).A continuación se analizaron tres posibles sistemas de detoxificación enzimáticos gracias al uso de sinergistas para estudiar el mecanismo de resistencia que podría estar involucrado.De tal forma se observó como estos monoterpenoides inducían lentamente resistencias resultando ventajosos en un futuro para el control de estas plagas.Esta Tesis ha englobado un estudio íntegro y profundo de estos insecticidas ecológicos desde la extracción de los aceites esenciales, seguido de la identificación de compuestos puros (CG-EM) con actividad insecticida junto con el estudio de un modo de acción de estos insecticidas, la selección de resistencia en las poblaciones de insectos y los posibles mecanismos de resistencia que pudieran estar implicados en este proceso. / Some pests cause serious damage to stored grains and stored products and consequently production and quality losses in these products.The rice weevil, Sitophilus oryzae L. (Coleoptera: Curculionidae), the lesser grain borer, Rhyzopertha dominica Fabricius (Coleoptera: Bostrichidae) and Cryptolestes pusillus Schönherr (Coleoptera: Cucujidae) were the main damaging pests found in stored rice in Spain.S.oryzae and R.dominica are primary pests attacking directly the intact grain and are quite destructive because their larvi feed and develop inside the grain whereas C.pusillus is a secondary pest which benefits from grains previously damaged.At the present time, organic synthetic pesticides are still the main method to control stored grain pests, however, recently (due to restriction in agrochemicals use) there is a great interest in using other altenatives such as biological control, storage at low-temperatures, or heat treatment.Likewise, many products obtained mainly from plants and derived from secondary metabolism have insecticidal activity against insects, such as monoterpenoids, which present a broad variety of bioinsecticide products which could turn out to be an ecologic alternative to synthetic pesticides since the majority of alternative products are not harmful for the human healthy and they become less environmentally damaging, exhibiting a low impact on the environment.In addition, it cannot be ignored the different difficulties related to resistance due to several active compounds from organic pesticides found in some species of insects.With this work, we have considered remarkable to study the insecticide activity of essential oils extracted from three plants: Coriandrum sativum L. (Umbelliferae), Carum carvii L. (Umbelliferae) and Ocimum basilicum L. (Labiatae) and carry out a bioassay-guided fractionation of their essential oils to identify which compounds were responsible for the volatile toxicity shown on three stored rice pests (S. oryzae, R. dominica and C. pusillus).Linalool, S-carvone and estragole turned out to have a high insecticide activity on these pests. Nevertheless E-anetol was more selective to R. dominica and C. pusillus, being only active on C. pusillus limonene, γ-terpinene, geraniol and eucalyptol.Some monoterpenoids were found as synergists, increasing the activity of the other ones, like for instance, camphor, geranyl acetate and E-anethole with linalool in R.dominica and C.pusillus or metoxycinnamaldehyde, p-anysaldehyde and linalool which could activate to estragole.In this work, the inhibition of acetylcholinesterase as a posible mode of action was studied as well, to reach a clear understanding about the action of these products inside the insects.The majority of monoterpenoids inhibited the enzyme acetylcholinesterase being fenchone, S-carvone and linalool the monoterpenoids that produced a higher inhibition.Furthermore, it was observed how fenchone, γ-terpinene, geraniol and linalool showed a competitive inhibition whereas S-carvone, estragole and camphor produced a mixed inhibition for this enzyme. However the enzyme acetylcholinesterase was not inhibited by E-anethole.To finish up this work, the selection for monoterpenoid resistance on these pests as well as the metabolic mechanisms implicated was studied.The resistant strains were selected from susceptible insect populations and survivors were reared separately for each monoterpenoid and successive generations were treated with higher concentrations. These populations were selected until seven times.As a result we could calculate the resistance factor on each pest (comparing lethal concentration 50 values of susceptible and resistant strains).Next, three enzymatic systems detoxifying these monoterpenoids were analysed to study the metabolic mechanism implicated.In this way we could realize that all monoterpenoids induced resistance slowly, concluding that these pesticides will be appropriated to control these pests in the future.This Thesis has concerned a study in depth about ecological insecticides from extraction of essential oils, identification of compounds (GC-MS) with insecticide activity, mode of action and study of resistance and mechanism of insecticide resistance involved in this process.

