Browsing by Issue Date, starting with "2017-03-30"
Now showing 1 - 7 of 7
Results Per Page
Sort Options
- Optimization of a bioassay to evaluate Escherichia coli stress responsesPublication . Ribeiro Da Cunha, Bernardo; Russo, Ana; Fonseca, Luís; Calado, CecíliaDuring the genomics era, great emphasis was placed on sequencing microbial genomes and understanding their global gene expression. Later in the post-genomic era, focus shifted towards building on said information, mainly towards deeper knowledge on the relationship of genomics and transcriptomics with the biomolecular composition of microbes, and ultimately their metabolism. To that end, exploring microbial adaptation, e.g. to adverse conditions, is an integral part of current research, for which Escherichia coli is a favored model organism, as its metabolic pathways share pronounced similarities to mainstream pathways across other microorganisms. As such, E. coli has been extensively studied regarding general stress and specific metabolic responses, inclusively for establishing bioassays for detecting and characterizing the mode of action of antibacterial agents. For that, as an example, the global responses of E. coli to diverse stress conditions has been previously compared by transcriptomic and Fourier-transform infrared (FTIR) spectroscopy analysis, highlighting the importance of combining multiple techniques to profoundly understand the holistic cell response. In this work, a high-throughput FTIR spectroscopy based bioassay was developed for E. coli, and optimized regarding multiple parameters as inoculum growth phase, growth media composition and stress agent exposure time. The approach described allowed to identify the impact of each parameter on the metabolic resolution of the biomolecular fingerprint acquired with FTIR spectroscopy, and consequently the optimized bioassay maximizes the ability of FTIR spectroscopy to specifically characterize the impact of diverse stress agents on the E. coli metabolism.
- Mid-infrared spectroscopy: a groundbreaking tool for monitoring mammalian cells processesPublication . Rosa, Filipa O.P.; Ribeiro Da Cunha, Bernardo; Carmelo, Joana G.; Fernandes-Platzgummer, Ana; da Silva, Claudia; Calado, CecíliaMammalian cells are extensively used in cell biology studies, e.g. as a model system of human pathologies, or as a major source of very high-value biopharmaceuticals (that can be the cells itself or their products e.g. heterologous proteins). As such, it is highly pertinent to develop monitoring methods for mammalian cultivations capable of delivering detailed bioprocess information in a rapid and economic way. It is relevant to acquire information concerning the conventional critical variables (as cell growth, consumption of nutrients, production and consumption of by-products and the bioproduct production), and the cell metabolism towards a better understanding of the culture process and consequently for more efficient optimization and control procedures. In the present work, Mid-infrared (MIR) spectroscopy was evaluated as a monitoring technique enabling the acquisition of said bioprocess information in a simple (single step of dehydration), rapid (minutes), economic (without reagent consumption), label-free and high-throughput mode (using 96-wells microplates). The new method was evaluated across a highly diverse set of mammalian culture processes: The monitoring of ex vivo expansion of human mesenchymal stem/stromal cells (MSC) conducted under diverse culture strategies, where it was possible to accurately predict glucose, lactate and ammonia concentrations. The monitoring of recombinant human embryonic kidney cells producing green fluorescent protein, which enabled the estimation of transfection efficiency and the metabolic impact of protein production on the host cell metabolism. Finally, the monitoring of infected gastric cell lines with Helicobacter pylori, which enabled to identify spectral biomarkers for defining the status of infection (infected vs non infected) and to characterize the infection conducted by virulent strains, usually associated to severe gastric diseases as peptide ulcer and gastric cancer. In resume, high-throughput MIR spectroscopy enabled to adequately monitor diverse mammalian cell cultures, thus allowing to attain meaningful information concerning said bioprocesses, from traditional critical variables of the process, to the metabolic status of mammalian host cells and even to define disease biomarkers in a groundbreaking way.
- Biopharmaceuticals process monitoring based on infrared spectroscopy according to quality by designPublication . Sales, Kevin; Ribeiro Da Cunha, Bernardo; Calado, CecíliaEscherichia coli, the most common host microorganism to produce high value biopharmaceuticals, presents, however a natural variability due to high sensitivity towards small fluctuations of the cultivation environment. Therefore, to speed up the development process and to control this critical manufacturing step it is relevant to develop monitoring techniques allowing simultaneous characterization of the heterologous product synthesis and of the physiologic cell behavior under a variety of culture conditions. In the present work high-throughput mid-infrared (MIR) spectral analysis of dehydrated E. coli cell pellets and in situ near infrared (NIR) spectral analysis of the whole culture broth were compared to monitor the bioproduction of a model plasmid along recombinant Escherichia coli cultures. Good partial least square regression models were built, either based on MIR or on NIR spectral data. Besides being conducted in situ, NIR spectroscopy allowed building regression models as accurate as those obtained from the MIR setup, and even better models for estimating biomass and glycerol. However, MIR spectroscopy allows retrieving valuable biochemical and metabolic information along the cell culture, e.g. lipids, RNA, protein synthesis and turnover metabolism. This information contributed to better understand the complex interrelationships between the recombinant cell metabolism and the bioprocess environment. In resume, since NIR spectroscopy enables the in situ and real time monitoring without the risk of culture contamination, thus represents an appealing tool for control purposes specially at industrial (i.e. large-scale) productions. On the other hand, MIR spectroscopy is better suited for optimization processes, as it enables the analysis of hundreds of samples at ounce, and to acquire off line historical information along industrial production processes. Both monitoring techniques are therefore complementary, empowering optimization and control procedures more efficiently and quicker, according to the new regulatory framework based on Quality by Design.
