Dissertations / Theses on the topic 'Butanoate'

L'auteur decrit les spectres raman de l'ion butanoate, associe au cation potassium et de trois isotopomeres, 4-d#3, 3-d#2 et 2-d#2, en solution aqueuse et a l'etat solide. L'influence de la temperature et de la concentration sur ces spectres raman a ete etudiee. Utilisant un champ de force approximatif, et s'appuyant sur l'evolution des spectres en fonction de la temperature, l'auteur propose une attribution des bandes observees pour les conformeres gauche et trans. Puis par application d'une methode originale de restitution des spectres des constituants a partir de ceux de leurs melanges, l'auteur a pu isoler les spectres des formes gauche et trans pour les quatre varietes isotopiques, en utilisant le fait que l'equilibre gauche-trans se deplace par variation de la concentration. En definitive, entre 100 et 1350 cm##1, 55 frequences sont attribuees avec certitude pour la forme trans de l'ensemble des isotopomeres et 59 pour la forme gauche. Enfin, on montre que dans le conformere trans, le groupement carboxylate s'oriente dans le plan de la chaine, alors que dans le conformere gauche l'angle diedre que fait le groupement carboxylate avec le plan des noyaux de carbone 1, 2 et 3 est superieur a 0, ce qui confirme la formation d'agregats aux concentrations elevees

Ce travail porte sur l'étude de l'impact environnemental des esters méthyliques utilisés comme biodiesel et concerne plus particulièrement la cinétique de formation des oxydes d'azote (NOX). Les objectifs de ce travail de thèse visent (i) à étudier la cinétique d’oxydation d’un ester méthylique saturé, le Butanoate de Méthyle (MB), afin de disposer une base de données expérimentales en condition de flamme laminaire de pré-mélange, (ii) et tester des mécanismes cinétiques détaillés de l'oxydation du MB disponibles dans la littérature sur la formation du NO précoce. Pour prendre en compte la chimie de l’azote, nous avons ajouté à ces mécanismes un sous-mécanisme de formation du NO récemment validée au laboratoire PC2A. Cinq flammes CH4/MB/O2/N2 ont été stabilisées à basse pression (5,3 kPa) avec des quantités connues d'ester (0%, 20% et 50% dans le mélange combustible). Les mélanges étudiés sont caractérisés de manière à évaluer l'effet du facteur de richesse et du rapport C/O sur la formation de NO. Les profils d’espèces ont été mesurés par couplage de techniques in situ de spectroscopie laser (Fluorescence Induite par Laser, LIF) et de techniques analytiques après prélèvement des gaz (Chromatographie en Phase Gazeuse (CPG), spectroscopie Infra Rouge à Transformée de Fourrier (IRTF)). Les résultats expérimentaux montrent que la substitution du CH4 par le MB dans la flamme CH4/air diminue la fraction molaire de NO. Cette diminution est plus importante lorsque la richesse diminue par rapport à la flamme de CH4/air. Les profils expérimentaux ont été confrontés aux profils simulés issus de trois modèles cinétiques détaillés, indiquant des variations notables d’un modèle à l’autre. Il a été observé que le modèle de Dooley et al. (2008) donne des accords assez satisfaisants en comparaison avec les résultats expérimentaux. L’analyse des voies réactionnelles a permis de mettre en évidence les réactions prépondérantes de la consommation du MB et celles impliquées dans la formation du NO précoce<br>This work is focused on the study of the environmental impact of methyl esters used as biodiesel and concerns more particularly the kinetic of nitrogen oxides formation in flame conditions. The aim of this PhD is (i) to study the kinetics of oxidation of a methyl ester saturated, as Methyl Butanaote (MB), in order to have an experimental database on condition of laminar premixed flame, (ii) to test detailed kinetic mechanisms of oxidation of MB available in the literature on the formation of prompt-NO. To account for the nitrogen chemistry, we added these mechanisms a sub-mechanism of NO formation recently validated in PC2A laboratory. Five flames CH4/MB/O2/N2 have been stabilized at low pressure (P = 5.3 kPa) with known amounts of ester (0%, 20% and 50% in the fuel mixture). The mixtures studied are characterized so as to evaluate the effect of the equivalence ratio and the C/O ratio on NO formation. The species profiles were measured by coupling laser spectroscopy techniques in situ (Laser Induced Fluorescence (LIF)) and analytical techniques after gas probe sampling through a quartz microprobe (Gas Chromatography (GC), Fourier Transform Infrared Spectroscopy (IRTF)). The experimental results show that the substitution of CH4 by MB in the CH4/air flame decreases the mole fraction of NO. This reduce is higher when the equivalence ratio decreases compared to the stoichiometric CH4/air flame. The experimental profiles were compared with profiles modeled from three detailed kinetic models, showing significant variations from one model to another. It was observed that the model of Dooley et al. (2008) gives quite satisfactory agreements compared with experimental results. The analysis of reaction pathways allowed to highlight the predominant reactions in consumption of MB and those involved in the formation of prompt NO

En raison de la complexité de leur composition, l’étude de l’oxydation des gazoles et des carburants biodiesel nécessite de choisir des molécules modèles représentant ces mélanges. Dans ce contexte nous avons sélectionné deux molécules pouvant représenter les gazoles : le n-décane, souvent considéré comme molécule modèle des paraffines contenues dans les gazoles, et le n-hexadécane, molécule de référence pour l’estimation de l’indice cétane, ainsi que deux molécules représentant les carburants biodiesel : le palmitate de méthyle (C17H34O2, ester méthylique saturé) et l’oléate de méthyle (C19H36O2, ester méthylique insaturé). L’étude de l’oxydation de ces molécules a été menée en réacteur auto-agité par jets gazeux, à une richesse de 1, des températures comprises entre 550 et 1100 K, à pression atmosphérique et à un temps de passage constant de 1,5 s. La formation d’un nombre important d’espèces a été observée parmi lesquelles figurent des oléfines, des diènes, des esters méthyliques insaturés, des éthers cycliques avec différentes tailles de cycle, des cétones et des aldéhydes. Grâce à deux nouvelles versions du logiciel EXGAS, des mécanismes cinétiques détaillés de l’oxydation des molécules étudiées ont été générés et validés par comparaison avec les résultats expérimentaux. Enfin, une comparaison de la réactivité du n-décane, du n-hexadécane, du palmitate de méthyle et de l’oléate de méthyle et des quantités de produits formées (dont certains polluants) a été effectuée<br>Because of the complexity of their compositions, the study of the oxidation of diesel and biodiesel fuels requires choosing model molecules (surrogates) representing the real mixtures. In this context, we have selected two molecules to represent the diesel fuel: n-decane, usually considered as model molecule of paraffin contained in diesel fuel, and n-hexadecane, molecule of reference for the estimation of the cetane number, and two molecules representing biodiesel fuel: methyl palmitate (C17H34O2, a saturated methyl ester) and methyl oleate (C19H36O2, an unsaturated methyl ester). The study of oxidation of these molecules has been conducted in a jet-stirred reactor, with an equivalence ratio of 1, temperatures between 550 and 1100 K, at atmospheric pressure and for a constant residence time of 1.5 sec. The formation of a large number of species has been observed which includes olefins, dienes, unsaturated methyl esters, cyclic ethers with different size of ring, ketones and aldehydes. Using two new versions of EXGAS software, detailed kinetic mechanisms for the oxidation of the studied molecules were generated and validated by comparaison with experiemental results. Finally, a comparison of the reactivity of n-decane, n-hexadecane, methyl palmitate and methyl oleate and amounts of formed products (including some pollutants) has been performed

The focus of this thesis is the role of the solvent and scale-up upon rate and selectivity in heterogeneous catalysed hydrogenations, which are ubiquitous in the production of fine chemicals and pharmaceuticals. A kinetic method has been developed based on rigorous statistical methods and sensitivity analysis for the catalytic hydrogenation of 4-phenyl-2- butanone in stirred tank reactors at two different scales using Pt/TiO2 and Pt/SiO2 catalysts. In this thesis, modelling carried out for a 100 mL scale reactor was validated against experimental data supplied by Queens University Belfast (QUB). Experimental measurements of rate and selectivity and model validation at a larger 3000 mL scale were both carried out as part of this study. The models were evaluated over a wide range of operational conditions at both scales for Pt/TiO2 catalyst, and by using a systematic kinetic methodology it was possible to identify the dominant reaction route, derive physiochemically meaningful kinetic data and a reduced kinetic model that was applicable to the scale-up. Comprehensive kinetic analysis made it possible to gain some insight into the shift in reaction mechanism upon scale-up. Kinetic investigation of solvent effects was also carried out at the 100 mL scale for a range of solvents (protic, aprotic polar, apolar, ethers, and halogens) and both catalysts, again tested against experimental data supplied by QUB. The dominant effects of solvent on rate and selectivity of the chosen reaction system were identified as the degree of active site availability imposed by competitive adsorption of solvent on catalyst and the extent of which the solvents assist the product desorption from catalyst surface. The solvent effects upon scale-up give the remarkable result of a significant shift in the selectivity of the catalyst towards phenyl ring and ketone hydrogenation groups of 4-phenyl-2-butanone.

La synthèse de dérivés de 4-(méthylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) que nous décrivons se divise en deux parties. La première consiste en la synthèse de dérivés de NNK portant une chaîne alkyl oméga-fonctionnalisée sur la fonction nitrosamine. Pour cela, nous avons synthétisé la 3-[2-(3-pyridinyl)-1,3-dioxolan-2-yl]propylamine, intermédiaire clef dans notre synthèse, et qui nous offre de nombreuses possibilités quant à la fonctionnalisation de l'amine terminale. La seconde partie consiste à fonctionnaliser le cycle pyridinique de la NNK. Nous avons imaginé différentes méthodes de synthèse, utilisant des pyridines 2,5-disubstituées mono ou dihalogénées ainsi que des dérivés du type 6-chloronicotinate. Seule cette dernière méthode nous a permis d'obtenir la 1-(6-chloro-3-pyridinyl)-4-(1-méthyl-2-oxohydrazino)-1-butanone. Le chlore présent sur le cycle pourra être utilisé par la suite pour diverses fonctionnalisations, telles que l'insertion de chaînes aliphatiques oméga-fonctionnalisées<br>AIn this work we report the synthesis of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone derivatives (abbreviated NNK), which was divided in two parts. The first one was about the synthesis of NNK derivatives linked with an omega-functionalized aliphatic chain on the nitrosamine function. The key intermediate in this synthesis was the 3-[2-(3-pyridinyl)-1,3-dioxolan-2-yl]propylamine. The primary aliphatic amine was monoalkylated to introduce various omega-functionalized chains. The second one used functionalization of the pyridine ring. We studied 2 ways, one using 2,5-disubstituted mono or dihalogenated pyridines which didn't afford the expected 1-(6-chloro-3-pyridinyl)-4-(1-methyl-2-oxohydrazino)-1-butanone. The other way using ethyl 6-chloronicotinate derivatives was a good alternative to synthesize the target molecule. The chlorine atom present on the molecule may be used to introduce an aliphatic omega-functionalized chain

This thesis focuses on the asymmetric reduction of N-protected derivatives of (3S)-3-amino-1-chloro-4-phenyl-2-butanone to their corresponding diastereomeric alcohol products, which are key intermediates in the synthesis of HIV protease inhibitors. Although the stereoselective synthesis of the (S,S) alcohol product is easily achieved, preparing the (R,S) diastereomer is much more challenging. I investigated three diastereoselective reduction processes: 1) Meerwein-Ponndorf-Verley (MPV) reduction, 2) asymmetric transfer hydrogenation, and 3) boron reducing agents. The diastereoselectivity of the MPV reduction still favored the (S,S) product; however, I discovered a significant rate enhancement when the standard catalyst (aluminum isopropoxide) was replaced with aluminum tert-butoxide. Many reaction variables were investigated in the asymmetric transfer hydrogenation reaction and the diastereoselectivity was improved to give a ratio of the desired (R,S) diastereomer to the undesired (S,S) alcohol of 9.5:1. Using chiral oxazaborolidine catalysts, an unprecedented (R,S) to (S,S) ratio of 9.5:1 was achieved. Finally, I investigated the effect of the N-protecting group on the stereoselectivity of the reduction. When the original boc-protecting group was replaced with a phthalimide group, the diastereoselectivity of the MPV reduction was reversed to favor the desired (R,S) product.

