Nuevos mecanismos moleculares de tolerancia a sequía y otros tipos de estrés abiótico en especies arbóreas de interés económico

Las masas forestales tienen una importancia colosal para nuestra sociedad y el conjunto de la biosfera. Estudios recientes a escala mundial indican que la sequía es el factor abiótico que más afecta a su crecimiento y supervivencia, seguida por las temperaturas extremas y la salinidad. Aunque comprender los mecanismos con que las especies arbóreas toleran estas formas de estrés tiene un interés aplicado evidente, dichos mecanismos se han estudiado mucho más en especies herbáceas modelo o de interés agronómico. Existen sin embargo diferencias notables entre ellas, como se demuestra en esta tesis y en otros trabajos recientes. Nuestro estudio se centra concretamente en la respuesta molecular del chopo –el sistema modelo forestal más desarrollado– al estrés abiótico, con particular énfasis en la sequía. Utilizando una estrategia proteómica y tratamientos controlados, hemos identificado componentes mayoritarios de dicha respuesta. Su participación en la misma se ha validado mediante análisis transcripcionales detallados utilizando tecnología qRT-PCR (PCR cuantitativa en tiempo real). Hemos identificado proteínas cuyo nexo funcional con mecanismos de tolerancia ya era conocido, como chaperonas moleculares sHSP o enzimas que atenúan el estrés oxidativo, pero también proteínas cuya relación funcional con el estrés es menos clara o incluso novedosa, como polifenol oxidasas (PPO), deshidrogenasas/reductasas de cadena corta (SDR), o bicupinas (BIC), entre otras. El cuerpo central de la tesis consiste en la caracterización detallada de una PPO inusual, cuya inducción por estrés hídrico se describe por vez primera. Estas enzimas están ampliamente distribuidas en plantas, si bien su número es muy variable de unas especies a otras. Algunas, como nogal, tienen un único gen, mientras que Arabidopsis no tiene ninguno. En la última versión del genoma de chopo hemos identificado un total de 12 miembros bona fide, corrigiendo trabajos previos, y hemos caracterizado su expresión individual ante diferentes situaciones de estrés controlado y tratamientos hormonales. La isoforma antedicha es el único miembro de la familia que responde claramente a la deshidratación. También responde a salinidad y a la mayor parte de tratamientos hormonales ensayados, pero no a daño mecánico o tratamientos con metil jasmonato. Esto la diferencia de enzimas homólogas presentes en otras especies de plantas, que se han relacionado experimentalmente con estrés biótico. Los patrones de acumulación de transcritos en árboles adultos son compatibles con un papel protector frente a la sequía. La integración de nuestros estudios funcionales y filogenéticos sugiere que la familia ha sufrido un proceso reciente de diversificación y neofuncionalización, siendo la protección frente a deshidratación su papel primigenio. Aunque se conoce la actividad bioquímica in vitro de este tipo de enzimas, sus sustratos naturales son esencialmente una incógnita. Mediante expresión heteróloga en Escherichia coli BL21(DE3) hemos detectado que la enzima de chopo es capaz de oxidar L-DOPA a dopaquinona, siendo menos activa frente a otros sustratos. Por otra parte, hemos demostrado su localización cloroplástica mediante transformación transitoria de protoplastos con fusiones a la proteína fluorescente YFP. Mediante la obtención de plantas transgénicas de A. thaliana hemos demostrado que la enzima de chopo aumenta considerablemente la tolerancia in vivo frente a la deshidratación y al estrés salino. El análisis fenotípico detallado de las líneas transgénicas, combinando múltiples metodologías, nos ha permitido sustanciar que la tolerancia tiene una base compleja. Esta incluye una mayor protección del sistema fotosintético, una capacidad antioxidante muy incrementada y la acumulación de solutos osmoprotectores como la prolina. Los análisis metabolómicos nos han permitido asociar la expresión de la proteína a la síntesis de un flavano no descrito previamente en A. thaliana, vinculando la enzima de chopo con la síntesis de fenilpropanoides. También hemos observado alteraciones en los niveles hormonales que podrían subyacer a efectos pleiotrópicos con interés aplicado, como un aumento consistente del tamaño de la planta o el acortamiento del ciclo de crecimiento. Además de aportar datos novedosos sobre la funcionalidad