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The creation of enzymes displaying desired substrate specificity is an important objective of enzyme engineering. To help achieve this goal, computational protein design (CPD) can be used to identify sequences that can fulfill interactions required to productively bind a desired substrate. Standard CPD protocols find optimal sequences in the context of a single state, for example an enzyme structure with a single substrate bound at its active site. However, many enzymes catalyze reactions requiring them to bind multiple substrates during successive steps of the catalytic cycle. The design of multi-substrate enzyme specificity requires the ability to evaluate sequences in the context of multiple substrate-bound states because mutations designed to enhance activity for one substrate may be detrimental to the binding of a second substrate. Additionally, many enzymes undergo conformational changes throughout their catalytic cycle and the equilibrium between these conformations can have an impact on their substrate specificity. In this thesis, I present the development and implementation of two multistate computational protein design methodologies for the redesign of multi-substrate enzyme specificity and the modulation of enzyme conformational equilibrium. Overall, our approaches open the door to the design of multi-substrate enzymes displaying tailored specificity for any biocatalytic application.

Submitted by Ramon Santana (ramon.souza@ufpe.br) on 2015-03-04T12:32:53Z No. of bitstreams: 2 DISSERTAÇAO Luís Henrique Sette.pdf: 1478834 bytes, checksum: 70e3b9b20b2337f99852592e164c098a (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-04T12:32:53Z (GMT). No. of bitstreams: 2 DISSERTAÇAO Luís Henrique Sette.pdf: 1478834 bytes, checksum: 70e3b9b20b2337f99852592e164c098a (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013 / Pacientes com doença renal crônica (DRC) apresentam níveis séricos das enzimas ALT e AST diminuídas em relação aos indivíduos saudáveis o que compromete o diagnóstico, seguimento e tratamento dos pacientes com DRC e doenças hepáticas crônicas. Tem-se postulado que a gama glutamil transferase (GGT) poderia ser marcador disponível e barato na avaliação do estresse oxidativo (EO) que, associado com a inflamação e desnutrição, determinariam maior morbimortalidade em pacientes com DRC. O objetivo deste estudo foi avaliar os níveis séricos das enzimas ALT, AST e GGT, de marcadores de inflamação e de EO em pacientes com DRC em tratamento conservador. No período de setembro de 2011 a maio de 2012, foi realizado estudo com pacientes atendidos no ambulatório de DRC do Hospital das Clínicas da Universidade Federal de Pernambuco. Os pacientes foram classificados em estágios da DRC, de acordo com a definição das diretrizes do Kidney Disease Outcomes Quality Initiative (K/DOQI). Foram dosados ALT, AST e GGT; marcadores de inflamação: proteína C reativa, ferritina e albumina e marcadores de EO: TBARS, Tiol, Catalase e Carbonil. Foram avaliados 142 pacientes com DRC apresentando idade média de 64,4 anos sendo 51,4% do sexo masculino. A média da TFG foi de 28,8 mL/min/1,73m2. Os pacientes apresentaram a seguinte distribuição conforme os estágios da DRC: 16 (11,4%) pacientes estágio 5; 69 (49,2%) estágio 4 e 55 pacientes estágio 3 (39,2%). Os níveis séricos de ALT e AST apresentaram redução proporcional à diminuição da TFG (p = 0,006 e p = 0,049, respectivamente). Houve diminuição dos níveis séricos de ALT entre os estágios iniciais quando comparados com os mais avançados da DRC (p < 0,03). Não houve associação entre os níveis séricos de GGT ou dos marcadores de EO com a TFG ou com os estágios da DRC. Entre os marcadores de inflamação, observou-se correlação direta da albumina (r = 0,26 e p < 0,001) e inversa da ferritina (r = -0,23 e p < 0,005) com a TFG, que acompanhou a progressão da DRC de acordo com os estágios.Portanto, as aminotransferases apresentaram redução dos seus níveis séricos conforme a diminuição da TFG, no entanto, os níveis de GGT não se alteraram com a evolução da DRC. Os marcadores de EO avaliados não apresentaram correlação com a TGF, mas os níveis séricos de ferritina aumentaram e de albumina diminuíram de acordo com a progressão da DRC.

