Made available in DSpace on 2014-10-09T12:42:41Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:35Z (GMT). No. of bitstreams: 1 05027.pdf: 12073377 bytes, checksum: 07fdd3a7ed9e60cb7be90d8745f24034 (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:45:38Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:05Z (GMT). No. of bitstreams: 1 07307.pdf: 7122491 bytes, checksum: c6f55b3c5e36e13004a54501231c20a4 (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Proposição: O objetivo deste estudo foi avaliar como a contração volumétrica (CV), o grau de conversão (GC), a taxa máxima de polimerização (RPmax) e a tensão de polimerização (TPmax) de dois compósitos experimentais são influenciados pela concentração de fotoiniciadores na matriz e pela irradiância aplicada na fotopolimerização. Material e métodos: Duas séries de monômeros foram formuladas, com partes iguais em peso dos seguintes monômeros: série B= Bis-GMA/ TEGDMA; série U= Bis-GMA/ UDMA / TEGDMA. Essas séries foram combinadas a três níveis de fotoiniciadores, num total de seis materiais experimentais, com a concentração em porcentagem de peso de uma amina terciária (EDMAB) e canforoquinona (CQ), respectivamente: alta (H)= 0,8 / 1,6; intermediária (M)= 0,4 / 0,8 e baixa (L)= 0,2 / 0,4. A todas as misturas, 75% e peso de vidro de estrôncio e 5% em peso de OX-50 (sílica coloidal) foram adicionados. O estudo foi dividido em três módulos: no primeiro, os seis compósitos foram testados quanto aos parâmetros descritos acima para a mesma dose de energia aplicada. No segundo, os mesmos materiais foram testados com a dose de energia ajustada de acordo com a concentração de fotoiniciadores de modo que todos os níveis atingissem graus de conversão semelhantes. No terceiro módulo, os materiais com a concentração \"M\" de fotoiniciadores foram submetidos a uma de três irradiâncias: 200, 400 ou 600 mW/cm2, com o tempo de exposição ajustado para que a dose de energia fosse mantida constante. CV foi registrada em um dilatômetro de mercúrio, GC foi avaliado através de FTIR, RPmax foi obtida através de um DSC 7 e a TP foi avaliada em um aparato de compliance controlado, descrito por Sakaguchi et al., 2004. Resultados: Em todos os módulos, não houve interação entre os fatores e assim, as médias foram agrupadas por série de monômeros e nível de fotoiniciadores (módulos 1 e 2) ou nível de irradiância (módulo 3). No módulo 1, as concentrações \"H\" e \"M\" apresentaram GC semelhante, ambas estatisticamente superiores à apresentada pelo grupo com o menor conteúdo de CQ/amina. A série U apresentou GC superior à da série B. RPmax aumentou significantemente com a concentração de fotoiniciadores entre cada um dos níveis estudados (p<0,001), e foi maior para a série U (p<0,05). O grupo de concentração \"H\" apresentou valores de TPmax maiores do que os grupos de concentração \"L\", enquanto que \"M\" apresentou valores de TPmax semelhantes aos dos outros dois (p<0,001). As séries B e U apresentaram TPmax equivalente (p>0,05). CV foi maior para \"L\", estatisticamente diferente das outras duas. A série B apresentou maior CV (p<0,05 para ambos os fatores). No módulo 2, o GC não foi influenciado pela concentração de fotoiniciadores (p>0,05). A série U apresentou maior GC comparado com a série B (p<0,001). RPmax aumentou com a concentração de fotoiniciadores entre cada um dos níveis estudados e foi maior para a série U (p<0,001 para ambos). TPmax não foi influenciada pela concentração de fotoiniciadores (p>0,05). A série B apresentou maior TPmax comparado com a série U (p<0,001). CV foi maior para \"L\", estatisticamente diferente das outras duas. A série B apresentou maiores valores de CV (p<0,001 para ambos os fatores). No módulo 3, GC variou significantemente apenas com a irradiância (p<0,001), sendo que os grupos de alta irradiância apresentaram valores estatisticamente mais baixos comparados aos níveis de irradiância médio e baixo. RPmax, aumentou com a irradiância e foi maior para a série U (p<0,001 para ambos). A série B apresentou valores de TPmax do que os da série U. CV não foi influenciada pela irradiância (p=0,442). A série B apresentou