Lo Sze Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 100-113). / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.iv / List of Figures and Tables --- p.vii / Abbreviations --- p.xii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Edible mushrooms --- p.1 / Chapter 1.1.1 --- Pleurotus sajor-caju --- p.1 / Chapter 1.1.2 --- Flammulina velutipes --- p.2 / Chapter 1.2 --- Lignocellulose and phenolic monomers --- p.4 / Chapter 1.2.1 --- Sources of phenolic monomers --- p.4 / Chapter 1.2.2 --- Toxicity of phenolic monomers --- p.10 / Chapter 1.3 --- Fungal laccases --- p.13 / Chapter 1.3.1 --- Occurrence --- p.13 / Chapter 1.3.2 --- Laccase reaction --- p.14 / Chapter 1.3.3 --- Physiological functions --- p.18 / Morphogenesis --- p.18 / Pathogenicity --- p.19 / Lignin degradation --- p.20 / Chapter 1.4 --- Purpose of study --- p.22 / Chapter 2. --- Materials and Methods --- p.24 / Chapter 2.1 --- General --- p.24 / Chapter 2.1.1 --- Organisms --- p.24 / Chapter 2.1.2 --- Culture medium --- p.24 / Chapter 2.1.3 --- Addition of phenolic compounds --- p.24 / Chapter 2.2 --- Effect of phenolic monomers on the growth of mushroom mycelium on agar plates --- p.25 / Chapter 2.3 --- Effect of phenolic monomers on the production of fungal biomass in liquid culture --- p.25 / Chapter 2.4 --- Effect of phenolic monomers on the extracellular laccase produced by P. sajor-caju and F. velutipes --- p.26 / Chapter 2.5 --- Assay of laccase activity --- p.26 / Chapter 2.6 --- Electrophoresis patterns of laccase proteins --- p.27 / Chapter 2.6.1 --- Non-denaturing polyacrylamide gel electrophoresis --- p.27 / Chapter 2.6.2 --- Localization of laccase activity --- p.27 / Chapter 2.7 --- Purification of extracellular laccases from P. sajor-caju --- p.28 / Chapter 2.7.1 --- Inoculum preparation --- p.28 / Chapter 2.7.2 --- Culture conditions --- p.28 / Chapter 2.7.3 --- Concentration of culture supernatant --- p.29 / Chapter 2.7.4 --- Ammonium sulphate fractionation --- p.29 / Chapter 2.7.5 --- Anion exchange chromatography --- p.29 / Chapter 2.7.6 --- Preparative polyacrylamide gel electrophoresis --- p.30 / Chapter 2.7.7 --- Protein detection and quantification --- p.30 / Chapter 2.8 --- Characterization of Laccase Protein --- p.31 / Chapter 2.8.1 --- "Effect of pH, temperature and substrate concentration" --- p.31 / Chapter 2.8.2 --- Effect of inhibitors --- p.32 / Chapter 2.8.3 --- Determination of isoelectric point --- p.32 / Chapter 2.8.4 --- Determination of molecular weight --- p.33 / Chapter 3. --- Results --- p.34 / Chapter 3.1 --- Effect of phenolic monomers on the growth of P. sajor-caju and F. velutipes --- p.34 / Chapter 3.1.1 --- P. sajor-caju --- p.34 / Chapter 3.1.2 --- F. velutipes --- p.38 / Chapter 3.2 --- Effect of phenolic monomers on laccase production by P. sajor-caju and F. velutipes --- p.41 / Chapter 3.2.1 --- P. sajor-caju --- p.45 / Chapter 3.2.2 --- F. velutipes --- p.49 / Chapter 3.3 --- Electrophoretic patterns of extracellular laccase --- p.53 / Chapter 3.3.1 --- P. sajor-caju --- p.53 / Chapter 3.3.2 --- F. velutipes --- p.56 / Chapter 3.4 --- Purification of laccase protein from P. sajor-caju --- p.58 / Chapter 3.4.1 --- Separation of laccase proteins --- p.58 / Chapter 3.4.2 --- Purification of laccase IV --- p.59 / Chapter 3.5 --- Characterization of laccase IV from P. sajor-caju --- p.64 / Chapter 3.5.1 --- Optimum temperature and thermostability --- p.64 / Chapter 3.5.2 --- Optimum pH and pH stability --- p.67 / Chapter 3.5.3 --- Substrate concentration --- p.70 / Chapter 3.5.4 --- Effect of inhibitors --- p.74 / Chapter 3.5.5 --- Isoelectric point --- p.74 / Chapter 3.5.6 --- Molecular weight --- p.74 / Chapter 4. --- Discussion --- p.78 / Chapter 4.1 --- Phenolic monomers and