8 leaves of plates in pocket / Bibliography: leaves 148-154 / 154 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--Dept. of Organic Chemistry, University of Adelaide, 1983

Orientador: Gláucia Maria Pastore / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-25T05:52:01Z (GMT). No. of bitstreams: 1 Molina_Gustavo_D.pdf: 62790925 bytes, checksum: aac4f7324e48e27f7a1b9fdc0102ecd7 (MD5) Previous issue date: 2014 / Resumo: Este trabalho de doutorado teve como principal objetivo estudar a produção biotecnológica de compostos de aroma a partir da biotransformação de terpenos, analisando potenciais biocatalisadores, processos bioquímicos diferenciados e a otimização das condições de processo envolvidas na produção destes aromas naturais para possíveis aplicações industriais. Inicialmente, o trabalho prático foi direcionado para uma série de estudos com o fungo Fusarium oxysporum 152B, visando complementar os recentes avanços alcançados pelo grupo de pesquisa na caracterização e otimização da bioconversão de R-(+)-limoneno em ?-terpineol. Desta forma, os resultados foram promissores, demonstrando o grande potencial e versatilidade deste fungo para a área, sendo que este biocatalisador foi capaz de produzir diferentes metabólitos a partir dos substratos S-(?)-limoneno, ?-pineno, ?-terpineno e linalol. Na sequência, o trabalho visou avaliar a bioconversão de S-(?)-limoneno para limoneno-1,2-diol em maiores detalhes. A produção de limoneno-1,2-diol, em condições não otimizadas, chegou a 1,2 g.L-1, sendo que foi detectada a continuação desta via metabólica quando este produto foi posteriormente metabolizado a 1-hidroxi-2- oxolimoneno, sugerindo que o fungo Fusarium oxysporum 152B possui uma via de degradação de limoneno recentemente descoberta. O trabalho prático visou também a otimização da produção de limoneno-1,2-diol por meio de uma estratégia sequencial de experimentos. Com base nas análises estatísticas, a produção deste composto a partir da bioconversão de S-(?)-limoneno pelo fungo Fusarium oxysporum 152B chegou a 3,7 g.L-1, utilizando pH 6,5, 5 g.L-1 de substrato, a 28 oC e 250 rpm de agitação. Além disso, esta foi a primeira descrição da utilização do resíduo agroindustrial conhecido como manipueira, originada ao longo do processamento da mandioca, para a produção de biomassa deste fungo, bem como uma das maiores concentrações de limoneno-1,2-diol reportadas na literatura específica. Além disso, o trabalho conduzido visou realizar uma série de comparações práticas entre a biotransformação de R-(+)-limoneno a ?-terpineol e a bioconversão de S-(?)-limoneno a limoneno-1,2-diol, pela mesma linhagem. Adicionalmente, este foi o primeiro trabalho que analisou as diferenças ultraestruturais causadas no biocatalisador ao longo do processo de bioconversão destes substratos, por meio de microscopia eletrônica de varredura e transmissão. Finalmente, foi estudada a otimização da produção de ?-terpineol, a partir da biotransformação de limoneno, utilizando o biocatalisador reconhecido como Sphingobium sp. Após o trabalho prático e análise estatística dos dados, observou-se que as melhores condições para desenvolvimento deste processo foram pH 7,0, concentração de limoneno de 350 g.L-1, agitação de 200 rpm e 28 oC. Nestas condições, a produção deste álcool monoterpênico chegou a 500 g.L-1, que pode ser considerada como a maior concentração de ?-terpineol já relatada na bibliografia de processos biotecnológicos. / Abstract: This work aimed to study the biotechnological production of aroma compounds from the biotransformation of terpenes, analyzing potential biocatalysts, biochemical processes and optimization of process conditions involved in the production of natural flavors for industrial applications. Initially, the practical work was directed to a series of studies with the fungal strain Fusarium oxysporum 152B, aiming to enhance the knowledge obtained in recent advances achieved by our research group in the characterization and optimization of the biotransformation of R-(+)-limonene into ?-terpineol. Thus, the results were promising, showing the great potential and versatility of this fungus to the specific area, and this biocatalyst was able to produce new metabolites from substrates S-(?)-limonene, ?-pinene, ?-terpinene and linalool. In the sequence, this study aimed at assessing the bioconversion of S-(?)