<p>A screening method was developed for identification of catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferase (GST) derivatives expressed in <i>E. coli</i>. The method is based on spraying monochlorobimane (MCB) directly over bacterial colonies growing on agar. The substrate MCB become fluorescent under UV light, when the bacterial colony contains active GSTs catalyzing the conjugation with endogenous glutathione. Eleven out of twelve GSTs investigated where active with MCB. This method can be used to screen libraries generated from most cytosolic GSTs in the search for proteins with altered functions and structures. Azathioprine (Aza), a thiopurine that has been used clinically for 40 years was investigated with 14 GSTs. Three enzymes showed prominent catalytic activities with Aza and all of them are highly expressed in the liver. We estimated the contribution of the three enzymes GSTs A1-1, A2-2 and M1-1 bioactivation of Aza in the liver and concluded that it was about 2 orders of magnitude more effective than the uncatalyzed reaction. GST bioactivation of Aza could clarify aspects of idiosyncratic reactions observed in some individuals. Two other thiopurine prodrugs, cis-acetylvinylthiopurine (cAVTP) and trans-acetylvinylthioguanine (tAVTG), were investigated with the same 14 GSTs. The results displayed diverse catalytic activities. A mechanism of consecutive reactions was proposed. The studies contribute to knowledge under what conditions the drug should optimally be administered. A study of the same prodrugs with several mutants from the Mu class characterized by a point mutation of a hypervarible residue. We conclude that the effects of the mutations were qualitatively parallel for cAVTP and tAVTG, but they vary significantly in magnitude; steric hindrance may interfere with transition-state stabilization. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.</p>

This work presents a method for screening synchrophasor data to search for power system events of interest. The method employs prony algorithm to perform modal analysis and estimate mode amplitude, frequency, and damping ratio on the data obtained from the Texas Synchrophasor Network. The procedure uses seven different Linear Prediction Model (LPM) orders, plus a 10 second window width that slides in steps of 1 second, to minimize the possibility of overlooking events of interest. Further, the algorithm is extended to include user defined modal characteristics thresholds, window length and step size to capture specific power system events. / text

A screening method was developed for identification of catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferase (GST) derivatives expressed in E. coli. The method is based on spraying monochlorobimane (MCB) directly over bacterial colonies growing on agar. The substrate MCB become fluorescent under UV light, when the bacterial colony contains active GSTs catalyzing the conjugation with endogenous glutathione. Eleven out of twelve GSTs investigated where active with MCB. This method can be used to screen libraries generated from most cytosolic GSTs in the search for proteins with altered functions and structures. Azathioprine (Aza), a thiopurine that has been used clinically for 40 years was investigated with 14 GSTs. Three enzymes showed prominent catalytic activities with Aza and all of them are highly expressed in the liver. We estimated the contribution of the three enzymes GSTs A1-1, A2-2 and M1-1 bioactivation of Aza in the liver and concluded that it was about 2 orders of magnitude more effective than the uncatalyzed reaction. GST bioactivation of Aza could clarify aspects of idiosyncratic reactions observed in some individuals. Two other thiopurine prodrugs, cis-acetylvinylthiopurine (cAVTP) and trans-acetylvinylthioguanine (tAVTG), were investigated with the same 14 GSTs. The results displayed diverse catalytic activities. A mechanism of consecutive reactions was proposed. The studies contribute to knowledge under what conditions the drug should optimally be administered. A study of the same prodrugs with several mutants from the Mu class characterized by a point mutation of a hypervarible residue. We conclude that the effects of the mutations were qualitatively parallel for cAVTP and tAVTG, but they vary significantly in magnitude; steric hindrance may interfere with transition-state stabilization. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.

Yes / New multicomponent solid forms of sildenafil have been discovered by means of a combined virtual/experimental cocrystal screening. Coformer selection of candidates was conducted based on an in silico screening method from a database of more than 2000 organic compounds, and the intensive experimental screen produced 23 new solid forms. Since the 12 coformers chosen have a combination of phenol and carboxylic acid groups, a variety of cocrystals, salts, and hybrid salt-cocrystals were discovered and characterized.