the growth of P. sajor-caju and F. velutipes --- p.78 / Chapter 4.2 --- Phenolic monomers and laccase production by P. sajor- caju and F. velutipes --- p.81 / Chapter 4.3 --- Electrophoretic patterns of laccase proteins --- p.83 / Chapter 4.4 --- Physiological functions of laccase --- p.85 / Chapter 4.5 --- Purification of selected laccase protein from P. sajor-caju --- p.88 / Chapter 4.6 --- Properties of laccase IV from P. sajor-caju --- p.89 / Chapter 4.6.1 --- Optimum temperature and thermostability --- p.89 / Chapter 4.6.2 --- Optimum pH and pH stability --- p.90 / Chapter 4.6.3 --- Effect of inhibitors --- p.92 / Chapter 4.6.4 --- Km --- p.93 / Chapter 4.6.5 --- Isoelectric point --- p.94 / Chapter 4.6.6 --- Molecular weight --- p.94 / Chapter 4.7 --- Future works --- p.96 / Chapter 5. --- Conclusion --- p.98 / Chapter 6. --- References --- p.100

Water diffusion is widely believed to be a driving factor in the breakdown of corrosion protection by polymer coatings. However, in the epoxy-phenolic system examined, water absorption into more cured, electrically resistive coatings is shown to increase, contradicting the common perception that hydrophobic coatings yield improvements in corrosion protection. Water uptake into epoxy-phenolic coatings was estimated as a function of time using the dielectric and resistive properties of the coating measured using electrochemical impedance spectroscopy (EIS). Bulk water uptake through the coating surface and the localised nature of corrosive failure was then confirmed using localised electrochemical impedance (LEIS). This technique also allowed comparison of resistance for coatings with different curing degrees. The greater degree of water absorption into highly cured coatings was attributed to diffusion into the polymer free volume, which increases with prolonged crosslinking. Evidence for this mechanism was found in the decreasing density of epoxy-phenolic coatings as a function of cure time. The effect of coating volume was therefore evaluated with respect to the dry coating dielectric properties. Cathodic delamination of the epoxy-phenolic coatings was achieved on tinplated steel substrates, whereas this was not possible from tin-free steel (ECCS). The electrochemical behaviour of these two substrates was examined under the same NaCl concentration, their surfaces were analysed by XPS prior to immersion, and XRD after potentiostatic polarisations. The electrochemical behaviour of the bare substrates was found to be unchanged by heating to the coating cure temperature (within the accuracy of polarisation tests carried out). Nonetheless, the delamination rate of epoxy-phenolic coatings deposited on them was dependent on the coating crosslinking (cure time). The reason coating adhesion selectively failed on tinplate substrates is believed to be the lower concentration of hydroxides on its relatively flat surface, allowing less bonding to take place at the interface with the coating. The impact of the substrate on coating resistivity was further investigated by volume resistivity measurements in the presence and absence of a metallic substrate. The resistivity of free standing films was tested in permeation cells, and showed a direct correlation between the polymer resistivity and the resistivity of the solution in which it was immersed. In comparison, attached films remained highly resistive at all examined concentrations. To conclude, the results presented here demonstrate that water uptake is not as critical to the breakdown in the protection offered by epoxy-phenolics as bonding at the metal-polymer interface. These results suggest that the complex interaction between these two dissimilar materials determines the overall coating electrochemical behaviour.