Dans ce travail, l'oxydation totale du toluène et de la butanone sur des catalyseurs à base d'or et de palladium supportés sur un oxyde de titane macro-mésoporeux a été étudiée. Nous avons tout d'abord discuté l'effet bénéfique du dopage (V, Nb, Fe, Ce, Ni) du support TiO₂ macro-mésoporeux sur l'activité catalytique de ce matériau. Une interaction existant entre le dopant et le support a joué un rôle important dans l'augmentation de l'activité des matériaux dopés par rapport à celle du titane pure. Une phase active constituée d'or et/ou de palladium a été ensuite déposée sur les solides dopés et la performance des catalyseurs préparés a été suivie dans l'oxydation du toluène et de la butanone. La présence de la phase active a assuré une sélectivité totale pour le CO₂ avec la formation de sous-produits en particulier dans l'oxydation de la butanone. Par ailleurs, l'oxydation du mélange des deux COVs a montré l'existence d'une compétition entre les molécules en terme d'adsorption sur le support, ce qui a favorisé leur éliminationà de basses températures. Une étude operando DRIFT réalisée dans l'oxydation du mélange toluène/butanone a vérifié la présence de cette compétition.

Dans le cadre du projet DISPATMO (étude de la prévision des risques de pollution liés à la dispersion atmosphérique de produits chimiques), des études de risques liés aux incendies et explosions dus aux produits chimiques stockés sur deux sites tests ont été menées. Le but est d’identifier les produits de combustion de certains composés cibles définis au début du projet, ainsi que d’estimer leur concentration. Les composés tests sont l’éthanol, le 2-butanone, le toluène et le solvant TIFLEX. Ces composés sont susceptibles, surtout à richesse élevées, de former des quantités non-négligeables d’isomères du butène, composés chimiques connus pour être d’importants intermédiaires de la combustion d’hydrocarbures. Après une étude bibliographique sur les isomères du butène, de l’éthanol, de la 2-butanone et du toluène, un mécanisme cinétique détaillé pour simuler l’oxydation de ces composés a été proposé. Une étude expérimentale de l’oxydation de 4 butènes (1-butène, trans-2-butène, cis-2-butène et iso-butène) a été réalisée en réacteur auto-agité (T = 900-1440 K, p = 1 atm, = 0,25, 0,5, 1 et 2, = 70 ms) et en chambre de combustion sphérique (Ti = 300 K, pi = 1, 2, 3 et 5 atm, = 0,8-1,4). Les résultats obtenus ont été confrontés à la simulation. Des données expérimentales issues de la littérature ont été utilisées afin de valider le modèle pour l’oxydation de l’éthanol, de la 2-butanone, du toluène et des isomères du butène. Enfin, une étude expérimentale de l’oxydation du solvant TIFLEX a été menée en réacteur auto-agité (T = 740-1310 K, p = 1 atm, = 0,5, 1 et 2) pour en connaître la composition ainsi que pour identifier et quantifier les produits d’oxydation. Le mécanisme cinétique proposé comporte un coeur C0-C4 robuste, en faisant un outil prédictif fiable, pouvant servir de base à des mécanismes plus étendus capables de représenter la combustion de nombreuses autres espèces (alcanes, alcènes, alcools, aldéhydes ou cétones), par ajout de sous-mécanismes<br>In the context of the DISPATMO project (study of the forecast of the risks of pollution related to the atmospheric dispersal of chemicals), risk studies linked to the fires and the explosions due to chemical storage were conducted. The purpose is to identify the combustion products of certain target compounds defined at the beginning of the project, as well as to estimate their concentration. The target compounds include ethanol, 2-butanone, toluene and the solvent TIFLEX. These compounds lead, especially in fuel-rich conditions, to the formation of high quantities of butene isomers, compounds known as important intermediates of hydrocarbon combustion. After a bibliographical study on butene isomers, ethanol, 2- butanone and toluene, a detailed kinetic mechanism for the simulation of the oxidation of these compounds was proposed. An experimental study of the oxidation of the butene isomers was performed in a jet-stirred reactor (T = 900-1440 K, p = 1 atm, = 0.25, 0.5, 1 and 2, = 70 ms) and in a spherical combustion chamber (Ti = 300 K, pi = 1, 2, 3 and 5 atm, = 0.8-1.4). Experimental results were compared with their simulations. Experimental data from the literature were used to validate the model for the oxidation of ethanol, 2-butanone, toluene and butene isomers. Finally, an experimental study of the oxidation of the solvent TIFLEX was performed in the jet-stirred reactor (T = 740-1310 K, p = 1 atm, = 0.5, 1 and 2) in order to know the composition as well as to identify and quantify of the oxidation products. The proposed kinetic mechanism contains a strong C0-C4 base, resulting in a reliable predictive tool, which can be used as a base in larger mechanisms simulating the combustion other species (alkanes, alkenes, alcohols, aldehydes or ketones), by addition of sub-mechanisms

Tobacco-specific nitrosamines (TSNA) are carcinogenic constituents derived from alkaloids in tobacco. Researchers are actively exploring several avenues to reduce TSNA levels in tobacco products like moist snuff tobacco. The focus of the research presented within is the quantitative analysis of TSNA in tobacco, specifically N’-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone (NNK), N’-nitrosoanatabine (NAT), and N’-nitrosoanabasine (NAB). Tobacco alkaloids and nitrosamines in tobacco are currently analyzed by different instrumentation due to orders of magnitude difference in their concentrations, chromatographic separation challenges due to structural similarities, and similar mass fragmentation patterns. An analytical column using silica and 1,2-bis(siloxy)ethane hybrid particles of 1.7 µm size is the foundation of a chromatographic separation of NNN, NNK, NAT, NAB, nicotine, nornicotine, anatabine, and anabasine. This is the first rapid and robust quantitative method for the TSNA and their alkaloid precursors using high pH mobile phase conditions with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The suitability of the method is demonstrated by its application to the analysis of reference tobacco materials for cigarettes and moist snuff. In addition, a novel TSNA analytical method was developed using TSNA-specific molecularly imprinted polymers (MIP) as the selective extraction element from tobacco extract. The affinity mechanisms between MIP and TSNA were found to have extensive cross-reactivity to structurally similar alkaloids present in tobacco extract. TSNA-specific MIP was demonstrated to have stronger retention for the alkaloids than for the TSNA substrate. The MIP-TSNA interaction was optimized to create the first analytical method to quantify underivatized NNN and NNK from tobacco extracts by HPLC-UV.

La fumée de tabac contient la nitrosamine 4-(méthylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) qui serait impliquée dans la cancérogenèse pulmonaire. L’α-hydroxylation de la NNK génère deux métabolites réactifs qui mènent à la méthylation et à la pyridyloxobutylation de l’ADN, et qui peuvent être générés respectivement par la N-acétoxynitrosométhylméthylamine et par la 4-(acétoxyméthylnitrosamino)-1-(3-pyridyl)-1-butanone. Nous avons observé que la fréquence et la distribution des dommages induits à l’ADN par la méthylation et la pyridyloxobutylation au niveau des gènes p53, ras et c-jun ne sont pas identiques. La fréquence et la distribution des dommages par la méthylation de l’ADN varient avec la classe et la structure chimique des agents méthylants. La pyridyloxobutylation de l’ADN induit des cassures monocaténaires caractérisées par un groupement hydroxyle à l’extrémité 5’ et par un groupement bloquant à l’extrémité 3’. La formation de dommages causée par la NNK et la fréquence élevée de dommages à certaines positions nucléotidiques qui correspondent à des positions fréquemment mutées dans le cancer pulmonaire chez le fumeur constitueraient des arguments en faveur d’un rôle de la NNK dans la cancérogenèse pulmonaire associée à la fumée de tabac.

La fumée de tabac contient la nitrosamine 4-(méthylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) qui serait impliquée dans la cancérogenèse pulmonaire. L'?-hydroxylation de la NNK génère deux métabolites réactifs qui mènent à la méthylation et à la pyridyloxobutylation de l'ADN, et qui peuvent être générés respectivement par la N-acétoxynitrosométhylméthylamine et par la 4-(acétoxyméthylnitrosamino)-1-(3-pyridyl)-1-butanone. Nous avons observé que la fréquence et la distribution des dommages induits à l'ADN par la méthylation et la pyridyloxobutylation au niveau des gènes p53, ras et c-jun ne sont pas identiques. La fréquence et la distribution des dommages par la méthylation de l'ADN varient avec la classe et la structure chimique des agents méthylants. La pyridyloxobutylation de l'ADN induit des cassures monocaténaires caractérisées par un groupement hydroxyle à l'extrémité 5' et par un groupement bloquant à l'extrémité 3'. La formation de dommages causée par la NNK et la fréquence élevée de dommages à certaines positions nucléotidiques qui correspondent à des positions fréquemment mutées dans le cancer pulmonaire chez le fumeur constitueraient des arguments en faveur d'un rôle de la NNK dans la cancérogenèse pulmonaire associée à la fumée de tabac.

