Diversos estudos epidemiológicos têm demonstrado que isoprenóides presentes em frutas e hortaliças possuem ação protetora contra o desenvolvimento de câncer. Objetivou-se, no presente trabalho, avaliar as eventuais atividades quimiopreventivas dos isoprenóides geraniol (GO) e beta-ionona (BI) quando administrados a ratos Wistar, isoladamente ou em associação, continuamente durante as etapas de iniciação e pós-iniciação de modelo de carcinogênese de cólon induzida por dimetilhidrazina. A atividade quimiopreventiva desses compostos foi observada microscopicamente por meio da análise de Focos de Criptas Aberrantes (FCAs) em cólons corados com azul de metileno (0,02%). Verificou-se que, em comparação ao grupo controle apenas os ratos tratados com GO, mas não com BI, apresentaram redução (p≤0,05) do número de lesões pré-neoplásicas (FCAs), no cólon distal, tanto em relação ao número de focos totais quanto aqueles com número maior ou igual do que quatro criptas. Por outro lado, somente tratamento com BI foi capaz de reduzir a concentração de colesterol plasmático total. Posteriormente, raspagem da mucosa colônica foi utilizada para a avaliação de danos no DNA pelo método do \"Cometa\". Em comparação ao grupo controle, tratamentos com GO, BI e GO+BI reduziram (p≤O,05) o comprimento dos cometas indicando, dessa forma, diminuição de danos no DNA. Avaliou-se, ainda, a expressão de Bcl-2, em nível de proteína, em mucosas colônicas dos animais dos diferentes grupos pela técnica Western Blot. Foi possível notar uma redução nos valores da expressão pelos tratamentos com GO (p≤0,05). Embora somente o tratamento com GO tenha inibido a formação de FCAs, tratamentos com BI e GO+BI podem ter apresentado efeitos protetores por inibirem os danos do DNA que não foram suficiente para a inibição da formação e crescimento de lesões pré-neoplásicas. / Several epidemiologic studies have shown consistently that isoprenoids presents in fruits and vegetables have a protective action against cancer. The objective of this study was to evaluate the chemopreventive effects of isoprenoids geraniol (GO) and beta-ionone (BI) during the initiation and pós-initiation phases of in rats submitted to model of chemical colon carcinogenesis induced by dimethylhydrazine (DMH). Aberrant Crypt Focus (ACFs) were detected using a light microscope in colons stained with 0,02% methylene blue. Treatment with GO significantly suppressed the total number of ACF and ACFs ≥ 4 crypts per colon compared with the control group com oil (p≤0,05), mainly in the distal colon. In other hand, only treatment with BI reduced total plasma cholesterol levels compared with the control group (p≤0,05). Subsequently colonic mucosa scraping was used for asses DNA damage through comet assay and expressed as comet length. DNA damage was inhibited in our study by all treatments (p≤0,05). Analysis of Bcl-2 protein from colonic mucosa scraping by Western Blot showed that treatment with GO reduced the values of expression de Bcl-2 (p≤0,05). Although the results of this study suggest that treatment with GO has chemopreventive effects, treatments with BI and GO+BI has initial protective action that maybe wasn\'t sufficient to reduce formation ACF.

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

Le principal objectif de ce travail de thèse était d’étudier l’influence de la fonctionnalisation des protéines par des sucres ou des polyphénols de raisin dans la formulation et le comportement de microsphères de vitamine A. La formulation de différents conjugués issus soit de la réaction de Maillard soit de la complexation des polyphénols sur les protéines a été effectué sur trois matières premières protéiques : les protéines de pois, le caséinate de sodium de lait de vache et la gélatine de type A porcine. Dans une première partie, les caractéristiques et le pouvoir émulsifiant des conjugués ont été étudiés, et ont confirmé le potentiel de stabilisation d’une huile dans le temps. Une seconde partie s’est concentrée sur les observations au microscope électronique à balayage des microsphères et sur une méthodologie d’observation spécifique à ce genre d’échantillon. Une troisième partie a étudié l’influence des fonctionnalisations sur la stabilité de la vitamine A dans le temps, sur sa libération dans des milieux de digestion gastriques et entériques simulés, et sur la libération de géraniol co-encapsulé. La dernière étude a porté sur le potentiel muco-adhésif des microsphères en utilisant une technique d’analyse originale. / The main objective of this thesis was to study the influence of functionalization of proteins by sugars or grape polyphenols in the vitamin A microsphere formulation and behavior. The formulation of different conjugated stemming either from the Maillard reaction or from the complexation of polyphenols on proteins was made on three proteins : pea proteins, sodium caseinate and type A gelatin. In a first part, the characteristics and the emulsifying power of the combined were studied, and confirmed the potential of stabilization of oil in the time. A second part was on the observations with scanning electron microscope of microspheres and on the methodology of specific observation in this kind of sample. The third part studied the influence of functionalization on vitamin A stability in the time, liberation on gastric or enteric digestion media, and liberation of co-encapsulated geraniol. The last study concerned the muco-adhesive potential of microspheres by using an original analysis.