Ao contrário de muitos polímeros, as resinas fenólicas se caracterizam por possuir um grande número de aplicações por conta de sua superior resistência ao fogo e baixa emissão de fumos e ao seu excelente nível de resistência térmica e química, além de seu baixo custo. No entanto, devido à sua estrutura tridimensional, caracterizada pelo alto grau de reticulação, este tipo de resina apresenta baixas tenacidade e resistência à fratura. Com isso, para garantir seu bom desempenho, faz-se necessário promover modificações em sua formulação ou acrescentar agentes de reforço de modo a compensar essas deficiências resultantes de sua estrutura. São inúmeros os materiais que, usualmente, são incorporados à matriz fenólica. Além da preparação de blendas poliméricas em que uma das fases consiste em um elastômero ou um termoplástico, a introdução de agentes de reforço como materiais fibrosos também são utilizados. São exemplos de agentes de reforço: fibras vegetais, fibras de vidro e de carbono, negro de fumo, argilas entre outros. A mistura de polímeros e argilas pode levar à formação de nanocompósitos de modo a obter excelentes combinações de resistência à fratura, tenacidade, condutividade, resistência ao calor e redução da permeabilidade a gases e líquidos quando comparados com o polímero puro. Neste trabalho, argilas do tipo montmorilonita modificadas foram utilizadas como agente de reforço na preparação de filmes de nanocompósitos de matriz fenólica com o objetivo de melhorar as propriedades mecânicas deste material polimérico quando comparado com o material puro. A resina fenólica utilizada neste trabalho é a do tipo resol, utilizada comercialmente para a formulação de vernizes para revestimento de embalagens metálicas. No estudo foram utilizadas três argilas comerciais: Cloisite® sódica (Na) e argilas Cloisite® modificadas com sais quaternários de alquilamônio, de códigos 15A e 30B, objetivando verificar qual apresentaria melhor compatibilidade com a matriz fenólica. Para a preparação de filmes poliméricos uniformes, isentos de defeitos como bolhas, foi feita, de forma preliminar, a seleção de um ciclo de cura apropriado. Como a formação de bolhas é intrínseco à cura da resina fenólica resol, a definição de um esquema de tratamento térmico de cura apropriado constituiu-se em etapa crítica no processo de preparação dos compósitos de resina fenólica/argila montmorilonita modificada. Os filmes curados isentos de defeitos foram caracterizados por difração de raios X, análise termomecânica dinâmica (DMA) e calorimetria exploratória diferencial (DSC). Os resultados das análises de DRX mostraram para os compósitos preparados com as argilas modificadas 15A e 30B manutenção e até mesmo redução do espaçamento basal da estrutura cristalina das argilas, indicando não ter ocorrido intercalação do polímero nessas argilas; enquanto que para a argila Cloisite® Na ocorreu aumento do espaçamento basal. Os resultados de DMA mostraram para a maioria das amostras aumento no módulo de armazenamento em baixa e alta temperatura. Por último, a análise de DSC mostrou redução na temperatura de transição vítrea nos compósitos preparados com as argilas modificadas 15A e 30B, e elevação na composição de 5% da argila Cloisite® Na. Os resultados indicam o potencial de reforço mecânico de resinas fenólicas com argilas lamelares do tipo montmorilonita sem a necessidade de modificação química. / Unlike most polymers, phenolic resins are characterized by having a large number of applications because of its superior fire resistance and low emission of smoke and its excellent level of thermal and chemical resistance, and low cost. However, due to its three dimensional structure, characterized by a high degree of crosslinking, this type of resin has low toughness and fracture resistance. Thus, to ensure their good performance, it is necessary to make enhancements in their formulation or adding strengthening agents so as to compensate for these deficiencies due to its structure. There are numerous materials that usually are incorporated into the phenolic matrix. Besides the preparation of polymer blends in which one phase consists of an elastomer or a thermoplastic, the introduction of agents such as fibrous reinforcement is also used. Examples of reinforcing agents: vegetable fibers, glass and carbon fibers, carbon black, clay and others. The mixture of polymer clays may cause the formation of nanocomposites in order to obtain excellent combination of fracture strength, toughness, conductivity, heat resistance and reduced permeability to gases and liquids when compared with the pure polymer. In this work, modified montmorillonite clays were used as a reinforcing agent in the preparation of nanocomposite films of phenolic matrix with the aim of improving the mechanical properties of polymer materials when compared with the pure material. The phenolic resin used in this work is that of the resol type, used commercially for the formulation of varnish for coating metal containers. The study used three types of commercial clay: Cloisite® sodium (Na) and Cloisite® clays modified with quaternary alkylammonium salts, codes 15A and 30B in order to verify which present better compatibility with the phenolic matrix. For the preparation of uniform polymeric films, free of defects such as bubbles, was preliminarily selected a suitable cure cycle. As the bubble formation is intrinsic to the cure of resol phenolic resin, the definition of a heat treatment scheme is a critical step in the process of preparing composites of phenolic resin/montmorillonite clay. The cured films free of defects were characterized by X-ray diffraction (DRX), dynamic mechanical analysis (DMA) and differential scanning calorimetry (DSC). The results of XRD analysis showed the composites prepared with the modified clays 15A and 30B maintained and even reduced the basal spacing within the clay crystal structure, indicating not having occurred polymer intercalation of the clay; for the Cloisite® Na clay DRX has shown increased basal spacing. DMA results showed for most of the samples increase in storage modulus at low and high temperatures. Finally, DSC analysis showed a reduction in glass transition temperature of the composites prepared with the modified clays 15A and 30B, and an increase in the composition of 5% in the Cloisite® Na clay. The results indicate the potential of enhancement of phenolic resins mechanical properties with layered clays of montmorillonite without chemical modification.