-limonene into limonene-1,2-diol in greater detail. Thus, chapters 3, 4 and 5 were designed to a better characterization of this pathway. The production of this compound under non-optimized conditions, reached 1.2 gL-1, and the continuation of this pathway has been detected when this product was subsequently metabolized to 1-hydroxy-2-oxolimonene, suggesting that the fungus Fusarium oxysporum 152B might have a limonene degradation pathway only recently discovered. The practical work also conducted an extensive optimization of the production of limonene-1,2-diol by means of a sequential strategy. Based on statistical analyzes, the production of this compound from the bioconversion of S-(?)-limonene by the fungus Fusarium oxysporum 152B reached 3.7 gL-1, using pH 6.5, 5 gL-1 of substrate at 28 ° C and 250 rpm agitation. Moreover, this is the first description of the use of agroindustrial residue known as cassava wastewater, for the production of this fungal biomass, as well as one of the highest concentration of biotechnological limonene-1,2-diol reported in the specific literature. In addition, the research conducted aimed to perform a series of comparisons between practical conditions involved in the biotransformation of R-(+)-limonene into ?-terpineol, and the bioconversion of S-(?)-limonene into limonene-1,2-diol, for the same strain. Additionally, this is the first study that reported the ultrastructural differences along the bioconversion process of these substrates by means of scanning and transmission electron microscopy. Finally, this work evaluated the optimization of the production of ?-terpineol from the biotransformation of limonene using the biocatalyst recognized as Sphingobium sp. After the practical work and statistical analysis, it was observed that the best conditions for developing this process were pH 7.0, concentration of limonene 350 g L-1, agitation at 200 rpm and 28 oC. Accordingly, the production of the monoterpe alcohol reached 500 g.L-1, which can be considered as the highest concentration of ?-terpineol already reported in the literature for biotechnological processes. / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos

Orientador: Marcos José Salvador / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T12:16:35Z (GMT). No. of bitstreams: 1 Feltran_GabrielPrimini_M.pdf: 2195659 bytes, checksum: ca442329b76d3b823e9a0079209f4252 (MD5) Previous issue date: 2014 / Resumo: Os monoterpenos são uma importante classe de produtos naturais com odor e sabor intenso e que contém muitas moléculas que apresentam atividade biológica, incluindo a atividade antimicrobiana, e aplicações na indústria farmacêutica, cosmética e alimentícia. Entretanto, são substâncias lipofílicas, voláteis e apresentam certa instabilidade química. Ciclodextrinas (CDs) são carreadores macrocíclicos, capazes de complexar, aumentar a solubilidade em água e estabilizar um largo espectro de substâncias e que, atualmente, têm sido objeto de pesquisas e de desenvolvimento de novos produtos pela indústria farmacêutica. Neste trabalho procedeu-se um estudo comparativo da complexação de quatro monoterpenos (citral, ?-mirceno, limoneno e 1,8-cineol) em ?CD e HP-?CD, bem como a avaliação biológica desses compostos, complexados ou não. Após essas avaliações realizou-se o desenvolvimento de um enxaguante bucal contendo o monoterpeno mais promissor no combate ao biofilme dentário. Para a caracterização físico-química do complexo de inclusão monoterpeno-CD foram utilizadas técnicas espectroscópicas (UV-Vis), cromatográficas (CG-EM) e calorimétricas (DSC), avaliando a solubilidade dos complexos em água e porcentagem de inclusão dos monoterpenos em CD. Foi realizado um screening da atividade antimicrobiana dos quatro monoterpenos em estudo frente à quatro cepas de bactérias (Staphylococcus aureus ATCC 6538; Pseudomonas aeruginosa ATCC 27853; Escherichia coli ATCC 10538 e Streptococcus mutans ATCC 25175) e quatro cepas de fungos (Candida albicans ATCC 10231; Candida dubliniensis ATCC 22019; Candida tropicalis ATCC 157 e Candida parapsilosis ATCC 22019). Essa avaliação foi realizada através da determinação da Concentração Inibitória Mínima (concentração que inibiu 50% do desenvolvimento microbiano, CIM) e Concentração Biocida Mínima (concentração que inibiu em 100% o desenvolvimento microbiano, CBM). O monoterpeno citral mostrou-se mais efetivo (CBM citral = 2,5µL/mL) frente à cepa de S. mutans (ATCC 25175). A partir desses resultados o citral foi o monoterpeno escolhido para dar continuidade aos estudos propostos no projeto. Avaliou-se o efeito do citral isoladamente, complexado e incorporado em formulações de enxaguante bucal, frente ao crescimento e