in vivo de esta familia de oxidasas, los resultados de esta tesis demuestran que los árboles son sistemas de estudio interesantes para caracterizar nuevas estrategias de tolerancia al estrés abiótico con potencial aplicado. ABSTRACT Forests masses have an extraordinary importance for our society and the biosphere. Recent worldwide studies indicate that drought is the abiotic factor that affects more their growing and survival, followed by extreme temperatures and salinity. The understanding of how the arboreal species tolerate the stress has an evident practical interest, but their mechanisms have been studied much more in herbaceous species or with agronomic interest. However, considerable differences exist between them, as this thesis and recent studies show. Our study is focused on the molecular response of the poplar –the more developed forestry model system- to abiotic stress, specifically focused in the drought. Using a proteomic strategy and controlled treatments, we have identified main components in such response. Its participation has been validated through transcriptional analysis using qRT-PCR technology. We have identified proteins whose functional connection with tolerance mechanisms were already known, as molecular chaperones sHSP or enzymes that attenuate the oxidative stress, but also some proteins whose functional relationship with the stress is less clear or even novel, as polifenol oxidases (PPO), short chain deshidrogenases/reductases (SDR), or bicupines (BIC), among others. The central body of the thesis consists of the detailed characterization of an unsual PPO, whose induction due to drought stress is first described. These enzymes are thoroughly distributed in plants, but their number of members is very variable among species. Some of them, as the walnut tree, have a single gene, while Arabidopsis has none. We have identified a total of 12 members in the last version of the poplar genome, correcting previous works, and have characterized their individual expression against different situations of controlled stress and hormone treatments. The aforementioned isoform is the only member of the family that responds clearly to the drought. It also reacts to salinity and the majority of hormonal treatments tested, but it does not respond to mechanical damage or treatments with methyl jasmonate. This is the difference with homologue enzymes present in other plant species, which have been related experimentally with abiotic stress. The accumulation patterns of transcripts in adult trees are compatible with a protector role against drought. The integration of our functional and phylogenetic studies suggests that the family has suffered a recent process of diversification and neofunctionalization, being the protection against drought their original role. Although the in vitro biochemistry activity of this kind of enzymes is already known, their natural substracts are essentially a mystery. By means of heterologous expression of Escherichia coli BL21(DE3) we have detected that the enzyme of poplar is able to oxidize L-DOPA to dopaquinone, being less active against other substrates. Additionally, we have proven its chloroplastic location with transitory transformation of protoplasts with YFP protein fusion. By means of getting transgenic plants of A. thaliana, we have demonstrated that the poplar enzyme increases notably the in vivo tolerance against the drought and salinity stresses. The phenotypic analysis of the transgenic lines, and the use of multiple methodologies, allowed us to test the complexity of the tolerance. This includes a major protection of the photosynthetic system, a very increased antioxidant capacity and the accumulation of osmoprotectant solutes as the proline. The metabolic analysis has allowed to associate the protein expression with the synthesis of a Flavan non described previously in A. thalaiana, linking the enzyme of poplar with the synthesis of phenylpropanoids. We have observed alterations in the hormonal levels that could underlie pleiotropic effects with applied interest, as a consistent increase of the size of the plant and the reduction of the growth cycle. The results of this thesis, in addition to provide novel data about the in vivo functionality of the oxidase family, demonstrate that the trees are interesting systems of study to characterize new strategies of tolerance against abiotic stress with applied potential.