La biosynthèse de phénol-glycolipides (PGL) par Mycobacterium tuberculosis et M. leprae favorise l’invasion des macrophages via l'interaction de la partie saccharidique des PGL avec le domaine lectine du récepteur cellulaire au complément CR3. Les PGL inhibent également la production de cytokines inflammatoires par la cellule hôte, par un mécanisme inconnu. J’ai observé que des bactéries BCG transgéniques exprimant les PGL de M. tuberculosis ou M. leprae avaient une capacité de survie accrue dans les macrophages. Cette persistance intracellulaire était dépendante de CR3 et associée à une diminution de la production d'oxyde nitrique dans les cellules infectées. L’addition de PGL purifié suffisait à inhiber la production d’oxyde nitrique par des macrophages stimulés avec LPS/IFN-γ. J’ai montré que la liaison de PGL-1 à CR3 provoquait la dégradation post-transcriptionnelle de TIR-domain-containing adapter-inducing interféron-β (TRIF) dans les macrophages, ce qui entraînait une réduction de la signalisation TRIF-dépendante. Dans les macrophages stimulés avec LPS/IFN-γ, la dégradation de TRIF réduisait la production d’oxyde nitrique synthase, et la production TRIF dépendante de cytokines inflammatoires et des chimiokines. Mes résultats ont donc permis d’identifier un nouveau mécanisme de virulence développé par les mycobactéries pathogènes pour réprimer conjointement les réponses inflammatoires et antimicrobiennes de l’hôte / Biosynthesis of phenolic glycolipids (PGL) by Mycobacterium tuberculosis and M. leprae promotes macrophage invasion, which proceeds through the interaction of the PGL sugar moieties with the lectin domain of cell-displayed complement receptor (CR3). PGL also limit host cell production of inflammatory cytokines by an unknown mechanism. I observed that transgenic BCG that express PGL specific to M. tuberculosis or M. leprae displayed enhanced survival within macrophages. Increased intracellular persistence of PGL-expressing BCG was CR3-dependent and correlated with the decreased production of nitric oxide in infected cells. Notably, the addition of soluble PGL to macrophages was sufficient to induce a reduction in nitric oxide production upon stimulation with LPS/IFN-γ. I showed that PGL-1 binding to CR3 causes the post-transcriptional degradation of TIR-domain-containing adapter-inducing interferon-β (TRIF) in macrophages, resulting in impaired TRIF-dependent signaling. Functionally, PGL-1-mediated degradation of TRIF resulted in the decreased induction of nitric oxide synthase, and TRIF-dependent inflammatory cytokines and chemokines in LPS/IFN-γ-stimulated macrophages. My results thus identified a virulence mechanism evolved by pathogenic mycobacteria to suppress both the inflammatory and antimicrobial responses of infected host cells

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

The major objectives of this research were to produce a phenolic rich isolate from two locally grown Saskatchewan fruits, chokecherries and saskatoons, develop an encapsulation system for the phenolic isolate, and test this system for the delivery of the phenolic isolate in an animal (rat) model. Natural phenolic compounds present in plants such as fruits have antioxidant and free radical scavenging activities, which have been proposed to have health benefits. The extraction of these compounds from plants is commonly performed using methanol despite being toxic to both humans and animals. As such, ethanol was investigated for its ability to extract phenolics from plants as a food safe alternative to methanol. Phenolic extraction from chokecherries with ethanol:formic acid:water (EFW) resulted in higher concentrations (9.83 mg gallic acid equivalents (GAE)/g fresh weight) than with methanol:formic acid:water (MFW) (7.97 mg GAE/g fresh weight). Results from saskatoons showed similar phenolic levels of 4.26 and 4.21 mg GAE/g fresh weight with MFW and ethanol (EFW), respectively. These results showed that EFW was a suitable substitute for MFW in phenolic compound isolation from chokecherries and saskatoons, and could be used to produce extracts that were safe for use in foods and feeds. High performance liquid chromatography with photodiode array detection (HPLC-PDA) was used to determine the phenolic compound composition of the raw fruits and their phenolic rich isolates. Chlorogenic acid was identified in both chokecherry and saskatoon samples, and rutin was also shown to be present in saskatoons. These identifications were based on the relative retention time and ultra violet-visual spectra comparisons to standards. Solid phase extraction (SPE) using Amberlite XAD-16 was employed to produce phenolic isolates from chokecherries and saskatoons. HPLC-PDA results determined that there was a ~2.7x and ~1.6x increase in peak area for chokecherries and saskatoons, respectively when SPE was employed. The antioxidant activity of the extracts and isolates was determined using in vitro radical scavenging tests including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2´-azinobis-3-ethylbenzthiazoline-sulphonic acid (ABTS). The EFW chokecherry extract and isolate had the highest overall free radical scavenging activity. Crude fruit extracts exhibited lower free radical scavenging values compared to the isolate samples in both of the assays performed. The fruit phenolic isolates were