aderência de S. mutans (ATCC 25175) em superfície sólida. Avaliou-se, também, a citotoxicidade do citral e de algumas formulações de enxaguante bucal verificando-se baixa citotoxicidade frente a cultura de queratinócitos humanos (HaCat). Tanto o citral em sua forma livre como complexado em CDs apresentou atividade frente a cepa indicadora de S. mutans (ATCC 25175), impedindo a formação do biofilme in vitro em concentração sub-inibitória (CBM/2 = 1,25µL/mL). Ao avaliar o citral e seus complexos de inclusão incorporados nas formulações de enxaguante bucal, observou-se manutenção do efeito antibacteriano in vitro frente a cepa de S. mutans (ATCC 25175) e uma melhor solubilidade do complexo de inclusão citral-HP?CD se comparado ao monoterpeno não complexado em CD / Abstract: Monoterpenes are an important class of natural products with intense flavor and odor and can exhibit biological activity including antimicrobial activity, and applications in the pharmaceutical, cosmetic and food industry. However, monoterpenes are lipophilic and volatile substances which demonstrate some chemical instability. Cyclodextrins (CDs) are macrocyclic carriers capable of complexing a wide range of substances and increase their water solubility currently been the subject of research and development of new products for the pharmaceutical industry. In this project a comparative study was carried complexing four monoterpenes (citral, ?-myrcene, limonene and 1,8-cineole) in ?-cyclodextrin and hydroxypropyl-?-cyclodextrin, and biological evaluation of such monoterpenes, complexed or not. After these assessments was performed to develop a mouthwash containing the most promising monoterpene to combat dental biofilm. For physico-chemical characterization of the inclusion complex monoterpene-CD spectroscopic (UV-Vis), chromatographic (GC-MS) and calorimetric (DSC) were used to evaluate the solubility of the complex in water and percentage of inclusion of these monoterpenes in cyclodextrin. Antimicrobial activity screening for the monoterpenes was conducted across four strains of bacteria (Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 10538 and Streptococcus mutans ATCC 25175) and four strains of fungi (Candida albicans ATCC 10231, Candida dubliniensis ATCC 22019, Candida tropicalis ATCC 157 and Candida parapsilosis ATCC 22019). This evaluation was performed by determining the minimum inhibitory concentration (concentration that inhibited 50% of microbial growth, MIC) and Minimum Biocide Concentration (concentration that inhibited 100% microbial development, MBC). The monoterpene citral was more effective (MBC citral = 2.5?L/mL) against the strain of S. mutans (ATCC 25175). From these results the monoterpene citral was chosen to continue the studies in the proposed project. Were evaluated the effect of citral alone, complexed and incorporated into mouthwash formulations, to the growth and adherence of S. mutans (ATCC25175) on a solid surface. It was also assessed the cytotoxicity of citral and some mouthwash formulations afford low cytotoxicity against human keratinocytes (HaCat) cell culture. Both citral and citral complexed with CDs presented activity against the indicator strain of S. mutans (ATCC25175), preventing the formation of biofilms in vitro in the sub-inhibitory concentration (MBC/2 = 1.25?L/mL). When evaluating citral and their inclusion complexes embedded in mouthwash formulations, the in vitro antibacterial effect against S. mutans (ATCC 25175) was maintained and an increased solubility of the inclusion complex citral-HP?CD compared to monoterpene uncomplexed with CD / Mestrado / Fármacos, Medicamentos e Insumos para Saúde / Mestre em Ciências

Accurate simulations of the global radiative impact of anthropogenic emissions must employ a tropospheric chemistry model that predicts realistic distributions of aerosols of all types. The need for a such a comprehensive yet computationally efficient tropospheric chemistry model is addressed in this research via systematic development of the various sub-models/mechanisms representing the gas-, aerosol-, and cloud-phase chemistries. The gas-phase model encompasses three tropospheric chemical regimes - background and urban, continental rural, and remote marine. The background and urban gas-phase mechanism is based on the paradigm of the Carbon Bond approach, modified for global-scale applications. The rural gas-phase chemistry includes highly condensed isoprene and a-pinene reactions. The isoprene photooxidation scheme is adapted for the