Contribuições para modelagem e predição do metabolismo de bactérias expostas à ação de antibióticos

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Elétrica, Florianópolis, 2016.Neste trabalho estudou-se o metabolismo do Mycobacterium tuberculosis com ferramentas computacionais. Compreender a fisiologia e o metabolismo dos organismos patogênicos é de primordial importância na pesquisa e desenvolvimento de antibióticos, pois a compreensão do mecanismo de ação de uma nova droga passa pela compreensão de como a exposição do organismo ao antibiótico afeta e altera o seu metabolismo. Esse conhecimento se torna ainda mais importante em situações em que o organismo desenvolve resistência aos compostos. É sabido que o Mycobacterium tuberculosis possui um grande poder de adaptação e capacidade de desenvolver resistência a muitos dos compostos atualmente utilizados para o tratamento da tuberculose. As linhagens resistentes adaptam-se e desenvolvem a capacidade de sobreviver mesmo em presença de um composto a que antes eram susceptíveis. A sobrevivência do organismo está associada diretamente com a capacidade de manter o seu metabolismo em um estado funcional após exposto ao composto bactericida. A sobrevivência das cepas resistentes, portanto, depende de um uso diferenciado dos caminhos metabólicos. Estudar o metabolismo permite compreender a gama de variações metabólicas executadas pelo organismo para manter a sua sobrevivência. O estudo computacional do metabolismo em escala genômica apresenta entretanto diversos desafios. Três principais problemas são (1) o reduzido número de amostras em grande parte dos experimentos de bancada, devido ao alto custo da implementação e complexidade dos métodos de obtenção de dados (2) a inerente alta dimensionalidade dos dados e (3) como incorporar conhecimento biológico existente em modelos computacionais. Este trabalho busca soluções para contornar esses três problemas, com um estudo sobre geração de dados sintéticos, técnicas de estimação, e propostas de modelos com dimensionalidade reduzida e uso de informação biológica a priori em modelos computacionais. Os resultados mostram que a aplicação de técnicas computacionais para estudo de metabolismo de organismos expostos à ação de antibióticos é de fundamental importância na identificação de caminhos metabólicos de sobrevivência, além de produzir uma maior compreensão do mecanismo de ação de compostos antibióticos sobre um organismo patogênico.Abstract : In this work the metabolism of the Mycobacterium tuberculosis (Mtb) was studied with computational biology tools. To understand the physiology and metabolism of pathogenic organisms is of paramount importance in antibiotics R & D, because in order to understand the mechanism of action of a new drug it is necessary to understand how exposing an organism to the antibiotics affects and alters its metabolism. Even more important is this knowledge in situations when the organism may develop resistance to the compound. It is known that the Mycobacterium tuberculosis is highly adaptable and is able to develop resistance to a great number of the anti-tubercular compounds used for tuberculosis (TB) treatment today. The resistant strains adapt and develop the ability to survive even in the presence of a bactericidal compound for which they were once susceptible. The survival of the organism is directly linked to its ability to keep metabolism in a functional state after drug exposure. Therefore, the survival of Mtb resistant strains hinges on being able to use its metabolic mechanisms in a different way. By studying metabolism one may be able to understand the large spectrum of metabolic variations performed by the organism in order to remain alive. A computational study of metabolism in a genomic-scale however presents a number of challenges. Three main difficulties that arise are: (1) the small number of samples available in the majority of wetlab experiments, due to high cost and complexity of methods used for experimental data gathering, (2) the high dimensionality of the data and (3) how to incorporate available biological knowledge in computational models. This work has searched for solutions to overcome these three problems with a study on synthetic data generation, estimation tools, models with reduced dimensionality, and with inclusion of a priori biological knowledge. The results show that the use of computational techniques to study the metabolism of organisms exposed to antibiotics is of fundamental importance for identification of metabolic pathways used for survival and also for producing a better understanding of the mechanism of action of antibiotic compounds on a pathogenic organism