CV estatisticamente maior que a série U (p<0,001). Conclusões: De maneira geral, podemos dizer que CV, GC e RPmax mostraram uma interação bastante complexa e, nas condições deste estudo, não foi possível determinar a contribuição relativa de cada um destes fatores no desenvolvimento da tensão de polimerização. No entanto, parece haver uma tendência de TPmax ser influenciada mais pronunciadamente pelo GC ou CV do que pela RPmax, como demonstrado nos módulos 2 e 3. / Proposition: The objective of this study was to evaluate how the volumetric shrinkage (VS), degree of conversion (DC), maximum rate of reaction (RPmax), polymerization stress (PSmax) and maximum rate of stress development (RSmax) of two experimental composites are influenced by the photoinitiator concentration ([photo]) and by the irradiance. Methods and materials: Bis-GMA/TEGDMA (B series) and Bis GMA/UDMA/TEGDMA (U series) were mixed in equal parts in weight. A tertiary amine (EDMAB) and camphoroquinone (CQ), respectively, were added in three concentrations: high (H)= 0.8/1.6; intermediate (M)= 0.4/0.8 and low (L)= 0.2/0.4 (in wt%). In all mixtures, 80 wt% filler was added. The study was divided in three sections: I) composites were photoactivated with 13 J/cm2. II) radiant exposure was adjusted so that all [photo] would achieve similar DC. III) [photo] \"M\" materials were submitted to three irradiances: 200 (L), 400 (I) or 600 (H) mW/cm2, with exposure time adjusted to convey the same radiant exposure. VS was registered in a mercury dilatometer, DC was evaluated by FTIR spectroscopy, RPmax was obtained in a DSC 7 and PSmax was evaluated in a controlled compliance device, described previously (SAKAGUCHI; WILTBANK; MURCHISON, 2004a). Results: Because no interaction between the factors was observed (except for RSmax in sections I and III), means were pooled for monomer series and [photo] (sections I and II) or irradiance (section III). I) concentrations \"H\" and \"M\" presented similar DC, both statistically superior to \"L\" (p<0.001). U series presented higher DC compared to B series. RPmax increased significantly with photoinitiator concentration between each of the studied levels (p<0.001), and was higher for U series (p<0.05). VS was higher for \"L\", statistically different than the other two (p<0.05). B series presented higher VS (p<0.05). The group with \"H\" concentration presented PSmax higher than \"L\", while \"M\" presented values similar to both (p<0.001). B and U series presented equivalent PSmax (p=0.284). For B series, RSmax increased 100% between the lower e intermediate [photo]. For U series, this increase was only around 50%. II) DC was not influenced by [photo] (p=0.388). U series presented higher DC compared to B series (p<0.001). RPmax increased with [photo] between all studied levels and was higher for U series (p<0.001 for both). VS was higher for \"L\", statistically different from the other two (p<0.001). B series presented higher VS (p<0.001). PSmax was not influenced by the photoinitiator concentration (p=0119). B series presented higher PSmax compared to U series (p<0.001). RSmax increased with [photo] and was higher for U series (p<0.001). III) High irradiance groups presented statistically lower DC compared to the other irradiance levels (p<0.001). Monomer series did not influence DC (p=0.793). RPmax increased with the irradiance and was higher for U series (p<0.001 for both). VS was not influenced by the irradiance (p=0.442). B series presented VS statistically higher than U series (p<0.001). \"H\" groups presented higher PSmax compared to \"L\". \"M\" presented PSmax similar to both (p<0.001). B series presented PSmax higher than U series. While no difference in RSmax between irradiance leves was observed for B series, RSmax increased with the irradiance for U series. Conclusions: There seems to be a trend for PSmax to be influenced more markedly by DC or VS than by RPmax. However, VS, DC and RPmax showed a quite complex interaction and, in the conditions of the present study, it was not possible to determine the relative contribution of each of these factors on stress development.

