Thesis (Ph.D.)--University of Adelaide, Department of Organic Chemistry, 1977. / Includes bibliographical references.

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 105-110). Also available via the World Wide Web.

Thesis (Ph. D.)--University of Chicago, Pritzker School of Medicine, Department of Biochemistry and Molecular Biology, June 2002. / Includes bibliographical references. Also available on the Internet.

CAPES / A carne e seus derivados representam uma parte importante da dieta humana. Porém, devido a sua riqueza na composição química são suscetíveis a alterações de ordem físico-química como a oxidação lipídica, tornando necessário o uso de aditivos para sua conservação. A tendência é utilizar produtos naturais como alternativas aos aditivos químicos. Entre os produtos naturais, a manjerona e o pólen apícola apresentam, em sua composição, metabólitos secundários com alta atividade antioxidante. O objetivo deste estudo foi avaliar as propriedades bioativas de dois produtos, pólen apícola orgânico e manjerona (Origanum majorana L.), bem como sua aplicação como antioxidantes naturais em hambúrgueres. No pólen apícola desidratado, foi realizada caracterização palinológica e determinação da composição centesimal. O pólen apícola e a manjerona foram extraídos individualmente com etanol 80% em shaker, a 40 ºC/60 minutos. Os compostos fenólicos e flavonoides totais dos extratos etanólicos de pólen apícola e manjerona foram quantificados e a identificação feita por cromatografia líquida de alta eficiência em fase reversa (CLAE-FR). A atividade antioxidante dos extratos foi determinada e comparada à de antioxidantes comerciais por quatro métodos distintos. Após os extratos serem evaporados e liofilizados, foram aplicados separadamente na concentração de 0,3% em formulações de hambúrgueres de carne bovina, também foi elaborada uma formulação padrão, com 0,01% de butilhidroxitolueno; além da formulação controle, sem antioxidantes. Os hambúrgueres foram caracterizados através da determinação da composição centesimal. A oxidação lipídica foi determinada pelo índice de peróxido a cada 14 dias, e pelo índice de substâncias reativas ao ácido tiobarbitúrico (TBARS) a cada 7 dias. Após avaliação microbiológica foi realizada a análise sensorial. Verificou-se que o pólen apícola foi classificado como heterofloral, com predominância da família Asteraceae. Os resultados obtidos na composição centesimal do pólen apícola, com exceção do teor de umidade, estão em conformidade com a legislação brasileira. O extrato de manjerona apresentou o maior teor de compostos fenólicos e flavonoides totais em relação ao extrato de pólen apícola. Pela técnica de CLAE foi possível identificar e quantificar os flavonoides rutina, miricetina, quercetina e canferol, e os ácidos fenólicos, cafêico, p-cumárico, ferúlico e trans-cinâmico. Os extratos apresentaram alta atividade antioxidante in vitro pelos quatro métodos avaliados, sendo esta atividade atribuída à presença de compostos fenólicos. Os resultados obtidos para composição centesimal dos hambúrgueres indicam que com exceção do conteúdo de carboidratos, os demais parâmetros estão de acordo com a legislação brasileira. Com relação à oxidação lipídica, não houve a formação de peróxidos em nenhuma formulação, durante todo o período em que os produtos permaneceram congelados. Os extratos de manjerona e pólen apícola demonstraram resultados satisfatórios pelo TBARS, portanto, a concentração de antioxidantes naturais, foi suficiente para manter a estabilidade oxidativa. Sob os aspectos microbiológicos, os hambúrgueres atenderam à legislação vigente. Os índices de aceitabilidade para a avaliação global foram superiores a 70% para as formulações controle, padrão e adicionada de pólen apícola, no entanto a aplicação de manjerona interferiu negativamente nas características sensoriais. / The meat and its derivatives represent an important part of the human diet. However, due to its richness in chemical composition are susceptible to changes in physicalchemical and lipid oxidation, requiring the use of additives for its preservation. The trend is to use natural products as alternatives to the chemical additives. Among the natural products, marjoram and bee pollen present in its composition, secondary metabolites with high antioxidant activity. The aim of this study was to evaluate the bioactive properties of two products, organic bee pollen and marjoram (Origanum majorana L.) and its application as natural antioxidants in burgers. Dehydrated in bee pollen, pollen characterization was performed and determination of chemical composition. The bee pollen and marjoram were individually extracted with 80% ethanol in a shaker at 40 °C/60 minutes. Phenolic compounds and total flavonoid of ethanol extracts of pollen and marjoram were quantified and the identification made by high performance liquid chromatography on reversed phase (HPLC-RP). The antioxidant activity of the extracts was determined and compared to that of commercial antioxidants by four different