The questionnaire DACH was created within the pilot survey of the dissertation project of PhDr. Kateřina Thorová, Ph.D. Since it was put into practice, the questionnaire has become a relatively common tool used in the diagnosis of Autism Spectrum Disorders. The theoretical section of my dissertation introduces the reader to the wider context of Autism Spectrum Disorders - from ethiology to treatment (therapy). The empirical section then focuses on editing, validation and standardization of the DACH questionnaire created by Thorová. Empirical testing of the DACH was conducted on a sample of 240 people. Statistical analysis of the collected data demonstrated a relatively high level of reliability, sensitivity and specificity of the method, along with a high predictive value of positive results. The correlation between the questionnaire results and the results of the diagnostic assessment with the use of the methods of the "golden standard" for autism was lower, but can be considered as satisfactory. The main goal of my dissertation is to develop a standardized Czech method for screening early symptoms of autism in children. For wider access of the practical applications of this project, an electronic version of the DACH questionnaire is available, including scoring and general recommendations. The...

A genética forense tem grande importância na geração de provas em casos de violência sexual, paternidade criminal, identificação de cadáveres e investigação de evidências de locais de crime. A análise de STRs apresenta grande poder de discriminação, mas é uma metodologia multi-etapas, trabalhosa, cara e em muitos casos a análise genética é prejudicada pela baixa quantidade e qualidade das evidências coletadas. Este estudo teve como objetivo desenvolver e caracterizar uma metodologia de triagem de amostras forenses através da análise de perfis de dissociação em alta resolução (HRM) de regiões do DNA mitocondrial, o qual está presente em maior número de cópias e mais resistente a degradação. Para tanto, foram extraídos DNAs de 68 doadores. Estas amostras foram sequenciadas e analisadas por HRM para sete alvos no DNA mitocondrial. Também foram realizados ensaios para determinar a influência do método de extração, da concentração e nível de degradação do DNA no perfil de HRM obtido para uma amostra. Os resultados demonstraram a capacidade da técnica de excluir indivíduos com sequências diferentes da referência comparativa em cinco regiões amplificadas. Podem ser analisadas em conjunto, amostras de DNA com variação de concentração de até a ordem de 100 vezes e extraídas por diferentes metodologias. Condições de degradação de material genético não prejudicaram a obtenção de perfis de dissociação em alta resolução. A sensibilidade da técnica foi aprimorada com a análise de produtos de amplificação de tamanho reduzido. A fim de otimizar o ensaio foi testada a análise de HRM em reações de PCR duplex. Um dos pares de amplificação forneceu perfis de HRM compatíveis com resultados obtidos de reações com amplificação de apenas um dos alvos. Através da análise conjunta das cinco regiões, esta metodologia visa a identificação de indivíduos não relacionados com as referências comparativas, diminuindo o número de amostras a serem analisadas por STRs, reduzindo gastos e aumentando a eficiência da rotina de laboratórios de genética forense. / The forensic genetics has an important role in the generation of evidence in cases of sexual assault, criminal paternity, identification of corpses and crime scenes investigation. The analysis of STRs has great power of discrimination, but it is a multi-stage methodology, complex, expensive and in many cases the genetic analysis is hampered by the low quantity and quality of evidence collected. This study aimed to develop and characterize a forensic samples screening methodology to examine high resolution melting profiles (HRM) of regions of the mitochondrial DNA, which is present in more copies and more resistant to degradation. Thus, we extracted DNA from 68 donors. These samples were sequenced and analyzed by HRM to seven mitochondrial DNA targets. Tests were also conducted to determine the influence of extraction method, concentration and DNA degradation level of HRM profile obtained for a sample. The results demonstrated the technical ability to exclude individuals with different sequences of comparative reference amplified in five regions. Can be analyzed together samples with varying concentration to the order of 100 times and extracted by different methods. Genetic material degradation conditions did not prevent obtaining high resolution melting profiles. The sensitivity of the technique was improved with the analysis of reduced size amplification products. In order to optimize the assay HRM analysis was tested in duplex PCR reactions. A pair of amplification provided HRM profiles consistent with results from amplification in reactions with only one of the targets. Through the joint analysis of the five regions, this approach aims to identify individuals not related to comparative references, reducing the number of samples to be analyzed by STRs, reducing costs and increasing the efficiency of the routine of forensic genetics laboratories.