encapsulated in chitosan (CH) sodium tripolyphosphate (TPP) nanoparticles at a ratio of 4.0:1.0 (CH:TPP). HPLC-PDA was used to determine the entrapment efficiency of phenolic isolates to be 15.9 ± 2.7% and 23.0 ± 7.1% for chokecherries and saskatoons, respectively. Characteristics such as the size, surface potential and phenolic release were determined for the two fruit isolate containing nanoparticles. The size of the nanoparticles were 527.90 ± 74.57 nm and 443.03 ± 15.79 nm for chokecherries and saskatoons, respectively. Both of the nanoparticle systems had positive surface charges at 52.70 ± 2.93 mV and 54.43 ± 1.27 mV for chokecherries and saskatoons, respectively. The release properties of the CH:TPP nanoparticles containing fruit phenolics were examined in enzymatic simulated intestinal fluid and resulted in ~23% and ~28% release of chokecherry and saskatoon phenolics, respectively. Saskatoon phenolic isolates and isolates encapsulated in CH:TPP were gavage fed to rats (six animals in each of the two groups) at a dosage rate of 276.36 ± 9.74 mg/kg body weight. The saskatoon isolate contained 12.44 ± 0.44 mg/kg body weight anthocyanins (~3.30 mg anthocyanin per rat). These animals were sacrificed after 1 h and all stomach tissue samples in each of the treatment groups contained detectable levels of anthocyanins. In the small intestine tissues all six of the saskatoon isolate and three of the encapsulated isolate groups had detectable amounts of anthocyanins, while in the large intestine tissue, only one sample from the isolate group showed detectable amounts of anthocyanins. Although other tissues were tested (brain, heart, kidney and liver), anthocyanins were not detected. Therefore anthocyanins were detected in the gastrointestinal tract of both of the treatment groups. The research performed therefore illustrated that phenolic compounds can be extracted from fruit sources using EFW and can be successfully encapsulated in chitosan tripolyphosphate capsules allowing for targeted delivery in an animal model.

In Canada, colorectal cancer is the second leading cause of death from cancer in men and the third leading cause of death from cancer in women. Several factors contribute to the development of cancer. Genetic predisposition, diet, and lifestyle habits are some of the major factors for colorectal cancer development. In the diet related factors, epidemiological studies suggest that consumption of whole grains rich in dietary fiber are associated with low incidence of human colon cancer. Recent studies have also shown that, in addition to dietary fiber, the type of dietary fiber and other compounds such as phenolic acids present in cereal grain bran may also have a role to play in colon cancer prevention. In a recent study, eleven major phenolic acids which differed in anti-oxidant activity were identified in wheat bran from wheat varieties belonging to six different market classes. The main objective of this study was to develop an in vitro cell culture based assay system to study the effect of phenolic acids on colon cancer development. Another objective was to study the effect of phenolic acids on selected molecular markers associated with cell proliferation, apoptosis and inflammation. Two well established colon carcinoma cell lines HT-29 and HCT 116 were treated with varying concentrations of fourteen phenolic acids to study their effect on cell survival and proliferation. In addition, immunohistochemical assays were performed on treated cells for two cell proliferation markers (Cyclin D1 and Ki67), an apoptosis marker (Bax) and three inflammatory markers (Beta-catenin, COX-2 and iNOS). Treatment of phenolic acids inhibited the growth of both the cell lines, however the effects varied with phenolic acid and cell line used in the assay. As determined by IC50, the growth of HCT 116 cells was inhibited the most by caffeic, ellagic, and gallic acids with IC50 of 0.22 mM, 0.17 mM, and 0.15 mM, respectively. On the other hand, caffeic, chlorogenic, and gallic acids are most effective in preventing the growth of HT-29 cells, with IC50 at 0.06 mM, 0.28 mM, and 0.30 mM, respectively. Immunohistochemical and Western Blotting studies revealed that phenolic acids differentially affected markers for cell proliferation, apoptosis and cell inflammation. In most of the cases, phenolic acid treatments up-regulated the pro-apoptosis marker Bax, while it down-regulated cell proliferation marker Cyclin D1. The results clearly show that a cell culture based assay can be used to study the effect of phenolic acids or other chemical constituents isolated from plants to study their effect on colon cancer cell lines. Statistical analysis revealed that only in very limited cases, results of molecular markers correlated to cell growth and proliferation. Therefore, to draw firm conclusions, more detailed and extensive studied need to be completed using different phenolic acids, the two cell lines and more replications. However, this study has developed the necessary protocols and provided some indicative results such as most of the phenolic acids induced pro-apoptosis pathway in both the colon cancer lines. Future studies with extracted phenolic acids from wheat bran using the cell culture system optimized in this study can be used to define the role of different wheat varieties in colon cancer prevention.

