Spectroscopic and mechanistic investigation of two flavin-dependent enzymes: nitronate monooxygenase and choline oxidase

Propionate 3-nitronate (P3N) is a natural toxin that irreversibly inhibits mitochondrial succinate dehydrogenase. P3N poisoning leads to a variety of neurological disorders and even death. Nitronate monooxygenase (NMO) from Cyberlindnera saturnus (CsNMO) and Pseudomonas aeruginosa PAO1 (PaNMO) serve as paradigms for Class I NMO, which catalyze the oxidation of P3N involving single electron transfer. In this dissertation, the crystallographic structure of CsNMO was solved and demonstrated a highly conserved three-dimensional structure and active site with respect to NMO from PaNMO. The role of conserved residues in the active site of Class I NMO, e.g. Y109, Y254, Y299, Y303, and K307 in PaNMO in substrate binding and catalysis were investigated using site-directed mutagenesis, steady-state kinetics and pH effects on the UV-visible absorption spectrum. The study revealed that a protonated tyrosine is required for binding of the negatively charged P3N substrate. We also report that PaNMO can stabilize both the neutral and anionic semiquinones anaerobically for hours, providing a constant protein environment to study their photochemical and photophysical properties. Choline oxidase catalyzes two-step oxidation of choline to glycine betaine with betaine aldehyde as an intermediate. The FAD cofactor is covalently attached to the choline oxidase via H99 through an 8α-N3-histidyl linkage. In the active site of choline oxidase, S101 and H466 are located on two extent loops, ~ 4 Å from the flavin C4a atom. In this dissertation, a charge-induced, reversible C4a-S-cysteinyl-8α-N3-histidyl FAD was engineered by replacing S101 with a cysteine. The mechanistic rationale for the stabilization of de novo C4a-S-cysteinyl-flavins was illustrated with rapid kinetics, pH, kinetic isotope effects and proton inventory. A photoinduced transient C4a-N-histidyl-8α-N3-histidyl FAD in choline oxidase wild-type was also observed with the aid of fluorescence excitation spectroscopy. Site-directed mutagenesis, solvent equilibrium isotope effects and pH effects on the stoke shifts of flavin in choline oxidase wild-type demonstrated H466 as the adduct on the C4a atom of flavin upon excitation, and provided a mechanistic rationale involving photoinduced electron transfer (PET) for the formation of the novel photoinduced transient flavin C4a adduct

Use of hydrogen bonds to control molecular aggregation : self-assembly of three-dimensional networks with large chambers

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.La liaison hydrogène est omniprésente dans la nature et joue un rôle capital dans la chimie de la matière vivante. Cette interaction non covalente, sélective, forte et directionnelle peut induire l'auto-assemblage de molécules. Les tectones sont définies comme étant des molécules dont les interactions sont dominées par des forces attractives spécifiques qui induisent la formation d'agrégats de géométrie contrôlée. La tectonique moléculaire est l'art et la science de la construction de supramolécules en utilisant des sous-unités tectoniques. Les liens hydrogène intermoléculaires présentent une force particulièrement efficace pour promouvoir la formation d'agrégats tectoniques et de telles tectones pouvant participer dans un vaste réseau de liens hydrogène peuvent être synthétisée simplement en ajoutant des sous-unités 2-pyridones sur une molécule support appropriée. Cette thèse décrit la conception et la synthèse de tectones comprenant quatre sous-unités 2-pyridones orientées suivant un tétraèdre de telle sorte qu'elles sont prédisposées à générer un réseau diamantoïde ou de géométrie comparable. Nous avons mis en évidence que de telles tectones tétraédriques peuvent s'associer par des liaisons hydrogène pour former un réseau diamantoïde contenant de larges cavités. Ces cavités générées par le réseau sont comblées par un entrelacement de réseaux diamantoïdes indépendants et autres molécules hôtes. Nous avons aussi mis en évidence que des tectones tétraédrique de différentes grandeurs peuvent engendrer des réseaux tridimensionnels autre que diamantoïdes. Ces résultats laissent entrevoir que l'application astucieuse de la tectonique moléculaire peut être employée à l'élaboration de molécules organiques dont l'assemblage tridimensionnel offrirait les propriétés remarquables des zéolites et autre substances inorganiques semblables comme la microporosité, la présence de cavités et la stabilité structurelle

