Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-17T20:36:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_VivianePedrosoFausto.pdf: 2096881 bytes, checksum: c58d1ae11d22b250146714c3cf084b16 (MD5) / Made available in DSpace on 2018-08-17T20:36:49Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_VivianePedrosoFausto.pdf: 2096881 bytes, checksum: c58d1ae11d22b250146714c3cf084b16 (MD5) Previous issue date: 2017-04-28 / Listeria monocytogenes is a food-borne bacterium that causes an infection called listeriosis, Although rare, this infection can be lethal for immunocompromised, pregnant, and stillborn. The objective of this work was to develop, characterize, evaluate the stability, anti-Listeria monocytogenes activity, cytotoxicity and genotoxicity of nanostructures (nanocapsules and nanoemulsion) containing Geranium essential oil (OEG), as well as verify synergistic effects with the essential oils of Cinnamon, Lemongrass And Thyme. The characterization of the essential oils was performed by GCMS. The nanocapsules (NC) were prepared by the interfacial deposition method of the preformed polymer while Geranium nanoemulsion (NEG) was prepared by homogenization using an Ultra-Turrax T18. The analysis of the stability indicators was performed by the evaluation of particle size, polydispersity index, zeta potential and pH for 60 days. The bactericidal effect of the different concentrations of the essential oils, pure or in combination and the synergism were determined by the serial microdilution technique, the cytotoxicity was determined by the MTT assays and determination of the LDH in VERO cells and the genotoxicity was evaluated by the GEMO test. Results: The NCGs produced had mean nanometer diameters of (232.6 nm), polydispersity indices below 0.053, pH 3.5 and zeta potentials of about -20.7 mV. The study demonstrated that the stability of the formulations is temperature dependent, remaining more stable at refrigeration temperatures (4 °C) or at stable temperatures (25 ° C). The NEG had mean nanometer diameters of (136.2.6 nm), polydispersity indices below 0.039, pH 3.4 and zeta potentials of about -8.1 mV. The MICs obtained for Geranium, Cinnamon, Campi-lemon and Thyme oils were 10975 mg / ml, 0.05 mg / ml, 0.18 mg / ml and 0.35 mg / ml, respectively. The CIM of NEG was 0.0625 mg / ml and the nanocapsules did not demonstrate antimicrobial activity in the test performed. It was possible to observe at least one point of synergism between the OE of geraniums and the tested EOs.the nanostructures did not show synergistic effect with OE. In the MTT assay OEG and NEG showed increased cell viability at concentrations of 10, 100 and 1000 μg / μl as compared to the control, the LDH assay shows that both OEG and NEG at concentrations of 0,1, 1, 10, 100 and 1000 μg / μl were not cytotoxic. In the GEMO test the OEG had a genoprotective effect on lymphocytes at concentrations of 10, 100 and 1000 μg / μl. The study demonstrated that both OG and NEG do not present tocicity 7 and genotoxicity but present very high MIC values to be used in food, and the synergism and nanostructuring were a good alternative to reduce the amount of oil required for an antimicrobial response. / A Listeria monocytogenes é um microrganismo contaminante de alimentos causador da listeriose, uma infecção que embora rara pode ser letal para indivíduos imunodeficientes, gestantes e neonatos. O objetivo deste trabalho foi desenvolver, nanoestruturas contendo óleo essencial de Gerânio (OEG), caracterizar, avaliar a atividade contra L. monocytogenes, a cito e genotoxicidade, bem como verificar efeitos sinérgicos com os óleos essenciais de Canela, Capim-limão e Tomilho. A caracterização dos óleos essenciais foi realizada por GC-MS. As nanocápsulas (NC) foram preparadas pelo método de deposição interfacial do polímero pré-formado