A case-control study was performed to evaluate the Swedish population-based symphysis fundus (SF) reference curves. The study included 242 small for gestational age (SGA) neonates (169 term and 73 preterm infants) as cases and 296 non-SGA infants as controls. Two Swedish SF curves were evaluated. In term pregnancies they showed a sensitivity of 32 % and 51 % and a specificity of 90 % and 83 %, respectively, at a cut-off level of &lt; - 2 SD from the mean according to the SF reference curve. The sensitivity for SGA was higher in preterm pregnancies (49 % and 58 %, respectively) and the first alarm below – 2 SD was noted before 32 weeks in 37 % and 43 % of the preterm pregnancies, respectively. (Study I) A study of self-administered SF measurements was designed to achieve more regular and frequent SF measurements. Thirty-three women with singleton, ultrasound dated pregnancies performed SF measurements on average 14 weeks from gestational week 20 to 25 until delivery. Self-administered SF measurements were higher and had higher variance than midwives’ measurements. Four consecutive SF measurements on each occasion can compensate for higher variance. Reliable self-administered SF measurements can be obtained. (Study II) Self-administered SF measurements from 191 women were used to construct absolute and relative SF growth references. The influence of fetal sex, maternal obesity and parity was assessed in regression models. The lnSF growth was statistically influenced by maternal obesity, and a borderline significance was recorded for fetal sex and parity. Statistical analysis and graphical displays show no evidence that the relative lnSF growth should be dependent on these variables. (Study III) To improve detection of infants with intrauterine growth restriction (IUGR) rather than SGA a new statistical model (the SR method) was used. The SR method was evaluated with SF measurements from 1122 pregnant women. The sensitivity for neonatal morbidity and SGA was low, between 6 and 36 % for SGA (&lt; -2SD). Neonates classified as SGA (&lt; -2SD and &lt; 10th percentile) had increased morbidity compared with the total study group. Neonates suspected to be SGA before delivery by the population-based SF measurement method had lower morbidity than those not suspected. The SR method was found not to improve detection of fetuses with increased morbidity or SGA neonates in this study. Better screening methods to detect IUGR and SGA prior to delivery are needed. (Study IV)

A genética forense tem grande importância na geração de provas em casos de violência sexual, paternidade criminal, identificação de cadáveres e investigação de evidências de locais de crime. A análise de STRs apresenta grande poder de discriminação, mas é uma metodologia multi-etapas, trabalhosa, cara e em muitos casos a análise genética é prejudicada pela baixa quantidade e qualidade das evidências coletadas. Este estudo teve como objetivo desenvolver e caracterizar uma metodologia de triagem de amostras forenses através da análise de perfis de dissociação em alta resolução (HRM) de regiões do DNA mitocondrial, o qual está presente em maior número de cópias e mais resistente a degradação. Para tanto, foram extraídos DNAs de 68 doadores. Estas amostras foram sequenciadas e analisadas por HRM para sete alvos no DNA mitocondrial. Também foram realizados ensaios para determinar a influência do método de extração, da concentração e nível de degradação do DNA no perfil de HRM obtido para uma amostra. Os resultados demonstraram a capacidade da técnica de excluir indivíduos com sequências diferentes da referência comparativa em cinco regiões amplificadas. Podem ser analisadas em conjunto, amostras de DNA com variação de concentração de até a ordem de 100 vezes e extraídas por diferentes metodologias. Condições de degradação de material genético não prejudicaram a obtenção de perfis de dissociação em alta resolução. A sensibilidade da técnica foi aprimorada com a análise de produtos de amplificação de tamanho reduzido. A fim de otimizar o ensaio foi testada a análise de HRM em reações de PCR duplex. Um dos pares de amplificação forneceu perfis de HRM compatíveis com resultados obtidos de reações com amplificação de apenas um dos alvos. Através da análise conjunta das cinco regiões, esta metodologia visa a identificação de indivíduos não relacionados com as referências comparativas, diminuindo o número de amostras a serem analisadas por STRs, reduzindo gastos e aumentando a eficiência da rotina de laboratórios de genética forense. / The forensic genetics has an important role in the generation of evidence in cases of sexual assault, criminal paternity, identification of corpses and crime scenes investigation. The analysis of STRs has great power of discrimination, but it is a multi-stage methodology, complex, expensive and in many cases the genetic analysis is hampered by the low quantity and quality of evidence collected. This study aimed to develop and characterize a forensic samples screening methodology to examine high resolution melting profiles (HRM) of regions of the mitochondrial DNA, which is present in more copies and more resistant to degradation. Thus, we extracted DNA from 68 donors. These samples were sequenced and analyzed by HRM to seven mitochondrial DNA targets. Tests were also conducted to determine the influence of extraction method, concentration and DNA degradation level of HRM profile obtained for a sample. The results demonstrated the technical ability to exclude individuals with different sequences of comparative reference amplified in five regions. Can be analyzed together samples with varying concentration to the order of 100 times and extracted by different methods. Genetic material degradation conditions did not prevent obtaining high resolution melting profiles. The sensitivity of the technique was improved with the analysis of reduced size amplification products. In order to optimize the assay HRM analysis was tested in duplex PCR reactions. A pair of amplification provided HRM profiles consistent with results from amplification in reactions with only one of the targets. Through the joint analysis of the five regions, this approach aims to identify individuals not related to comparative references, reducing the number of samples to be analyzed by STRs, reducing costs and increasing the efficiency of the routine of forensic genetics laboratories.