Le fluor a démontré sa capacité à modifier les propriétés physico-chimiques et biologiques des molécules par rapport à leurs analogues hydrogénés, expliquant son utilisation fréquente en chimie médicinale et agrochimie. Des groupes fluorés émergents (GFE) sont actuellement à l'étude afin de diversifier la nature des motifs fluorés utilisés. Nous avons donc cherché à développer la synthèse d'une brique polyvalente, à savoir une cétone, permettant l'introduction du motif -OCHF2. Celui-ci possède en effet des propriétés intrinsèques intéressantes, mais souffre d’un manque de méthodes d'introduction efficaces, notamment sur les chaînes alkyles. La synthèse de la cétone difluorométhoxylée a d'abord été optimisée. Son potentiel synthétique a ensuite été mis à profit pour l’accès à des composés tels que des amidines, des imidates et des thioimidates via une céténimine comme espèce réactive. La cétone a ensuite démontré son utilité pour accéder à des hétérocycles difluorométhoxylés encore rarement rencontrés, d'abord via un intermédiaire énaminone, et finalement directement par une cyclisation de type Fischer<br>Fluorine has demonstrated its ability to modify the physico-chemical and biological properties of molecules compared to their hydrogenated analogues, explaining its widespread use in medicinal- as well as agrochemistry. So-called Emerging Fluorinated Groups (EFG) are currently under investigation, in order to diversify the nature of the used fluorinated moieties. Therefore, we aimed to develop the synthesis of a versatile building block, namely a ketone, allowing the introduction of the OCHF2 motif. As a matter of fact, the latter possesses some interesting inherent properties, but suffers a lack of efficient introduction methods, especially on alkyl chains. First the synthesis of the difluoromethoxylated ketone was optimized. Its synthetic potential was then exploited, as it was used to access compounds such as amidines, imidates and thioimidates via a ketenimine as highly reactive specie. Subsequently, the ketone demonstrated its usefulness in accessing very rarely encountered difluoromethoxylated heterocycles, first via an enaminone intermediate, and finally directly through a Fischer-type cyclization

La fumée de tabac contient plusieurs substances carcinogènes qui mènent à la formation constante de petites quantités de dommages à l'ADN dans les poumons des fumeurs ainsi que des non-fumeurs exposés à la fumée environnementale de tabac. La 4-(méthylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) est l'une de ces substances et elle semble plus particulièrement associée avec le développement des adénocarcinomes, la forme de cancer pulmonaire dont l'incidence progresse le plus rapidement ces dernières années. Dans les cellules pulmonaires, la NNK est bioactivée via des cytochromes P450 en intermédiaires réactifs capables de méthyler ou de pyridyloxobutyler l'ADN. Les dommages résultant de ces deux modes d'activation de la NNK peuvent être investigués séparément en utilisant des analogues qui génèrent sélectivement l'un ou l'autre type d'intermédiaires réactifs. Le test des comètes est une technique simple, très sensible et couramment utilisée pour étudier au niveau cellulaire les dommages à l'ADN qui sont peu fréquents. Les travaux présentés dans cette thèse montrent que certains des dommages résultant de l'activation de la NNK peuvent être investigués de manière spécifique à l'aide de cette technique, et ce à des fréquences de dommages qui se rapprochent de celles correspondant à une exposition réelle à la fumée de tabac. Parmi ces dommages, un type d'adduits encore inconnu associé à la pyridyloxobutylation de l'ADN a pu être mis indirectement en évidence. Il s'agit vraisemblablement de la forme formamidopyrimidine (fapy) d'une lésion primaire formée dans les cellules. La vitesse de réparation d'un type de dommage influe sur le risque qu'il a d'être impliqué dans la transformation maligne des cellules. La disparition des dommages dans le temps a pu être suivie avec le test des comètes afin d'investiguer la réparation dans des cellules capables ou pas de bioactiver la NNK. Le suivi post-traitement des dérivés fapy associés à la pyridyloxobutylation de l'ADN, a montré un phénotype ne dépendant pas du type cellulaire mais plutôt du statut de p53 dans les cellules. En effet, au lieu de diminuer après la fin du traitement, la fréquence des adduits fapy dans les fibroblastes augmente dans un premier temps et ce, seulement dans les cellules ayant une protéine p53 fonctionnelle. La nature de ce phénotype particulier n'est pas clairement identifiée, mais elle est vraisemblablement liée à la réparation des dommages à l'ADN.<br>Tobacco smoke contains several carcinogens that lead to the frequent formation of rare DNA damage in lungs of smokers. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of these substances that seems more particularly associated with the development of adenocarcinoma. During the last 30 years, the frequency of this lung cancer type has increased significantly. In lung cells, cytochromes P450 can bioactivate NNK into reactive species capable of either methylating or pyridyloxobutylating DNA. The use of analogs capable of generating only one type of NNK-associated reactive species allows to investigate methylation and pyridyloxobutylation separately. The comet assay is a simple and sensitive technique that is commonly used to investigate low frequency DNA damage at the cellular level. The work presented here show how some of the NNK-related DNA damage can be investigated specifically with this technique at damage frequencies that are relevant to a real exposure to cigarette smoke. One of the adduct type resulting of DNA pyridyloxobutylation that we studied here had never been demonstrated before. It corresponds likely to the formamidopyrimidine (fapy) form of a lesion primarily formed in cells. The repair rate of a damage type influences the probability that it has to be implicated in mutagenesis. The time course of different damage types was documented with the comet assay in order to investigate the repair of NNK-related damage in different cell types that can either bioactivate NNK or not. When the fapy adducts associated with pyridyloxobutylation were investigated post-treatment, their time course did not depend on the cell type but showed a p53-dependant phenotype. In fact, instead of decreasing because of repair, the frequency of these fapy adducts in fibroblasts first increased post-treatment and this increase seemed associated with p53 proficiency. The cause of this phenotype is not clearly elucidated but it should be related to DNA damage repair.

Le potentiel synthétique des énolates de potassium de cétones dissymétriques, contrairement à leurs homologues lithiés, n'a pas été, jusqu'alors, vraiment exploité faute de méthode efficace de préparation régiospécifique. Cette étude décrit l'accès, à partir d'éthers d'énols silylés, à des énolates de potassium (par action de tertiobutylate de potassium) ou de lithium (par action de bromure de lithium sur les énolates de potassium) ainsi que l'influence du cation alcalin sur la régiosélectivité des réactions d'alkylation et d'aldolisation de tels énolates. Cette étude montre que la nature du cation associé est capitale puisque les énolates de lithium conduisent après addition d'un agent électrophile (halogénure d'alkyle ou aldéhyde) à une réaction sur le site attendu de l'énolate alors que les énolates de potassium conduisent aux cétones les plus encombrées après alkylation via une équilibration des énolates et aux aldols les moins encombrés après aldolisation via un équilibrage des aldolates. Cette étude décrit également l'utilisation de la réaction de rétroaldolisation dans des synthèses asymétriques d'alcools benzyliques. En effet des aldols optiquement purs (produits par aldolisation diastéréosélectives de cétones issues du pool chiral avec le benzaldéhyde), placés dans des conditions où la réaction de rétroaldolisation est permise, sont susceptibles de donner un équivalent synthétique du benzaldéhyde (dont une face est masquée) pouvant réagir avec un agent nucléophile. Ainsi la réaction d'organométalliques sur un aldol dérivé de la dihydrocarvone permet d'accéder aux alcools benzyliques de configuration R majoritaire avec des excès énantiomériques significatifs.

La turbidité du lait rendant impossibles les dosages spectrophotométriques, certains auteurs ont mis au point des réactifs de transparisation du lait. Etude d'un de ces réactifs (butanone-triton x 100-eau). Propriétés analytiques des milieux transparisés afin de mieux adapter les réactions chimiques aux nouveaux milieux ou de modifier le réactif.

Compostos &#945;-aminocarbonilícos como ácido 5-aminolevulínico (ALA) e aminoacetona (AA) apresentam um grande potencial pró-oxidante, pois sofrem reações de enolização e subseqüente oxidação aeróbica, com a formação de espécies radicalares de oxigênio, íons NH4+ e &#945;-oxoaldeídos potencialmente citotóxicos. A &#945;-aminocetona 1,4-diamino-2-butanona (DAB), um análogo da putrescina, é um agente microbicida de vários parasitas incluindo Trypanosoma cruzi. Acredita-se que o mecanismo de morte desencadeado por DAB nos parasitas seja por meio da inibição competitiva da ornitina descarboxilase (ODC), importante enzima do metabolismo de poliaminas, muito embora tenha sido observado de igual forma danos oxidativos nestes parasitas quando tratados com DAB. O objetivo deste trabalho é esclarecer o mecanismo de oxidação química de DAB e sua ação pró-oxidante à cultura de células de mamíferos (LLC-MK2 e RKO), assim como sua atividade microbicida contra tripomastigotas de Trypanosoma cruzi. Demonstramos aqui que DAB, quimicamente similar ao ALA e AA, sofre reação de oxidação catalisada por íons fosfato, e por íons de metais de transição como Fe(II) e Cu(II), resultando na formação de radicais de oxigênio, H2O2, NH4+, 2-oxo-4-aminobutanal como produto principal da oxidação de DAB e de compostos ciclicos de caracter pirrólico. Danos oxidativos observados em ferritina, apotransferrina e liposomos de cardiolipina e fosfatidilcolina (20:80) contribuem para a nossa hipótese de ação pró-oxidante de DAB. O tratamento de células de mamíferos das linhagens LLC-MK2 (IC50 1,5 mM, tratamento de 24 h) e RKO (IC50 0,3 mM, tratamento de 24 h) com DAB levou à alteração do balanço redox celular, à ativação de resposta antioxidante e ao desencadeamento de morte celular via apoptose e parada de ciclo celular. Em culturas de tripomastigotas de T. cruzi o tratamento com DAB culminou na redução da motilitidade e viabilidade destes parasitas (IC50 0,2 mM, tratamento de 4 h), assim como depleção do conteúdo tiólico acompanhado pelo aumento da atividade de TcSOD. Além do mais, DAB mostrou-se eficiente em limitar a invasão de tripomastigotas às células hospedeiras (LLC-MK2) e reduzir a proliferação de amastigotas intracelulares, contudo fortemente relacionada à necrose das células hospedeiras infectadas, uma vez que são alvos mais susceptíveis de ação oxidativa. Estes resultados suportam nossa hipótese que DAB exerce ação pró-oxidante e contribui deste modo com o mecanismo já descrito de morte celular associada à inibição da biossíntese de poliaminas em vários microorganismos.<br>&#945;-Aminocarbonyl componds such as 5-aminolevunilic acid (ALA) and aminoacetone (AA) have been shown to exhibit pro-oxidant properties. These compounds undergo phosphate-catalyzed enolization in physiological pH and subsequent aerobic oxidation, yielding reactive oxygen species, NH4+ ions and an &#945;-oxoaldehyde highly cytotoxic. The &#945-aminoketone 1,4-diamino-2-butanone (DAB) is a putrescine analogue and a microbicidal agent to various parasites including Trypanosoma cruzi. The mechanism of DAB toxicity to these parasites is attributed to DAB competitive inhibition of ornithine decarboxylase (ODC), a key enzyme on polyamine biosynthesis, although it has also been shown DAB isto implicated in oxidative damage to these parasites. Our aim is to clarify the mechanism of DAB aerobic oxidation and of its putative pro-oxidant activity to mammalian cell cultures (LLC-MK2 and RKO cell linages) and to Trypanosoma cruzi trypomastigotes. Here we show that, similar to ALA and AA, DAB undergoes aerobic oxidation in presence of phosphate ions and of transition metal ions such as Fe(II) and Cu(II), yielding oxygen radicals, H2O2, NH4+ and 2-oxo-4-aminobutanal accompanied by its condensation cyclic products displaying pyrrolic characteristics. Oxidative alterations to ferritin, apotransferrin and liposomes of cardiolipin and phosphatidylcholine (20:80) were observed under DAB treatment strongly supporting our hypothesis of DAB pro-oxidative activity. DAB treatment of mammalian cultured cells LLC-MK2 (IC50 1.5 mM, 24 h incubation) and RKO (IC50 0.3 mM, 24 h incubation) resulted in redox imbalance, induction of antioxidant response, activation of apoptosis pathway and cell cycle arrest. DAB is shown here to trigger Trypanosoma cruzi trypomastigotes decreased parasite motility and viability (IC50 0.2 mM, 4 h incubation), as well as redox thiol imbalance parallel to increase TcSOD activity. In addition, DAB efficiently hampered host cell (LLC-MK2) invasion by trypomastigotes. In addition, intracellular amastigotes showed to be susceptible to DAB toxicity, although strongly related to necrosis of infected host cells, which are more vulnerable to oxidative stress. Altogether, these data support our hypothesis that oxidative stress contributes to DAB cytotoxicity.