Synergistic Interfacial Properties of Casein and Small Molecule Surfactants for Fabrication of Essential Oil Nanoemulsions

Nanoemulsions of essential oils are important for delivery of flavors and antimicrobial preservatives in food systems. The overall goal of this work was to study the formation and properties of essential oil nanoemulsions fabricated with sodium caseinate (NaCas) or its hydrolysates and small molecule surfactants (SMSs). The first group of lemon oil nanoemulsion was prepared with NaCas and Tween 20 using a phase inversion temperature (PIT) method. The combination of NaCas and Tween 20 reduced the turbidity and droplet dimension of emulsions than using them individually. Heating at 90 °C for \u3e1 h resulted in transparent nanoemulsions for samples with 1.5% lemon oil. Negative and positive effects on nanoemulsion formation were observed with 0.2-0.4 and 0.6-0.8mM NaCl, respectively. The nanoemulsion prepared with 2% NaCas, 0.4-1.2% Tween 20 and 1.5% lemon oil had a volume-area mean diameter of around 100 nm that was optically stable during 15-day storage at room temperature, while creaming occurred after longer time storage. Protein hydrolysates have a higher diffusion rate and flexibility than proteins. In order to improve the emulsification efficiency and emulsion stability, the second group of thymol nanoemulsions fabricated with combinations of casein hydrolysates (CH) and sucrose stearate (SS) was studied. For NaCas hydrolyzed by pancreatin for different durations, the product hydrolyzed for 10 min resulted in the most transparent and stable emulsions due to limited reduction of casein molecular weight. Thermal treatment further improved the emulsion stability because of the improved solubility of SS and the strengthened interactions between CH and SS during heating. Addition of 0.25-1% SS improved the emulsification capacity of 2% CH and the emulsion clarity, while excess (2%) SS significantly increased turbidity and particle dimension of emulsions. With the increased thymol concentration (0-3%), stability of emulsions improved. Emulsions prepared with 0.25% SS and 1.5-3% thymol, or 1% SS and 3% thymol, had stable droplet dimensions during ambient storage for over two months. The combination of SS and CH also enabled stable emulsions at pH 5. Therefore, combinations of SMSs with NaCas and its hydrolysate can be used as novel approaches to prepare essential oil nanoemulsions for various applications

The worldwide impact of institutional funds on flow-performance relationship

This thesis uses data from 15 countries, over the 1998-2010 period, to examine and compare differences in the flow-performance sensitivity between institutional and retail investors. Our results show that the flow-performance relationship is convex, consistent with previous research, but the impact of institutional funds on flow-performance relationship outside the US is marked different from the US. Compared to retail investors, institutional funds sell more poor performance funds and buy less top performance funds outside the US while institutional investor act the same way as retail investors to past performance in the US. We then split our sample into more and less sophisticated countries, investors buy more top performers but only in less sophisticated countries. When it comes to bottom performance, the reactions are similar for both more sophisticated and less sophisticated countries. Our finds provide evidence that institutional investors are more sophisticated than retail investors in less sophisticated countries and retail investors are sophisticated as institutional investors in more sophisticated countries, like the US.Esta tese usa dados de 15 países, durante o período 1998-2010, para examinar e comparar as diferenças na sensibilidade dos fluxos monetários à performance entre investidores institucionais e investidores retalhistas. Os resultados mostram que a relação fluxoperformance é convexo, consistente com a literatura, mas o impacto dos fundos institucionais nesta relação nos EUA é marcadamente diferente da que existe noutros países. Em comparação com os investidores retalhistas, os investidores institucionais vendem mais fundos com desempenho baixo e compram menos fundos com elevado desempenho fora dos EUA, enquanto não há diferença entre investidores institucionais e retalhistas nos EUA. Em seguida, dividimos nossa amostra em países mais e menos sofisticados. Os investidores compram mais fundos com elevado desempenho, mas apenas em países menos sofisticados. Quando se trata de fundos com desempenho inferior, as reações são semelhantes para países mais sofisticados e menos sofisticados. Os nossos resultados permitem concluir que os investidores institucionais são mais sofisticados do que investidores retalhistas em países menos sofisticados e o nível de sofisticação é semelhante nos países mais sofisticados, como os EUA