Health monitoring of a new hysteretic damper subjected to earthquakes on a shaking table

This paper presents the experimental results obtained by applying frequency-domain structural health monitoring techniques to assess the damage suffered on a special type of damper called Web Plastifying Damper (WPD). The WPD is a hysteretic type energy dissipator recently developed for the passive control of structures subjected to earthquakes. It consists of several I-section steel segments connected in parallel. The energy is dissipated through plastic deformations of the web of the I-sections, which constitute the dissipative parts of the damper. WPDs were subjected to successive histories of dynamically-imposed cyclic deformations of increasing magnitude with the shaking table of the University of Granada. To assess the damage to the web of the I-section steel segments after each history of loading, a new damage index called Area Index of Damage (AID) was obtained from simple vibration tests. The vibration signals were acquired by means of piezoelectric sensors attached on the I-sections, and non-parametric statistical methods were applied to calculate AID in terms of changes in frequency response functions. The damage index AID was correlated with another energy-based damage index-ID- which past research has proven to accurately characterize the level of mechanical damage. The ID is rooted in the decomposition of the load-displacement curve experienced by the damper into the so-called skeleton and Bauschinger parts. ID predicts the level of damage and the proximity to failure of the damper accurately, but it requires costly instrumentation. The experiments reported in this paper demonstrate a good correlation between AID and ID in a realistic seismic loading scenario consisting of dynamically applied arbitrary cyclic loads. Based on this correlation, it is possible to estimate ID indirectly from the AID, which calls for much simpler and less expensive instrumentation.

De novo assembly and annotation of a transcriptome during xylogenesis in Pinus canariensis Chr. Sm. ex DC

Nowadays, there is a great amount of genomic and transcriptomic data available about forest species, including ambitious projects looking for complete sequencing and annotation of different gymnosperm genomes [1, 2]. Pinus canariensis is an endemic conifer of the Canary Islands with re-sprouting capability and resilience against fire and mechanical damage, as result of an adaptation to volcanic environments. Additionally, this species has a high proportion of axial parenchyma compared with other conifers, and this tissue connects with radial parenchyma allowing transport of reserves. The most internal tracheids stop accumulating water [3], and get filled of resins and polyphenols synthesized by the axial parenchyma; this is the so-called ?torch-heartwood? [4], which avoids decay. This wood achieves very high prices due to its particular resistance to rot. These features make P. canariensis an interesting model species for the analysis of these developmental processes in conifers. In this study we aim to perform a complete transcriptome annotation during xylogenesis in Pinus canariensis, using next-generation sequencing (NGS) -Roche 454 pyrosequencing-, in order to provide a genomic resource for further analysis, including expression profiling and the identification of candidate genes for important adaptive features.

Hypercapnia induces cleavage and nuclear localization of RelB protein, giving insight into CO2 sensing and signaling

Carbon dioxide (CO(2)) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO(2) (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO(2) or associated acidosis to this response remain poorly understood. Recently, a role for the non-canonical NF-?B pathway has been postulated to be important in signaling the cellular transcriptional response to CO(2). In this study, we demonstrate that in cells exposed to elevated CO(2), the NF-?B family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3ß or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO(2)-sensitive NF-?B family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung.

Estudo da variação de propriedades de geomembranas de PVC devido a danos mecânicos

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Tecnologia, Departamento de Engenharia Civil e Ambiental, 2015.

Susceptibility and Interactions of Drosophila suzukii and Zaprionus indianus (Diptera: Drosophilidae) in Damaging Strawberry.

Resumo: Drosophila suzukii (Matsumura) foi recentemente detectado causando danos idade para morangos no Brasil. Infestação na cultura de morango tem frequentemente foi observado conjuntamente com a presença de Zaprionus indianus Gupta. este estudo investigou a suscetibilidade de morangos em três amadurecimento estágios para infestação de D. suzukii e Z. indianus e sua interação. Abstracts: Drosophila suzukii (Matsumura) has been recently detected causing damage to strawberries in Brazil. Infestation in strawberry culture has often been observed jointly with the presence of Zaprionus indianus Gupta. This study investigated the susceptibility of strawberries at three ripening stages to infestation of D. suzukii and Z. indianus and their interaction. In the laboratory, strawberries cv. Albion at different ripening stages (green, semi-ripe and ripe) were exposed to D. suzukii and Z. indianus for 24 h in choice and no-choice bioassays. Additionally, we evaluated the effects of mechanical damage incurred artificially or by D. suzukii ovi-position on Z. indianus infestation. In no-choice bioassay, there were no significant differences in fruit susceptibility to D. suzukii infestation at different ripening stages. However, in choice bioassay, D. suzukii adults preferred to oviposit on R fruit. The presence of mechanical damage did not increase susceptibility of fruit to D. suzukii oviposition. For Z. indianus , there was greater susceptibility of R fruit in relation to SR and G fruit in both the choice and no-choice bioassays. There was a significant and positive interaction of mechanical damage and damage caused by D. suzukii to R fruit and infestation by Z. indianus , which was not observed in SR and G fruit. Although infestation of Z. indianus is related to attack damaged or decaying fruit, this work shows that this species has the ability to oviposit and develop in healthy strawberry fruit with and increased infestation level when the fruit has damage to its epidermis.