Le 3-méthyl-2,2,6,6-tétrachlorocyclohexanone a été cyclisé en milieu basique pour conduire à deux époxydes régioisomères avec un rapport 88/12. L'époxyde majoritaire est séparé de son régioisomère par chromatographie éclair sur silice. L'ouverture de l'époxyde majoritaire par l'éthérate de trifluorure de bore conduit de façon régio- et stéréosélective a une cis fluorhydrine. Une succession de cyclisation puis ouverture par les trihalogénures de bore permet la préparation d'halohydrines méthyles en position 3. Par oxydation et déshydrohalogénation des alcools, on accède aux crésols ortho, ortho' dihalogénés correspondants. Des cresols mono-halogénés (chloré ou fluoré) sont préparés à partir des époxydes intermédiaires. La coupure de la liaison oxygène-silicium des énoxysilanes tri- et tétrasubstitués de la 2-méthylcyclopentanone par t-BuOK conduit aux énolates tri- et tétrasubstitués correspondants. La méthylation de ces énolates conduit majoritairement a la 2,2-diméthylcyclopentanone. Ces énolates engagés dans des réactions d'aldolisation conduisant à des réactions en position 5. La 2-benzylidène-5,5-diméthylcyclopentanone a été ainsi préparée par coupure de l'énoxysilane tétrasubstitué de la 2-méthylcyclopentanone par t-BuOK suivie d'une aldolisation avec le benzaldéhyde. Après déprotonation par t-BuOK et alkylation par MeI, le composé a été obtenu avec un rendement global de 49% par rapport à l'énoxysilane de départ qui peut être constitué d'un mélange de régioisomères

Une introduction generale sur la spectrometrie de masse precede une premiere partie portant sur l'interpretation des fragmentations de la dimethyl-3,3 hydroxy-4 butanone-2 a basse energie. Une seconde partie est reservee aux fragmentations de plusieurs beta ceto-esters diversement substitues. Dans une troisieme partie sont examinees les decompositions unimoleculaires d'ions acylium beta carbonyles de faible energie interne. L'accent est mis, tout au long de ce travail, sur le role essentiel des atomes d'oxygene dont la presence induit des ruptures caracteristiques mais aussi sert de relais aux transferts d'hydrogenes

L'étude des différents paramètres affectant l'énantiosélectivité de la protonation a permis d'obtenir des enrichissements optiques élevés et de dégager l'influence préponderante de la structure de l'espèce amionique intermédiaire (solvant, cation associé, amine libre de l'amidure). Dans le cas de la benzoïne, la protonation conduit initialement à l'ènediol qui s'isomérise lentement en benzoïne optiquement active. Ce résultat constitue le premier exemple expérimental de tautomérisation énantiosélective d'une forme énolique en cétone

On met au point une méthode générale de synthèse de composés delta-dicarbonylés et delta-dicarbonylés α -halogénés par action respectivement d'un éther d'énol silyle ou d'un éther d'énol silyle β-halogéné sur des carbocations fonctionnels. En milieu basique, les composés delta-dicarbonylés sont transformés en cyclohexénones, tandis que leurs anologues halogénés conduisent, selon la structure du composé de départ, à des cyclopropanes disubstitués trans ou à des spiranes hétérocycliques. Ces méthodes ont été appliquées à la synthèse d'alpha -cypérones. On a également préparé des synthons de bis-annélation et obtenu ainsi la homo-D deshydrotestostérone

碩士<br>國立中山大學<br>機械與機電工程學系研究所<br>103<br>Kinetic modeling for biodiesel fuel surrogates is presently of great interest due to the role as a renewable energy source. Understanding how to generate reduced skeletal model without losing predicting accuracy is a challenge to establish computational dynamic fluid model coupled with reacting flow simulations. The main purpose of this study is to present a strategy to generate reduced skeletal models for methyl butanoate (MB) kinetic modeling. The original MB mechanism composed of 275 species and 1549 reactions has been reduced based on pyrolysis and oxidation conditions using multi-generation path flux analysis (PFA) method. The complexity of the reduced mechanisms is also analyzed. A trade-off reduced mechanism for MB pyrolysis composed of 81 species and 817 reactions is performed over temperatures range from 1250 K to 1700 K in shock tube simulations. For the MB oxidation study, a trade-off reduced mechanism consists of 46 species and 207 reactions has been validated in shock tube and opposed-flow diffusion flame experiment over wide range of temperatures, and equivalence ratios. CPU time analysis also shows one of the strengths in using the generated skeletal mechanisms for computational cost reduction. This thesis also reports for the first time that experimental kinetics from MB co-flow flame have been well validated. The rate of production analysis and sensitivity analysis are also performed to investigate chemical kinetic details for skeletal mechanisms. The reduced MB pyrolysis and oxidation mechanism can be served as a starting point of biodiesel fuel surrogate kinetic modeling with computational fluid dynamics.

碩士<br>逢甲大學<br>化學工程學系<br>103<br>Due to pressure in work, irregular living, and unbalance diet, leads to various gastrointestinal symptoms such as gastralgia and gastric ulcer. In case of late diagnosis and treatment, gastric carcinoma might evolve, which is negative to the national economic output and involved community and family. Therefore, it has been an important research topic for contemporary and family. The study aims at developing a new urea breath technique to detect the infection of H. pylori. The new technique replaces the traditional 13C reagent, and analytical instruments such as gas chromatography (GC) and mass spectroscopy (MS) with a low-cost and portable sensor. It would reduce the overall processing time of test while keeping the accuracy and sensitivity to enhance the market competition. 2-butanone is a bio-maker detected in breath of people who have H. pylori in stomach. Thus, the goal of this research is to establish a 2-butanone sensing system. The graphene, ZnO NDs and graphene modified ZnO NDs electrode as sensing films have been successfully prepared and characterized. The sensors for detecting 2-butanone have been developed too. The ZnO NDs electrode has higher sensitivity toward 2-butanone than graphene and graphene modified ZnO NDs electrode. Results show that it has potential to work as a new method for detecting H. pylori.

碩士<br>國立高雄第一科技大學<br>環境與安全衛生工程系碩士班<br>105<br>The study was conducted for the evaluation of Membrane bioreactor(MBR), Used in the process of Methyl Ethyl Ketone(MEK) concentration whether it is reduce effectiveness,Using the activated sludge for domesticated,Get on batch concentration load test and semi-continuous injection,The former mainly to explore whether the activated sludge for the study of the target MEK with domestication,The latter is closer to the real plant application,Use semi-continuous injection as test method for the effectiveness of MBR. Initially for the domestication of activated sludge results to get on batch test,Low concentration of inoculum MEK 10mg/L reduce the concentration of up to 4.3-4.7mg/L；High concentration of inoculum MEK 150mg/L reduce the concentration of up to 12mg/L,The results of the test were used to verify the source of the sludge selected in this study has domestication characteristics with MEK。 And MBR batch concentration test results show low concentration of 30mg/L to a high concentration of 200mg/L with MEK, Each stage of the experiment in the concentration trend of the map can be seen in the concentration of time decreases,Test results can be seen that the domesticated activated sludge has the ability to reduce the concentration of MEK,And it can stable biodegradation。Another semi-continuous injection can know at each unit/time,Maintained at the same concentration,The biodegradation rate constant(K value)obtained by semi-continuous injection was between 0.05 and 0.13,It similar to the previous batch injection(between 0.06 and 0.11) ,And in the semi-continuous injection set the hydraulic retention time (HRT) to consider the reference batch test decrement rate trend,To find the HRT,The concentration of MEK at 60mg/L HRT was set at 4hr,100mg/L was set at 6hr,and 150mg/L was set at 8hr。 This study results showed that the tested activated sludge could be effectively degraded by MEK,In the other batch and semin-continuous different injection mode,the sluge can be stabilized to achieve the effect of reducing MEK, And the K value of the batch and semi-continuous injection, the correlation coefficient of the design condition under the condition of the matching result can be used as the basis and reference for the future application.

碩士<br>國立臺中教育大學<br>科學教育與應用學系碩士在職專班<br>105<br>The equilibrium geometries, harmonic vibrational frequencies, and normal modes of propanone, butanone, and their cations were calculated by using the density functional theory (B3LYP and M06-2X functionals) with the basis set aug-cc-pVTZ. The Franck-Condon factors were computed by using the method developed by our group and the photoelectron spectra of propanone and butanone were simulated. The adiabatic ionization energies were also calculated by extrapolating the CCSD(T) energies to the complete basis set limit from the basis sets aug-cc-pVXZ (X = D, T, Q, 5). The vibrational structures in the photoelectron spectra of propanone and butanone were analyzed. The simulated photoelectron spectra of both molecules are in harmony with experiements. The computed adiabatic ionization energies are also in agreement with experiment, with deviations of 0.03 and -0.06 eV for propanone and butanone, respectively.