In silico studies of the effect of phenolic compounds from grape seed extracts on the activity of phosphoinositide 3-kinase (PI3K) and farnesoid X receptor (FXR)Montserrat Vaqué Marquès En aquesta tesis es pretén aplicar metodologies computacionals (generació de farmacòfors i docking proteïna lligand) en l'àmbit de la nutigenòmica (ciència que pretén entendre, a nivell molecular, com els nutrients afecten la salut). S'aplicaran metodologies in silico per entendre a nivell molecular com productes naturals com els compostos fenòlics presents en la nostra dieta, poden modular la funció d'una diana comportant un efect en la salut. Aquest efecte es creu que podria ser degut a la seva interacció directa amb proteïnes de vies de senyalització molecular o bé a la modificació indirecta de l'expressió gènica. Donat que el coneixement de l'estructura del complex lligand-receptor és bàsic per entendre el mecanisme d'acció d'aquests lligands s'aplica la metodologia docking per predir l'estructura tridimensional del complex. En aquest sentit, un dels programes de docking és AutoGrid/AutoDock (un dels més citats). No obstant, l'automatització d'AutoGrid/AutoDock no és trivial tan per (a) la cerca virtual en una llibreria de lligands contra un grup de possibles receptors, (b) l'ús de flexibilitat, i (c) realitzar un docking a cegues utilitzant tota la superfície del receptor. Per aquest motiu, es dissenya una interfície gràfica de fàcil ús per utilitzar AutoGrid/AutoDock. Blind Docking Tester (BDT) és una aplicació gràfica que s'executa sobre quatre programes escrits en Fortran i que controla les condicions de les execucions d'AutoGrid i AutoDock. BDT pot ser utilitzat per equips d'investigadors en el camp de la química i de ciències de la vida interessats en dur a terme aquest tipus d'experiments però que no tenen suficient habilitats en programació. En la modulació del metabolisme de la glucosa, treballs in vivio i in vitro en el nostre grup de recerca s'han atribuït els efectes beneficiosos de l'extracte de pinyol de raïm en induir captació de glucosa (punt crític pel manteniment de l'homeostasis de la glucosa). No obstant alguns compostos fenòlics no tenen efecte en la captació de la glucosa, d'altres l'inhibeixen reversiblement. En alguns casos aquesta inhibició és el resultat de la competició dels compostos fenòlics amb ATP pel lloc d'unió de l'ATP de la subunitat catalítica de la fosfatidil inositol 3-kinasa (PI3K). Estudis recents amb inhibidors específics d'isoforma han identificat la p110α (la subunitat catalítica de PI3Kα) com la isoforma crucial per la captació de glucosa estimulada per insulina en algunes línies cel·lulars. Els programes computacionals han estat aplicats per tal de correlacionar l'activitat biològica dels compostos fenòlics amb informació estructural per obtenir una relació quantitativa estructura-activitat (3D-QSAR) i obtenir informació dels requeriments estructura-lligand per augmentar l'afinitat i/o selectivitat amb la diana (proteïna). Tot hi haver-se demostrat que l'adició d'extractes de compostos fenòlics en l'aliment pot tenir en general un benefici per la salut, s'ha de tenir en compte que l'estudi 3D-QSAR (construït a partir d'inhibidors sintètics de p110α) prediu que algunes d'aquestes molècules poden agreujar la resistència a la insulina en individus susceptibles dificultant la capatació de glucosa en múscul i teixit adipós i, per tant, produir un efecte secundari indesitjat. Resultats en el nostre grup de recerca han demostrat que compostos fenòlics presents en extractes de llavor de raïm incrementen l'activitat del receptor "farnesoid x receptor" (FXR) de manera dosi depenent quan el lligand natural de FXR (CDCA) és present. Les metodologies in silico, docking i 3D-QSAR, han estat aplicades juntament amb dades biològiques d'agonistes no esteroidals de FXR que s'uneixen a un lloc d'unió proper però diferent al lligand esteroidal 6CDCA. Els resultats han mostrat que els compostos fenòlics no són capaços d'activar FXR per ells mateixos però poden afegir noves interaccions que estabilitzarien la conformació activa de FXR en presència del lligand natural CDCA. Els compostos fenòlics podrien induir canvis conformacionals específics que augmentarien