present model from an available mechanism in the literature, while an a-pinene photooxidation mechanism, capable of predicting secondary organic aerosol formation, is developed for the first time from the available kinetic and product formation data. The remote marine gas- phase chemistry includes a highly condensed dimethylsulfide (DMS) photooxidation mechanism, based on a comprehensive scheme available in the literature. The proposed DMS mechanism can successfully explain the observed latitudinal variation in the ratios of methanesulfonic acid to non-sea-salt sulfate concentrations. A highly efficient dynamic aerosol growth model is developed for condensing inorganic gases. Algorithms are presented for calculating equilibrium surface concentrations over dry and wet multicomponent aerosols containing sulfate, nitrate, chloride, ammonium, and sodium. This alternative model is capable of predictions as accurate for completely dissolved aerosols, and more accurate for completely dry aerosols than some of the similar models available in the literature. For cloud processes, gas to liquid mass-transfer limitations to aqueous-phase reactions within cloud droplets are examined for all absorbing species by using the two-film model coupled with a comprehensive gas and aqueous-phase reaction mechanisms. Results indicate appreciable limitations only for the OH, HO₂, and NO₃ radicals. Subsequently, an accurate highly condensed aqueous-phase mechanism is derived for global-scale applications. / Ph. D.

Les réactions de cycloisomérisation d’énynes catalysées par les métaux de transition sont de puissants outils en synthèse organique: elles donnent accès, en une seule étape économe en atomes, à une variété de structures intéressantes mono- ou bicycliques, comportant des motifs 1,4-diène, 1,3-diène, cyclobutène ou cyclopropane. Afin d’accéder à de nouvelles molécules dérivées de monoterpènes, des 1,6- et 1,7-énynes à pont oxygène ont été préparés à partir de l’alcool péryllique, du nérol et de l’isopulégol, et leur réactivité dans les réactions de cycloisomérisation catalysées par des sels ou des complexes des métaux de transition a été étudiée. De nouvelles molécules bi- et tricycliques contenant des motifs cyclopropane, 1,3- ou 1,4-diènes ont ainsi été synthétisées. Toutes ces molécules ont été purifiées et caractérisées par RMN techniques. Différents systèmes catalytiques décrits dans la littérature ont été comparés : PtCl2, AuCl3, [AuCl(PPh3)/AgPF6] et Rh2Cl2(CO)4. Nous avons ainsi pu montrer que la nature du produit de la réaction dépendait surtout de la structure de l’ényne initial mais aussi du catalyseur mis en jeu. De plus nous avons mis en évidence que la cinétique et la sélectivité pouvaient être modifiées lorsque les réactions étaient faites sous une atmosphère de CO. Dans un second temps, une version asymétrique de la réaction a été explorée dans le cas de l’1,6- ényne dérivé de l’alcool péryllique, en préparant des complexes du platine ou de l’or portant des ligands phosphorés chiraux. L’excès diastéréoisomérique observé sur les deux cyclopropanes obtenus a ainsi pu être augmenté dans le cas des complexes de l’or (I), mais les valeurs restent encore modestes. Enfin, nous avons entrepris une étude préliminaire de la réaction de cycloisomérisation en milieu liquide ionique dans le but de concevoir un système efficace de recyclage du catalyseur. Différents liquides ioniques et différents solvants d’extraction ont été testés pour la réaction avec l’1,6-ényne dérivé de l’alcool péryllique. L’activité et la sélectivité des différents catalyseurs se sont avérées modifiées par rapport à la réaction dans le solvant toluène et des problèmes de reproductibilité sont apparus. Cette étude devra être poursuivie afin d’optimiser les conditions de réaction et de recyclage. / The transition metals catalyzed cycloisomerization reactions of enynes are a powerful tool in organic synthesis: they give access, in only one atom-economic step, to a variety of interesting mono- or bicyclic structures, comprising 1,4-diene, 1,3-diene, cyclobutene or cyclopropane moieties. In order to reach new molecules derived from monoterpenes, O-tethered 1,6- and 1,7- enynes were prepared starting from perillyl alcohol, nerol and isopulegol, and their reactivity in cycloisomerization reactions catalyzed by transition metals salts or complexes was studied. New bi- and tricyclic molecules containing cyclopropane, 1,3- or 1,4-dienes moieties were thus synthesized. All these molecules were purified and characterized