Objetivo: Evaluar la asociación entre los niveles elevados de transaminasemia y resistencia a la insulina en una población de individuos sin alteraciones laboratoriales previas de glicemia, insulinemia, ni tiroideos. Métodos: Realizamos un modelo lineal generalizado crudo y ajustado de la familia Poisson con una varianza robusta, para evaluar la asociación entre los niveles elevados de transaminasemia y resistencia a la insulina. Las asociaciones se presentaron como razón de prevalencia (RP) con sus respectivos intervalos de confianza al 95%. Resultados: Se incluyeron 261 participantes. La mediana de edad fue de 39 años (31-45) y el 23,7% de los participantes eran hombres. La prevalencia de transaminasas séricas elevadas para TGO y TGP fue de 13.8% y 26.1%, respectivamente. La prevalencia de resistencia a la insulina fue del 34,1%. En el análisis en bruto encontramos significancia estadística entre TGP y TGO elevados y resistencia a la insulina (RP = 3,18; IC del 95%: 2,33-4,34 y RP = 2,44; IC del 95%: 1,88 a 3,30; respectivamente). Sin embargo, en el análisis multivariado ajustado, la asociación entre el nivel elevado de transaminasas séricas y la resistencia a la insulina permaneció estadísticamente significativa con TGP, pero se perdió con TGO; un PR = 1.90; CI95%: 1.31-2.77 y un PR = 1.23; CI95%: 0,93-1,61; respectivamente. Conclusión: niveles séricos elevados de TGP se asociaron con resistencia a la insulina. TGP podría usarse en la práctica clínica como una herramienta adicional para evaluar la resistencia a la insulina en personas sin alteraciones laboratoriales previas de glicemia, insulinemia, ni tiroideos. / Aim: To evaluate the association between elevated serum transaminase levels and insulin resistance in a population of individuals without alterations in their laboratorial values of glycemia, insulinemia and thyroid panel. Methods: We performed a crude and adjusted generalized linear model of the Poisson family with robust variance, in order to evaluate the association between elevated serum transaminase levels and insulin resistance. The associations were presented as prevalence ratio (PR) with their respective 95% confidence intervals (95% CI). Results: We included 261 participants in the study. The median age was 39 years (31-45) and 23,7% of the participants were men. The prevalence of elevated serum transaminase for TGO and TGP were, 13.8% and 26.1%, respectively. The prevalence of insulin resistance was 34,1%. In the crude analysis we found statistical significance between elevated TGP and TGO and insulin resistance (PR=3,18; 95% CI: 2,33-4,34 and PR=2.44; 95% CI: 1.88-3.30; respectively). However, in the multivariate analysis adjusted for age, sex, body mass index and thyroid hormones, the association between the elevated serum transaminase level and insulin resistance remained statistically significance with TGP, but lost its significance with TGO; a PR = 1.90; CI95%: 1.31-2.77 and a PR = 1.23; CI95%: 0.93-1.61; respectively. Conclusion: Elevated serum levels of TGP were associated with insulin resistance. TGP could be used in clinical practice as an additional tool to assess insulin resistance in people without laboratorial alterations in glycemia, insulinemia and thyroid panel. / Tesis

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Orientador: Guilherme de Camargo Ferraz / Banca: Ruthnea Aparecida Lázaro Muzzi / Banca: Aureo Evangelista Santana / Resumo: As principais alterações fisiológicas ou patológicas induzidas pelo exercício que ocorrem na musculatura esquelética ou cardíaca podem ser identificadas por meio de biomarcadores séricos. Consolidados em atletas da espécie humana e equina, são praticamente inexistentes em cães estudos sobre a dinâmica destes biomarcadores relacionados com a prática de exercício máximo e treinamento. Objetivou-se determinar a cinética dos biomarcadores séricos musculares creatina quinase (CK), aspartato aminotransferase (AST), mioglobina; e cardíacos, troponina cardíaca I (cTnI) e creatina quinase isoenzima MB (CK-MB) de cães submetidos a esforço intenso e condicionamento aeróbio. A ação da venopunção sobre os biomarcadores também foi avaliada. Foram utilizados 18 cães hígidos da raça Beagle distribuídos em três grupos: sedentário (S), não treinado (NT) e treinado (T). Os cães foram submetidos a dois testes de esforço incremental (TEI-1 e TEI-2) para obtenção da curva lactato-velocidade (CLV). Verificou-se se a CLV teve modelo exponencial. O programa de treinamento foi realizado em esteira por 8 semanas na velocidade relacionada a 70-80 % do limiar de lactato (VLL). Determinou-se a velocidade correspondente a frequência cardíaca no momento da fadiga (VFCFadiga). Os biomarcadores séricos foram quantificados nos momentos basal, antes e 1, 6, 12, 24, 48 e 72 h após os TEIs. Aplicou-se análise de variância de dois fatores para amostras repetidas no tempo seguida por teste de Tukey e correlação de ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The main physiological or pathological alterations induced by exercise on skeletal or cardiac musculature can be identified using serum biomarkers. While studies on the dynamics of such biomarkers are consolidated in humans and horses, they are virtually inexistent on maximum exercise and training among dogs. This study aimed to determine the kinetics of the muscle serum biomarkers creatine kinase (CK), aspartate aminotransferase (AST), and myoglobin and of cardiac biomarkers cardiac troponin I (cTnI) and creatine kinase isoenzyme MB (CK-MB) of dogs subjected to intense effort and aerobic conditioning. The effect of venipuncture on the biomarkers was also assessed. Eighteen healthy Beagle dogs were assigned to three groups: sedentary (S), untrained (U), and trained (T). The dogs were subjected to two incremental effort tests (IET-1 and IET-2) so that their lactate vs. velocity curve (LVC) could be obtained. It was verified whether LVC followed an exponential model. The eight-week training program was carried out on a treadmill with speed set to 70-80% of the velocity at lactate threshold (VLT). The velocity corresponding to the heart rate at the moment of fatigue (VHRFatigue) was determined. Serum biomarkers were quantified at the baseline, before, and 1, 6, 12, 24, 48, and 72 h after the IETs. Two-factor analysis of variance was applied for samples repeated over time followed by Tukey's test and Pearson correlation (P≤0.05). The increase (P≤0.05) in the velocyties corresponding to VLT and VHRFatigue indicated an improvement in aerobic fitness of group T. Serum activity of CK and AST increased (P≤0.05), reached maximum values after 6 h in both IETs, and returned to baseline levels after 12-24 h. As a whole, the assessment of the behavior of muscle biomarkers showed recovery of muscle tissues after the IETs. Lev ... (Complete abstract click electronic access below) / Mestre