by Leung Yuet Kin.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 1998.<br>Includes bibliographical references (leaves 86-100).<br>Abstract also in Chinese.<br>List of Figures --- p.ix<br>List of Tables --- p.xii<br>List of Abbreviations --- p.xiii<br>Chapter Chapter One: --- Introduction<br>Chapter 1.1 --- Carcinogenicity of the tobacco products --- p.1<br>Chapter 1.2 --- Biochemical Pathway involved in NNK metabolism --- p.5<br>Chapter 1.2.1 --- Metabolic activation of NNK --- p.6<br>Chapter 1.2.2 --- Detoxification of NNK --- p.8<br>Chapter 1.3 --- Modulation of NNK carcinogenesis --- p.9<br>Chapter 1.4 --- Mediation of NNK oxidation : chemoprevention by isothiocyanates --- p.11<br>Chapter 1.5 --- Aim of study --- p.13<br>Chapter 1.5.1 --- Experimental approaches --- p.15<br>Chapter Chapter Two : --- Modulation of α-carbon hydroxylations in NNK metabolism<br>Chapter 2.1 --- Background --- p.17<br>Chapter 2.2 --- Materials and Methods --- p.17<br>Chapter 2.2.1 --- Chemicals --- p.17<br>Chapter 2.2.2 --- Methods --- p.18<br>Chapter 2.2.2.1 --- Preparation of rat microsomes --- p.18<br>Chapter 2.2.2.2 --- Assay of NNK metabolism --- p.18<br>Chapter 2.2.2.3 --- Determination of solvent extraction --- p.19<br>Chapter 2.2.2.4 --- Determination of detergent effects --- p.19<br>Chapter 2.2.2.5 --- HPLC analysis of NNK metabolites --- p.20<br>Chapter 2.2.2.6 --- Study of strain differences between SD rats and F344 rats --- p.20<br>Chapter 2.3 --- Results --- p.21<br>Chapter 2.3.1 --- HPLC analysis of NNK metabolites --- p.21<br>Chapter 2.3.2 --- Determination of solvent extraction --- p.22<br>Chapter 2.3.3 --- Effects of detergents --- p.25<br>Chapter 2.3.4 --- Study of strain differences using F344 rats and SD rats --- p.26<br>Chapter 2.4 --- Discussion --- p.27<br>Chapter Chapter Three : --- Modulation by potentiation of detoxification process in NNK metabolism<br>Chapter 3.1 --- Background --- p.30<br>Chapter 3.2 --- Materials and Methods --- p.31<br>Chapter 3.2.1 --- Chemicals --- p.31<br>Chapter 3.2.2 --- Methods --- p.32<br>Chapter 3.2.2.1 --- Preparation of rat microsomes --- p.32<br>Chapter 3.2.2.2 --- Analysis of NNK metabolism --- p.32<br>Chapter 3.2.2.3 --- UDP-glucuronosyltransferase Assay --- p.33<br>Chapter 3.2.2.4 --- Cytochrome P450 2E1 Assay --- p.34<br>Chapter 3.3 --- Results --- p.34<br>Chapter 3.3.1 --- Screening tests of the effects of vitamins and drugs --- p.34<br>Chapter 3.3.1.1 --- Male liver microsomes --- p.35<br>Chapter 3.3.1.2 --- Female liver microsomes --- p.37<br>Chapter 3.3.1.3 --- Male lung microsomes --- p.39<br>Chapter 3.3.1.4 --- Female lung microsmes --- p.41<br>Chapter 3.3.2 --- Kinetic analysis of vitamin C-palmitate on NNK reduction --- p.43<br>Chapter 3.3.3 --- Effects of vitamin C-palmitate on UDP- glucuronosyltransferase --- p.47<br>Chapter 3.3.4 --- Effects of vitamin C-palmitate on cytochrome P4502E1 (CYP2E1) --- p.48<br>Chapter 3.4 --- Discussion --- p.50<br>Chapter Chapter Four: --- Purification of carbonyl reductase from rat liver microsomes<br>Chapter 4.1 --- Background --- p.58<br>Chapter 4.2 --- Materials and Methods --- p.58<br>Chapter 4.2.1 --- Chemicals --- p.58<br>Chapter 4.2.2 --- Methods --- p.59<br>Chapter 4.2.2.1 --- Preparation of male rat liver microsomes --- p.59<br>Chapter 4.2.2.2 --- Purification of carbonyl reductase from rat liver microsomes --- p.59<br>Chapter A. --- Solubilization of microsomes --- p.59<br>Chapter B. --- Chromatographic separation by octyl-Sepharose CL-4B --- p.60<br>Chapter C. --- Chromatographic separation by DEAE-cellulose --- p.60<br>Chapter 4.2.2.3 --- SDS polyacrylamide gel electrophoresis --- p.61<br>Chapter 4.3 --- Results --- p.61<br>Chapter 4.4 --- Discussion --- p.63<br>Chapter Chapter Five : --- Summary and Discussion<br>Chapter 5.1 --- Summary --- p.66<br>Chapter 5.2 --- Discussion --- p.67<br>Chapter 5.2.1 --- Anticarcinogenesis of vitamin C in NNK-induced cancer through smoking --- p.67<br>Chapter 5.2.2 --- Future studies on microsomal carbonyl reductase --- p.68<br>Chapter 5.2.3 --- Future studies on UDP-glucuronidation of NNAL --- p.72<br>Appendix A Effects of atropine on tolbutamide metabolism --- p.74<br>Appendix B Recovery of keto acid and keto alcohol from the assay mixture ( without solvent extraction ) --- p.75<br>Appendix C Calibration Curve of NNAL --- p.76<br>Appendix D Effects of methanol on NNK carbonyl reduction --- p.77<br>Appendix E Time course study of NNAL production at various concentrations of vitamin C-palmitate --- p.78<br>Appendix F Time course assay of UDP-glucuronosyltransferase at various concentrations of vitamin C-palmitate --- p.80<br>Appendix G Calculation of specific activity of UDP-Glucuronosyl transferase --- p.82<br>Appendix H Calculation of specific activity of cytochrome P450 2E1 --- p.84<br>References --- p.86

Studies described in this thesis were at aimed at characterizing the mechanisms involved in the pulmonary carcinogenicity of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by addressing two critical determinants of carcinogenicity; biotransformation and DNA repair. The contributions of cytochrome P450 (CYP) 2A13 and CYP2A6 to NNK biotransformation in human lung microsomes were investigated. Based on total bioactivation and detoxification of NNK and its keto-reduced metabolite, 4 (methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), subjects could be classified as either high or low bioactivators and detoxifiers. Data from all of 29 individuals revealed no correlations between levels of CYP2A mRNA, enzyme activity or immunoinhibition and the degree of total NNK bioactivation or detoxification. However, subgroups were identified for whom CYP2A13 mRNA correlated with total NNK and NNAL bioactivation (n=4) and NNAL detoxification (n=5). Although results do not support CYP2A13 or CYP2A6 as predominant contributors to NNK metabolism in lung of all individuals, CYP2A13 appears to be important in some. The involvement of nucleotide excision repair (NER) in the repair of NNK-induced DNA pyridyloxobutylation was assessed. Extracts from NER-deficient cells were less active at repairing pyridyloxobutyl (POB) adducts on plasmid DNA than were extracts from normal cells, and NER-deficient cells were more susceptible to 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc)-induced cytotoxicity, demonstrating the participation of NER in the repair of POB-DNA adducts. The role of DNA repair in contributing to inter-organ susceptibility to NNK-induced carcinogenesis was investigated. POB adduct repair was greater in extracts from mouse liver than lung, and activities in lungs of NNK-treated mice were lower than those of saline-treated mice, while repair was 3 times higher in livers of NNK-treated mice relative to control. NNK treatment decreased incision of POB adducts by 92 % in lung extracts and increased incision by 169 % in liver extracts. In addition, NNK altered the levels and binding to POB damage of key incision proteins. These results suggest that lower NER incision activity and NNK-mediated alterations in levels and activities of incision proteins contribute to the relative susceptibility of mouse lung to NNK-induced carcinogenesis.<br>Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2008-11-13 14:10:01.603

碩士<br>義守大學<br>化學工程學系暨生物技術與化學工程研究所<br>107<br>Butanone (MEK) and 3-methyl-2-butanone (MIPK) are volatile organic compounds (VOCs), and often used in factories as solvents, dewaxing agents and cleaning agents, which will lead to water and air pollution. Ozone can effectively decompose organic compounds due to its highly oxidizing properties. This research was applied the high oxidation capacity of ozone to study the products of reaction of ozone with methyl ethyl ketone and 3-methyl-2-butanone. The results will helpful for understanding the oxidation mechanism of oxidation reaction by ozone. This study was applied GC/MS and IR to analyze and identify the products of reaction of methyl ethyl ketone and 3-methyl-2-butanone with ozone. The products of the ozone reaction with MEK include acetaldehyde, acetic acid, 3-hydroxy-2-butanone and butanedione. After the reaction of ozone with MIPK, the products detected were acetaldehyde, acetone, 3,3-dimethyl-2-butanone, vinyl acetate, 3-hydroxy-3-methyl-2-butanone and 2-methyl-3-pentanone. When ozone reacts with ketones, ozone will react with the position of α-CH. MEK has secondary and primary α-CH, while MIPK has tertiary and primary α-CH. The results show that ozone does not react with the position of primary α-CH, and tertiary α-CH is easier to react with ozone than secondary α-CH. The reaction mechanism is also discussed.

碩士<br>中山醫學院<br>毒理學研究所<br>87<br>As a crowd living place, Taiwan has little tolerance toward environmental hazard. Therefore, emissions of the pollutant have not only disrupted the balance of environment but also caused severe impact to human being. Human exposure to organic solvents is widespread and occurs regularly. The principal routes of direct exposure to organic solvents are skin and inhalation. Organic solvent like toluene and methyl ethyl ketone(MEK) is widely used in industry and is well known to be neurotoxic and naphrotoxic. It is often used with other pollutants. Toluene and methyl ethyl ketone (MEK) can cause many of the pathophysiological effects. But they have been proposed that there have no mutagenic, carcinogenic and teratogenic effect for both solvents. Currently no suitable instrument can assay the biological effects of volatile organic chemicals (VOCs) in cell culture system, but the in vitro system like culture cell can tell some details about the mechanism of biological response than experimenting animal does. A cell-incubation system that can introduce different concentration of volatile organic solvent mimic the occupational of environmental exposure condition has established and presented in this work. The concentration of organic solvents in the exposed chamber has been evaluated by GC measurement of samples from air sampling of charcoal tube, passive monitor or CS2 extraction of medium. The effect of the cell cycle performance of V79 , a normal lung cell line, exposed in organic solvents has been assayed by flow cytometric determination of DNA histograms. Different organic solvents produce various types of V79 cell cycle performance and its effect has been observed in this study. The design of exposure system, stability of analytical results, and the application of this system on toxicity evaluation of volatile substance, are discussed. Recently, the concentration of organic solvents in most of workplaces has been reduced below the permissible exposure limit (PEL), however, the phenomenon of human exposed to multiple environmental hazard waste is often observed. Therefore, it is important to study the influence of organic solvent only, or coexistence with other heavy metal such as Cadmium, for human body. To understand the additive effect of the toxic chemical mixtures to biological system, as well as to estimate the cytotoxicity or cell cycle inhibition the in vitro assay has been performed in this work. It is devoted to establish the use of V79 cells as an in vitro screening tool for the cytotoxicity or cell cycle inhibition of volatile chemical agents. In this study MTT has been used to assay in vitro additive toxic effects of Cd2+ and solvents on V79 cells. As to elucidate the details of the combine effect of cell growth, the cell cycle with or without the chemical treatment has been assessed by propdium iodine stain in flow cytometric instrument.

In the event of a loss-of-coolant accident (LOCA) in a water cooled reactor, a break in the primary cooling circuit followed by fuel failure could release a significant fraction of the core inventory of radioactive iodine into containment. Non-volatile aqueous iodide may be converted into volatile I$\sb2$ and subsequently released into the environment. This process has been shown to be affected by the radiolytic breakdown of dissolved organic compounds that may be present in containment. In this study, the effect of a model organic compound, 2-butanone (methylethylketone, MEK), on iodine chemistry was investigated. In this work, MEK was $\gamma$-irradiated in aqueous solutions and the formation of decay products were followed by a various analytical methods (HPLC, GC, GC/MS and MS) to establish a decay profile. For aerated solutions, MEK degradation produced products, of continually decreasing size. In contrast, in non-aerated solutions where hydroxyl radical was not scavenged evidence of dimerization was observed. Under aerated conditions the main pathway for radiolysis is the abstraction of a hydrogen atom by hydroxyl radical attack to give MEK radical which undergoes reaction with dissolved oxygen, producing an organic peroxide. This species recombines to form a tetroxide intermediate that subsequently dissociates to form carbonyl compounds. Based on this understanding and literature data, a hypothetical decay pathway for the breakdown of MEK under aerated conditions was postulated and used to create a computer model of MEK decay. (Abstract shortened by UMI.)

