José Manuel Marques Martins de Almeida

Plants of Zea mays L. Developed under Enhanced UV-B Radiation. I. Some Ultrastructural and Biochemical Aspects

Summary Zea mays L. cv. LG 12 plants were grown under controlled conditions in growth chambers with and without UV-B radiation. After 10 days of irradiation it was found that UV-B provoked an increase in flavonoid content and even the synthesis of some new ones as observed by chromatography. Total protein content was decreased when expressed on a dry weight basis; however, SDS-PAGE showed that UV-B promoted the increase of some soluble proteins and also interfered with some insoluble ones. Chlorophyllievel was also affected as well as the anatomy and ultrastructure of the leaves. Adaxial epidermal cells collapsed and cytoplasm of chlorophyllous cells showed abundant vesiculation, increased rough endoplasmic reticulum and dictyosome associated vesicles in mesophyll cells. The fractional volume of chloroplasts was decreased while the opposite was observed for fractional volume of starch relative to these organelles. The timing of flowering was not affected but the heigth of plants, the leaf area and the leaf thickness were reduced by UV-B radiation. The results of this study showed that despite the interference of the UV-B radiation on the physiology and morphology of the plants they did attain the reproductive stage and reached maturity.

OCT-1 is over-expressed in intestinal metaplasia and intestinal gastric carcinomas and binds to, but does not transactivate, CDX2 in gastric cells

Intestinal metaplasia (IM) is a preneoplastic lesion of the stomach in which there is transdifferentiation of the gastric mucosa to an intestinal phenotype. The caudal‐related homeobox gene CDX2 encodes an intestine‐specific transcription factor crucial for the regulation of proliferation and differentiation of intestinal cells. In addition, CDX2 is involved in the induction of IM in the stomach. The aim of this study was to access the putative involvement of OCT‐1 in the induction of CDX2 expression de novo in gastric mucosa leading to the onset of IM. OCT‐1 protein expression was evaluated by immunohistochemistry in 31 biopsies with chronic gastritis, 15 biopsies with foci of IM and adjacent gastric mucosa and 42 gastric carcinomas. Furthermore, we evaluated OCT‐1 binding by electrophoretic mobility shift assay and activation of the CDX2 promoter by co‐transfecting a CDX2 promoter/reporter construct with an OCT‐1 expression vector in two gastric carcinoma cell lines, GP220 and MKN45. Our results show that OCT‐1 is expressed in chronic gastritis, particularly when it is adjacent to IM and is expressed in 87% of IM foci. Furthermore, 74% of the gastric carcinomas were positive for OCT‐1 and a strong association was observed between OCT‐1 expression and intestinal‐type carcinoma. We identified that OCT‐1 binds to the CDX2 promoter, although we could not see a transactivation effect in gastric carcinoma cell lines. In conclusion, we observed increased OCT‐1 expression in IM and in intestinal gastric carcinomas and identified the capacity of OCT‐1 to bind to the CDX2 promoter, although we could not demonstrate a direct effect of OCT‐1 in the transactivation of CDX2. Copyright

Discrimination and characterisation of extra virgin olive oils from three cultivars in different maturation stages using Fourier transform infrared spectroscopy in tandem with chemometrics.

A methodology based on Fourier transform infrared (FTIR) spectroscopy, combined with multivariate analysis methods, was applied in order to monitor extra virgin olive oils produced from three distinct cultivars on different maturation stages. For the first time, this kind of methodology is used for the simultaneous discrimination of the maturation stage, and different cultivars. Principal component analysis and discriminant analysis were utilised to create a model for the discrimination of olive oil samples. Partial least squares regression was employed to design calibration models for the determination of chemical parameters. The performance of these models was based on the multiple coefficient of determination (R(2)), the root mean square error of calibration (RMSEC) and root mean square error of cross validation (RMSECV). The prediction models for the chemical parameters resulted in a R(2) ranged from 0.93 to 0.99, a RMSEC ranged from 1% to 4% and a RMSECV from 2% to 5%. It has been shown that this kind of approach allows to distinguish the different cultivars, and to clearly discern the different maturation stages, in each one of these distinct cultivars. Furthermore, the results demonstrated that FTIR spectroscopy in tandem with chemometric techniques allows the creation of viable and accurate models, suitable for correlating the data collected by FTIR spectroscopy, with the chemical composition of the EVOOs, obtained by standard methods.

