Browsing by Author "Costa, Joana"
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- Electrochemical magnetoassay coupled to PCR as a quantitative approach to detect the soybean transgenic event GTS40-3-2 in foodsPublication . Manzanares-Palenzuela, C. Lorena; Mafra, Isabel; Costa, Joana; Barroso, M. Fátima; de-los-Santos-Álvarez, Noemí; Delerue-Matos, Cristina; Oliveira, M. Beatriz P. P.; Lobo-Castañón, M. Jesus; López-Ruiz, BeatrizSimple, cost-effective and reliable tools for the quantification of genetically modified organisms (GMO) in food and feed are highly demanded to enforce labelling legislation in the EU. Herein, we report a novel method for quantitative analysis of genetically modified soybean with the event GTS-40-3-2, also known as Roundup Ready (RR) soybean, using magnetoassays with electrochemical detection, coupled to DNA amplification by end-point polymerase chain reaction (PCR). For the proposed work, two DNA sequences were targeted via hybridisation onto magnetic beads, one specific for the transgenic event and the other for the taxon or species-specific lectin gene. Enzymatic labelling was performed to obtain an electrochemically active product measured by chronoamperometry. By optimising the number of PCR cycles, among other parameters, two magnetoassays coupled to PCR were successfully accomplished and linearity was obtained in the ranges of 53–4425 and 1093–88,496 DNA copies for the event-specific and lectin sequences, respectively. The proposed method provides accurate and precise RR soybean quantitative results, being effectively compared to those obtained by real-time PCR, as the reference method. These findings confirm the suitability of the method as an alternative tool for GMO quantification.
- Proteomic analysis of the influence of the adipocyte secretome on Glioma Gl261 cellsPublication . Costa, Joana; Fernandes, Rúben; Sala, Carlo; Almeida, JoanaGlioma is the most frequent form of malignant brain tumor in the adults and childhood. There is a global tendency toward a higher incidence of gliomas in highly developed and industrialized countries. Simultaneously obesity is reaching epidemic proportions in such developed countries. It has been highly accepted that obesity may play an important role in the biology of several types of cancer. We have developed an in vitro method for the understanding of the influence of obesity on glioma mouse cells (Gl261). 3T3-L1 mouse pre-adipocytes were induced to the maturity. The conditioned medium was harvested and used into the Gl261 cultures. Using two-dimension electrophoresis it was analyzed the proteome content of Gl261 in the presence of conditioned medium (CGl) and in its absence (NCGl). The differently expressed spots were collected and analyzed by means of mass spectroscopy (MALDI-TOF-MS). Significantly expression pattern changes were observed in eleven proteins and enzymes. RFC1, KIF5C, ANXA2, N-RAP, RACK1 and citrate synthase were overexpressed or only present in the CGl. Contrariwise, STI1, hnRNPs and phosphoglycerate kinase 1 were significantly underexpressed in CGl. Aldose reductase and carbonic anhydrase were expressed only in NCGl. Our results show that obesity remodels the physiological and metabolic behavior of glioma cancer cells. Also, proteins found differently expressed are implicated in several signaling pathways that control matrix remodeling, proliferation, progression, migration and invasion. In general our results support the idea that obesity may increase glioma malignancy, however, some interesting paradox finding were also reported and discussed.
- Soil MicroorganismsPublication . Costa, Joana; Oliveira, Rui S.; Tiago, Igor; Ma, Ying; Galhano, Cristina; Freitas, Helena; Castro, PaulaThis handbook covers the most commonly used techniques for measuring plant response to biotic and abiotic stressing factors, including: in vitro and in vivo bioassays; the study of root morphology, photosynthesis (pigment content, net photosynthesis, respiration, fluorescence and thermoluminiscence) and water status; thermal imaging; the measurement of oxidative stress markers; flow cytometry for measuring cell cycle and other physiological parameters; the use of microscope techniques for studying plant microtubules; programmed-cell-death; last-generation techniques (metabolomics, proteomics, SAR/QSAR); hybridization methods; isotope techniques for plant and soil studies; and the measurement of detoxification pathways, volatiles, soil microorganisms, and computational biology.