- Composition and in vitro antioxidants activity of Chamaerops humilis L.Publication . Coelho, Jose; Veiga, Jerson G.; Elvas Leitao, Ruben; Brigas, Amadeu F.; Dias, Ana M.; Oliveira, Maria ConceiçãoThe aim of this work is to evaluate the polyphenols composition and the antioxidant activity of methanol extracts of Chamaerops humilis L. Methanol extracts from dried leaves of Chamaerops humilis were prepared using Soxhlet extraction and examined as potential sources of phenolic compounds. Different methods were used to evaluate antioxidant activity of the extracts, including colored (ABTS*+), DPPH radical scavenging assay and reducing power. Total phenolic and flavonoid contents of the extracts were evaluated by Folin-Ciocalteu and aluminum chloride (AlCl3) methods, respectively. Phenolic compositions of the methanol extracts were elucidated by high performance liquid chromatography coupled on-line with tandem mass spectrometry (HPLC-MS/MS). The extract was mainly composed of C- and O-flavones and its O-methylated derivatives. The results suggest that methanol extracts have good potential as sources of bioactive compounds and presents an important antioxidants capacity, which can ensures its potential recommendation for application in the pharmaceutical and nutraceutical sectors.
- High-throughput bioassay for mechanism of action determination of antibacterial drugsPublication . Ribeiro Da Cunha, Bernardo; Fonseca, Luís P. P.; Calado, CecíliaWhile the ‘war’ on infectious diseases has been considered won, antibiotic-resistant bacteria are currently responsible for 25,000 death’s yearly in Europe. No new broadspectrum antibiotic has been introduced since the 1960s, and the last new class was discovered in 1986. As the antibiotic pipeline is clearly exhausted, new tools to advance antibiotic research are required. The current work explored Fourier-transform infrared spectroscopy to classify the mechanism of action of 13 antibiotics, acting by 3 distinct Mode-Of-Action (MOA) and belonging to 7 different classes. After optimization of a biological assay and pre-processing techniques, principal component analysis and partial least squares discriminant analysis were applied in a multi-level approach, including the MOA, antibiotic class and ultimately individual antibiotics acting on very specific molecular targets. Overall results indicate that the proposed method presents metabolic resolution to identify antibiotics at three levels of classification (i.e. different MOA, classes and even acting on specific targets). Interestingly, the resolution capacity obtained at these three levels of classification depended on the antibiotic type, which highlights the importance of the multilevel approach taken. Ultimately the present work reinforces the applicability of the method has a metabolic fingerprinting tool for antibiotic discovery.
- Non-invasive ECG recording for zebrafishPublication . Vaz da Silva, V; Patricia Napoleão; Geraldes, Vera; Rocha, IsabelMyocardial infarction (MI) is a high prevalence cardiovascular disease with a tendency to increase. Although MI therapy has increased its effectiveness, the loss of myocardial function in consequence of MI is still responsible for major subsequent complications as rhythm changes and heart failure. In human hearts, MI is characterized by cardiomyocyte death and fibrin deposition, which is later replaced by scar tissue. In opposition, zebrafish heart undergoes minimal scaring and cardiac tissue can undergo full functionally regeneration in 60 to 120 days. Therefore, this animal model has been used in cardiovascular research as a model for studying mechanisms of cardiac regeneration. The hearts of zebrafish is anatomically different from the human heart, though the electrocardiogram (ECG) pattern and the critical conduction pathways are comparable to the human’s. The objective of our full project is the implementation of na ECG recording system with 3 orthogonal electrodes in 2 planes, in order to create a 4D recording of the zebrafish cardiac electrical activity. Here we present our preliminary work related to a non-invasive ECG monitoring with two electrodes.
- Identification of contact properties of the foot in the stance phase of walkingPublication . Barbosa, InêsIn the study of biped walking, when using forward dynamics analysis, the description of the contact between the feet and the ground is of fundamental importance. The aim of this work is to build a foot model that is capable of describe the foot-ground contact during the stance phase in a simulation. Hyper ellipsoids are used as the contact elements between the foot and the ground and an optimization procedure is used to identify the material and geometric properties of the ellipsoids for the model to simulate human walking. The optimal problem is the minimization of a function relating the predicted ground reaction forces with the forces measured experimentally for a given trial and their application points. Several optimization methods were tested. In the end, a combination of a genetic algorithm and the Sequential Quadratic Programming method was used. There is a good correlation between the solution obtained by the model and experimental data acquired. The model presented is able to simulate the same task as the one captured experimentally and reliable enough to be used in a biomechanical model of the human locomotor system.