In primary rat hepatocyte cultures, growth hormone was shown to depress tyrosine aminotransferase levels induced with hydrocortisone. Both induction by glucocorticoid and repression by growth hormone could be demonstrated in cultures several days old.

In malting barley, rapid germination is desirable and linked to end use quality. Modern malting varieties have been bred for low seed dormancy leading to issues with pre-harvest sprouting in wetter growing regions. To maintain malting capacity while minimizing germination on the maternal plant requires in-depth understanding of the genetic regulation of dormancy in malting barley. Currently, the major effect QTLs SD1 and SD2 have been shown to influence dormancy across multiple populations of barley, though the physiological mechanisms involved remain unclear. To search for novel genetic regions that influence primary dormancy, three mapping populations were assessed including two Canadian biparental populations (Synch and Legci) as well as a diversity panel sourced from multiple locations worldwide (ICARDA AM-14). The SD2 locus had a major effect in the Synch population while the SD1 locus had a major effect in the Legci population and neither SD1 nor SD2 were linked to dormancy in the diversity panel. Instead, 14 additional marker trait associations were identified in AM-14 suggesting that investigating a broader range of genetic regulation of dormancy outside of North American varieties may provide solutions to regulate this trait. Additional testing on SD1 revealed that variation at this locus did not affect ABA sensitivity during germination or GA or ABA-regulated gene expression during grain fill. Indeed, lines containing the non-dormant SD1 allele germinate at a similar rate as the dormant SD1 seeds when the glumella was removed from the embryo. This indicated that the effect of the alanine aminotransferase gene underlying the SD1 allele is dependent on physical restriction on the embryo or the hypoxic effects produced by the glumella. Imposing a hypoxic (5% oxygen) environment on exposed embryos revealed an association between non-dormancy at SD1 and reduced sensitivity to the suppressive effects of hypoxia on germination. This suggests that alanine aminotransferase regulates dormancy release during barley germination at least in part through regulation of the seed’s response to hypoxia.

Mit Aminotransferasen können chirale Amine auf biotechnologischem Weg hergestellt werden. Diese besitzen große Bedeutung als Bausteine für weitere Synthesen in der pharmazeutischen und agrochemischen Industrie. Da natürlich vorkommende Enzyme oft nicht die gewünschte Substratspezifität für bestimmte industrielle Anwendungen besitzen, ist eine Optimierung durch Mutagenese notwendig. Solche Entwicklungen sind jedoch oft mit hohem Zeit- und Kostenaufwand verbunden. Die Optimierung kann entweder ungezielt durch empirische Methoden oder gezielt unter Einbeziehung von Informationen über das Enzym erfolgen. Die notwendigen Daten können als Vorbereitung zu konkreten Produktentwicklungen durch Untersuchungen an potentiell geeigneten Enzymen gewonnen werden. Um einen solchen rationalen Ansatz bei der einer speziellen Amin-Pyruvat-Aminotransferase zu ermöglichen, war es Ziel der vorliegenden Arbeit die Grundlagen für die Veränderung der Substratspezifität dieses Enzyms zu erarbeiten. Zunächst wurden strukturelle Informationen durch ein Homologie-Modell gewonnen und später durch eine experimentell bestimmte Struktur ergänzt. Mit dieser Struktur wurden die Substrat-bindenden Reste identifiziert und zunächst der Einfluss auf die Substratbindung durch ortsgerichtete Mutagenese überprüft. Es konnte gezeigt werden, dass alle acht ausgewählten Aminosäurereste an der Substratbindung beteiligt sind. Zudem wurde unter diesen Positionen nach Mutanten gesucht, die neue Substrate umsetzen können. Eine Reihe von Mutanten wurde identifiziert, die verschiedene neue Substrate umsetzen. Für zwei Positionen konnten eine Reihe von Mutanten identifiziert werden, die neue Substrate akzeptieren. Durch die Art der Seitenketten, die Position der Aminosäuren und der chemischen Struktur der akzeptierten Substrate konnten eine Reihe von Aussagen über den Mechanismus der Substratbindung für diese Amin-Pyruvat-Aminotransferase gemacht werden. Außerdem wurde die Zweckmäßigkeit der eingesetzten theoretischen und experimentellen Methoden für die Anwendung bei Entwicklungen mit Enzymen dieser Klasse gezeigt.