In this thesis, on the basis of the asymmetrical charge distribution of E. coli topoisomerase I, I developed a new rapid procedure to purify E. coli DNA topoismoerase I in the milligram range. The new procedure includes using both cation- and anion-exchange columns, i.e., SP-sepharose FF and Q-sepharose FF columns. E. coli topoisomerase I purified here is free of nuclease contamination. The kinetic constants of the DNA relaxation reaction of E. coli DNA topoisomerase I were determined as well. I also used isothermal titration calorimetry to investigate the energetics of DNA supercoiling by using the unwinding properties of DNA intercalators, ethidium and daunomycin. After comparing the enthalpy changes of these DNA intercalators binding to supercoiled and nicked or relaxed plasmid DNA pXXZ06, I determined the DNA supercoiling enthalpy is about 12 kcal/mol per turn of DNA supercoil, which is in good agreement with the previously published results.

By the everyday increasing enthusiasm for using renewable-sustainable sources in energy production area, focusing on one and optimizing it in the best possible way should be of much interest. Biogas production from anaerobic digestion of wastes is a well known energy source which could be applied more efficiently if the CO2portion of it would be upgraded to CH4as well. There is a methanation reaction which could convert carbon dioxide to methane with the use of hydrogenation. In this report, the effort is to simulate this methanation reactor which is a catalytic bed of ruthenium on alumina base. The temperature change and its’ effect on reaction kinetics and equilibrium, also deriving designing parameters for the catalyst bed are different tasks which was tried to be covered in this thesis work. Based on calculations, the reactor can operate isothermally or adiabatically. The point is that each method has its own cons and pros. For the isothermal case finally the optimum temperature to run the reaction is decided to be 600 K in 10 bar total pressure. In adiabatic case then it is understood to work on interstage cooling strategy which in given conditions came to the number of 6 for reactors and 5 for interstage cooling devices. Afterwards it is thought to apply some technical changes to conventional adiabatic method and recycle some part of the product to the entrance of the reactor and assist the conversion. In this method number of reactors would be reduced to 2 and one heat exchanger in the middle. Selecting the best process in large scale treatment, needs lots of economical analysis and detail design while in small scale condition the most preferred method to run the reaction is isothermal.

The 2-His-1-carboxylate facial triad is a common metal binding motif among nonheme iron(II) enzymes. Made up of two histidine side chain residues, and one carboxylate side chain of either a glutamate or aspartate residue occupying one face of the iron(II) octahedral coordinating sphere, the 2-His-1-carboxylate motif provides proximity of substrate(s) and molecular oxygen for important oxidation reactions in biological chemistry. Computational, structural, and kinetic analyses have afforded mechanistic details on how these enzymes control the oxidation reactions they catalyze; from the oxidation state of the metal center to the supporting interactions from secondary sphere amino acid residues. However, the extensive literature on the 2-His-1-carboxylate facial triad enzymes currently contains deficiencies in the area of fundamental, experimental thermodynamic analyses of metal and substrate binding in these systems. The focus of this study is to determine the energetics of substrate and metal binding to two representative enzymes of the 2-His-1-carboxylate facial triad-containing family. More specifically, we examine iron(II) binding to the alpha-ketoglutarate- dependent model system alpha-ketoglutarate/taurine dioxygenase, and substrate binding to a well-known extradiol dioxygenase, homoprotocatechuate 2,3-dioxygenase. Using isothermal titration calorimetry, we are able to determine equilibrium constants, enthalpies, entropies and Gibbs free energies for the binding reactions, affording new insight into what drives the reactions forward at the 2-His-1-carboxylate facial triad active site center.