El reactor anaerobio de membranas sumergidas (SAnMBR) está considerado como tecnología candidata para mejorar la sostenibilidad en el sector de la depuración de aguas residuales, ampliando la aplicabilidad de la biotecnología anaerobia al tratamiento de aguas residuales de baja carga (v.g. agua residual urbana) o a condiciones medioambientales extremas (v.g. bajas temperaturas de operación). Esta tecnología alternativa de tratamiento de aguas residuales es más sostenible que las tecnologías aerobias actuales ya que el agua residual se transforma en una fuente renovable de energía y nutrientes, proporcionando además un recurso de agua reutilizable. SAnMBR no sólo presenta las principales ventajas de los reactores de membranas (i.e. efluente de alta calidad, y pocas necesidades de espacio), sino que también presenta las principales ventajas de los procesos anaerobios. En este sentido, la tecnología SAnMBR presenta una baja producción de fangos debido a la baja tasa de crecimiento de los microorganismos implicados en la degradación de la materia orgánica, presenta una baja demanda energética debido a la ausencia de aireación, y permite la generación de metano, el cual representa una fuente de energía renovable que mejora el balance energético neto del sistema. Cabe destacar el potencial de recuperación de nutrientes del agua residual bien cuando el efluente es destinado a irrigación directamente, o bien cuando debe ser tratado previamente mediante tecnologías de recuperación de nutrientes. El objetivo principal de esta tesis doctoral es evaluar la viabilidad de la tecnología SAnMBR como núcleo en el tratamiento de aguas residuales urbanas a temperatura ambiente. Por lo tanto, esta tesis se centra en las siguientes tareas: (1) implementación, calibración y puesta en marcha del sistema de instrumentación, control y automatización requerido; (2) identificación de los parámetros de operación clave que afectan al proceso de filtración; (3) modelación y simulación del proceso de filtración; y (4) desarrollo de estrategias de control para la optimización del proceso de filtración minimizando los costes de operación. En este trabajo de investigación se propone un sistema de instrumentación, control y automatización para SAnMBR, el cual fue esencial para alcanzar un comportamiento adecuado y estable del sistema frente a posibles perturbaciones. El comportamiento de las membranas fue comparable a sistemas MBR aerobios a escala industrial. Tras más de dos años de operación ininterrumpida, no se detectaron problemas significativos asociados al ensuciamiento irreversible de las membranas, incluso operando a elevadas concentraciones de sólidos en el licor mezcla (valores de hasta 25 g·L-1 ). En este trabajo se presenta un modelo de filtración (basado en el modelo de resistencias en serie) que permitió simular de forma adecuada el proceso de filtración. Por otra parte, se propone un control supervisor basado en un sistema experto que consiguió reducir el consumo energético asociado a la limpieza física de las membranas, un bajo porcentaje de tiempo destinado a la limpieza física respecto al total de operación, y, en general, un menor coste operacional del proceso de filtración. Esta tesis doctoral está integrada en un proyecto nacional de investigación, subvencionado por el Ministerio de Ciencia e Innovación (MICINN), con título ¿Modelación de la aplicación de la tecnología de membranas para la valorización energética de la materia orgánica del agua residual y la minimización de los fangos producidos¿ (MICINN, proyecto CTM2008-06809- C02-01/02). Para obtener resultados representativos que puedan ser extrapolados a plantas reales, esta tesis doctoral se ha llevado a cabo utilizando un sistema SAnMBR que incorpora módulos comerciales de membrana de fibra hueca. Además, esta planta es alimentada con el efluente del pre-tratamiento de la EDAR del Barranco del Carraixet (Valencia, España). / Robles Martínez, Á. (2013). Modelling, simulation and control of the filtration process in a submerged anaerobic membrane bioreactor treating urban wastewater [Tesis doctoral]. Editorial Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34102 / Premios Extraordinarios de tesis doctorales