enquanto a nanoemulsão de Gerânio (NEG) foi preparada por homogeneização utilizando um T18 Ultra-Turrax. A análise dos indicadores de estabilidade foi realizada pela avaliação do tamanho de partícula, índice de polidispersão, potencial zeta e pH por 60 dias. O efeito bactericida das diferentes concentrações dos óleos essenciais e o sinergismo foram determinados pela técnica de microdiluição seriada, a citotoxicidade foi determinada pelos ensaios de MTT e LDH e a genotoxicidade foi avaliada pelo teste GEMO. Resultados: As NCG produzidas apresentaram diâmetros nanométricos médios de (232,6 nm), índices de polidispersão abaixo de 0,053, pH de 3,5 e potenciais zeta de cerca de -20,7 mV. O estudo demonstrou que a estabilidade das formulações são mais estáveis em temperatura ambiente (25 ºC). A NEG apresentou diâmetros nanométricos médio de (136,2 nm), índices de polidispersão abaixo de 0,039, pH de 3,4 e potenciais zeta de cerca de -8,1 mV. As CIM obtidas para os óleos de Gerânio, Canela, Campi-limão e Tomilho foram de 10975 mg/ml, 0,05 mg/ml, 0,18 mg/ml e 0,35 mg/ml respectivamente. A CIM da NEG foi de 0,0625 mg/ml e as nanocápsulas não demonstraram atividade antimicrobiana no teste realizado. Foi possível observar pelo menos um ponto de sinergismo entre o OEG os OE testados. As nanoestruturas não apresentaram efeito sinérgico com os OE. No ensaio de MTT o OEG e a NEG apresentaram um aumento da viabilidade celular nas concentrações de 10, 100 e 1000 μg/μl quando comparado com o controle. O ensaio de LDH mostrou que tanto o OEG quanto a NEG nas concentrações de 0,1, 1, 10, 100 e 1000 μg/μl não foram citotóxicas. No teste GEMO o OEG apresentou um efeito genoprotetor sobre os linfócitos nas concentrações de 10, 100 e 1000 μg/μl. O presente estudo demonstrou que tanto o OEG quanto a NEG não apresentaram toxicidade e genotoxicidade porém apresentam valores de CIM muito altos para poder serem utilizados em alimentos, podendo ser o sinergismo e a nanoestruturação uma boa alternativa para reduzir a quantidade de óleo necessária para uma resposta antimicrobiana.

Philosophiae Doctor - PhD / African medicinal plants are commonly used throughout Africa to treat a variety of ailments including wounds and ulcers, cough and chest complaints, gingivitis, fever and gonorrhoea, indication all related to infection and inflammation. In screening several plant species from an inventory of common medicinal plants from both South and West Africa for diverse medicinal purposes, 6 plants were selected because of their interesting and useful ethnomedicinal values. / South Africa

The western North American species of the genus Geranium have never been satisfactorily clarified. The perennial, indigenous species of this interesting group have been particularly confusing. One important reason for confusion in this group is the fact that the taxonomic probems of the perennial species have never been studied from the genetic point of view. A sound delimitation of specific and subspecific boundaries and phylogenetic relationships cannot be achieved without the application of cyto-genetic principles. In respect to the cytological phase of this study, the author has placed emphasis on chromosome numbers in order to show evidences for and possible origin of polyploidy. Detailed chromosome morphology has not been attempted because of the very small size of the chromosomes. This study has been limited to the Wasatch region. this area respresents a natural geographical unit which is small enough to permit a detailed field study of the group and yet provide a wide range of habitats, both ecologic and geographic. This region forms the western front of the Rocky Mountain province and extendes 200 miles south from the great bend in the Bear river at Soda springs, Idaho, to the pass south of Mt. Nebo and east of Nephi, Utah.