Recombinant tags are used extensively in protein expression systems to allow purification through IMAC (Immobilized Metal Affinity Chromatography), identification through Western blot, and to facilitate crystal formation for structural analysis. While widely used, their role in enzyme characterization has raised concerns with respect to potential impact on activity. In this study, a flavonol-specific 3-O glucosyltransferase (Cp3GT) from grapefruit (Citrus paradisi) was expressed in Pichia pastoris, and was assayed in its untagged form and with a C-terminal c-myc/6x His tag under various conditions to determine the effect of tags. Prior characterization of pH optima for Cp3GT obtained through expression in Escherichia coli, containing an N-terminal thioredoxin/6x His tag, indicated an optimal pH of 7–7.5, which is indicative of a normal physiological pH and agrees with other glucosyltransferase (GT) pH optima. However, characterization of Cp3GT expressed using P. pastoris with a C-terminal c-myc-6x His tag showed a higher optimal pH of 8.5–9. This suggests a possible tag effect or an effect related to physiological differences between the cell expression systems. Results testing recombinant Cp3GT expressed in Pichia with and without C-terminal tags showed a possible tag effect with regard to substrate preference and interactions with metals, but no apparent effect on enzymatic kinetics or pH optima.

N5-deoxyribosyl derivatives of 2,5-diaminoimidazolone formed by oxidative damage to the guanine bases in 2-deoxyguanosine and highly polymerized DNA readily undergo nucleophilic substitution at C5 in reaction with primary amines in neutral aqueous solutions at 37–70 °C, as it was found in a kinetic study using reverse-phase HPLC. The reaction of 2-amino-5-[(2′-deoxy-β-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) with excess of ethanolamine, alanine and γ-aminobutyric acid (0.2–1 M) is a pseudo-first-order process that proceeds with 45–80 % yields depending on the nature of the amine, its concentration, and the reaction temperature. In the case of ethanolamine, the corresponding bimolecular rate constant has a pre-exponential factor and activation energy of 1.1 × 105 s−1 and 47 kJ mol−1, respectively. The reaction is highly competitive with the previously described hydrolysis of dIz into 2,2-diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone under biologically relevant conditions. A similar reaction with the same lesion in polymeric DNA results in the release of a low-molecular-weight analog of dIz, presumably producing an abasic site as the second reaction product. Kinetic characteristics of this process make it a potentially important source of abasic sites in oxidatively damaged DNA, formed through the reaction of 2,5-diaminoimidazolone lesions with naturally abundant DNA-affinic amines and proteins. The release of low-molecular-weight analogs of dIz can potentially be employed for quantification of imidazolone lesions in oxidized DNA. The half-life of imidazolone lesions in double-stranded DNA evaluated using this approach was found to be 154 min at 37 °C.

No description available.

No description available.

Lignocellulosic biomass is a promising alternative to non-renewable fossil fuel resources for the sustainable production of fuels and chemicals. Levulinic acid (LA) is considered to be a versatile platform chemical which can be derived from biomass and further upgraded to high-value products like levulinate esters. In this work, the direct chemical conversion of lignocellulosic biomass to ethyl levulinate (EL) through LA was studied in collaboration with Gascon Biomass Research Inc. in an effort to valorize their surplus of lignocellulosic feedstock obtained from biomass recycling activities. A novel one-pot, biphasic, acid-catalyzed hydrolysis-esterification process developed by Prof. Tom Baker's lab (uOttawa Chemistry) was used. Kinetic analyses of the overall reaction pathway and major reaction steps were performed to investigate the effect of reaction conditions such as time, temperature, alcohol concentration, and catalyst concentration and evaluate the kinetic and thermodynamic parameters. Reactions were well modeled by power law rate equations with applicability of the Arrhenius expression and exhibited nearly first-order dependence with respect to each of the corresponding reactants and catalyst, as expected. Kinetic studies of LA esterification to various levulinate esters showed that the reaction is endothermic, endergonic, non-spontaneous, and non-rate-limiting. Increased alcohol alkyl chain length and branching reduced the thermodynamic favourability of LA esterification, likely due to steric effects. Process considerations such as reactor pressure, LA and EL partitioning, solvent:feedstock ratio, solvent recycling, and alternative feedstocks were investigated through various lab-scale studies and simulation case studies with UniSim® Design. The results of experimental studies informed the development of a preliminary potential process flow diagram consisting of eleven batch slurry reactors under agitation in parallel. The product streams feed into a continuous separation process composed of flash vessels and distillation columns to separate and purify the various products, intermediates, unconsumed reactants, and recoverable catalyst and solvent. This work ultimately informs decisions regarding future pilot-scale experiments, process design, and optimization for the envisioned large-scale biorefinery to produce EL.