by NMR techniques. Various catalytic systems described in the literature were compared: PtCl2, AuCl3, [AuCl(PPh3)/AgPF6] and Rh2Cl2(CO)4. We thus could show that the nature of the final product depended especially on the starting enyne structure but also on concerned catalyst. Moreover we highlighted that kinetics and selectivity could be modified under CO atmosphere. Analogously, an asymmetrical version of the reaction was explored in the case of the 1,6-enyne derived from perillyl alcohol by preparing platinum or gold complexes with chiral phosphorated ligands. The observed diastereoisomeric excess on two obtained cyclopropanes thus could be increased in the case of gold (I) complexes, but the values remain still modest. Finally, we undertook a preliminary study of the cycloisomerization reaction in ionic liquid with an aim of conceiving an effective system for catalyst recycling. Various ionic liquids and different extraction solvents were tested for 1,6-enyne derived from perillyl alcohol. The activity and the selectivity of various catalysts proved, resulted modified compared to the reaction in toluene and reproducibility problems appeared. This study will have to be continued in order to optimize the conditions of reaction and the catalysts recycling.

La rose est l'une des plantes ornementales les plus populaires, dont les composés volatils sont non seulement impliqués dans les interactions des fleurs avec l’environnement au sens large, mais aussi largement utilisés dans l’industrie des arômes et parfums. Le chapitre 1 décrit l'histoire de la culture de la rose, les usages de son parfum, les connaissances actuelles sur la biosynthèse des composés de ce parfum, ainsi que les voies de biosynthèse des composés volatils qui ont été récemment élucidées chez différentes plantes. Les chapitres expérimentaux 2 et 3 analysent les fonctions de deux gènes exprimés dans les pétales de rose. Ils codent pour des protéines Nudix hydrolase 1 (NUDX1). Le gène NUDX1-1 (nommé RhNUDX1 dans la publication) a été découvert en comparant les transcriptomes de deux cultivars de rose, Rosa x hybrida cv. 'Papa Meilland' (PM) très parfumé et R. x hybrida cv. 'Rouge Meilland' (RM), dépourvu de parfum. Le gène RhNUDX1-1 n'est exprimé que chez PM et son expression est corrélée avec la production de monoterpènes dans les pétales, en particulier de géraniol. Lors de l'étude d'une descendance issue du croisement de R. chinensis cv. ‘Old Blush’ (OB) et de R. x wichurana (Rw), le gène orthologue RcNUDX1-1a, présentant la même fonction, a été caractérisé chez OB. Un gène paralogue, RwNUDX1-2, a été découvert chez Rw et il a été démontré que son expression présentait une corrélation avec la production sesquiterpènes, en particulier de E,E-farnesol. Une série d'analyses in vitro et in vivo ainsi qu'une analyse de corrélation ont permis de vérifier la fonction de RhNUDX1-1, qui hydrolyse le géranyl diphosphate (GPP) en géranyl monophosphate (GP). Une phosphatase non identifiée pourrait catalyser la transformation du GP en géraniol. Des expériences de fusion avec la Green Fluorescent Protein (GFP), suivies de transformation transitoire de feuilles de tabac, ont révélé que RhNUDX1-1 était localisée dans le cytoplasme. Les mêmes approches (analyses QTL, essais enzymatiques et expression transitoire) ont également été appliquées à RwNUDX1-2, démontrant sa fonction dans la production de E,E-farnesol. La cartographie de RwNUDX1-2 et la localisation subcellulaire de la protéine sont encore à l'étude. De plus, la cristallographie des protéines et la modélisation ont été employées pour étudier le mécanisme de l'interaction NUDX1-substrat et les acides aminés potentiellement importants pour la reconnaissance du substrat. Collectivement, ces données révèlent une voie alternative pour la biosynthèse des terpènes, en particulier le géraniol et E,E-farnesol, via l'hydrolyse des prényl diphosphates par les enzymes NUDX1. Nos résultats montrent que la production de composés volatils dans les pétales est fortement corrélée avec l’expression des gènes des voies de biosynthèse. Par conséquent, la régulation transcriptionnelle de RcNUDX1-1a et RwNUDX1-2 joue probablement un rôle important dans la production de parfum. Les promoteurs de RcNUDX1-1a, RcNUDX1-1b, et RwNUDX1-2 et deux facteurs de transcription (FT), RcbHLH79 (OB TF) et RwbHLH79 (Rw TF) ont ainsi été isolés et testés (Chapitre 4). Les FT candidats ont été choisis lors d’une analyse RNA-Seq (Chapitre 5). En utilisant des tests d'expression transitoire avec le