no / Pterin-4a-carbinolamine dehydratase (PCD) is an essential component of the phenylalanine hydroxylase (PAH) system, catalyzing the regeneration of the essential cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin [6(R)BH4]. Mutations in PCD or its deactivation by hydrogen peroxide result in the generation of 7(R,S)BH4, which is a potent inhibitor of PAH that has been implicated in primapterinuria, a variant form of phenylketonuria, and in the skin depigmentation disorder vitiligo. We have synthesized and separated the 7(R) and 7(S) diastereomers confirming their structure by NMR. Both 7(R)- and 7(S)BH4 function as poor cofactors for PAH, whereas only 7(S)BH4 acts as a potent competitive inhibitor vs. 6(R)BH4 (Ki=2.3–4.9 μM). Kinetic and binding studies, as well as characterization of the pterin-enzyme complexes by fluorescence spectroscopy, revealed that the inhibitory effects of 7(R,S)BH4 on PAH are in fact specifically based on 7(S)BH4 binding. The molecular dynamics simulated structures of the pterin-PAH complexes indicate that 7(S)BH4 inhibition is due to its interaction with the polar region at the pterin binding site close to Ser-251, whereas its low efficiency as cofactor is related to a suboptimal positioning toward the catalytic iron. 7(S)BH4 is not an inhibitor for tyrosine hydroxylase (TH) in the physiological range, presumably due to the replacement of Ser-251 by the corresponding Ala297. Taken together, our results identified structural determinants for the specific regulation of PAH and TH by 7(S)BH4, which in turn aid in the understanding of primapterinuria and acute vitiligo. —Pey, A. L., Martinez, A., Charubala, R., Maitland, D. J., Teigen, K., Calvo, A., Pfleiderer, W., Wood, J. M., Schallreuter, K. U. Specific interaction of the diastereomers 7(R)- and 7(S)-tetrahydrobiopterin with phenylalanine hydroxylase: implications for understanding primapterinuria and vitiligo

RNA-cleaving DNAzymes (RCDs) are a class of functional nucleic acids that can bind various targets ranging in size from small molecules to large proteins, which results in activation of cleavage activity. The activation of RCDs results in the cleavage of a ribonucleotide site in an otherwise all-DNA substrate, leading to two cleavage fragments. In this work, a previously identified DNAzyme that binds to a protein biomarker endogenous to Helicobacter pylori (J99) crude extracellular matrix was evaluated for coupling to an isothermal amplification method termed rolling circle amplification (RCA) as a way to improve the originally reported detection limit. Three RCD constructs were designed with the goal of generating a cleavage fragment that could act as a primer to initiate RCA. The first method used the original HP DNAzyme, which liberated a short cleavage fragment that could be used as a primer. However, the primer fragment was rapidly digested by the bacterial matrix, preventing RCA. A second method evaluated use of a circularized substrate and separate RCD to generate a primer, however this system was not capable of generating a cleavage fragment. A final method redesigned the original RCD to move the substrate region from the 3’ to the 5’ end of the RCD, causing the longer RCD-containing fragment to be the primer for RCA. In this case, target-triggered cleavage was observed and the resulting primer was sufficiently resistant to digestion to allow its use as a primer for RCA. Preliminary characterization of the rearranged RCD showed that it retained selectivity similar to the original RCD, but that the cleavage rate was slower. In addition, the RCA based reaction, while successful, did not produce improved detection sensitivity relative to unamplified assays. Methods to further improve RCA performance are discussed for future work. / Thesis / Master of Science (MSc)

Fluorinated polymers of substantial high performance such as perfluorocyclobutyl (PFCB) and fluorinated aryl vinyl ether (FAVE) polymers can readily be synthesized by thermal [2+2] cyclopolymerization as a melt or by classical polycondensation. These fluoropolymers naturally possess high thermal and chemical resistance, low conductivity properties, and other mechanical properties. In this work, a method using 0th order kinetics is proposed and thermal degradation studies were conducted on six different aromatic ether-based polymers to gauge trends in activation energy barrier and differences in thermal stability by 0th order degradation kinetics. The activation barrier (E_a) obtained can give accurate insight into the stability of the polymer based only on structure for external applications. Activation energies ranging from 17 to 41 kcal/mol were obtained for the various polymers. Overall, this study provides an established method using TGA for thermal stability studies through 0th order kinetics that can be potentially used for future lab applications.

No description available.