Secondary structure dynamics of biopolymers play a vital role in many of the complex processes within a cell. However, due to the substantial number of atoms in the involved biopolymers along with the multitude of interactions that occur between the molecules, understanding these processes in detail is challenging and often involves computationally demanding simulations. In this thesis, the secondary structure dynamics of three different biopolymer systems were modeled using a single approach, which is based on intuitive principles that facilitate the interpretation. To this end, the kinetic behavior of each system was experimentally determined, and described by simplified reaction schemes, which were then connected to Markov chain models encompassing all principal secondary structural conformations. Firstly, we investigated the toehold-mediated strand displacement reaction, which is widely applied in nanotechnology to create DNA-based nano-devices and biochemical reaction networks. Our model correctly described the impact of base pair mismatches on the kinetics of these reactions, as measured by bulk fluorescence experiments. Additionally, it revealed that incumbent dissociation, base pair fraying, and internal loop formation are important processes during strand displacement. Furthermore, we established two dissipative elements to enhance temporal control over toehold-mediated strand displacement reactions. The first element allowed a reversible and repeatable incumbent strand release, whereas the second element provided the possibility to start the displacement reaction after a programmable temporal delay. Secondly, we studied the target recognition by the CRISPR-Cas effector complex Cascade, a highly promising protein for applications in genome engineering. Our model successfully reproduced all aspects of the torque- and mismatch-dependent R-loop formation time by Cascade obtained by single-molecule torque and bulk fluorescence measurements. Furthermore, we demonstrated that the seed effect observed for Cascade results from DNA supercoiling, rather than a structural property of the protein complex. Lastly, we explored the folding/unfolding of α-helices, which plays a critical role in the folding and function of proteins. Our model accurately described α-helix unfolding kinetics obtained by fast triplet-triplet energy transfer. Moreover, we showed that the complex α-helix unfolding does not follow a simple Einstein-type diffusion but is a combination of the sub-diffusive boundary diffusion and the rather peptide-length-independent coil nucleation. The presented models enabled access to the diverse timescales of the characterized processes, which are generally difficult to access experimentally, despite utilizing just a single approach. In particular, we obtained: tens of microseconds for the branch migration step time of the toehold-mediated strand displacement, hundreds of microseconds for the R-loop formation steps by Cascade, and tens of nanoseconds for folding or unfolding of an α-helix by a single residue. Given the simplicity and accessibility of the established models, we are confident that they will become useful tools for researchers to analyze the dynamics of biomolecules, and anticipate that similar modeling approaches can be applied to other biopolymer systems, being well-described by probabilistic models. / Die Sekundärstrukturdynamik von Biopolymeren spielt eine entscheidende Rolle bei vielen komplexen Prozessen innerhalb einer Zelle. Aufgrund der beträchtlichen Anzahl von Atomen in den beteiligten Biopolymeren und der Vielzahl an Wechselwirkungen zwischen den Molekülen ist es jedoch eine Herausforderung diese Prozesse im Detail zu verstehen, und erfordert oft rechenintensive Simulationen. In dieser Arbeit wurde die Sekundärstrukturdynamik von drei verschiedenen Biopolymersystemen mit einem einzigen Ansatz modelliert, welcher auf intuitiven Prinzipien beruht und somit eine erleichterte Interpretation der Ergebnisse ermöglicht. Hierzu wurde das kinetische Verhalten jedes Systems experimentell bestimmt und durch vereinfachte Reaktionsschemata beschrieben. Diese wurden anschließend mit Markov-Kettenmodellen verknüpft, welche alle wichtigen Konformationen der Sekundärstruktur abbilden. Als erstes System untersuchten wir die DNA Strangaustauschreaktion, welche in der Nanotechnologie häufig zur Herstellung von DNA-basierten Nanomaschinen und biochemischen Reaktionsnetzwerken eingesetzt wird. Unser Modell beschrieb die durch