gène rapporteur GUS (β-glucuronidase) dans les pétales de rose, il a été montré que les trois promoteurs pouvaient entraîner l'expression de GUS. Les deux FT ont ensuite été introduits dans des feuilles de tabac avec les promoteurs testés, pour voir s'ils étaient capables d'activer ces promoteurs. Aucune transactivation significative n'a été détectée, même si Rw TF semblait pouvoir activer une construction témoin (promoteur du gène de la tomate TPS5. Les transcriptomes de quatre cultivars de rose, dont deux produisent du géraniol mais pas de E,E-farnesol et deux autres produisent du E,E-farnesol mais pas de géraniol, ont été analysés (Chapitre 5) et ont abouti à une liste de FT putatifs pour une étude plus approfondie / Roses are one of the most popular ornamental plants, whose volatiles are not only involved in environmental interactions but also widely used for industries. Chapter 1 describes the cultivation history of roses, usages of rose fragrance, knowledge on the biosynthesis of rose scent compounds, as well as non-canonical biosynthesis pathways of other plant volatiles. Experimental chapters (Chapter 2 and 3) analyse the functions of two genes expressed in rose petals, both encoding Nudix hydrolase 1 (NUDX1) protein. NUDX1-1 gene (named RhNUDX1) was first discovered by comparing the transcriptomes of two rose cultivars, the scented Rosa x hybrida cv. ‘Papa Meilland’ (PM) and the unscented R. x hybrida cv. ‘Rouge Meilland’ (RM). RhNUDX1-1 was only expressed in scented PM and its expression exhibited a positive correlation with the monoterpenoid production in petals, especially geraniol. When studying a rose progeny of R. chinensis cv. ‘Old Blush’ (OB) and R. x wichurana (Rw), an orthologous gene RcNUDX1-1a was found in OB, whose expression also had positive correlation with geraniol emission. A paralogous gene in Rw, RwNUDX1-2, was discovered and it was shown that its expression displayed a correlation with the sesquiterpenoid production, especially E,E-farnesol. A series of in vitro and in vivo assays as well as correlation analyses verified the function of RhNUDX1-1, which hydrolysed geranyl diphosphate (GPP) to geranyl monophosphate (GP). The transformation of GP into geraniol is supposed to be processed by an, as yet, unidentified phosphatase. The prediction of the localisation together with green fluorescent protein (GFP) fusion experiments revealed that RhNUDX1-1 was located in the cytosol. A series of approaches (QTL analyses, enzymatic assays and transient expression studies) were also applied to RwNUDX1-2, demonstrating its function in the production of E,E-farnesol. Mapping of RwNUDX1-2 and subcellular localization of the protein are still under investigation. Furthermore, protein crystallography and protein modelling illustrated the NUDX1-substrate interaction and proposed several residues that may be important for substrate recognition, although further experimental and computational data are required to gain more insight into the enzymatic mechanism. Collectively, these data revealed an alternative pathway for the biosynthesis of terpenoids, especially geraniol and E,E-farnesol, in rose, via the hydrolysis of prenyl diphosphates by NUDX1 enzymes. Transcriptional regulation of RcNUDX1-1a or RwNUDX1-2 probably plays an important role in the scent production by rose petals. Therefore, three promoters, pOB1a (promoter of RcNUDX1-1a), pOB1b (promoter of RcNUDX1-1b, not expressed in rose petals), pRw (promoter of RwNUDX1-2) were cloned and tested (Chapter 4). In addition, two transcription factors (TFs), RcbHLH79 (OB TF) and RwbHLH79 (Rw TF) candidates were chosen via RNA-Seq analysis as their expression correlated with expression of RcNUDX1-1a or RwNUDX1-2, respectively (Chapter 5). Using transient expression assays with a reporter gene, β-glucuronidase (GUS) in rose petals, it was shown that all three promoters could drive the expression of GUS, suggesting that all of them are active. However, quantification of promoter activities is still needed. OB TF and Rw TF were introduced into Nicotiana benthamiana leaves together with the promoters driving GUS , to determine if they were able to activate these promoters. However, no significant transactivation was detected in any promoter-TF combination. The expression of the TF in the progeny was also analysed but, due to the similarity of the sequences of family members, no conclusive data were obtained. Transcriptomes of the petals four roses, two of which produce geraniol but not E,E-farnesol and two that produce E,E-farnesol but not geraniol, were analysed (Chapter 5) and this resulted in a list of putative scent related genes and transcription factors for further study