The dioxygenase-cytochrome P450 fusion proteins (DOX-CYP) comprise a heme-containing enzyme family that shares structural and catalytic properties with mammalian prostaglandin H (PGH) synthases. 7,8-Linoleate diol synthase (7,8-LDS) of Gaeumannomyces graminis was first characterized, and DOX-CYP enzymes are of mechanistic and biological interest. The growing number of fungal genome sequences has revealed DOX-CYP homologues in medically and economically important species. The aim of this thesis was to identify novel members of the DOX-CYP fusion protein family. The devastating rice pathogen Magnaporthe oryzae contains two DOX-CYP genes. The fungus synthesizes 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) by dioxygenation of linoleic acid to 8R-hydroperoxyoctadecadienoic acid (8R-HPODE), and subsequent isomerisation to the diol. 7,8-LDS of M. oryzae was identified by gene deletion, but the infection and reproduction processes of the Δ7,8-LDS strain were not altered. A mutant with constitutive protein kinase A activity profoundly changed the oxygenation profile, possibly due to post-translational modification. The human pathogens Aspergillus fumigatus and A. clavatus contain three DOX-CYP, designated psi producing oxygenase A (ppoA), ppoB, and ppoC, and form three oxylipins: 5S,8R-DiHODE, 8R,11S-DiHODE, and 10R-hydroxyoctadecadienoic acid.  PpoA was identified as 5,8-LDS, and ppoC as 10R-DOX. The 8,11-linoleate hydroperoxide isomerase activity was reduced by two imidazole-containing P450 inhibitors, miconazole and 1-benzylimidazole. PpoB could not be linked to the biosynthesis of 8,11-DiHODE for the following reasons: First, the 8,11-hydroperoxide isomerase activity was retained in A. fumigatus ΔppoB strains. Second, the P450 domain of the deduced ppoB of A. clavatus lacks a heme-thiolate cysteine ligand, presumably essential for hydroperoxide isomerase activity. Linoleate 9R-DOX activities of Aspergillus terreus and Lasiodiplodia theobromae were discovered. 9R-HPODE was further converted into unstable allene oxides, as judged by the accumulation of their hydrolysis products, α- and γ-ketols. These allene oxide synthase activities were specific for 9R-hydroperoxides. The 9R-DOX and AOS were found to have unique characteristics. In conclusion, novel DOX-CYP enzymes were identified in human and plant pathogenic fungi. These enzymes might be involved in biological processes, and show interesting catalytic similarities to human PGH synthase and thromboxane synthase (CYP5A).

Microbes live in complex environments where competitive and cooperative interactions occur that dictate their success and the status of their environment. By furthering our understanding of the interactions between microbes, questions into the evolution of cooperation, disease virulence and biodiversity can be addressed. This will help develop strategies to overcome problems concerning disease, socioeconomics and conservation. We use an approach that combines evolutionary ecology theory with genetics and molecular biology to establish and develop model microbial ecological systems to examine feeding strategies, in what has been termed synthetic ecology. Using the model fungal plant pathogen system of rice blast disease, we generated less virulent gene deletion mutants to examine the sociality of feeding strategies during infection and test a nascent virulence reduction strategy based on competitive exclusion. We revealed that the success of the pathogen is unexpectedly enhanced in mixed strain infections containing the virulent wild-type strain with a less virulent gene deletion mutant of the metabolic enzyme invertase. Our finding is explained by interference between different social traits that occur during sucrose feeding. To test the generality of our result, gene deletion mutants of putative proteases were generated and characterised. We found that if virulence related genes acted ‘privately’, as predicted by social theory, the associated mutants would not make viable strains to use for this virulence reduction strategy by competitive exclusion. Our study then went on to study the fitness of digesting resources extracellularly, as many microbes do, given that this strategy is exposed to social exploitation by individuals who do not pay the metabolic costs. This was investigated by developing an experimental system with Saccharomyces cerevisiae. Though internalising digestion could suppress cheats, the relative fitness of opposing strategies was dependent upon the environmental and demographic conditions. Using this polymorphic system, the influence of competitors on the stability of cooperation, and the influence of cheats on the maintenance of diversity were assessed. To test the fitness of internal versus external digestion in a more natural setting, we generated an internally digesting strain of the rice blast fungus. In addition to suppressing cheats, the strain had enhanced fitness and virulence over the wild-type. We propose that this is caused by a shift in a trade-off between yield and rate. We show how a synthetic ecology approach can capture details of the biology underlying complex ecological processes, while having control over the factors that drive them, so that the underlying mechanisms can be teased apart.

No description available.