Ensemble-Fluoreszenz-Experimente gemessenen Auswirkungen von Basenfehlpaarungen auf die Kinetik dieser Reaktionen korrekt. Des Weiteren zeigte sich, dass die vorzeitige Strangablösung, das Ausfransen von Basenpaaren und die Bildung interner Schleifen wichtige Prozesse während des Strangaustausches sind. Darüber hinaus konnten wir zwei dissipative Elemente etablieren, um die zeitliche Kontrolle über die Strangaustauschreaktionen zu verbessern. Das erste Element ermöglicht eine reversible und wiederholbare Strangablösung, während das zweite Element die Möglichkeit bietet die Strangaustauschreaktionen nach einer programmierbaren zeitlichen Verzögerung zu starten. Zweitens untersuchten wir den Zielerkennungsprozess durch den CRISPR-Cas Komplex Cascade, ein vielversprechendes Protein für Anwendungen in der Genomtechnologie. Unser Modell reproduzierte erfolgreich alle Aspekte der torsions- und fehlpaarungs-abhängigen R-Schleifenbildung durch Cascade, welche durch Einzelmolekül-Torsions- und Ensemble-Fluoreszenz-Messungen ermittelt wurden. Zusätzlich konnten wir nachweisen, dass der für Cascade beobachtete „seed“-Effekt auf DNA-Verdrehung und nicht auf eine strukturelle Eigenschaft des Proteinkomplexes zurückzuführen ist. Schließlich untersuchten wir die Faltung/Entfaltung von α-Helices, welche eine entscheidende Rolle bei der Faltung und Funktion von Proteinen spielen. Unser Modell beschrieb die durch schnelle Triplett-Triplett-Energietransfer Experimente ermittelte α-Helix-Entfaltungskinetik exakt. Darüber hinaus konnten wir zeigen, dass die komplexe α-Helix-Entfaltung nicht einer einfachen Diffusion vom Einstein-Typ folgt, sondern eine Kombination aus subdiffusiver Grenzdiffusion und der eher peptidlängenunabhängigen Coil-Nukleation ist. Obwohl nur ein einziger Ansatz verwendet wurde, ermöglichten die vorgestellten Modelle den Zugang zu den vielschichtigen Zeitskalen der charakterisierten Prozesse, welche im Allgemeinen experimentell schwer zugänglich sind. Insbesondere konnten die folgenden zeitlichen Bereiche bestimmt werden: Dutzende von Mikrosekunden für die Schrittzeit der Strangaustauschreaktion, Hunderte von Mikrosekunden für die Schritte der R-Schleifenbildung durch Cascade, und Dutzende von Nanosekunden für die Faltung oder Entfaltung einer α-Helix um ein einzelnes Segment. Angesichts der Simplizität und Zugänglichkeit der etablierten Modelle sind wir zuversichtlich, dass sie zu nützlichen Werkzeugen für Forscher werden, um die Dynamik von Biomolekülen zu analysieren. Zusätzlich gehen wir davon aus, dass ähnliche Modellierungsansätze auf andere Biopolymersysteme angewendet werden können, sofern sie gut durch probabilistische Modelle beschrieben werden.

Interest in non-traditional fuel sources, carbon dioxide sequestration, and cleaner combustion has brought attention on gasification to supplement fossil fueled energy, particularly by a fluidized bed. Developing tools and methods to predict operation and performance of gasifiers will lead to more efficient gasifier designs. This research investigates bed fluidization and particle decomposition for fluidized materials. Experimental methods were developed to model gravimetric and energetic response of thermally decomposing materials. Gravimetric, heat flow, and specific heat data were obtained from a simultaneous thermogravimetric analyzer (DSC/TGA). A method was developed to combine data in an energy balance and determine an optimized heat of decomposition value. This method was effective for modeling simple reactions but not for complex decomposition. Advanced method was developed to model mass loss using kinetic reactions. Kinetic models were expanded to multiple reactions, and an approach was developed to identify suitable multiple reaction mechanisms. A refinement method for improving the fit of kinetic parameters was developed. Multiple reactions were combined with the energy balance, and heats of decomposition determined for each reaction. From this research, this methodology can be extended to describe more complex thermal decomposition. Effects of particle density and diameter on the minimum fluidization velocity were investigated, and results compared to empirical models. Effects of bed mass on pressure drop through fluidized beds were studied. A method was developed to predict hydrodynamic response of binary beds from the response of each particle type and mass. Resulting pressure drops of binary mixtures resembled behavior superposition for individual particles. / Master of Science