Seasonal dynamics and operational monitoring of hedgerow olive tree transpiration in response to applied water

We used 2012 sap flow measurements to assess the seasonal dynamics of daily plant transpiration (ETc) in a high-density olive orchard (Olea europaea L. cv. ‘Arbequina’) with a well-watered (HI) control treatment A to supply 100 % of the crop water needs, and a moderately (MI) watered treatment B that replaced 70% of crop needs. To assure that treatment A was well-watered, we compared field daily ETc values against ETc obtained with the Penman-Monteith (PM) combination equation incorporating the Orgaz et al. (2007) bulk daily canopy conductance (gc) model, validated for our non-limiting conditions. We then tested the hypothesis of indirectly monitoring olive ETc from readily available vegetation index (VI) and ground-based plant water stress indicator. In the process we used the FAO56 dual crop coefficient (Kc) approach. For the HI olive trees we defined Kcb as the basal transpiration coefficient, and we related Kcb to remotely sensed Soil Adjusted Vegetation Index (SAVI) through a Kcb-SAVI functional relationship. For the MI treatment, we defined the actual transpiration ETc as the product of Kcb and the stress reduction coefficient Ks obtained as the ratio of actual to crop ETc, and we correlated Ks with MI midday stem water potential (ψst) values through a Ks-ψ functional relationship. Operational monitoring of ETc was then implemented with the ETc = Kcb(SAVI)Ks(ψ)ETo relationship stemmed from the FAO56 approach and validated taking as inputs collected SAVI and ψst data reporting to year 2011. Low validation error (6%) and high goodness-of-fit of prediction were observed (R2 = 0.94, RSME = 0.2 mm day-1, P = 0.0015), allowing to consider that under field conditions it is possible to predict ETc values for our hedgerow olive orchards if SAVI and water potential (ψst) values are known.

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

Long term performance evaluation of small-diameter vascular grafts based on polyvinyl alcohol hydrogel and dextran and MSCs-based therapies using the ovine pre-clinical animal model.

The functional and structural performance of a 5 cm synthetic small diameter vascular graft (SDVG) produced by the copolymerization of polyvinyl alcohol hydrogel with low molecular weight dextran (PVA/Dx graft) associated to mesenchymal stem cells (MSCs)-based therapies and anticoagulant treatment with heparin, clopidogrel and warfarin was tested using the ovine model during the healing period of 24 weeks. The results were compared to the ones obtained with standard expanded polyetetrafluoroethylene grafts (ePTFE graft). Blood flow, vessel and graft diameter measurements, graft appearance and patency rate (PR), thrombus, stenosis and collateral vessel formation were evaluated by B-mode ultrasound, audio and color flow Doppler. Graft and regenerated vessels morphologic evaluation was performed by scanning electronic microscopy (SEM), histopathological and immunohistochemical analysis. All PVA/Dx grafts could maintain a similar or higher PR and systolic / diastolic laminar blood flow velocities were similar to ePTFE grafts. CD14 (macrophages) and α-actin (smooth muscle) staining presented similar results in PVA/Dx/MSCs and ePTFE graft groups. Fibrosis layer was lower and endothelial cells were only detected at graft-artery transitions where it was added the MSCs. In conclusion, PVA/Dx graft can be an excellent scaffold candidate for vascular reconstruction, including clinic mechanically challenging applications, such as SDVGs, especially when associated to MSCs-based therapies to promote higher endothelialization and lower fibrosis of the vascular prosthesis, but also higher PR values.