l'activitat de FXR. In silico studies of the effect of phenolic compounds from grape seed extracts on the activity of phosphoinositide 3-kinase (PI3K) and farnesoid X receptor (FXR)Montserrat Vaqué Marquès This thesis was written with the aim of applying computational methods that have already been developed for molecular design and simulation (i.e. pharmacophore generation and protein-ligand docking) to nutrigenomics. So, in silico tools that are routinely used by the pharmaceutical industry to develop drugs have been used to understand, at the molecular level, how natural products such as phenolic compounds (i.e. molecules that are commonly found in fruits and vegetables) can improve health and prevent diseases. Therefore, we first focused on predicting the structure of protein-ligand complexes. The docking algorithms can use the individual structures from receptor and ligand to predict (1) whether they can form a complex and (2) if so, the structure of the resulting complex. This prediction can be made, for instance, with AutoGrid/AutoDock, the most cited docking software in the literature. The automation of AutoGrid/AutoDock is not trivial for tasks such as (1) the virtual screening of a library of ligands against a set of possible receptors; (2) the use of receptor flexibility and (3) making a blind-docking experiment with the whole receptor surface. Therefore, in order to circumvent these limitations, we have designed BDT (i.e. blind-docking tester; http://www.quimica.urv.cat/~pujadas/BDT), an easy-to-use graphic interface for using AutoGrid/AutoDock. BDT is a Tcl/Tk graphic front-end application that runs on top of four Fortran programs and which controls the conditions of the AutoGrid and AutoDock runs. As far as the modulation of the glucose metabolism is concerned, several in vivo and in vitro results obtained by our group have shown that grape seed procyanidin extracts (GSPE) stimulate glucose uptake in 3T3-L1 adipocytes and thus help to maintain their glucose homeostasis. In contrast, it is also well known that although some phenolic compounds do not affect glucose uptake, others reversibly inhibit it in several cell lines. Moreover, for at least some of these phenolic compounds, this inhibition is the result of their competition with ATP for the ATP-binding site in p110α (i.e. the α isoform of the catalytic subunit of phosphoinositide 3-kinase or PI3Kα). Furthermore, recent studies with isoform-specific inhibitors have identified p110α as the crucial isoform for insulin-stimulated glucose-uptake in some cell lines. Therefore, although it has been proved that the addition of phenolic compound extracts to food can have an overall benefit on health, it should be taken into account that some of these molecules may exacerbate insulin resistance in susceptible individuals via impaired glucose uptake in muscle and adipose tissues and, therefore, produce an undesirable side effect. In this context, we have applied computational approaches (i.e. protein-ligand docking and 3D-QSAR) to predict the IC50 (i.e. the concentration that reduces the p110α activity to 50%). Our results agree with previous experimental results and predict that some compounds are potential inhibitors of this enzyme. Recent results in our research group have demonstrated that the phenolic compounds in GSPE increase the activity of the farnesoid X receptor (i.e. FXR) in a dose-dependent way when the natural ligand of FXR (i.e. CDCA) is also present. The phenolic compounds might induce specific conformational changes that increase FXR activity and then contribute to cardioprotection through mechanisms that are independent of their intrinsic antioxidant capacities but that involve direct interaction with FXR to modulate gene expression. Taking into account this hypothesis a 3D-QSAR analysis was made in an attempt to understand how phenolic compounds activate FXR. So, our results explain why phenolic compounds cannot activate FXR by themselves and how they can add new interactions to stabilize the active conformation of FXR when its natural ligand (i.e. CDCA) is present. Therefore, we proposed a mechanism of FXR activation by dietary phenolic compounds in which they may enhance bile acid-bound FXR activity.

