Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity. The wild-type North American firefly(Photinus pyralis)luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C. The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible. Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c7dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS). Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions. A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU. Key words:bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c7dATP, dATPαS. / <p>QCR 20161027</p>

A normative experimental study was undertaken to establish whether engaging in positive, negative, and neutral mental imagery affected the reaction rate of participants to positive, negative, and neutral word stimuli. The sample consisted of computer literate, English speaking participants with no history of clinical disorders. A total of 80 participants took part in the study, with 40 participants from either gender. The results of a factorial ANOVA indicated that the type of mental imagery engaged in had a significant effect on the rate at which participants responded to stimuli (p=.00023, F=8.4057), whilst the emotional valence of the stimuli did not have a significant effect (p=.30503, F=1.1877). However, the interaction between the type of mental imagery and the emotional valence of the stimuli was highly significant (p=.00794, F=3.4576), thereby indicating that engaging in positive or negative mental imagery did bias participants towards a faster reaction rate to positive or negative stimuli respectively. / M. A. (Psychology) / Psychology

Este trabalho visa determinar as razões espectrais e o espectro de energia de nêutrons no interior do combustível do Reator Nuclear IPEN/MB-01. Estes parâmetros são de grande importância para determinar com precisão parâmetros físicos de reatores nucleares, como taxas de reação, tempo de vida do combustível e também parâmetros de segurança, tais como reatividade. Para o experimento, utilizou-se detectores de ativação na forma de finas folhas metálicas, introduzidas em uma vareta combustível experimental desmontável. Em seguida, a vareta foi colocada na posição central do núcleo, que tem uma configuração retangular padrão de 26x28 varetas combustível. Foram utilizados detectores de ativação de diferentes elementos como 197Au, 238U, 45SC, 58Ni, 24Mg, 47Ti e 115In para cobrir grande parte do espectro de energia dos nêutrons. Após a irradiação, os detectores de ativação foram submetidos a espectrometria gama utilizando um sistema de contagem com Germânio hiper-puro, afim de se obter a taxa de reação (atividade de saturação) por núcleo alvo. As razões espectrais foram comparadas com valores obtidos através do método de Monte Carlo utilizando o código MCNP-4C. O espectro de energia de nêutrons foi obtido no interior da vareta combustível utilizando o código SANDBP com um espectro de entrada obtido pelo código MCNP-4C, a partir dos valores de atividade de saturação por núcleo alvo dos detectores de ativação irradiados. / This study aims to determine the spectral ratios and the neutron energy spectrum inside the fuel of IPEN/MB-01 Nuclear Reactor. These parameters are of great importance to accurately determine spectral physical parameters of nuclear reactors like reaction rates, fuel lifetime and also security parameters such as reactivity. For the experiment, activation detectors in the form of thin metal foils were introduced in a collapsible fuel rod. Then the rod was placed in the central position of the core which has a standart rectangular configuration of 26x28 fuel rods. There were used activation detectors from different elements such Au-197, U-238, Sc-45, Ni-58, Mg-24, Ti-47 and In-115 to cover a large range of the neutrons energy spectrum. After the irradiation, the activation detectors were submitted to gamma spectrometry using a counting system with high purity Germanium, to obtain the reaction rates (saturation activity) per target nucleus. The spectral ratios were compared with calculated values obtained by the Monte Carlo method using the MCNP-4C code. The neutron energy spectrum was obtained inside the fuel rod using the SANDBP code with an input spectrum obtained by the MCNP-4C code, based on the saturation activity per target nucleus values of the activation detectors irradiated.