In the event of a loss-of-coolant accident (LOCA) in a water cooled reactor, a break in the primary cooling circuit followed by fuel failure could release a significant fraction of the core inventory of radioactive iodine into containment. Non-volatile aqueous iodide may be converted into volatile I$\sb2$ and subsequently released into the environment. This process has been shown to be affected by the radiolytic breakdown of dissolved organic compounds that may be present in containment. In this study, the effect of a model organic compound, 2-butanone (methylethylketone, MEK), on iodine chemistry was investigated. In this work, MEK was $\gamma$-irradiated in aqueous solutions and the formation of decay products were followed by a various analytical methods (HPLC, GC, GC/MS and MS) to establish a decay profile. For aerated solutions, MEK degradation produced products, of continually decreasing size. In contrast, in non-aerated solutions where hydroxyl radical was not scavenged evidence of dimerization was observed. Under aerated conditions the main pathway for radiolysis is the abstraction of a hydrogen atom by hydroxyl radical attack to give MEK radical which undergoes reaction with dissolved oxygen, producing an organic peroxide. This species recombines to form a tetroxide intermediate that subsequently dissociates to form carbonyl compounds. Based on this understanding and literature data, a hypothetical decay pathway for the breakdown of MEK under aerated conditions was postulated and used to create a computer model of MEK decay. (Abstract shortened by UMI.)

碩士<br>國立陽明大學<br>藥理學研究所<br>93<br>Abstract Epidemiological studies revealed that the incidence of oral cancer was 89-fold higher in people who smoked and chewed betel quid than those without such habits. Therefore, this study was designed to test whether alkaline environment generated by chewing betel quid containing lime facilitate the absorption of cigarette smoke containing carcinogens and consequently contribute to the synergistic potential. 4-(Methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) is a potent tobacco-specific carcinogen in animals and is rapidly reduced to its main metabolite, 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanol (NNAL). NNK is also metabolically activated by cytochromes P450 to produce DNA damage. In this study, NNK was applied to hamster buccal pouch at different pH (pH 7.0 and 8.7) and utilized the liquid chromatography/ tandem mass spectrometry (LC/MS/MS) to quantify urinary NNAL and try to test the absorption of NNK. Urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG) and 7-methylguanine（N7-MeG） were also determined, which are biomarkers for oxidative DNA damage and methylation lesion, respectively. NNAL and 8-OHdG were isolated by solid-phase extraction (SPE), separated by liquid chromatography and quantified by electrospray ionization mass spectrometry. The recovery of NNAL, 8-OHdG and N7-MeG were in the range of 94 to 116 ％. The linearity of these three analytes ranged from 0.05 to 50 ngml-1, 0.05 to 20 ngml-1 and 0.5 to 20 ngml-1, respectively . Limit of detection (LOD) was 0.01 ngml-1 and that of quantification (LOQ) was 0.05 ngml-1 for NNAL and 8-OHdG. LOD and LOQ of N7-MeG approached 0.01 and 0.5 ngml-1, respectively. This accurate LC/MS/MS method has potential to determine three biomarkers, such as NNAL, 8-OHdG and N7-MeG. These data implied that urinary NNAL was significantly higher under the alkaline condition (pH 8.7) than that in normal pH (pH 7.0), indicating that the absorption of NNK is facilitated in alkaline environment. Moreover, the level in urinary 8-OHdG and N7-MeG were also higher at pH 8.7 than at pH 7.0, demonstrating that NNK might induce more oxidative DNA damage and methylation lesion under the alkaline condition. Overall, these findings indicated that alkaline environment generated by chewing betel quid containing lime enhances the absorption of tobacco carcinogen NNK.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is believed to play a role in human lung cancer induced by tobacco smoking. NNK biotransformation may involve the enzymes prostaglandin H synthase (PHS)-1, PHS-2 and cytochrome 450 (CYP) 2F. PHS activity is thought to be important in extrahepatic tissues, where CYP activity is low. The CYP2F subfamily contains a single functional enzyme in humans (CYP2F1) and goats (CYP2F3); these enzymes are preferentially expressed in the lung, with little or no expression in other organs. The role of these enzymes in the pulmonary biotransformation of NNK was investigated. 4.2 µM [5-3H]NNK was incubated with human lung microsomes under NADPH-dependent and arachidonic acid-dependent conditions. Metabolites reflective of NNK α-carbon hydroxylation, N-oxidation and carbonyl reduction were detected in the presence of NADPH, and metabolite levels for all three biotransformation pathways were lower in the presence of arachidonic acid compared with NADPH (p<0.05, N=4). Incubation of microsomes with the PHS-1 selective inhibitor SC-560 and the PHS-2 selective inhibitor NS-398 did not change NNK biotransformation either in the presence of NADPH or in the presence of arachidonic acid (p>0.05, N=4). Incubation of [5-3H]NNK with ovine PHS-1 or PHS-2 did not result in formation of α-carbon hydroxylation or N­-oxidation metabolites; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was measurable only in the presence of PHS-2. Incubation of goat recombinant CYP2F3 with [5-3H]NNK resulted in formation of keto acid, keto alcohol and NNK-N-oxide (65.0%, 17.5% and 30.0% (µmol enzyme)-1 minute-1, respectively). Metabolite formation was inhibited by 3-methylindole (3-MI), a mechanism-based inactivator of CYP2F3. Based on an N value of 3, incubation of human lung microsomes with 3-MI inhibited N-oxidation (p<0.05) but did not alter NNK bioactivation or carbonyl reduction (p>0.05). However, when metabolite formation was examined in lung microsomes from different individuals, decreases in NNK biotransformation (ranging from 19.6 to 68.5%) were observed and were more pronounced in some patients than others, suggesting inter-individual variability in CYP2F1 activity. These studies demonstrate the ability of CYP2F to biotransform NNK and suggest inter-individual variability in the importance of CYP2F1 for this activity in human lung. They also strongly argue against the involvement of PHS enzymes.<br>Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2007-12-30 16:12:58.228

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen found in unburned tobacco and tobacco smoke. To exert its carcinogenic effect, NNK is metabolically activated to reactive intermediates that can damage DNA by alkylation or pyridyloxobutylation. NNK also has the ability to induce DNA oxidation and alter DNA repair activities that can result in deficient repair and potentially exacerbate carcinogenesis. Base excision repair (BER) is a ubiquitous DNA repair system that mainly repairs oxidative DNA damage. The goal of this study was to determine the effect of NNK on DNA oxidation status and BER activity in A/J mouse lung and liver. Female mice were treated with 10 µmol of NNK i.p. and lung and liver were isolated 1, 2 and 24 hours post administration. DNA was isolated from lung and liver, and the formation of 8-hydroxydeoxyguanosine (8-OHdG, a biomarker of DNA oxidation) was assessed by high-performance liquid chromatography with electrochemical detection. At 1, 2 and 24 hours in both murine lung and liver, there was no statistically significant difference in 8-OHdG levels (n = 4, P > 0.05) between control and NNK-treated mice. To assess BER, cell-free whole tissue nuclear protein extracts from liver and lung were prepared and incubated with a plasmid substrate containing oxidative DNA damage. In vivo treatment with NNK did not alter BER activity in lung or liver compared to control mice (n=3 or 4, P > 0.05). These experiments indicate that acute treatment with a tumourigenic dose of NNK does not significantly stimulate oxidative DNA damage or significantly alter BER activity in murine lung and liver.<br>Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2011-04-28 17:42:08.172

The interest in supercritical fluid extraction (SFE) to obtain bioactive compounds has risen over the years. This interest derives from a lower environmental impact of supercritical fluids (SFC), compared to the conventional solvents, and due to the quality and purity of the final products, which in turn, leads to the need of knowledge about the compounds transport properties for the accurate design of industrial processes. The initial objective of this dissertation was the measurement and modelling of binary diffusion coefficients (𝐷12) of bioactive compounds in supercritical mixtures of CO2 (SC-CO2) and ethanol. However, due to problems in the experimental installation, it was not possible to operate with SC-CO2 so 𝐷12 was measured in ethanol. These data will be important for modelling the properties of CO2 and ethanol mixtures. The 𝐷12 of five ketones (acetone, 2-butanone, 2-pentanone, 2-hexanone and 3-hexanone) were measured in ethanol, in the range of 1 − 150 𝑏𝑎𝑟 and 303.15 −333.15 𝐾, using the Chromatographic Peak Broadening (CPB) method. The obtained results were in the range of [1.77− 2.89]∙ 10−5 (𝑐𝑚2 𝑠 −1 ) for acetone, [1.53 − 2.63]∙ 10−5 (𝑐𝑚2 𝑠 −1 ) for 2-butanone, [1.39 − 2.41]∙ 10−5 (𝑐𝑚2 𝑠 −1 ) for 2-pentanone, [1.29 − 2.18]∙ 10−5 (𝑐𝑚2 𝑠 −1 ) for 2-hexanone and [1.28 − 2.17]∙ 10−5 (𝑐𝑚2 𝑠 −1 ) for 3-hexanone. Experimental data were analysed using models based on the free volume theory (e.g. Dymond-Hildebrand-Batschinski, DHB), on the hydrodynamic theory, hybrid models (e.g. Tracer Liu-Silva-Macedo, TLSM) and empirical correlations. The models were assessed in terms of the mean absolute relative deviation (𝐴𝐴𝑅𝐷). The most suitable models to represent 𝐷12 were: DHB for acetone, 2-butanone, 2-pentanone, 2- hexanone and 3-hexanone (𝐴𝐴𝑅𝐷 = 2.22 %, 2.43 %, 2.37 %, 2.33 % and 3.65 %, respectively), TLSMd for acetone, 2-butanone, 2-pentanone, 2-hexanone and 3-hexanone (𝐴𝐴𝑅𝐷 = 0.90 %, 1.82 %, 1.91 %, 1.56 % and 2.63 %, respectively) and Magalhães et al. correlation for acetone, 2-butanone, 2-pentanone, 2-hexanone and 3-hexanone (𝐴𝐴𝑅𝐷 = 0.42 %, 0.89 %, 0.45 %, 0.49 % and 1.13 %, respectively).<br>O interesse em extração de fluídos supercríticos (SFE), para obter compostos bioativos, tem vindo a aumentar ao longo dos anos. Este interesse deve-se ao menor impacto ambiental dos fluídos supercríticos (SCF), em relação aos solventes convencionais, e ao facto de proporcionarem produtos finais de elevada pureza. o que por sua vez, leva à necessidade do conhecimento sobre as propriedades de transporte nos solventes supercríticos para o dimensionamento preciso dos processos industriais. O objetivo inicial desta dissertação era a medição e modelação de coeficientes de difusão binários (𝐷12) de componentes bioativos em misturas supercríticas de CO2 e etanol. No entanto, devido a problemas na instalação experimental não foi possível operar com o CO2 supercrítico pelo que se mediram 𝐷12 em etanol. Estes dados serão importantes para a modelação das propriedades em misturas de CO2 e etanol. Mediram-se os coeficientes de difusão de cinco cetonas (acetona, 2- butanona, 2-pentanona, 2-hexanona e 3-hexanona) em etanol, no intervalo de 1 −150 𝑏𝑎𝑟 e 303.15 − 333.15 𝐾, usando o método cromatográfico de abertura de pico (CPB). Os resultados obtidos encontram-se no intervalo de [1.77 − 2.89] ⋅ 10−5 (𝑐𝑚2 𝑠 −1 ) para acetona, [1.53 − 2.63] ⋅ 10−5 (𝑐𝑚2 𝑠 −1 ) para 2-butanona, [1.39 − 2.41] ∙ 10−5 (𝑐𝑚2 𝑠 −1 ) para 2-pentanona, [1.29 − 2.18] ∙ 10−5 (𝑐𝑚2 𝑠 −1 ) para 2-hexanona e [1.28 − 2.17] ∙ 10−5 (𝑐𝑚2 𝑠 −1 ) para 3- hexanona. Os dados experimentais foram analisados usando modelos baseados na teoria de volume livre (p.e. Dymond-Hildebrand-Batschinski, DHB), na teoria hidrodinâmica, nos modelos híbridos (p.e. Tracer Liu-Silva Macedo, TLSM) e em correlações empíricas. Os modelos foram avaliados em termos de desvios relativos absolutos médios (𝐴𝐴𝑅𝐷) concluindo-se que os modelos mais adequados para representar 𝐷12 são: DHB para acetona, 2-butanona, 2-pentanona, 2-hexanona e 3- hexanona (𝐴𝐴𝑅𝐷 = 2.22 %, 2.43 %, 2.37 %, 2.33 % e 3.65 %, respetivamente), TLSMd para acetona, 2-butanona, 2-pentanona, 2- hexanona e 3-hexanona (𝐴𝐴𝑅𝐷 = 0.90 %, 1.82 %, 1.91 %, 1.56 % e 2.63 %, respetivamente) e correlação de Magalhães et al. Para acetona, 2-butanona, 2-pentanona, 2-hexanona e 3-hexanona (𝐴𝐴𝑅𝐷 = 0.42 %, 0.89 %, 0.45 %, 0.49 % e 1.13 %, respetivamente).<br>Mestrado em Engenharia Química