Susceptibility and Interactions of Drosophila suzukii and Zaprionus indianus (Diptera: Drosophilidae) in Damaging Strawberry.

Resumo: Drosophila suzukii (Matsumura) foi recentemente detectado causando danos idade para morangos no Brasil. Infestação na cultura de morango tem frequentemente foi observado conjuntamente com a presença de Zaprionus indianus Gupta. este estudo investigou a suscetibilidade de morangos em três amadurecimento estágios para infestação de D. suzukii e Z. indianus e sua interação. Abstracts: Drosophila suzukii (Matsumura) has been recently detected causing damage to strawberries in Brazil. Infestation in strawberry culture has often been observed jointly with the presence of Zaprionus indianus Gupta. This study investigated the susceptibility of strawberries at three ripening stages to infestation of D. suzukii and Z. indianus and their interaction. In the laboratory, strawberries cv. Albion at different ripening stages (green, semi-ripe and ripe) were exposed to D. suzukii and Z. indianus for 24 h in choice and no-choice bioassays. Additionally, we evaluated the effects of mechanical damage incurred artificially or by D. suzukii ovi-position on Z. indianus infestation. In no-choice bioassay, there were no significant differences in fruit susceptibility to D. suzukii infestation at different ripening stages. However, in choice bioassay, D. suzukii adults preferred to oviposit on R fruit. The presence of mechanical damage did not increase susceptibility of fruit to D. suzukii oviposition. For Z. indianus , there was greater susceptibility of R fruit in relation to SR and G fruit in both the choice and no-choice bioassays. There was a significant and positive interaction of mechanical damage and damage caused by D. suzukii to R fruit and infestation by Z. indianus , which was not observed in SR and G fruit. Although infestation of Z. indianus is related to attack damaged or decaying fruit, this work shows that this species has the ability to oviposit and develop in healthy strawberry fruit with and increased infestation level when the fruit has damage to its epidermis.

Study of tissue damage during mechanical peeling of tough skinned vegetables

Peeling is an essential phase of post harvesting and processing industry; however the undesirable losses and waste rate that occur during peeling stage are always the main concern of food processing sector. There are three methods of peeling fruits and vegetables including mechanical, chemical and thermal, depending on the class and type of fruit. By comparison, the mechanical method is the most preferred; this method keeps edible portions of produce fresh and creates less damage. Obviously reducing material losses and increasing the quality of the process has a direct effect on the whole efficiency of food processing industry which needs more study on technological aspects of this industrial segment. In order to enhance the effectiveness of food industrial practices it is essential to have a clear understanding of material properties and behaviour of tissues under industrial processes. This paper presents the scheme of research that seeks to examine tissue damage of tough skinned vegetables under mechanical peeling process by developing a novel FE model of the process using explicit dynamic finite element analysis approach. In the proposed study a nonlinear model which will be capable of simulating the peeling process specifically, will be developed. It is expected that unavailable information such as cutting force, maximum shearing force, shear strength, tensile strength and rupture stress will be quantified using the new FEA model. The outcomes will be used to optimize and improve the current mechanical peeling methods of this class of vegetables and thereby enhance the overall effectiveness of processing operations. Presented paper aims to review available literature and previous works have been done in this area of research and identify current gap in modelling and simulation of food processes.

Process modelling and simulation of tissue damage during mechanical peeling of pumpkin as a tough skinned vegetable

Food waste is a current challenge that both developing and developed countries face. This project applied a novel combination of available methods in Mechanical, agricultural and food engineering to address these challenges. A systematic approach was devised to investigate possibilities of reducing food waste and increasing the efficiency of industry by applying engineering concepts and theories including experimental, mathematical and computational modelling methods. This study highlights the impact of comprehensive understanding of agricultural and food material response to the mechanical operations and its direct relation to the volume of food wasted globally.