Ivy geranium (Pelargonium peltatum) is a heat susceptible species with its heat tolerance varying among varieties. Reactive oxygen species (ROS) and in-vivo defense systems are related to plant heat damage and heat tolerance. Application of chelated-iron has also been reported to enhance ivy geranium heat tolerance; however, the correlation of ROS, relative enzyme stability, and iron content to differences in heat tolerance in ivy geraniums is unknown. Here we show that the H2O2 content and ROS scavenging enzyme stability in ivy geranium varies with varieties and active iron is not related to heat tolerance in ivy geranium. H2O2 content in mature leaves in both heat tolerant 'Beach' and sensitive 'Butterfly' increased under heat stress, but 'Butterfly' had a relatively greater increase of this toxic compound. Catalase (CAT) activities in young leaves in both varieties decreased. In young leaves of 'Butterfly', CAT activities decreased to a level significantly lower than that in old leaves while this did not occur in 'Beach'. Superoxide dismutase (SOD) activities in 'Butterfly' young leaves were also decreased. All these phenomenon coincided with the heat tolerance differences of the two varieties. Active iron content only changed with leaf age and did not vary between varieties or treatments. Our results demonstrated that ROS scavenging ability and relative enzyme stability may indicate heat tolerance in ivy geranium and that iron deficiency was not the cause of heat damage. Cell Membrane Themostability (CMT) and Triphenyl Tetrazolium Chloride (TTC) cell viability tests are alternative, laboratory-based screening methods for screening for heat-tolerance. Both CMT and TTC tests can represent the variance in heat tolerance observed in ivy geraniums. The results of both CMT and TTC tests correlated well with plant width and growth indexes although their correlations to plant chlorosis were low. Unlike TTC, CMT strongly correlated with plant width. CMT and TTC tests are complementary laboratory-based methods that can be applied to cultivar screening for heat tolerance in ivy geraniums.

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Field experiments (2007-2009) and laboratory bioassays (2009) tested the efficacy of insecticides with short pre-harvest intervals, caneberry cultivar susceptibility, and geranium toxicity for reducing Japanese beetle (JB) activity on primocane-bearing caneberries. Deltamethrin, chlorantraniliprole, bifenthrin, lime-alum, and thyme oil reduced JB activity in the field. Deltamethrin, chlorantraniliprole, acetamiprid, an azadirachtin and pyrethrin mixture, an azadirachtin and neem oil extract mixture, and an extract of Chenopodium ambrosioides reduced JB activity during the bioassays. "Prelude" had significantly more JB than "Anne", "Caroline", "Heritage", "Dinkum", or "Himbo Top" and "Prime-Jan" had significantly more JB than "Prime-Jim". Compared to certain cultivars, "Heritage", "Caroline", "Himbo Top", and "Prime-Jan" had higher percentages of injured fruit and "Autumn Bliss", "Heritage", and "Caroline" produced greater marketable and overall yields. "Prime-Jan" produced more overall yield than "Prime-Jim"; marketable yields from both blackberry cultivars were similar. Defoliation was significantly less for "Dinkum", "Caroline", "Heritage", and "Anne" than for "Prelude" in 2008 and significantly less for "Caroline" and "Anne" than "Prelude" or "Fall Gold" in 2009. In field tests, previous consumption of geraniums lessened raspberry defoliation by JB. Bioassays indicated that JB activity was only reduced if JB were continually exposed to geranium. Therefore, the efficacy of geranium as a trap crop for JB may be limited. The stink bug species within the caneberries were identified (2008-2009) and Euschistus servus (Say) made up 48.1 % of the overall species composition. Stink bug injury to ripening raspberries was identified as small holes between drupelets; stink bug excretions also ruined fruit. / Master of Science in Life Sciences

Thesis (M.S.)--Montana State University--Bozeman, 2004. / Typescript. Chairperson, Graduate Committee: Roger L. Sheley. Includes bibliographical references (leaves 26-30).