Este trabalho apresenta e consolida uma metodologia alternativa para a determinação de taxas de reação nuclear ao longo da direção radial das pastilhas combustíveis sem necessidade de intensos fluxos neutrônicos. Esta técnica se baseia na irradiação de um disco de UO2 inserido no interior de uma vareta combustível desmontável no núcleo do reator IPEN/MB-01. Após a irradiação são realizadas várias espectrometrias gama do disco utilizando um detector HPGe alternando sequencialmente 6 colimadores de chumbo com diâmetros diferentes. Consequentemente, as reações nucleares de captura radiativa que ocorrem nos átomos de 238U, juntamente com as fissões que ocorrem em ambos 235U e 238U são mensuradas em função de 6 regiões radiais distintas do disco combustível. As correções de eficiência geométrica devido à introdução dos colimadores no sistema de detecção HPGe são determinadas através do código MCNP-5. As medidas de taxa de fissão são realizadas utilizando o 99Mo como radionuclídeo traçador. Esse radionuclídeo foi estudado e provou-se ideal para estas medidas por possuir um comportamento linear de formação, alto rendimento de fissão e principalmente por emitir fótons de baixa energia. As medidas foram efetuadas irradiando discos de UO2 (com enriquecimento de 4,3%) na posição central do reator IPEN/MB-01 a potência de 100 Watts durante uma hora. Algumas medidas foram realizadas utilizando uma luva de cádmio envolta na vareta combustível para determinar as taxas de reação nuclear na faixa de energia epitérmica. Os resultados experimentais obtidos são comparados a cálculos de taxa de reação nuclear via MCNP-5 utilizando a biblioteca de dados ENDF/B-VII.0, os quais apresentaram discrepâncias de no máximo 9% para as taxas de captura no 238U e 14% para as taxas de fissão no U na faixa epitérmica. Foram obtidos valores máximos de 4,5% para incertezas relativas as taxas de captura total e epitérmica e para as taxas de fissão total e epitérmica valores máximos de 11,3%. / This research presents and consolidates an alternative methodology for determining nuclear reaction rates along the radial direction of the fuel pellets which does not require high neutron flux. This technique is based on irradiating a thin UO2 disk inserted into a removable fuel rod at the IPEN/MB-01 reactor core. Several gamma spectrometries are performed after irradiation using a HPGe detector. Six lead collimators with different diameters are sequentially alternated during this process, thus, the nuclear radioactive capture which occurs in 238U and the fissions which occur in both 235U and 238U are measured according to six different radial regions of the fuel disk. Geometric efficiency corrections due to the introduction of collimators in HPGe detection system are determined by MCNP-5 code. The fission rate measurements are performed using the 99Mo. This radionuclide was studied and proved ideal for these measurements because it is formed in linear behavior in the reactor core, have a high yield fission and emits low-energy photons. Measurements were performed irradiating UO2 disks (with 4.3% enrichment) in the central position of the IPEN/MB-01 core at 100 watts power level during one hour. Some measurements were performed using a cadmium glove wrapped in the fuel rod to determine the nuclear reaction rates in the epithermal energy range. The experimental results obtained are compared with nuclear reaction rate calculations by means of MCNP-5 with ENDF/B-VII.0 data library showing discrepancies of up to 9% in 238U capture rates and 14% for U fission rates for epithermal energies. Uncertainties regarding the nuclear capture rates have maximum values of 4.5% and the fission rates has maximum values of 11.3%.

<p>Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity.</p><p>The wild-type North American firefly<i>(Photinus pyralis)</i>luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C.</p><p>The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible.</p><p>Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c⁷dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS).</p><p>Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions.</p><p>A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU.</p><p><b>Key words:</b>bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c⁷dATP, dATPαS.</p>

Este trabalho visa determinar as razões espectrais e o espectro de energia de nêutrons no interior do combustível do Reator Nuclear IPEN/MB-01. Estes parâmetros são de grande importância para determinar com precisão parâmetros físicos de reatores nucleares, como taxas de reação, tempo de vida do combustível e também parâmetros de segurança, tais como reatividade. Para o experimento, utilizou-se detectores de ativação na forma de finas folhas metálicas, introduzidas em uma vareta combustível experimental desmontável. Em seguida, a vareta foi colocada na posição central do núcleo, que tem uma configuração retangular padrão de 26x28 varetas combustível. Foram utilizados detectores de ativação de diferentes elementos como 197Au, 238U, 45SC, 58Ni, 24Mg, 47Ti e 115In para cobrir grande parte do espectro de energia dos nêutrons. Após a irradiação, os detectores de ativação foram submetidos a espectrometria gama utilizando um sistema de contagem com Germânio hiper-puro, afim de se obter a taxa de reação (atividade de saturação) por núcleo alvo. As razões espectrais foram comparadas com valores obtidos através do método de Monte Carlo utilizando o código MCNP-4C. O espectro de energia de nêutrons foi obtido no interior da vareta combustível utilizando o código SANDBP com um espectro de entrada obtido pelo código MCNP-4C, a partir dos valores de atividade de saturação por núcleo alvo dos detectores de ativação irradiados. / This study aims to determine the spectral ratios and the neutron energy spectrum inside the fuel of IPEN/MB-01 Nuclear Reactor. These parameters are of great importance to accurately determine spectral physical parameters of nuclear reactors like reaction rates, fuel lifetime and also security parameters such as reactivity. For the experiment, activation detectors in the form of thin metal foils were introduced in a collapsible fuel rod. Then the rod was placed in the central position of the core which has a standart rectangular configuration of 26x28 fuel rods. There were used activation detectors from different elements such Au-197, U-238, Sc-45, Ni-58, Mg-24, Ti-47 and In-115 to cover a large range of the neutrons energy spectrum. After the irradiation, the activation detectors were submitted to gamma spectrometry using a counting system with high purity Germanium, to obtain the reaction rates (saturation activity) per target nucleus. The spectral ratios were compared with calculated values obtained by the Monte Carlo method using the MCNP-4C code. The neutron energy spectrum was obtained inside the fuel rod using the SANDBP code with an input spectrum obtained by the MCNP-4C code, based on the saturation activity per target nucleus values of the activation detectors irradiated.