methods. After the extracts were evaporated and lyophilized, were applied separately at a concentration of 0.3% in formulations of beef burgers, was also drafted a standard formulation with 0.01% butylhydroxytoluene; beyond the control formulation without antioxidants. The burgers were characterized by determining the chemical composition. Lipid oxidation was determined by peroxide value every 14 days, and the rate of thiobarbituric acid reactive substances (TBARS) every 7 days. After microbiological evaluation was performed sensory analysis. It was found that bee pollen was classified as heterofloral, predominantly family Asteraceae. The results on the chemical composition of bee pollen, with the exception of moisture content, the other parameters are in accordance with Brazilian law. The extract of marjoram had the highest content of total phenolics and flavonoids in relation to the extract of pollen. By HPLC technique was possible to identify and quantify flavonoids rutin, myricetin, quercetin and kaempferol, and phenolic acids, caffeic, p-coumaric, ferulic and transcinnamic acid. The extracts showed high antioxidant activity in vitro by the four methods evaluated, this activity being attributed to the presence of phenolic compounds. The results obtained for proximate composition of burgers indicate that with the exception of carbohydrate content, the other parameters are in accordance with Brazilian law. With respect to lipid oxidation, no formation of peroxides in any formulation during the period in which the products remained frozen. The extracts of bee pollen and marjoram demonstrated by the TBARS satisfactory results, therefore, the concentration of natural antioxidants was sufficient to maintain oxidative stability. Under the microbiological aspects, the burgers attended the law. The rate of acceptability for the overall evaluation was above 70% for the control formulations, standard and added bee pollen, however the application of marjoram negative influence on sensory characteristics.

CAPES / A carne e seus derivados representam uma parte importante da dieta humana. Porém, devido a sua riqueza na composição química são suscetíveis a alterações de ordem físico-química como a oxidação lipídica, tornando necessário o uso de aditivos para sua conservação. A tendência é utilizar produtos naturais como alternativas aos aditivos químicos. Entre os produtos naturais, a manjerona e o pólen apícola apresentam, em sua composição, metabólitos secundários com alta atividade antioxidante. O objetivo deste estudo foi avaliar as propriedades bioativas de dois produtos, pólen apícola orgânico e manjerona (Origanum majorana L.), bem como sua aplicação como antioxidantes naturais em hambúrgueres. No pólen apícola desidratado, foi realizada caracterização palinológica e determinação da composição centesimal. O pólen apícola e a manjerona foram extraídos individualmente com etanol 80% em shaker, a 40 ºC/60 minutos. Os compostos fenólicos e flavonoides totais dos extratos etanólicos de pólen apícola e manjerona foram quantificados e a identificação feita por cromatografia líquida de alta eficiência em fase reversa (CLAE-FR). A atividade antioxidante dos extratos foi determinada e comparada à de antioxidantes comerciais por quatro métodos distintos. Após os extratos serem evaporados e liofilizados, foram aplicados separadamente na concentração de 0,3% em formulações de hambúrgueres de carne bovina, também foi elaborada uma formulação padrão, com 0,01% de butilhidroxitolueno; além da formulação controle, sem antioxidantes. Os hambúrgueres foram caracterizados através da determinação da composição centesimal. A oxidação lipídica foi determinada pelo índice de peróxido a cada 14 dias, e pelo índice de substâncias reativas ao ácido tiobarbitúrico (TBARS) a cada 7 dias. Após avaliação microbiológica foi realizada a análise sensorial. Verificou-se que o pólen apícola foi classificado como heterofloral, com predominância da família Asteraceae. Os resultados obtidos na composição centesimal do pólen apícola, com exceção do teor de umidade, estão em conformidade com a legislação brasileira. O extrato de manjerona apresentou o maior teor de compostos fenólicos e flavonoides totais em relação ao extrato de pólen apícola. Pela técnica de CLAE foi possível identificar e quantificar os flavonoides rutina, miricetina, quercetina e canferol, e os ácidos fenólicos, cafêico, p-cumárico, ferúlico e trans-cinâmico. Os extratos apresentaram alta atividade antioxidante in vitro pelos quatro métodos avaliados, sendo esta atividade atribuída à presença de compostos fenólicos. Os resultados obtidos para composição centesimal dos hambúrgueres indicam que com exceção do conteúdo de carboidratos, os demais parâmetros estão de acordo com a legislação brasileira. Com relação à oxidação lipídica, não houve a formação de peróxidos em nenhuma formulação, durante todo o período em que os produtos permaneceram congelados. Os extratos de manjerona e pólen apícola demonstraram resultados satisfatórios pelo TBARS, portanto, a