Introducing effective irrigation management in arid and semi-arid regions, like most areas of South Africa, is an indispensable way of maximising crop yield and enhancing productivity of scarce freshwater resources. Holistic improvements in agricultural water management could be realised through integrating the knowledge of crop-specific water requirements. In order to develop effective irrigation schedules for rose-scented geranium (Pelargonium capitatum x P. radens), greenhouse and field experiments were conducted at the Hatfield Experimental Farm of the University of Pretoria, Pretoria, South Africa, from 28 October 2004 to 2006. Results from 20, 40, 60 and 80% maximum allowable depletion (MAD) levels of the plant available soil water (ASW) indicated that plant roots extracted most of the soil water from the top 40 cm soil layer, independent of the treatment. Both essential oil yield and fresh herbage mass responded positively to high soil water content. Increasing the MAD level to 60% and higher resulted in a significant reduction in herbage mass and essential oil yields. An increase in the degree of water stress apparently increased the essential oil concentration (percentage oil on fresh herbage mass basis), but its contribution to total essential oil yield (kg/ha oil) was limited. There was no significant relationship between MAD level and essential oil composition. For water saving without a significant reduction in essential oil yield of rose-scented geranium, a MAD of 40% of ASW is proposed. Response of rose-scented geranium to a one-month irrigation withholding period in the second or third month of regrowth cycles showed that herbage mass and oil yield were positively related. Herbage yield was significantly reduced when the water stress period was imposed during the third or fourth month of regrowth. A remarkable essential oil yield loss was observed only when the plants were stressed during the fourth month of regrowth. Essential oil content (% oil on fresh herbage mass basis) was higher in stressed plants, especially when stressed late, but oil yield dropped due to lower herbage mass. The relationship between essential oil composition and irrigation treatments was not consistent. Water-use efficiency was not significantly affected by withholding irrigation in the second or in the third month of regrowth. With a marginal oil yield loss, about 330 to 460 m3 of water per hectare per regrowth cycle could be saved by withholding irrigation during the third month of regrowth. The overall results highlighted that in water-scarce regions withholding irrigation during either the second or the third month of regrowth in rose-scented geranium could save water that could be used by other sectors of society. In greenhouse pot experiments, rose-scented geranium was grown under different irrigation frequencies, in two growth media. Irrigation was withheld on 50% of the plants (in each plot) for the week prior to harvesting. Herbage and essential oil yields were better in the sandy clay soil than in silica sand. Essential oil content (% oil on fresh herbage mass basis) apparently increased with a decrease in irrigation frequency. Both herbage and total essential oil yields positively responded to frequent irrigation. A one-week stress period prior to harvesting significantly increased essential oil content and total essential oil yield. Hence, the highest essential oil yield was obtained from a combination of high irrigation frequency and a one-week irrigation-withholding period. In the irrigation frequency treatments, citronellol and citronellyl formate contents tended to increase with an increase in the stress level, but the reverse was true for geraniol and geranyl formate. Leaf physiological data were recorded during the terminal one-week water stress in the glasshouse pot trial. Upon rewatering, stomatal conductance (Gs) and transpiration rate (Rt) were significantly lower in the less often irrigated than in the more often irrigated treatments, while leaf water potential (yw) and relative water content (RWC) were the same for all plants, indicating that water stress had an after-effect on Gs and Rt. At the end of the stress period, Gs, Rt, yw and RWC were lower in the plants from the more often irrigated than from the less often irrigated treatments. Irrespective of irrigation treatment, one type of non-glandular and two types (different in shape and size) of glandular trichomes were observed. In water stressed-conditions, stomata and trichome densities increased, while the total number of stomata and trichomes per leaf appeared to remain more or less the same. Water stress conditions resulted in stomatal closure. / Thesis (PhD)--University of Pretoria, 2009. / Plant Production and Soil Science / unrestricted

The work described in this thesis focuses on the effects that adsorption processes on catalytic surfaces pose in controlling key steps that can affect and control reaction pathways. To that, the development of Nuclear Magnetic Resonance (NMR) relaxometry methods and the comparison with traditional catalytic was performed with a series of C5 and C6 unsaturated hydrocarbons on two different alumina supports, γ- and θ-Al2O3. The developed techniques were applied in the study of liquid phase selective hydrogenation of citral on 5% Pt/SiO2. Infrared (IR) spectroscopy, volumetric adsorption isotherms, dynamic isotherms via a Tapered Element Oscillating Microbalance (TEOM), temperature programmed desorption (TPD) as well as 13C T1 NMR and 1H 2D T1-T2 relaxometry methods were employed. Energies of adsorption as a function of coverage were obtained via adsorption isotherms and the particular surface adsorbate interactions were described with IR spectroscopy. For example, 1-pentyne showed the strongest interaction with the alumina (94 kJ mol-1) while 1-pentene presented a weaker interaction (46 kJ mol-1) on θ-Al2O3. Desorption energies obtained from TPD ranged 85 – 130 kJ mol-1, irrespective of the adsorbate. Reactivity of the aluminas was captured with TPD, TEOM and NMR relaxometry. Interaction of adsorbates with hydrocarbon occurred predominantly on weak adsorption sites. 13C NMR T1 relaxometry provided in addition atom-specific adsorbate-adsorbent interaction strengths, showing the molecular geometry of adsorption, and applied in co adsorption measurements. The selective hydrogenation of citral as a model α,β-unsaturated aldehyde and the effect of different solvents on the activity and product distribution was studied at 298 and 373 K. A series of polar protic, polar aprotic and non polar solvents was investigated. Results showed higher initial reaction rates in non polar solvents but higher selectivities towards desired products on polar protic solvents. Solvent used also affected by product formation. The strong variations in reaction rates and selectivities reported were related with adsorbate catalyst interactions, as well as solvent reactant interactions. For example, adsorption isotherms showed that ethanol notably reduced the adsorption capacity of citral as compared with hexane, related with the rate differences observed. ATR-IR measurements indicated solvent citral interactions were solely present in polar protic solvents in line with higher yields of geraniol and nerol. Finally, 13C T1 NMR and 1H 2D T1-T2 correlation experiments determined that the geometry of adsorption of citral, influenced by solvent, affected product selectivity, and that product adsorption affected selectivity and deactivation.