Importance of cholesterol-rich microdomains in the regulation of Nox isoforms and redox signaling in human vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) function is regulated by Nox-derived reactive oxygen species (ROS) and redox-dependent signaling in discrete cellular compartments. Whether cholesterol-rich microdomains (lipid rafts/caveolae) are involved in these processes is unclear. Here we examined the sub-cellular compartmentalization of Nox isoforms in lipid rafts/caveolae and assessed the role of these microdomains in VSMC ROS production and pro-contractile and growth signaling. Intact small arteries and primary VSMCs from humans were studied. Vessels from Cav-1−/− mice were used to test proof of concept. Human VSMCs express Nox1, Nox4, Nox5 and Cav-1. Cell fractionation studies showed that Nox1 and Nox5 but not Nox4, localize in cholesterol-rich fractions in VSMCs. Angiotensin II (Ang II) stimulation induced trafficking into and out of lipid rafts/caveolae for Nox1 and Nox5 respectively. Co-immunoprecipitation studies showed interactions between Cav-1/Nox1 but not Cav-1/Nox5. Lipid raft/caveolae disruptors (methyl-β-cyclodextrin (MCD) and Nystatin) and Ang II stimulation variably increased O2− generation and phosphorylation of MLC20, Ezrin-Radixin-Moesin (ERM) and p53 but not ERK1/2, effects recapitulated in Cav-1 silenced (siRNA) VSMCs. Nox inhibition prevented Ang II-induced phosphorylation of signaling molecules, specifically, ERK1/2 phosphorylation was attenuated by mellitin (Nox5 inhibitor) and Nox5 siRNA, while p53 phosphorylation was inhibited by NoxA1ds (Nox1 inhibitor). Ang II increased oxidation of DJ1, dual anti-oxidant and signaling molecule, through lipid raft/caveolae-dependent processes. Vessels from Cav-1−/− mice exhibited increased O2− generation and phosphorylation of ERM. We identify an important role for lipid rafts/caveolae that act as signaling platforms for Nox1 and Nox5 but not Nox4, in human VSMCs. Disruption of these microdomains promotes oxidative stress and Nox isoform-specific redox signalling important in vascular dysfunction associated with cardiovascular diseases

Importance of cholesterol-rich microdomains in the regulation of Nox isoforms and redox signaling in human vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) function is regulated by Nox-derived reactive oxygen species (ROS) and redox-dependent signaling in discrete cellular compartments. Whether cholesterol-rich microdomains (lipid rafts/caveolae) are involved in these processes is unclear. Here we examined the sub-cellular compartmentalization of Nox isoforms in lipid rafts/caveolae and assessed the role of these microdomains in VSMC ROS production and pro-contractile and growth signaling. Intact small arteries and primary VSMCs from humans were studied. Vessels from Cav-1−/− mice were used to test proof of concept. Human VSMCs express Nox1, Nox4, Nox5 and Cav-1. Cell fractionation studies showed that Nox1 and Nox5 but not Nox4, localize in cholesterol-rich fractions in VSMCs. Angiotensin II (Ang II) stimulation induced trafficking into and out of lipid rafts/caveolae for Nox1 and Nox5 respectively. Co-immunoprecipitation studies showed interactions between Cav-1/Nox1 but not Cav-1/Nox5. Lipid raft/caveolae disruptors (methyl-β-cyclodextrin (MCD) and Nystatin) and Ang II stimulation variably increased O2− generation and phosphorylation of MLC20, Ezrin-Radixin-Moesin (ERM) and p53 but not ERK1/2, effects recapitulated in Cav-1 silenced (siRNA) VSMCs. Nox inhibition prevented Ang II-induced phosphorylation of signaling molecules, specifically, ERK1/2 phosphorylation was attenuated by mellitin (Nox5 inhibitor) and Nox5 siRNA, while p53 phosphorylation was inhibited by NoxA1ds (Nox1 inhibitor). Ang II increased oxidation of DJ1, dual anti-oxidant and signaling molecule, through lipid raft/caveolae-dependent processes. Vessels from Cav-1−/− mice exhibited increased O2− generation and phosphorylation of ERM. We identify an important role for lipid rafts/caveolae that act as signaling platforms for Nox1 and Nox5 but not Nox4, in human VSMCs. Disruption of these microdomains promotes oxidative stress and Nox isoform-specific redox signalling important in vascular dysfunction associated with cardiovascular diseases

Flux Balance Analysis with Objective Function Defined by Proteomics Data-Metabolism of Mycobacterium tuberculosis Exposed to Mefloquine