concentração de antioxidantes naturais, foi suficiente para manter a estabilidade oxidativa. Sob os aspectos microbiológicos, os hambúrgueres atenderam à legislação vigente. Os índices de aceitabilidade para a avaliação global foram superiores a 70% para as formulações controle, padrão e adicionada de pólen apícola, no entanto a aplicação de manjerona interferiu negativamente nas características sensoriais. / The meat and its derivatives represent an important part of the human diet. However, due to its richness in chemical composition are susceptible to changes in physicalchemical and lipid oxidation, requiring the use of additives for its preservation. The trend is to use natural products as alternatives to the chemical additives. Among the natural products, marjoram and bee pollen present in its composition, secondary metabolites with high antioxidant activity. The aim of this study was to evaluate the bioactive properties of two products, organic bee pollen and marjoram (Origanum majorana L.) and its application as natural antioxidants in burgers. Dehydrated in bee pollen, pollen characterization was performed and determination of chemical composition. The bee pollen and marjoram were individually extracted with 80% ethanol in a shaker at 40 °C/60 minutes. Phenolic compounds and total flavonoid of ethanol extracts of pollen and marjoram were quantified and the identification made by high performance liquid chromatography on reversed phase (HPLC-RP). The antioxidant activity of the extracts was determined and compared to that of commercial antioxidants by four different methods. After the extracts were evaporated and lyophilized, were applied separately at a concentration of 0.3% in formulations of beef burgers, was also drafted a standard formulation with 0.01% butylhydroxytoluene; beyond the control formulation without antioxidants. The burgers were characterized by determining the chemical composition. Lipid oxidation was determined by peroxide value every 14 days, and the rate of thiobarbituric acid reactive substances (TBARS) every 7 days. After microbiological evaluation was performed sensory analysis. It was found that bee pollen was classified as heterofloral, predominantly family Asteraceae. The results on the chemical composition of bee pollen, with the exception of moisture content, the other parameters are in accordance with Brazilian law. The extract of marjoram had the highest content of total phenolics and flavonoids in relation to the extract of pollen. By HPLC technique was possible to identify and quantify flavonoids rutin, myricetin, quercetin and kaempferol, and phenolic acids, caffeic, p-coumaric, ferulic and transcinnamic acid. The extracts showed high antioxidant activity in vitro by the four methods evaluated, this activity being attributed to the presence of phenolic compounds. The results obtained for proximate composition of burgers indicate that with the exception of carbohydrate content, the other parameters are in accordance with Brazilian law. With respect to lipid oxidation, no formation of peroxides in any formulation during the period in which the products remained frozen. The extracts of bee pollen and marjoram demonstrated by the TBARS satisfactory results, therefore, the concentration of natural antioxidants was sufficient to maintain oxidative stability. Under the microbiological aspects, the burgers attended the law. The rate of acceptability for the overall evaluation was above 70% for the control formulations, standard and added bee pollen, however the application of marjoram negative influence on sensory characteristics.

Les kinases dépendantes des Cyclines (Cdk) contrôlent le déroulement du cycle cellulaire, mais leur étude est difficile car des mécanismes de compensation se développent lorsqu'une Cdk est absente. Cdk2 est principalement impliquée dans la phase de réplication de l'ADN, cependant l'ablation génétique de Cdk2 chez la souris n'a pas d'effet sur leur développement: les fonctions de Cdk2 sont compensées par d'autres Cdk. L'inhibition chimique permet de bloquer une Cdk et de limiter les compensations. Pour étudier les rôles de Cdk2, nous l'avons inhibé avec NU6102 qui sélectif pour Cdk2 dans le modèle xénope. Nous avons aussi développé des mutants de Cdk2 résistants à NU6102 pour vérifier sa sélectivité et avons cherché à mieux comprendre les paramètres qui déterminent l'affinité entre Cdk2 et un ligand. D'autre part, nous déterminons in vitro que NU6102 serait plus sélectif pour Cdk2 que pour les autres Cdk chez l'humain, et avons décrit les phénotypes induits par cet inhibiteur dans les cellules humaines en cultures. Ces résultats ne permettent pas de confirmer la sélectivité de NU6102 mais montrent que NU6102 a des caractéristiques intéressantes pour être utilisé dans le traitement contre le cancer. L'activité des Cdk est essentielle à l'initiation de la réplication de l'ADN, mais aucun substrat essentiel n'a été identifié chez les métazoaires. Nous avons réalisé un crible des protéines chargées sur la chromatine en présence