肺癌是一個世界性的健康難題。大量研究證據顯示，煙草及其致癌物NNK對環氧酶（COX）-2及其下游產物具有促進效應。血栓素（TxA）2是COX-2的關鍵性下游產物之一，該論文闡述了TxA2在NNK導致的肺癌增長中的可能作用。<br>我們發現相对于非吸烟者，吸煙者肺癌組織表达更高水平的TxA2合酶（TxAS）。NNK可以刺激培養的肺癌細胞TxA2合成。用TxAS抑制劑和TxA2受體（TP）拮抗劑分別阻抑TxA2的合成與功能可以引起細胞凋亡，從而有效抑制NNK導致的細胞增殖效應。在TxA2合成受抑制的情況下，TP激動劑U46619幾乎可以重建NNK效應，說明TP在NNK效應中的重要作用。研究還顯示，激活的TP可以通過PI3K/Akt和ERK通路進一步激活CREB，從而參與NNK對肺癌細胞的促生長效應。<br>緊接著，我們的研究顯示TP 可以調節NNK對COX-2 和TxA2的誘導，而且發現NNK刺激的TxA2合成主要依賴於COX-2活性。COX-2和TxA2功能抑製劑對NNK的促細胞生長作用具有相似的抑制效用。考慮到TP是TxA2的功能受體，該資料說明TP在NNK處理的肺癌細胞中傳遞了上游因子COX-2的促腫瘤作用。在使用COX-2小干擾RNA（siRNA）抑制NNK作用的情況下，TP激動劑U46619幾乎可以恢復NNK的效應證實了TP的傳遞者角色。研究還發現 TPα而不是TPβ在培養的肺癌細胞系中廣泛表達，並且過表達TPα具有促進腫瘤生長的作用。在用NNK處理細胞的條件下，TPα還具有促COX-2表達和TxA2生成的作用。<br>我們的研究進一步發現，在吸煙者肺癌組織中TPα表達增高，這與TxAS的表達相似。与此结果相一致，在經NNK處理的A/J小鼠肺癌組織中，TxAS和TP表達水準也是明顯上升的。在細胞培養實驗中，NNK能夠提高TxAS蛋白和信使RNA（mRNA）的表達水準。但是，在TP的兩個亞型TPα和TPβ中， NNK僅能促進TPα的蛋白表達，對它們的mRNA均無影響。NNK對TxAS的促表達作用是核轉錄因數(NF)-κB依賴性的。其他的幾個關鍵轉錄因數，諸如特異性蛋白(SP)-1，CREB和活化受體 （PPAR）γ均未參與NNK對TxAS和TPα的表達促進作用。進一步的，轉錄後機理被證實參與了NNK對TPα的作用。TPα而不是TPβ經鑒別在NNK的促NF-κB 激活 和 促TxAS 表達效應中起正向調節作用。<br>總之， 我們的研究說明TxA2相關通路在NNK的促肺癌細胞生長效應中起正向調節作用。我們的研究揭示了TPα的自我激活環路。通過該環路，TxA2，或者說TxAS和TPα參與了NNK的肺癌促生長效應。因此，我們的研究為肺癌的防治了提供了一個新的方向，即靶向TxAS和TPα是一種可能有效的策略。<br>Lung cancer concerns a world-wide health problem. There is considerable evidence of that tobacco smoke and its carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) have the potential effects on the production of cyclooxygenase (COX)-2 and its downstream products in tumor cells. This thesis is constructed to describe the study focused on the role of thromboxane A2 (TxA2), one of the key downstream products of COX-2, in NNK-induced lung tumor growth.<br>We found that as compared to non-smokers, lung cancer tissues obtained from smokers tended to express more TxA2 synthase (TxAS). Moreover, NNK could stimulate TxA2 synthesis in lung cancer cells. Blockade of TxA2 synthesis and action by TxAS inhibitor and TxA2 receptor (TP) antagonist completely blocked NNK-promoted cell proliferation via inducing apoptosis. Moreover, TP agonist U46619 reconstituted a near full proliferative response to NNK when TxAS was inhibited, affirming the role of TP in NNK-induced cell growth. Furthermore, we revealed that the activated TP may then activate CREB through PI3K/Akt and ERK pathways, thereby contributing to the NNK-induced lung cancer cell growth.<br>We subsequently showed that TP could modulate the induction of COX-2 and TxA2 by NNK. The synthesis of TxA2 stimulated by NNK was found to be mainly dependent on COX-2 activity. Intriguingly, there are similar inhibitory effects on NNK-induced cell growth between pharmacological inhibition of COX-2 and the blockade of TxA2 synthesis and action. Because TP is the natural receptor of TxA2, these results suggest that TP may function as a mediator for the tumor-promoting effects of COX-2 upon NNK treatment, which was confirmed by the data showing that U46619 almost restored NNK effects in the presence of COX-2-siRNA. Importantly, TPα, but not TPβ was found to be widely expressed in lung cancer cells and be able to promote tumor growth, COX-2 expression and TxA2 synthesis upon NNK treatment.<br>We further demonstrated that in lung tumor tissues obtained from smoker, TPα protein was increased, which was similar to the change in TxAS protein. The increased levels of TxAS and TP proteins were also found in lung cancer tissues of A/J mice treated with NNK. In cell culture experiments, NNK could increase TxAS at both protein and mRNA levels. However, TPα rather than TPβ was increased by NNK at protein but not mRNA level. NNK-stimulated TxAS expression was dependent on nuclear factor (NF)-κB signaling. Other key transcriptional factors, such as specificity protein(SP)-1, CREB and peroxisome proliferator-activated receptor-gamma (PPARγ), were not involved in NNK-induced TxAS and TPα expression. Further experiments revealed that post-transcriptional mechanisms were responsible for NNK-induced TPα expression. TPα rather than TPβ was finally identified to have a positive role in NNK-induced NF-κB activation and TxAS expression.<br>Taken together, our study suggests that TxA2 pathway has a positive role in NNK-induced lung cancer cell growth. An auto-positive feedback loop of TPα activation to facilitate lung tumor growth in the presence of NNK is delineated by these results. Therefore, targeting TxAS or/and TPα may represent a promising strategy for prevention and treatment of lung cancer.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Huang, Runyue.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.<br>Includes bibliographical references (leaves 119-146).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract also in Chinese.<br>Abstract --- p.I<br>摘要<br>Publications<br>Acknowledgement<br>Abbreviations<br>Table of contents<br>Chapter Chapter 1 --- General introduction--Tobacco smoking, COX-2 pathway and cancer<br>Chapter 1.1 --- Abstract --- p.1<br>Chapter 1.2 --- Introduction --- p.2<br>Chapter 1.3 --- Cyclooxygenase and prostanoids --- p.5<br>Chapter 1.4 --- The effects of tobacco smoking on COX-2 pathway, and the related pathologies --- p.8<br>Chapter 1.4.1 --- Smoking, PGE2, inflammation and immunosupression --- p.8<br>Chapter 1.4.2 --- Smoking, TxA2, platelet activation, cell contraction and angiogenesis --- p.11<br>Chapter 1.4.3 --- Smoking and PGI2 --- p.16<br>Chapter 1.5 --- The role of cyclooxygenase-2 pathway in the progression of tobacco smoke-related cancers --- p.19<br>Chapter 1.5.1 --- Lung cancer --- p.19<br>Chapter 1.5.2 --- Gastrointestinal cancer --- p.23<br>Chapter 1.5.3 --- Bladder cancer --- p.24<br>Chapter 1.5.4 --- Head and neck squamous cell carcinoma --- p.25<br>Chapter 1.5.5 --- The signaling mechanisms underlying the induction of COX-2 by smoking in tumors --- p.26<br>Chapter 1.6 --- Summary, future directions and key questions --- p.28<br>Chapter Chapter 2 --- NNK induces lung cancer cell growth by stimulating TxA2 and its receptor<br>Chapter 2.1 --- Abstract --- p.32<br>Chapter 2.2 --- Introduction --- p.33<br>Chapter 2.3 --- Materials and Methods --- p.35<br>Chapter 2.3.1 --- Cell lines and cell culture --- p.35<br>Chapter 2.3.2 --- Chemicals and drug treatment --- p.35<br>Chapter 2.3.3 --- Thromboxane B2 EIA assay --- p.36<br>Chapter 2.3.4 --- MTT assay --- p.36<br>Chapter 2.3.5 --- BrdU cell proliferation assay --- p.37<br>Chapter 2.3.6 --- Flow cytometry for analysis of apoptosis --- p.37<br>Chapter 2.3.7 --- Transfection of cells with CREB siRNA --- p.38<br>Chapter 2.3.8 --- Western blot analysis and antibodies --- p.38<br>Chapter 2.3.9 --- Statistical analysis --- p.39<br>Chapter 2.4 --- Results --- p.41<br>Chapter 2.4.1 --- High expression of TxAS in lung cancer tissues of smoker --- p.41<br>Chapter 2.4.2 --- NNK stimulated TxA2 synthesis in lung cancer cells --- p.43<br>Chapter 2.4.3 --- Blockade of TxA2 synthesis and action prevented NNK-induced cell growth --- p.44<br>Chapter 2.4.4 --- TxA2 mimetic U46619 reconstituted NNK-enhanced cell proliferation under TxA2-inhibited condition --- p.47<br>Chapter 2.4.5 --- Blockade of TxA2 synthesis or action induced the apoptosis of the NNK-exposed cells --- p.47<br>Chapter 2.4.6 --- CREB is accountable for the key role of TxA2 in NNK-enhanced cell proliferation --- p.49<br>Chapter 2.4.7 --- PI3K/Akt and ERK rather than JNK and p38 pathways were mediated by TxA2 in the NNK-exposed cells --- p.52<br>Chapter 2.4.8 --- CREB is located downstream of the PI3K/Akt and ERK pathways in NNK-treated cells --- p.53<br>Chapter 2.5 --- Discussion --- p.55<br>Chapter Chapter 3 --- The positive role of TPα in the induction of COX-2, TxA2 and cell growth by NNK in human lung cancer cells<br>Chapter 3.1 --- Abstract --- p.62<br>Chapter 3.2 --- Introduction --- p.63<br>Chapter 3.3 --- Materials and methods --- p.65<br>Chapter 3.3.1 --- Cell culture and chemicals --- p.65<br>Chapter 3.3.2 --- Transient transfections --- p.66<br>Chapter 3.3.3 --- TxB2 measurement --- p.66<br>Chapter 3.3.4 --- Cell growth detection --- p.67<br>Chapter 3.3.5 --- Analysis of apoptosis --- p.67<br>Chapter 3.3.6 --- Western blot analysis and antibodies --- p.67<br>Chapter 3.3.7 --- Statistical analysis --- p.68<br>Chapter 3.4 --- Results --- p.70<br>Chapter 3.4.1 --- Examination of TP as the modulator for induction of COX-2 and TxA2 by NNK --- p.70<br>Chapter 3.4.2 --- The TxA2 generated in cells treated with NNK is mainly dependent on COX-2 activity --- p.72<br>Chapter 3.4.3 --- Examination of TP as the key mediator for the tumor-promoting effect of COX-2 --- p.72<br>Chapter 3.4.4 --- The expression and action of α and β isoforms of TP in human lung cancer cells --- p.77<br>Chapter 3.4.5 --- the identification of positive role of TPα in NNK-induced COX-2, TxA2 and cell growth in lung cancer cells --- p.79<br>Chapter 3.5 --- Discussion --- p.81<br>Chapter Chapter 4 --- TP-α facilitates lung tumor growth through an autoregulatory feedback mechanism<br>Chapter 4.1 --- Abstract --- p.88<br>Chapter 4.2 --- Introduction --- p.89<br>Chapter 4.3 --- Materials and methods --- p.91<br>Chapter 4.3.1 --- Human lung tissue and immunohistochemical analysis --- p.91<br>Chapter 4.3.2 --- Animal treatment --- p.91<br>Chapter 4.3.3 --- Cell culture and chemicals --- p.92<br>Chapter 4.3.4 --- Transient transfection --- p.93<br>Chapter 4.3.5 --- Real-time PCR --- p.93<br>Chapter 4.3.6 --- Western blot analysis and antibodies --- p.94<br>Chapter 4.3.7 --- Statistical analysis --- p.95<br>Chapter 4.4 --- Results --- p.96<br>Chapter 4.4.1 --- The effects of smoking on the expression of TP in human lung cancer tissue --- p.96<br>Chapter 4.4.2 --- The effects of NNK on the expression of TxAS and TP in lung tissues of A/J mice --- p.98<br>Chapter 4.4.3 --- The effects of NNK on the expression of TxAS and TPα in lung cancer cells --- p.99<br>Chapter 4.4.4 --- Identification of the roles of NF-κB, CREB and SP1 in NNK-induced TxAS and TPα expression --- p.101<br>Chapter 4.4.5 --- The negative role of PPARγ in NNK-induced TxAS and TPα expression --- p.104<br>Chapter 4.4.6 --- NNK-induced TPα expression via post-transcriptional mechanism --- p.105<br>Chapter 4.4.7 --- Examination of TPα auto-activation mechanism in lung cancer cells stimulated with NNK --- p.106<br>Chapter 4.5 --- Discussion --- p.109<br>Chapter Chapter 5 --- Conclusion and future works<br>Chapter 5.1 --- Conclusion --- p.114<br>Chapter 5.2 --- Future works --- p.115<br>Chapter 5.2.1 --- The possible role of miR-34c in the auto-regulatory loop of TxAS expression or TPα activation --- p.116<br>Chapter 5.2.2 --- The possible role of FOXO3a in the auto-regulatory loop of TxAS expression or TPα activation --- p.116<br>References --- p.119