H 2 Sensing Based on a Pd-Coated Tapered-FBG Fabricated by DUV Femtosecond Laser Technique

An optical fiber probe sensor based on a tapered-fiber Bragg grating (FBG) coated with 150-nm-thick Pd film is proposed for hydrogen detection. The FBG was written in a 50-μm-diameter tapered fiber by deep ultraviolet femtosecond laser technology. A second FBG was inscribed in the 125 μm-fiber section for temperature compensation. The sensing head was able to detect H2 concentration in the range 0%-1% (v/v) H2 at room temperature; a maximum sensitivity of 81.8 pm/% (v/v) H2 was attained with temperature compensation. The influence of the Pd coating over temperature sensitivity of standard and tapered-FBGs is also presented.

Differential responses of the antioxidant defence system and ultrastructure in a salt-adapted potato cell line

Changes in lipid peroxidation and ion content and the possible involvement of the antioxidant system in salt tolerance at the cellular level was studied in a potato (Solanum tuberosum L.) callus line grown on 150 mM NaCl (salt-adapted) and in a non-adapted line exposed to 150 mM NaCl (salt-stressed). Salinity reduced the growth rate and increased lipid peroxidation in salt-stressed line, which remained unaltered in the adapted line. Na⁺ and Cl⁻ content increased due to salinity in both lines, but the adapted line displayed greater K⁺/Na⁺ ratio than the stressed one. Total superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2) activities decreased in both salt-exposed lines; catalase (CAT, EC 1.11.1.6) activity did not change in the adapted line, but decreased in the stressed cell line. Salinity caused the suppression of one GR isoform, while the isozyme patterns of SOD, APX, and CAT were not affected. Ascorbate and reduced glutathione increased in both salt-exposed calli lines. α-Tocopherol increased as a result of salt exposure, with higher levels found in adapted calli. Electron microscopy showed that neither the structural integrity of the cells nor membrane structure were affected by salinity, but plastids from adapted cells had higher starch content. The results suggest that the enzymic and non-enzymic components of the antioxidant system are differentially modulated by salt. Different concentrations of antioxidant metabolites are more relevant to the adaptive response to salinity in potato calli than the differences in activity of the antioxidant enzymes.

Effect of hydrogen peroxide on catalase gene expression, isoform activities and levels in leaves of potato sprayed with homobrassinolide and ultrastructural changes in mesophyll cells

The effect of hydrogen peroxide (H2O2) on catalase (CAT) isoform activities and amounts and on mRNA levels was studied in leaves from potato plants untreated and treated with homobrassinolide (HBR). Northern blot analysis revealed that 100 mm H2O2 supplied through the leaf petiole for 4 h did not induce CAT expression. In contrast, CAT1 and CAT2 responded differently to longer treatment, as CAT2 transcript levels increased markedly whereas CAT1 transcript levels remained unchanged. Western blot analysis showed disparity between the level of CAT1 transcript and CAT1 amount, which actually decreased after 28 h. CAT2 amount correlated well with transcript accumulation and CAT2 activity as visualised by zymogram analysis. H2O2 modified the relative importance of CAT isoforms. After 4 h, CAT1 was prevalent in untreated and H2O2-treated leaves. After 28 h, CAT2 was prevalent in H2O2-treated leaves; therefore, the quantified increase in total CAT activity in these leaves was due to the rise in CAT2. HBR pre-treatment increased CAT2 basal level not changing the pattern of CAT responses to H2O2, only lowering its amplitude. Even so, ultrastructural studies showed that HBR significantly reduced H2O2 negative effects on cellular sub-structures, allowing better recovery of affected structures and reducing the macroscopic injury symptoms on leaves, thus data point to a HBR protective role.

