Browsing by Author "Couto, Andreia"
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- Characterization of gastric cells infection by diverse Helicobacter pylori strains through Fourier-transform infrared spectroscopyPublication . Marques, Vanda; Ribeiro Da Cunha, Bernardo; Couto, Andreia; Sampaio, Pedro; Fonseca, Luís P. P.; Aleixo, Sandra; Calado, CecíliaThe infection of Helicobacter pylori, covering 50% of the world-population, leads to diverse gastric diseases as ulcers and cancer along the life-time of the human host. To promote the discovery of biomarkers of bacterial infection, in the present work, Fourier-transform infrared spectra were acquired from adenocarcinoma gastric cells, incubated with H. pylori strains presenting different genotypes concerning the virulent factors cytotoxin associated gene A and vacuolating cytotoxin A. Defined absorbance ratios were evaluated by diverse methods of statistical inference, according to the fulfillment of the tests assumptions. It was possible to define from the gastric cells, diverse absorbance ratios enabling to discriminate: i) The infection; ii) the bacteria genotype; and iii) the gastric disease of the patients from which the bacteria were isolated. These biomarkers could fasten the knowledge of the complex infection process while promoting a platform for a new diagnostic method, rapid but also specific and sensitive towards the diagnosis of both infection and bacterial virulence.
- A comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event: estimating the transfection efficiency and extracting associated metabolic responsesPublication . Rosa, Filipa; Sales, Kevin C.; Cunha, Bernardo R.; Couto, Andreia; Lopes, Marta B.; Calado, CecíliaReporter genes are routinely used in every laboratory for molecular and cellular biology for studying heterologous gene expression and general cellular biological mechanisms, such as transfection processes. Although well characterized and broadly implemented, reporter genes present serious limitations, either by involving time-consuming procedures or by presenting possible side effects on the expression of the heterologous gene or even in the general cellular metabolism. Fourier transform mid-infrared (FT-MIR) spectroscopy was evaluated to simultaneously analyze in a rapid (minutes) and high-throughput mode (using 96-wells microplates), the transfection efficiency, and the effect of the transfection process on the host cell biochemical composition and metabolism. Semi-adherent HEK and adherent AGS cell lines, transfected with the plasmid pVAX-GFP using Lipofectamine, were used as model systems. Good partial least squares (PLS) models were built to estimate the transfection efficiency, either considering each cell line independently (R 2 ≥ 0.92; RMSECV ≤ 2 %) or simultaneously considering both cell lines (R 2 = 0.90; RMSECV = 2 %). Additionally, the effect of the transfection process on the HEK cell biochemical and metabolic features could be evaluated directly from the FT-IR spectra. Due to the high sensitivity of the technique, it was also possible to discriminate the effect of the transfection process from the transfection reagent on KEK cells, e.g., by the analysis of spectral biomarkers and biochemical and metabolic features. The present results are far beyond what any reporter gene assay or other specific probe can offer for these purposes.
- Modelling, monitoring and control of plasmid bioproduction in Escherichia coli culturesPublication . Lopes, Marta B.; Scholtz, Teresa; Silva, Daniel; Santos, Inês; Silva, Tito; Sampaio, Pedro; Couto, Andreia; Lopes, Vitor V.; Calado, CecíliaAn integrated approach for modelling, monitoring and control the plasmid bioproduction in Escherichia coli cultures is presented. In a first stage, by the implementation of a kinetic model for E. coli cultures, a better bioprocess understanding was reached, concerning the availability of nutrients and products along the bioprocess, and their effects on the plasmid production. Results presented may provide significant help for future modelling and monitoring implementation. In a second stage, FTIR spectroscopy coupled with chemometrics, namely PLS regression, shows its potential as a high-throughput technique for simultaneously estimating the key variables involved in the plasmid production process by E. coli cultures run under distinct conditions. Finally, owing to online monitoring and process control, an NIR fibre optic probe and chemometrics provided promising results concerning the control of biomass and carbon sources in E. coli cultures.
- The importance of Helicobacter pylori's genetic variability for the construction of an efficient vaccinePublication . Vaz, Filipa; Couto, Andreia; Calado, CecíliaHelicobacter pylori is responsible for several gastric diseases. The main constraints of vaccine trials against this pathogen are mainly due to the bacterium high antigenic variability and to down-regulation of the host immune responses. To counteract these factors we propose a DNA vaccine able to induce a balanced humoural and citotoxic specific immune responses, based on multi-antigens. The selection of the antigens NapA, HpaA, VacA and HomB were conducted based on immunoproteomic data and the protein role on infection and pathogenesis. A fragment of each target-antigen was selected by in silico methods based on the maximization of the gene conservation and antigenicity. The set of these small fragments will be presented as a vaccine based on several conserved epitopes of multi-antigenic targets, and consequently representative of the bacterium antigenic variability.