Abstract Most plants grow in association with arbuscular mycorrhizal (AM) fungi in their roots forming the so-called AM symbiosis. AM symbiosis is usually beneficial to the host as it improves plant survival and performance. However, AM symbiosis also entails a cost to the plant in terms of the carbon allocated to the fungus. In sexually dimorphic plants, more than one type of individual can be recognised with regard to their sexual expression or gender. The cost of reproduction in these plants will differ in relation to the relative investment in male versus female function, as the female and the male sexual functions incur different costs. This different cost of reproduction may be translated into differences in other plant functions between the sexes as all functions are connected through trade-offs. Therefore, since sexes differ in resource needs and allocation patterns, and AM mediate resource acquisition and allocation patterns through imposing both costs and benefits to the plant, the sexes of dimorphic plant species may possess, at least theoretically, a different relationship with their AM roots symbionts. In this thesis, I have investigated whether the sexes in sexually dimorphic plant species differ in their mycorrhizal relationship, and if so, in which ways. Several plant life history traits were studied in the dioecious species Antennaria dioica and also in the gynodioecious Geranium sylvaticum using greenhouse, common-garden and field experiments. Resource acquisition, resource allocation, and both plant and fungal benefits from AM symbiosis were considered. Mainly beneficial effects of AM symbiosis were observed in both sexes of the two dimorphic plant species for most of the studied plant life history traits. Overall, both partners benefited from the AM association. However, several sex-specific benefits were detected which were not uniformly present in all experiments for any given trait. Moreover, the responses observed in certain life history traits were dependent on both the AM fungal and plant species involved in the symbiosis. Remarkably, plants gained sex-specific benefits from the same species of AM fungi and the fungal benefit differed depending on the sex of the host plant. In addition, mycorrhizal benefits were lost under certain environmental conditions. To summarise, the results obtained in this study highlight the complexity of AM interactions. My results suggest that the plant-mycorrhizal fungus relationship may differ depending on the sex of the host plant. Through sex-specific effects on survival, growth and reproduction of the hosts, AM fungi may play a role in the evolution of the life histories in the studied species. In addition, sex-specific relationships between plants and their mycorrhizal symbionts may have potential important consequences for the population dynamics of the sexual morphs and the coevolution of the mycorrhizal relationship.

Pelargonium sp., appelé aussi « géranium » à odeur de rose ou « Géranium rosat » est l’une des plantes aromatiques et médicinales les plus cultivées au niveau international, essentiellement pour son huile essentielle (HE), utilisée par les industries des cosmétiques et de la parfumerie. Cette essence est extraite des feuilles par distillation vapeur et donne une HE riche de plusieurs centaines de molécules volatiles. Cette complexité est le résultat d’un long processus évolutif et de sélections variétales. Parmi ces composés volatils, les monoterpènes comme le géraniol, le citronellol et l’isomenthone, ou les sesquiterpènes comme le 10- γ-épi-eudesmol et le 6,9-guaiadiène, jouent un rôle prépondérant dans le parfum du Pelargonium. Les proportions relatives de ces différents composés sont d’ailleurs utilisées comme marqueurs de la qualité de l’HE et déterminent la typicité du parfum des différents cultivars et origines (P. cv. ‘rosat Bourbon’, P. cv. ‘rosat Chine’, P. cv. ‘rosat Égypte’ et P. cv. ‘rosat Grasse’). Malgré de très nombreux travaux portant sur la chimie de cette HE, il n’existe aucune information sur les voies de biosynthèse de ces molécules et aucun gène intervenant dans ces voies n’a été isolé. Durant cette thèse, nous avons cloné et caractérisé fonctionnellement par expression et purification des protéines recombinantes chez Escherichia coli des gènes codant les enzymes clés de ces voies de biosynthèse, les terpène synthases. Nous avons ainsi pu caractériser quatre terpène synthases, dont une géraniol synthase mono-produit. Nous avons isolé deux autres monoterpène synthases multi-produits, produisant pour l’une majoritairement du myrcène mais aussi trois autres monoterpènes, et pour l’autre majoritairement du 1,8-cinéole ainsi que 10 autres monoterpènes minoritaires. Enfin, une sesquiterpène multi-produit, la 10-γ-épi-eudesmol synthase, a été caractérisée. Nous avons ensuite analysé l’expression de la géraniol synthase et de la 