et en absence d'activité Cdk dans le modèle xénope, afin d'identifier des substrats qui pourraient être impliqués dans la réplication. Ces résultats suggèrent que l'activité Cdk, qui initie la réplication au niveau des origines de réplication de l'ADN, pourrait être impliquée dans d'autres fonctions cellulaires. / Cycline Dependant Kinases (Cdk) control cell cycle progression. The study of their roles is often difficult because of functional redundancy; when a given Cdk is absent, others may compensate. The main role of Cdk2 in the cell cycle is in the initiation of DNA replication, but absence of Cdk2 is compensated for by Cdk1. For example, mice with a genetic knockout of Cdk2 are viable. The chemical inhibition of Cdks may limit compensation by other Cdks. Therefore, to study Cdk2 roles, we have studied chemical inhibition by NU6102, which seems to be selective for Cdk2 in the Xenopus model. To verify the selectivity and study parameters that determine selectivity, we have designed and produced mutants of Cdk2 which are resistant to NU6102, allowing restoration of function in the presence of inhibitor. Moreover, we demonstrate in vitro that NU6102 is selective for Cdk2 compared to other human Cdks, and we describe phenotypes induced by NU6102 in cultured cells, which are interesting in the light of potential applications of NU6102 in cancer chemotherapy. Cdk activity is essential for initiation of DNA replication, but in metazoans no essential substrates are known. To identify potential Cdk substrates during DNA replication, we have performed a proteomics screen of the proteins loaded onto chromatin in the presence or absence of Cdk activity, in the Xenopus model. The results suggest that Cdk activity is not only required for assembling DNA replication complexes onto origins of replication, but may also be implicated in other cellular functions.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18181号 / 医博第3901号 / 新制||医||1004(附属図書館) / 31039 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩田 想, 教授 松田 道行, 教授 楠見 明弘 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Traditional bacterial disease therapies utilize compounds that ultimately kill the target bacteria but it exerts a strong selective pressure on the bacteria to develop multi-drug resistance mutants. The increasing occurrence of resistance in common pathogens has highlighted the need to identify new anti-microbials that target the control of bacterial pathogenicity in a non-extermination manner to reduce the incidence of bacteria resistance. One new strategy exploits the discrete signaling molecules that regulate the various bacterial signaling pathways, which are responsible for the expression of pathogenicity traits. Halogenated furanones (fimbrolides) from the marine red alga, Delisea pulchra have been shown to interfere with the key signaling pathway present in Gram-negative bacteria by competitively displacing the cognate signaling molecule from the transcription protein. This project focused on the design and synthesis of 1,5-dihydropyrrol-2-ones, a new class of fimbrolide derivatives capable of displaying strong antagonistic properties of the fimbrolides. Primary synthetic methodologies examined include the halolactamization of allenamides and the direct lactone-lactam transformation. No doubt, both methodologies yielded the lactam ring, the former failed to introduce the crucial C-5 bromomethylene group essential for bioactivity. A facile high yielding two-step lactone-lactam transformation method was developed and using this method, a wide range of substituted 5-bromomethyl- and 5-dibromomethylene-1,5-dihydropyrrol-2-ones were synthesized. Furthermore, a new class of tricyclic crown-ether type compounds with no literature precedent were discovered. To vary the diversity of the compounds, a related class of compounds, 5,6-dihydroindol-2-ones, were examined. A general versatile method for the synthesis of 7-substituted 5,6-dihydroindol-2-ones was developed. The synthetic strategy proceeds via the established Suzuki-Miyaura cross-coupling reaction of halogenated dihydroindol-2-ones with arylboronic acids/esters. The Suzuki methodology was found to be reliable in furnishing a wide range of 7-substituted products in high yields. A preliminary molecular modeling approach was used to assist in the design of new anti-microbials via the ligand-docking analyses of the TraR and LasR protein. A positive correlation was observed between the docking scores and biological activity and the methodology was further developed into an initial screening tool to filter potential active and non-active compounds. The newly synthesized compounds were analysed for their efficacy in reducing the expression of the Green Fluorescent Protein (GFP) in the presence of natural AHL signaling molecules in an AHL-monitor strain, indicative of the inhibition of bacterial phenotype expression. The dihydropyrrol-2-one class of compounds showed significant biological activity and this highlighted their potential for further development.

