Root ferric chelate reductase is regulated by iron and copper in straberry plants

In the present experiment, we studied the interaction between copper (Cu) and iron (Fe) in strawberry plants grown in nutrient solutions containing different concentrations of Fe. Plants grown in the absence of iron (Fe0) had the characteristic symptoms of Fe deficiency, with smaller chlorotic leaves, less biomass, acidification of the nutrient solution, and roots that were smaller and less ramified, while no symptoms of Fe deficiency were observed in plants grown with Fe. A greater amount of Cu was found in roots of chlorotic plants than in those grown with Fe, while plants grown with 20M of Fe (Fe20) in the nutrient solution had a greater amount of Fe compared with plants from the other treatments. Chlorotic plants (Fe0) and plants grown with the greatest level of Fe (Fe20) had a greater root ferric chelate reductase (FC-R; EC 1.16.1.17) activity compared with the other treatments with 5 or 10M Fe in the nutrient solution. The same pattern was obtained for relative FC-R mRNA concentration and for the sum of Fe and Cu contents in shoots (leaves plus crowns). The DNA obtained from amplification of the FC-R mRNA was cloned and several of the inserts analysed by single strand confirmation polymorphism (SSCP). Although there were different SSCP patterns in the Fe20 treatment, all the inserts that were sequenced were very similar, excluding the hypothesis of more than one FC-R mRNA species being present. The results suggest that Cu as well as Fe is involved in FC-R expression and activity, although the mechanism involved in this regulation is unknown so far. Both small contents of Fe and Cu in plants led to an over-expression of the FC-R gene and enhanced FC-R activity in strawberry roots.

Caracterização fisiológica e nutritiva da clorose férrica em citrinos

Tese dout., Ciências Agrárias, Produção Vegetal, Unidade de Ciências e Tecnologias Agrárias, Universidade do Algarve, 2000

Effects of active pharmaceutical ingredients in mussel mytilus galloprovincialis

More than 3000 types of active pharmaceutical ingredients (APIs) are applied in Human and veterinary medicine practice. These compounds are considered an emergent class of environmental contaminants with the ability to cause damage and unexpected effects to aquatic organisms, namely in species of high commercial value. APIs are ubiquitous in the environment being frequently detected in influents and effluents of waste water treatment plants (WWTPs), surface waters and more distressingly in the public tap water in concentrations ranging from ng to μg.L-1. Considering these premises, the present thesis focused on APIs detection in the Arade river water, the impact of summer period in APIs’ concentration alterations applying the passive sampler device, POCIS (polar organic compound integrative sampler), as well as, the assessment of the effects caused by non-steroidal anti-inflammatory drugs (NSAID) ibuprofen (IBU) and diclofenac (DCF) and antidepressant selective serotonin reuptake inhibitor (SSRI) fluoxetine as single and mixture exposures along with a classical contaminant copper (Cu) on a non-target species, mussel Mytilus galloprovincialis. For this purpose, a multibiomarker approach was applied namely including biomarkers of oxidative stress (antioxidant enzymes activities of superoxide dismutase – SOD, catalase – CAT, glutathione reductase – GR and Phase II glutathione-S-transferase), damage - lipid peroxidation (LPO), neurotoxic effects (through the activity of acetylcholinesterase enzyme - AChE) and endocrine disruption (through vitellogenin-like proteins measurement applying the indirect method of alkali-labile phosphate - ALP) after exposure of mussel species’ to selected APIs at environmental relevant concentrations. The main results highlighted the occurrence of 19 APIs in the river Arade from several distinct therapeutic classes. Stimulant caffeine, antiasthmatic theophylline, NSAID ibuprofen and analgesic paracetamol presented the highest concentrations. Summer impact was inconclusive due to each API transient concentration in each month. The multibiomarker results revealed distinct responses towards each selected API (as single exposure or as mixtures) that were tissue and time dependent. Several multistressor interactions were proposed for each biomarker. The results also revealed APIs potential to induce oxidative stress, LPO, neurotoxicity and endocrine disruption even at extremely low concentrations on a species extremely vulnerable to APIs presence highlighting the urgency on the development of methodologies able to prevent its entrance in the aquatic environment.

Identification of novel molecular determinants of tissue mineralization in fish

The identification of genes involved in signaling and regulatory pathways, and matrix formation is paramount to the better understanding of the complex mechanisms of bone formation and mineralization, and critical to the successful development of therapies for human skeletal disorders. To achieve this objective, in vitro cell systems derived from skeletal tissues and able to mineralize their extracellular matrix have been used to identify genes differentially expressed during mineralization and possibly new markers of bone and cartilage homeostasis. Using cell systems of fish origin and techniques such as suppression subtractive hybridization and microarray hybridization, three genes never associated with mechanisms of calcification were identified: the calcium binding protein S100-like, the short-chain dehydrogenase/reductase sdr-like and the betaine homocysteine S-methyltransferase bhmt3. Analysis of the spatial-temporal expression of these 3 genes by qPCR and in situ hybridization revealed: (1) the up-regulation of sdr-like transcript during in vitro mineralization of gilthead seabream cell lines and its specificity for calcified tissues and differentiating osteoblasts; (2) the up-regulation of S100-like and the down-regulation of bhmt3 during in vitro mineralization and the central role of both genes in cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish. While expression of S100-like and bhmt3 was restricted to calcified tissues, sdr-like transcript was also detected in soft tissues, in particular in tissues of the gastrointestinal tract. Functional analysis of gene promoters revealed the transcriptional regulation of the 3 genes by known regulators of osteoblast and chondrocyte differentiation/mineralization: RUNX2 and RAR (sdr-like), ETS1 (s100-like; bhmt3), SP1 and MEF2c (bhmt3). The evolutionary relationship of the different orthologs and paralogs identified within the scope of this work was also inferred from taxonomic and phylogenetic analyses and revealed novel protein subfamilies (S100-like and Sdr-like) and the explosive diversity of Bhmt family in particular fish groups (Neoteleostei). Altogether our results contribute with new data on SDR, S100 and BHMT proteins, evidencing for the first time the role for these three proteins in mechanisms of mineralization in fish and emphasized their potential as markers of mineralizing cartilage and bone in developing fish.

Effects of vitamin K deficiency and its mechanisms in vertebrate's early development: zebrafish as a model

Disertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Study of the association of Gla Rich Protein (GRP) to human blood cells

Dissertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Biomedicina, Universidade do Algarve, 2013