This study identified new compounds with pest control activities. The two sources of candidates that were followed here were the main heartwood extract of Alaska Yellow Cedar (AYC) constituents and several semi-synthetic counterparts. Five compounds were isolated and identified for the first time in AYC heartwood in this research: two monoterpenes, two sesquiterpenes, and one lignan. The two monoterpenes were (1S)-2-oxo-3-p-menthenol (41) and (4R)-4-hydroxy-4-isopropyl-cyclohex-1-enecarboxylic acid (63). The two sesquiterpenes were (5S,7R,10R,11R)-eudesm-4(14)-ene-11,12-diol (46) and (4R,5S,7R)-1(10)- eremohpilen-11,12-diol (59). The lignan was (1R,2S,5R,6S)-2,6-bis-(3,5- dimethoxy-4-hydroxyphenyl)-3,7-dioxabicyclo-[3.3.0]octane,(67). Structures for these compounds were confirmed on the basis of spectroscopic techniques such as 1- and 2-D NMR, high resolution MS and IR. The pest control activity studies of 15 compounds isolated or semi-synthesized from AYC heartwood were conducted at the Centers for Disease Control and Prevention (CDC). Two types of studies were conducted--short-term (24h) and residual (over 1-4 weeks) activity for application against three types of pests related to human health - nymphal I. scapularis ticks, adult X cheopis fleas and adult Ae. eagypti mosquitoes. The 24 h studies revealed that nootkatone, valencene-13-aldehyde and valencene-13-ol were the most active among the studied compounds against the three pests. They exhibited highly improved pest control activities compared to valencene. This suggests that oxidation on both positions C-2 and C-13 of the eremophilane ring structure has an important effect on the activity. For compounds where the conformation of the eremophilane bicyclic ring has been altered, the activity seemed to diminish greatly. The above mentioned three compounds can be good candidates as pest control lead compounds. The residual studies revealed that the most active compounds exhibited activity profiles that generally decreased with time. Although the long-term safety of these compounds has yet to be evaluated, the natural origin and the long history of use of these compounds suggest that they can be promising candidates. This study revealed that the three most promising compounds in the 24 h study exhibited reasonably promising behavior, which makes them even stronger as pest control candidates. / Graduation date: 2004

Lavenders (Lavandula) are widely grown for their essential oils, which have extensive applications in cosmetics, hygiene products and alternative medicine. The therapeutic and olfactory properties of lavender essential oils are attributed to monoterpenes, a class of low molecular weight (C₁₀) isoprenoids. Oil composition in these plants is primarily determined by plant genotype, but can also be influenced by developmental and environmental factors. In order to define some of the mechanisms that control monoterpene abundance in lavenders, I measured the abundance of quality-defining monoterpenes in several L. angustifolia and L. x intermedia cultivars grown in the Okanagan. Data obtained confirmed that essential oil yield, as well as the abundance of camphor, borneol, linalool, and limonene was species-specific. L. angustifolia cultivars contained high amounts of linalool but yielded little oil, whereas L. x intermedia cultivars were rich in camphor and total oil. Monoterpene abundance changed during flower development, and differed between vegetative and reproductive tissues indicating differential regulation of the biosynthetic pathways, or specialized ecological functions. The abundance of linalool correlated with the transcription of the linalool synthase gene, suggesting that linalool production is in part regulated transcriptionally. However, the degree of correlation between linalool abundance and linalool synthase transcription differed between L. angustifolia and L. x intermedia, suggesting additional, and differing mechanisms that control linalool abundance in these species. In addition, monoterpene abundances were subject to loss during storage and suboptimal detection, two factors that must be considered in future analyses. Results obtained in this study provide insight into the regulation of monoterpene production in lavenders, and build the basis for future research aimed at improving essential oil production in these plants.