Influences of CO2 laser annealing on mechanical and laser-induced damage properties of ZrO2 thin films

Zirconium dioxide (ZrO2) thin films were deposited on BK7 glass substrates by the electron beam evaporation method. A continuous wave CO2 laser was used to anneal the ZrO2 thin films to investigate whether beneficial changes could be produced. After annealing at different laser scanning speeds by CO2 laser, weak absorption of the coatings was measured by the surface thermal lensing (STL) technique, and then laser-induced damage threshold (LIDT) was also determined. It was found that the weak absorption decreased first, while the laser scanning speed is below some value, then increased. The LIDT of the ZrO2 coatings decreased greatly when the laser scanning speeds were below some value. A Nomarski microscope was employed to map the damage morphology, and it was found that the damage behavior was defect-initiated both for annealed and as-deposited samples. The influences of post-deposition CO2 laser annealing on the structural and mechanical properties of the films have also been investigated by X-ray diffraction and ZYGO interferometer. It was found that the microstructure of the ZrO2 films did not change. The residual stress in ZrO2 films showed a tendency from tensile to compressive after CO, laser annealing, and the variation quantity of the residual stress increased with decreasing laser scanning speed. The residual stress may be mitigated to some extent at proper treatment parameters. (c) 2007 Elsevier GmbH. All rights reserved.

An analysis of the changes to the mechanical and acoustical properties of paper caused by the damage mechanisms present in the fibre structure

This paper will analyse two of the likely damage mechanisms present in a paper fibre matrix when placed under controlled stress conditions: fibre/fibre bond failure and fibre failure. The failure process associated with each damage mechanism will be presented in detail focusing on the change in mechanical and acoustic properties of the surrounding fibre structure before and after failure. To present this complex process mathematically, geometrically simple fibre arrangements will be chosen based on certain assumptions regarding the structure and strength of paper, to model the damage mechanisms. The fibre structures are then formulated in terms of a hybrid vibro-acoustic model based on a coupled mass/spring system and the pressure wave equation. The model will be presented in detail in the paper. The simulation of the simple fibre structures serves two purposes; it highlights the physical and acoustic differences of each damage mechanism before and after failure, and also shows the differences in the two damage mechanisms when compared with one another. The results of the simulations are given in the form of pressure wave contours, time-frequency graphs and the Continuous Wavelet Transform (CWT) diagrams. The analysis of the results leads to criteria by which the two damage mechanisms can be identified. Using these criteria it was possible to verify the results of the simulations against experimental acoustic data. The models developed in this study are of specific practical interest in the paper-making industry, where acoustic sensors may be used to monitor continuous paper production. The same techniques may be adopted more generally to correlate acoustic signals to damage mechanisms in other fibre-based structures.

Testing equipment for the improvement of mechanical devices to minimize damage to fruit in commercial packing lines.

Damages -reduced in fruit packing lines is a major cause of grace reduction and quality loos in fresh marks: fruit. Fruit must be treated gently during in sir handling to improve their qualityin order to get a good price in a competitive market. The correct post-hardvest handling in fruit packing lines is a prerequisite to cut down the heavy post-harvest losses. Fruit packing lines must be evaluated, studying their design, the impacts applied to the fruits, the characteristics of the materials, etc. This study establishes the possibility of carrying out modifications and tests in a packing line during a long period of time. For this purpose, an experimental fruit packing line has been designed and located in the Agricultural Engineering Department of the Polythecnic University of Madrid with the aim of improving mechanical devices and fruit handling conditions to minimize damage to fruit. The experimental line consists of several transporting belts, one rollers transporter, one sizer, one elevator, one singularizer, and three trays to receive the calibrated fruit. The line has a length of 6.15 m and a width cf 1.9 m. Movement of the different components is regulated by electric motors with variable velocity electronically controlled. The height of the transfer points is variable and can be easily modified. The experimental line has been calibrated using two instrumented spheres IS 100 (8.8 cm Ø and6.2cm Ø). Average acceleration values obtained in all the transfers of the experimental line lay under 80 g's, although there is big variation for some of them being some values above 100 g's.