We present a study of the metabolism of the Mycobacterium tuberculosis after exposure to antibiotics using proteomics data and flux balance analysis (FBA). The use of FBA to study prokaryotic organisms is well-established and allows insights into the metabolic pathways chosen by the organisms under different environmental conditions. To apply FBA a specific objective function must be selected that represents the metabolic goal of the organism. FBA estimates the metabolism of the cell by linear programming constrained by the stoichiometry of the reactions in an in silico metabolic model of the organism. It is assumed that the metabolism of the organism works towards the specified objective function. A common objective is the maximization of biomass. However, this goal is not suitable for situations when the bacterium is exposed to antibiotics, as the goal of organisms in these cases is survival and not necessarily optimal growth. In this paper we propose a new approach for defining the FBA objective function in studies when the bacterium is under stress. The function is defined based on protein expression data. The proposed methodology is applied to the case when the bacterium is exposed to the drug mefloquine, but can be easily extended to other organisms, conditions or drugs. We compare our method with an alternative method that uses experimental data for adjusting flux constraints. We perform comparisons in terms of essential enzymes and agreement using enzyme abundances. Results indicate that using proteomics data to define FBA objective functions yields less essential reactions with zero flux and lower error rates in prediction accuracy. With flux variability analysis we observe that overall variability due to alternate optima is reduced with the incorporation of proteomics data. We believe that incorporating proteomics data in the objective function used in FBA may help obtain metabolic flux representations that better support experimentally observed features

Potencial reprodutivo de Spodoptera eridania (Stoll) (Lepidoptera: Noctuidae) em laboratório : efeito de múltiplos casais e do tamanho

Este estudo objetivou avaliar o efeito de confinar três casais em cada gaiola e o tamanho de adultos emergidos de pupas pequenas, medias e grandes (278,67 mg, 333,20 mg e 381,58 mg, respectivamente), sobre o potencial reprodutivo de S. eridania (Stoll, 1782), em condições controladas (25 ± 1 °C, 70% UR e 14 horas de fotofase). Avaliou-se a sobrevivência, o número de cópulas, fecundidade e fertilidade dos adultos. A sobrevivência não diferiu significativamente entre fêmeas provenientes de pupas de diferentes tamanhos, mas os machos oriundos de pupas grandes tiveram sobrevivência significativamente menor que os demais tamanhos. A fecundidade diferiu significativamente e correlacionou-se positivamente com o tamanho. O número de cópulas (espematóforos) e a fertilidade não variaram em função do peso pupal. Os resultados enfatizam a importância de indicar o número de cópulas e o tamanho dos insetos estudados para que comparações entre os parâmetros reprodutivos possam ser efetuadas.This study aimed to evaluate the effect of keeping three couples in the same cage, and the size of adults emerged from small, medium-sized and large pupae (278.67 mg; 333.20 mg and 381.58 mg, respectively), on the reproductive potential of S. eridania (Stoll, 1782) adults, under controlled conditions (25 ± 1 °C, 70% RH and 14 hour photophase). We evaluated the survival, number of copulations, fecundity and fertility of the adult females. The survival of females from these different pupal sizes did not differ statistically, but the survival of males from large pupae was statistically shorter than from small pupae. Fecundity differed significantly and correlated positively with size. The number of effective copulations (espematophores) and fertility did not vary significantly with pupal size. Our results emphasize the importance of indicating the number of copulations and the size of the insects when reproductive parameters are compared

NADPH oxidases: key modulators in aging and age-related cardiovascular diseases?

Reactive oxygen species (ROS) and oxidative stress have long been linked to aging and diseases prominent in the elderly such as hypertension, atherosclerosis, diabetes and atrial fibrillation (AF). NADPH oxidases (Nox) are a major source of ROS in the vasculature and are key players in mediating redox signalling under physiological and pathophysiological conditions. In this review, we focus on the Nox-mediated ROS signalling pathways involved in the regulation of 'longevity genes' and recapitulate their role in age-associated vascular changes and in the development of age-related cardiovascular diseases (CVDs). This review is predicated on burgeoning knowledge that Nox-derived ROS propagate tightly regulated yet varied signalling pathways, which, at the cellular level, may lead to diminished repair, the aging process and predisposition to CVDs. In addition, we briefly describe emerging Nox therapies and their potential in improving the health of the elderly population