This PhD thesis aims to improve the knowledge on the processes and chemical species in the gas- and particle-phases that are involved in the production of secondary organic aerosol (SOA) from monoterpene oxidation in the atmosphere. A denuder/filter technique that enabled the simultaneous sampling of gaseous and particulate compounds was applied in the present study. The sampling technique was comprehensively characterised and optimised using twelve atmospherically relevant carbonyl compounds. The present study improved the denuder coating procedure and the sampling performance. An additional coating with the derivatisation reagent, 2,4‑dinitrophenylhydrazine (DNPH), reduced the break-through potential (e.g., from 98% to 0.9% for methyl vinyl ketone) and the fraction of carbonyl compounds on the filter material (e.g., from 8.7% to 0% for acetone). Calibration experiments against an aerosol chamber were performed to reduce the relative standard deviation (RSD) of the calibration points in the denuder measurements. The RSDs were reduced by half for acetone, acetaldehyde, methyl vinyl ketone, glyoxal, benzaldehyde and campholenic aldehyde using a XAD‑4/DNPH denuder, and the quantification error was also reduced. This sampling technique was then applied to a series of α- and β-pinene ozonolysis experiments. The present study examined the influence of an OH radical scavenger (CO), and hence the HO2/RO2 ratio, on the SOA formation, product distribution and partitioning behaviour of selected oxidation products in conjunction with different seed particle acidities. It was shown that SOA yields increased by about 8% in α-pinene ozonolysis when CO and acidic seed particles co-existed, whereas only a marginal difference was observed (increase of 2%) for β-pinene compared to neutral seed particles. From the denuder/filter sample analysis, it was possible to tentatively identify a new compound from the α-pinene ozonolysis, i.e., terpenylic aldehyde. Gas- and particle-phase yields were estimated for the first time for this compound (i.e., 1% and 0.4%, respectively). The atmospheric relevance of terpenylic aldehyde was demonstrated based on ambient filter measurements and a possible formation pathway was suggested. Furthermore, the present study provided an additional explanation for enhanced SOA formation when acidic seed particles are used in monoterpene ozonolysis. It was demonstrated that the isomerisation of monoterpene oxides on acidic seed particles leads to the formation of highly reactive SOA precursors, whose subsequent reaction with ozone contributes significantly to SOA formation.

Lavenders (Lavandula) are widely grown for their essential oils, which have extensive applications in cosmetics, hygiene products and alternative medicine. The therapeutic and olfactory properties of lavender essential oils are attributed to monoterpenes, a class of low molecular weight (C₁₀) isoprenoids. Oil composition in these plants is primarily determined by plant genotype, but can also be influenced by developmental and environmental factors. In order to define some of the mechanisms that control monoterpene abundance in lavenders, I measured the abundance of quality-defining monoterpenes in several L. angustifolia and L. x intermedia cultivars grown in the Okanagan. Data obtained confirmed that essential oil yield, as well as the abundance of camphor, borneol, linalool, and limonene was species-specific. L. angustifolia cultivars contained high amounts of linalool but yielded little oil, whereas L. x intermedia cultivars were rich in camphor and total oil. Monoterpene abundance changed during flower development, and differed between vegetative and reproductive tissues indicating differential regulation of the biosynthetic pathways, or specialized ecological functions. The abundance of linalool correlated with the transcription of the linalool synthase gene, suggesting that linalool production is in part regulated transcriptionally. However, the degree of correlation between linalool abundance and linalool synthase transcription differed between L. angustifolia and L. x intermedia, suggesting additional, and differing mechanisms that control linalool abundance in these species. In addition, monoterpene abundances were subject to loss during storage and suboptimal detection, two factors that must be considered in future analyses. Results obtained in this study provide insight into the regulation of monoterpene production in lavenders, and build the basis for future research aimed at improving essential oil production in these plants.