10-γ-épi-eudesmol synthase dans différentes accessions de Pelargonium par RT-qPCR et nous avons montré la relation entre la capacité de production des différents composés volatils et le niveau d’expression dans les feuilles de ces deux terpène synthases. L’efficacité de la transformation génétique du Pelargonium par Agrobacterium tumefaciens étant élevée, des expériences de transgénèse ont aussi été réalisées afin de compléter la caractérisation fonctionnelle des gènes isolés. Dans une deuxième partie, nous avons réalisé l’analyse des essences produites par 64 espèces et cultivars de Pelargonium d’odeurs très diverses (citron, menthe, rose, abricot, pin, épices…). A l’aide d’analyses statistiques (ACP, analyse discriminante…), nous avons mis en évidence des relations entre la biochimie de ces cultivars, leurs odeurs et leurs proximités génétiques et cela afin de nous donner des pistes sur des croisements potentiellement intéressants. Enfin, un dernier chapitre est consacré à l’amélioration de la production d’HE en Égypte. Grâce à ce programme commencé il y a trois ans, nous améliorons chaque année la qualité et le rendement en HE de plus de 10 Ha de plantation de Pelargonium en Égypte. Un travail d’optimisation de la distillation ainsi que des améliorations des pratiques culturales, nous ont permis de produire une HE de qualité avec un rendement de plus de 60 kg.Ha-1 d’HE. D’autres expériences présentées dans ce chapitre soulignent l’influence de l’environnement et notamment de la température sur le ratio entre le citronellol et le géraniol ainsi que sur la biosynthèse de l’isomenthone, du 10-γ-épi-eudesmol et du 6,9-guaiadiène / Pelargonium sp, also named rose scented « geranium » or « Geranium rosat » is one of the the most cultivated aromatic and medicinal plant worldwide, especially for its essential oil (EO), which is used by cosmetic and perfumery industries. This essence is extracted from leaves by steam distillation and gives an EO containing several hundreds of organic volatile compounds (VOC). This complexity is the result of a long evolutive process and varietal selections. Among these VOC, the monoterpenes like geraniol, citronellol and isomenthone and the sesquiterpenes like 10-γ-epieudesmol and 6,9-guaiadiene, play an important role for the Pelargonium fragrance. The relative proportions of these compounds are used as EO quality markers and determine the different cultivars origins (P. cv. ‘rosat Bourbon’, P. cv. ‘rosat Chine’, P. cv. ‘rosat Egypt’ and P. cv. ‘rosat Grasse’). Despite the important researches on the chemistry of these EO, there is no information on the biosynthesis pathways for these molecules and no genes involved in the pathways have been isolated. During this PhD thesis, we have functionally characterized by recombinant proteins expression and purification in Escherichia coli, four genes, three monoterpene and one sesquiterpene synthases, coding for key enzymes in terpene biosynthesis pathway. The first enzyme is a mono-product geraniol synthase. The second enzyme is a multi-product enzyme with a major peak of myrcene and 3 minor peaks of other monoterpenes. The third enzymes is also a multi-product protein, producing 1,8-cineol as major product and 10 others monoterpenes. The last one is a multi-products sesquiterpene synthase producing mainly the 10-γ-epi-eudesmol and other sesquiterpenes. We have also analyzed the level of expression of the geraniol and 10 γ-epi-eudesmol synthases in several Pelargonium accessions by RT-qPCR and we have demonstrated the relationship between the level of expression of these two terpene synthases and the quantity of the related terpenes produced in leaves. Pelargonium transformation efficiency by Agrobacterium tumefaciens was tested in order to complete the functional characterization of the genes. In a second part, we have analyzed the essence of 64 species and cutivars of Pelargonium having very different fragrances like lemon, mint, rose, apricot, pine, spices… With different statistical tools (PCA, discriminant analysis…), we have highlighted the links between the biochemistry of these species and cultivars, their odors and their phylogenetic relationships. This worked gave us some interesting ideas for some new crossings. Finally, the last chapter concerns the EO production improvements in Egypt. Thanks to these researches, started 3 years ago, we are improving year after year our EO yield and quality in our 10 Ha R&D plantation. An important work was done to optimize the distillation process and improve the agricultural practices which abled us to reach a yield of 60 kg of EO per hectare. Some other experiments show the effect of the environmental factors such as the temperature on the biosynthesis of several important molecules like citronellol and geraniol, 6,9-guaiadiene and 10-γ-epi-eudesmol

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