Chromium diffusion in lithium niobate for active optical waveguides

A method to fabricate chromium‐doped lithium niobate substrates in which the active ions are introduced by thermal diffusion from a film is reported. Chromium concentration depth profiles have been obtained by secondary‐ion‐mass spectrometry and the relevant diffusion parameters have been derived. Fluorescence spectrum and upper laser level lifetime of chromium diffused proton‐exchanged and chromium/titanium‐diffused lithium niobate waveguides have been measured. A simple model has been used to estimate the performance of such structures as waveguide optical amplifiers and lasers.A method to fabricate chromium‐doped lithium niobate substrates in which the active ions are introduced by thermal diffusion from a film is reported. Chromium concentration depth profiles have been obtained by secondary‐ion‐mass spectrometry and the relevant diffusion parameters have been derived. Fluorescence spectrum and upper laser level lifetime of chromium diffused proton‐exchanged and chromium/titanium‐diffused lithium niobate waveguides have been measured. A simple model has been used to estimate the performance of such structures as waveguide optical amplifiers and lasers.

Evaluation of olive oil mill wastewater toxicity on the mitochondrial bioenergetics after treatment with Candida oleophila

In a previous work the ability of Candida oleophila to use phenolic compounds as sole carbon and energy source at high concentrations without an additional carbon source was reported. C. oleophila grown in bioreactor batch cultures in a diluted and sterilized olive oil mill wastewater (OMW) caused a significant decrease in the total tannins content but no significant alteration was observed in phenolic acid and fatty acid content. Both treated and untreated OMWs were tested to evaluate the capacity in interfering with mitochondrial bioenergetics. Mitochondrial respiration was not affected by treated OMW on the range of used concentrations, contrary to the untreated OMW. Furthermore, mitochondrial membrane potential and respiratory complexes were always significantly less affected by treated OMW in comparison with untreated OMW. However, supplementary treatment should be applied before OMW could be considered non-toxic.

Uptake and effects of the antimicrobial florfenicol, microplastics and their mixtures on freshwater exotic invasive bivalve Corbicula fluminea

Microplastics and antimicrobials are widely spread environmental contaminants and more research on their toxicity is needed. The uptake and effects of the antimicrobial florfenicol, microplastics, and their mixtures on Corbicula fluminea were investigated. Bivalves were exposed for 96h to florfenicol (1.8 and 7.1mg/l), microplastics (0.2 and 0.7mg/l), or mixtures of the two substances. After 96h, all bivalves exposed to antimicrobial treatments had florfenicol in their body (e.g. 2±1μg/g). Microplastics were found in the gut, lumen of the digestive gland, connective tissue, hemolymphatic sinuses, and gills surface of animals. Florfenicol caused a significant inhibition of cholinesterase (ChE) activity (~32%). Animals exposed to 0.2mg/l of microplastics showed ChE activity inhibition (31%), and no other significant alterations. Mixtures caused feeding inhibition (57-83%), significant ChE inhibition (44-57%) and of isocitrate dehydrogenase activity, and increased anti-oxidant enzymes activity and lipid peroxidation levels. Overall, the results indicate that C. fluminea take up florfenicol and microplastics from the water and accumulated or at least retained it in their body for some time; both florfenicol (low ppm range) and microplastics (ppb range) were toxic to C. fluminea, with mixtures containing florfenicol and microplastics being more toxic. Thus, the risk of exposure and toxic effects of florfenicol to C. fluminea and other bivalves, and its predators increase in ecosystems contaminated with the antimicrobial and microplastics, as well as to humans consuming contaminated species from these ecosystems.

Short wavelength Raman spectroscopy applied to the discrimination and characterization of three cultivars of extra virgin olive oils in different maturation stages.

Extra virgin olive oils produced from three cultivars on different maturation stages were characterized using Raman spectroscopy. Chemometric methods (principal component analysis, discriminant analysis, principal component regression and partial least squares regression) applied to Raman spectral data were utilized to evaluate and quantify the statistical differences between cultivars and their ripening process. The models for predicting the peroxide value and free acidity of olive oils showed good calibration and prediction values and presented high coefficients of determination (>0.933). Both the R(2), and the correlation equations between the measured chemical parameters, and the values predicted by each approach are presented; these comprehend both PCR and PLS, used to assess SNV normalized Raman data, as well as first and second derivative of the spectra. This study demonstrates that a combination of Raman spectroscopy with multivariate analysis methods can be useful to predict rapidly olive oil chemical characteristics during the maturation process.