碩士<br>國防醫學院<br>病理及寄生蟲學研究所<br>101<br>Aim: Cigarette smoking with the tobacco-specific components of 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) becomes one of the major risk factors in the carcinogenesis of many cancers, including head and neck squamous cell carcinoma (HNSCC). Recent studies also suggest that long-term NNK exposure also involved in progression of HNSCC. However, the underlying nicotine-mediated mechanism (s) in regulating cancer stem cell (CSC) and anti-apoptotic properties in HNSCC during tumor progression still remains unclear. Methods and Results: We used SCC-25 and Fadu cells with LT-NNK treatment for three months as study groups. A comparative analysis was performed between the control and LT-NNK cells focusing on the evaluation of proliferation, migration and invasion abilities, the expression of epithelial–mesenchymal transition (EMT)-associated markers, drug-resistance-related genes, CSC, and anti-apoptotic properties. As a result, LT-NNK stimulates cell proliferation in a dose dependent manner and also induces obvious morphological alterations showing EMT phenomenon with changes of representative markers and attenuates apoptosis in addition to an enhancement of migration and invasion. Moreover, LT-NNK also promotes the sphere-forming ability and expression of the drug resistant genes, ABCG2 and MDR1 in addition to upregulation of Snail. Knockdown of Snail or administration of anti-alpha 7 nicotinic acetylcholine receptor increases apoptosis, along with upregulation of RKIP confirming Snail-RKIP signaling pathway. Conclusion: Our data demonstrated that the present study is the first to demonstrate the critical role of LT-NNK exposure of HNSCC responsible for the enhancement of anti-apoptosis and therapeutic resistance via Snail- RKIP signaling pathway. These findings may also provide that administration of anti-alpha 7 nicotinic acetylcholine receptor rather than targeting Snail as a feasible and rational method for the treatment of HNSCC.

碩士<br>中山醫學大學<br>營養學研究所<br>98<br>Quercetin, a flavonoid, is found ubiquitously in vegetables and fruits. Several in vitro studies have shown that quercetin suppresses the harmful effects of cigarette smoking-associated carcinogens alone or in combination with β-carotene through several mechanisms, including antioxidative and anti-inflammatory activity. However, quercetin conjugated metabolites rather than quercetin aglycone is present in human plasma because of its efficient phase II metabolism. Whether those effects of quercetin observed in vitro are significant in vivo is unclear. Therefore, using Balb/c mice and Mongolian gerbils, the thesis work was divided into two parts to study the influence of quercetin in vivo. Part I We investigated the preventive effects of quercetin at 50 mg/kg wt body (LQ) or 500 mg/kg body wt (HQ) against benzo[a]pyrene (Bap)+ 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced oxidative stress and the secretion of proinflammatory cytokines in Balb/c mice. The animals were randomly assigned to following six groups (n = 6/group): (1)control, (2) LQ, (3) HQ, (4) Bap+NNK, (5) Bap+NNK+LO, (6) Bap+NNK+HQ. Quercetin was given to animals by gavage 2 times/week for 3 months; whereas Bap+ NNK exposure was performed by intraperitoneal injection 1 time/week. HPLC assay showed that at 1-2 h after oral administration of quercetin, the sum concentration of plasma glucuronidated and sulfated quercetin reached the maximum (about 2.3 and 4 μM at LQ, and HQ group, respectively). LQ significantly reduced Bap+NNK-induced endogenous DNA damage, whereas two doses of qeurcetin significantly reduced Bap+NNK -increased H2O2-lesion in a dose-dependent manner. The TBARs levels in Bap+NNK+LQ and +HQ group were also significantly lower than that of Bap+NNK. In addition, Bap+NNK significantly increased the levels of TNF-α and IL-1βin bronchoalveolar lavage fluid and liver, while quercetin significantly decreased the effects of Bap+NNK in a dose-dependent manner. Such effects of quercetin may be associated with the regulation of c-Jun and JNK activation, because quercetin also decreased the phosphorylation of c-Jun and JNK induced by Bap+NNK. These results suggest that quercetin attenuates the pro-oxidative and pro-inflammatory effects of Bap+NNK in Balb/c mice. Part II In this part, we investigated whether quercetin decrease the effects ofβ-carotene induced by Bap in Mongolian gerbils (gerbils). We also compared the effects of quercetin with that of vitamin C (C)+vitamin E (E). The gerbils were given β-carotene (BC; 10 mg/kg body wt) alone or in comebination with quercetin (50 mg/kg body wt for LQ, or 100 mg/kg body wt for HQ) or vitamin C (13 mg/kg body wt) +vitamin E(92 mg/kg body wt) by gavage 3 times/week for 6 months. At the firs 2 months, expect for control group, the gerbils were exposed to Bap by intratracheal instillation 1 time/week. As expected, Bap exposure significantly increased endogenous DNA damage and H2O2-lesion of while blood cells and TBRAs concentration in plasma. BC enhanced the effect of Bap on increase of H2O2-lesion, while LQ/HQ+BC significantly suppressed the effect of Bap mentioned above. The total antioxidant activity, the level of glutathione and the activity of superoxide dimutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in lungs of gerbils in Bap-exposed group were significantly lower than those of control group. Compared with Bap exposure group, BC increased the levels or activity of the parameters mentioned above except for SOD; the effects of LQ/HQ+BC were better than or similar to that of BC alone in these parameters. LQ/HQ+BC also significantly increased the activity of SOD. In addition, Bap exposure significantly or slightly increased the concentration of IL-6, IL-1β and TNF-α in plasma or bronchoalveolar lavage fluid, respectively. BC tended to enhance such effects of Bap while LQ/HQ+BC tended to decreased the effects of Bap. Compared with BC+C+E, LQ/HQ+BC had similar effects on suppressing the proinflammatory effect of BC. These results demonstrated that supplemental quercetin interacts withβ-carotene in vivo. The antioxidative and anti-inflammatory effects of quercetin metabolites may be contributed to such effect of quercetin. Further studies are warranted to investigate the precise mechanisms underlying these effects of quercetin in vivo.