Rare Earth Elements (REEs: La–Lu, Y, and Sc) are critical components for technological innovations, therefore more effective methods for the domestic extraction and purification of REEs are in ever-increasing demand. Metal-chelating polymers have great potential in these applications due to their relatively low cost and high affinity for target elements. However, while much research has focused on specific ligands attached to polymers, little is known about the effect of polymer architecture itself on metal chelation. This dissertation reports recent progress in the design, synthesis, and application of polymers for the chelation of various REEs. In addition to synthesizing a series of metal-chelating polymers, we elucidated the thermodynamics of binding using isothermal titration calorimetry (ITC) to gain insight into the specific relationship between polymer structure and metal binding. ITC enables the direct measurement of the binding affinity (Ka), enthalpy changes (ΔH), and stoichiometry of the interactions between macromolecules and metal ions in solution. The thermodynamics of metal chelation underpins many technologies for REE extraction. Consequently, elucidating these parameters enables the rational design of future materials. / Doctor of Philosophy / Rare-Earth Elements (REEs) are critical metals used in many modern technologies, therefore more effective methods for the recovery and purification of REEs are in ever-increasing demand. Metal-chelating polymers—materials that can bind metals—have great potential in these applications due to their relatively low cost and high affinity for target elements. However, while much research has focused on the specific metal-binding group attached to the polymer, little is known about the effect of polymer architecture itself on metal chelation. This dissertation reports recent progress in the design, synthesis, and application of materials that bind to various REEs. In addition to synthesizing a series of metal-binding polymers, we measured the heat absorbed or produced during metal-binding interactions. These experiments produced fundamental insights into the interactions between the polymers, metals ions, and water molecules in solution. Overall, this work produces a depiction of the polymer–metal binding process, which enables insight into each polymer's properties as a metal-binding material. Future researchers can use these guidelines to develop the next generation of materials for the extraction of these critical metals.

This work is an experimental study of the effect of impinging distance on the heat transfer from a cylinder to turbulent isothermal and non-isothermal jets. The isothermal jet is discharged horizontally at the same temperature as the ambient air while the non-isothermal jet is discharged vertically upwards and vertically downwards at a temperature colder than the ambient air. Temperature measurements are made on a heated cylinder using an infrared (IR) camera at five equal impinging distances ranging from Z/d =4 to Z/d=20 and the distributions of the local Frossling numbers are determined. The overall decrease in the average Frossling numbers of the cylinder impinged by the isothermal jet and the cold jets was 25 % and 40% respectively. The peak values of average Frossling number for the isothermal and the cold jets occurred at Z/d=8 and Z/d=4 respectively. The Stagnation Frossling number and the normalized jet centerline velocity for the isothermal and the cold jets were found to be very close to each other at all impinging distances indicating that the effect of buoyancy is negligible in the range of jet temperatures and distances used in the experiment. / Master of Science

O método convencional de ensaios para a obtenção das curvas de fluência de geossintéticos pode necessitar de períodos de até 10.000 horas. Entretanto, a utilização de ensaios acelerados têm se mostrado bastante eficiente, especialmente para avaliar rapidamente a qualidade do material. Estudos bem sucedidos realizados por diversos autores, utilizaram o método Stepped Isothermal Method (SIM) para acelerar a fluência nos geotêxteis. Neste trabalho, com base neste método foi estimada a fluência de dois geotêxteis com 300 g/m² do tipo não tecido de poliéster (PET) de fibra curta e contínua. Neste estudo, foi analisada a fluência causada por carregamentos de 5, 10, 20, 40 e 60% da carga que causa a ruptura do material. Com base nos resultados conclui-se que os valores de deformação por fluência obtidos são satisfatórios, pois as previsões de alcance de até 355 anos estão próximos aos valores encontrados na literatura internacional. Ainda, para o tempo de 100 anos ficou evidenciado que para o geotêxtil não tecido do tipo PET, de fibra curta ou contínua, o comportamento mecânico do geotêxtil é mais influenciado pela deformação inicial do que pela fluência. / The conventional method of tests to achieve the geosynthetic creep curves may require times of up to 10,000 hours. However, the use of accelerated tests have shown to be very effective, especially for rapidly assessing the quality of the material. Successful studies by various authors used the Stepped Isothermal Method Method (SIM) to accelerate creep in geotextiles. In this work, based on this method was estimated creep of two non-woven geotextiles of polyester with 300 g/m², short or continuous fiber. In this study, creep caused by loads of 5, 10, 20, 40 and 60% of the rupture load of the material was analyzed. Based on the results, it is concluded that the creep strain values obtained are satisfactory, because up to 355 years range forecasts are close to those found in the literature. Still, for the 100-year time, it became evident that for the nonwoven geotextile type PET with short or continuous fiber, the mechanical behavior of the geotextile is more influenced by the initial deformation than by creep.