Este trabalho apresenta e consolida uma metodologia alternativa para a determinação de taxas de reação nuclear ao longo da direção radial das pastilhas combustíveis sem necessidade de intensos fluxos neutrônicos. Esta técnica se baseia na irradiação de um disco de UO2 inserido no interior de uma vareta combustível desmontável no núcleo do reator IPEN/MB-01. Após a irradiação são realizadas várias espectrometrias gama do disco utilizando um detector HPGe alternando sequencialmente 6 colimadores de chumbo com diâmetros diferentes. Consequentemente, as reações nucleares de captura radiativa que ocorrem nos átomos de 238U, juntamente com as fissões que ocorrem em ambos 235U e 238U são mensuradas em função de 6 regiões radiais distintas do disco combustível. As correções de eficiência geométrica devido à introdução dos colimadores no sistema de detecção HPGe são determinadas através do código MCNP-5. As medidas de taxa de fissão são realizadas utilizando o 99Mo como radionuclídeo traçador. Esse radionuclídeo foi estudado e provou-se ideal para estas medidas por possuir um comportamento linear de formação, alto rendimento de fissão e principalmente por emitir fótons de baixa energia. As medidas foram efetuadas irradiando discos de UO2 (com enriquecimento de 4,3%) na posição central do reator IPEN/MB-01 a potência de 100 Watts durante uma hora. Algumas medidas foram realizadas utilizando uma luva de cádmio envolta na vareta combustível para determinar as taxas de reação nuclear na faixa de energia epitérmica. Os resultados experimentais obtidos são comparados a cálculos de taxa de reação nuclear via MCNP-5 utilizando a biblioteca de dados ENDF/B-VII.0, os quais apresentaram discrepâncias de no máximo 9% para as taxas de captura no 238U e 14% para as taxas de fissão no U na faixa epitérmica. Foram obtidos valores máximos de 4,5% para incertezas relativas as taxas de captura total e epitérmica e para as taxas de fissão total e epitérmica valores máximos de 11,3%. / This research presents and consolidates an alternative methodology for determining nuclear reaction rates along the radial direction of the fuel pellets which does not require high neutron flux. This technique is based on irradiating a thin UO2 disk inserted into a removable fuel rod at the IPEN/MB-01 reactor core. Several gamma spectrometries are performed after irradiation using a HPGe detector. Six lead collimators with different diameters are sequentially alternated during this process, thus, the nuclear radioactive capture which occurs in 238U and the fissions which occur in both 235U and 238U are measured according to six different radial regions of the fuel disk. Geometric efficiency corrections due to the introduction of collimators in HPGe detection system are determined by MCNP-5 code. The fission rate measurements are performed using the 99Mo. This radionuclide was studied and proved ideal for these measurements because it is formed in linear behavior in the reactor core, have a high yield fission and emits low-energy photons. Measurements were performed irradiating UO2 disks (with 4.3% enrichment) in the central position of the IPEN/MB-01 core at 100 watts power level during one hour. Some measurements were performed using a cadmium glove wrapped in the fuel rod to determine the nuclear reaction rates in the epithermal energy range. The experimental results obtained are compared with nuclear reaction rate calculations by means of MCNP-5 with ENDF/B-VII.0 data library showing discrepancies of up to 9% in 238U capture rates and 14% for U fission rates for epithermal energies. Uncertainties regarding the nuclear capture rates have maximum values of 4.5% and the fission rates has maximum values of 11.3%.