Laboratory and modeling studies were performed with a mixed-anaerobic-culture obtained from the Evanite site in Corvallis, Oregon. The culture completely transforms trichloroethene (TCE) to cis-dichloroethene (c-DCE), vinyl chloride (VC), and finally to ethene. Acetylene inhibition studies were used to examine the culture's microbial activities. Kinetic studies determined the half-saturated constant (K[subscript s]), the maximum utilization rate (k[subscript max]X), and inhibition constants (K[subscript I]). The kinetic constants were used to model the results of inhibition studies using competitive and uncompetitive inhibition models. Acetylene was found to function as a reversible inhibitor and was used to probe the activities of reductive dechlorination. Various acetylene concentrations were used to differentiate microbial processes, including methanogenesis, acetogenesis, and halorespiration. Acetylene concentrations of 48, 192, and 12 ��M, respectively, were required to achieve 90% inhibition in the rates of methanogenesis, TCE and VC transformation. H���-dependent acetate production was not inhibited by acetylene. K[subscript s] values for TCE and VC were 12 ��M and 63 ��M, respectively. Model fitting of acetylene inhibition constants (K[subscript IC]) for TCE and VC transformations yielded the same value (0.4 ��M) for a competitive inhibition model. However, for uncompetitive inhibition the estimated K[subscript IU] for TCE to c-DCE, TCE to 1,1-DCE and VC to ethene were 13.3, 14.1 and 2.2 ��M, respectively. Competitive and uncompetitive inhibition models simulated experimental data equally well for results obtained at high TCE and VC concentrations. The models were further verified to fit transient data of acetylene inhibition at lower TCE and VC concentrations, and competitive inhibition resulted in a better fit to the experimental data. 1-chloro-1-fluoroethene (1,1-CFE) was found to track the rate of VC transformation well, since VC and 1,1-CFE had similar maximum transformation rates and K[subscript s] values. A competitive inhibition model with the measured K[subscript s] values, 63 and 87 ��M. was used to predict the rates of VC and 1,1-CFE transformation, respectively. The similar rates and results of acetylene and compound inhibition studies indicated VC and 1,1-CFE were transformed by the same enzyme. 1,1-CFE transformation by three different cultures, clearly demonstrate that 1,1-CFE was an excellent surrogate to track rates of VC transformation. / Graduation date: 2004

Traditional bacterial disease therapies utilize compounds that ultimately kill the target bacteria but it exerts a strong selective pressure on the bacteria to develop multi-drug resistance mutants. The increasing occurrence of resistance in common pathogens has highlighted the need to identify new anti-microbials that target the control of bacterial pathogenicity in a non-extermination manner to reduce the incidence of bacteria resistance. One new strategy exploits the discrete signaling molecules that regulate the various bacterial signaling pathways, which are responsible for the expression of pathogenicity traits. Halogenated furanones (fimbrolides) from the marine red alga, Delisea pulchra have been shown to interfere with the key signaling pathway present in Gram-negative bacteria by competitively displacing the cognate signaling molecule from the transcription protein. This project focused on the design and synthesis of 1,5-dihydropyrrol-2-ones, a new class of fimbrolide derivatives capable of displaying strong antagonistic properties of the fimbrolides. Primary synthetic methodologies examined include the halolactamization of allenamides and the direct lactone-lactam transformation. No doubt, both methodologies yielded the lactam ring, the former failed to introduce the crucial C-5 bromomethylene group essential for bioactivity. A facile high yielding two-step lactone-lactam transformation method was developed and using this method, a wide range of substituted 5-bromomethyl- and 5-dibromomethylene-1,5-dihydropyrrol-2-ones were synthesized. Furthermore, a new class of tricyclic crown-ether type compounds with no literature precedent were discovered. To vary the diversity of the compounds, a related class of compounds, 5,6-dihydroindol-2-ones, were examined. A general versatile method for the synthesis of 7-substituted 5,6-dihydroindol-2-ones was developed. The synthetic strategy proceeds via the established Suzuki-Miyaura cross-coupling reaction of halogenated dihydroindol-2-ones with arylboronic acids/esters. The Suzuki methodology was found to be reliable in furnishing a wide range of 7-substituted products in high yields. A preliminary molecular modeling approach was used to assist in the design of new anti-microbials via the ligand-docking analyses of the TraR and LasR protein. A positive correlation was observed between the docking scores and biological activity and the methodology was further developed into an initial screening tool to filter potential active and non-active compounds. The newly synthesized compounds were analysed for their efficacy in reducing the expression of the Green Fluorescent Protein (GFP) in the presence of natural AHL signaling molecules in an AHL-monitor strain, indicative of the inhibition of bacterial phenotype expression. The dihydropyrrol-2-one class of compounds showed significant biological activity and this highlighted their potential for further development.