The major objectives of this research were to produce a phenolic rich isolate from two locally grown Saskatchewan fruits, chokecherries and saskatoons, develop an encapsulation system for the phenolic isolate, and test this system for the delivery of the phenolic isolate in an animal (rat) model. Natural phenolic compounds present in plants such as fruits have antioxidant and free radical scavenging activities, which have been proposed to have health benefits. The extraction of these compounds from plants is commonly performed using methanol despite being toxic to both humans and animals. As such, ethanol was investigated for its ability to extract phenolics from plants as a food safe alternative to methanol. Phenolic extraction from chokecherries with ethanol:formic acid:water (EFW) resulted in higher concentrations (9.83 mg gallic acid equivalents (GAE)/g fresh weight) than with methanol:formic acid:water (MFW) (7.97 mg GAE/g fresh weight). Results from saskatoons showed similar phenolic levels of 4.26 and 4.21 mg GAE/g fresh weight with MFW and ethanol (EFW), respectively. These results showed that EFW was a suitable substitute for MFW in phenolic compound isolation from chokecherries and saskatoons, and could be used to produce extracts that were safe for use in foods and feeds. High performance liquid chromatography with photodiode array detection (HPLC-PDA) was used to determine the phenolic compound composition of the raw fruits and their phenolic rich isolates. Chlorogenic acid was identified in both chokecherry and saskatoon samples, and rutin was also shown to be present in saskatoons. These identifications were based on the relative retention time and ultra violet-visual spectra comparisons to standards. Solid phase extraction (SPE) using Amberlite XAD-16 was employed to produce phenolic isolates from chokecherries and saskatoons. HPLC-PDA results determined that there was a ~2.7x and ~1.6x increase in peak area for chokecherries and saskatoons, respectively when SPE was employed. The antioxidant activity of the extracts and isolates was determined using in vitro radical scavenging tests including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2´-azinobis-3-ethylbenzthiazoline-sulphonic acid (ABTS). The EFW chokecherry extract and isolate had the highest overall free radical scavenging activity. Crude fruit extracts exhibited lower free radical scavenging values compared to the isolate samples in both of the assays performed. The fruit phenolic isolates were encapsulated in chitosan (CH) sodium tripolyphosphate (TPP) nanoparticles at a ratio of 4.0:1.0 (CH:TPP). HPLC-PDA was used to determine the entrapment efficiency of phenolic isolates to be 15.9 ± 2.7% and 23.0 ± 7.1% for chokecherries and saskatoons, respectively. Characteristics such as the size, surface potential and phenolic release were determined for the two fruit isolate containing nanoparticles. The size of the nanoparticles were 527.90 ± 74.57 nm and 443.03 ± 15.79 nm for chokecherries and saskatoons, respectively. Both of the nanoparticle systems had positive surface charges at 52.70 ± 2.93 mV and 54.43 ± 1.27 mV for chokecherries and saskatoons, respectively. The release properties of the CH:TPP nanoparticles containing fruit phenolics were examined in enzymatic simulated intestinal fluid and resulted in ~23% and ~28% release of chokecherry and saskatoon phenolics, respectively. Saskatoon phenolic isolates and isolates encapsulated in CH:TPP were gavage fed to rats (six animals in each of the two groups) at a dosage rate of 276.36 ± 9.74 mg/kg body weight. The saskatoon isolate contained 12.44 ± 0.44 mg/kg body weight anthocyanins (~3.30 mg anthocyanin per rat). These animals were sacrificed after 1 h and all stomach tissue samples in each of the treatment groups contained detectable levels of anthocyanins. In the small intestine tissues all six of the saskatoon isolate and three of the encapsulated isolate groups had detectable amounts of anthocyanins, while in the large intestine tissue, only one sample from the isolate group showed detectable amounts of anthocyanins. Although other tissues were tested (brain, heart, kidney and liver), anthocyanins were not detected. Therefore anthocyanins were detected in the gastrointestinal tract of both of the treatment groups. The research performed therefore illustrated that phenolic compounds can be extracted from fruit sources using EFW and can be successfully encapsulated in chitosan tripolyphosphate capsules allowing for targeted delivery in an animal model.

A series of immiscible crystalline-crystalline diblock copolymers, poly(ethylene oxide)-b-(£`-caprolactone) (PEO-b-PCL), were synthesized through ring-opening polymerization and then blended with phenolic resin. FT-IR analyses provide that the ether group of PEO is a stronger hydrogen bond acceptor than the carbonyl group of PCL with the hydroxyl group of phenolic. Phenolic after curing with hexamethylenetetramine (HMTA) results in the excluded and confined PCL phase based on differential scanning calorimeter (DSC) analyses. This effect leads to the formation of a variety of composition-dependent nanostructures, including disorder, gyroid and short cylinder. The self-organized mesoporous phenolic resin was only found at 40~60 wt% phenolic content by intriguing balance of the contents of phenolic, PEO, and PCL. In addition, the mesoporous structure was destroyed with the increasing the ratio of PCL to PEO in block copolymers by small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) analyses. In addition, the large and long-range order of bicontinuous gyroid-type mesoporous carbon was obtained from mesoporous gyroid phenolic resin calcined at 800 ¢XC under nitrogen.