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.

Cardiac arrest in rodents: Maximal duration compatible with a recovery of neuronal activity

We report here that during a permanent cardiac arrest, rodent brain tissue is “physiologically” preserved in situ in a particular quiescent state. This state is characterized by the absence of electrical activity and by a critical period of 5–6 hr during which brain tissue can be reactivated upon restoration of a simple energy (glucose/oxygen) supply. In rat brain slices prepared 1–6 hr after cardiac arrest and maintained in vitro for several hours, cells with normal morphological features, intrinsic membrane properties, and spontaneous synaptic activity were recorded from various brain regions. In addition to functional membrane channels, these neurons expressed mRNA, as revealed by single-cell reverse transcription–PCR, and could synthesize proteins de novo. Slices prepared after longer delays did not recover. In a guinea pig isolated whole-brain preparation that was cannulated and perfused with oxygenated saline 1–2 hr after cardiac arrest, cell activity and functional long-range synaptic connections could be restored although the electroencephalogram remained isoelectric. Perfusion of the isolated brain with the γ-aminobutyric acid A receptor antagonist picrotoxin, however, could induce self-sustained temporal lobe epilepsy. Thus, in rodents, the duration of cardiac arrest compatible with a short-term recovery of neuronal activity is much longer than previously expected. The analysis of the parameters that regulate this duration may bring new insights into the prevention of postischemic damages.

Transport of uncharged organic solutes in Xenopus oocytes expressing red cell anion exchangers (AE1s)

When expressed in Xenopus oocytes, the trout red cell anion exchanger tAE1, but not the mouse exchanger mAE1, elicited a transport of electroneutral solutes (sorbitol, urea) in addition to the expected anion exchange activity. Chimeras constructed from mAE1 and tAE1 allowed us to identify the tAE1 domains involved in the induction of these transports. Expression of tAE1 (but not mAE1) is known to generate an anion conductance associated with a taurine transport. The present data provide evidence that (i) the capacity of tAE1 and tAE1 chimeras to generate urea and sorbitol permeability also was associated with an anion conductance; (ii) the same inhibitors affected both the permeability of solutes and anion conductance; and (iii) no measurable water transport was associated with the tAE1-dependent conductance. These results support the view that fish red blood cells, to achieve cell volume regulation in response to hypotonic swelling, activate a tAE1-associated anion channel that can mediate the passive transport of taurine and electroneutral solutes.

Quantification of the hydrophobic interaction by simulations of the aggregation of small hydrophobic solutes in water

The hydrophobic interaction, the tendency for nonpolar molecules to aggregate in solution, is a major driving force in biology. In a direct approach to the physical basis of the hydrophobic effect, nanosecond molecular dynamics simulations were performed on increasing numbers of hydrocarbon solute molecules in water-filled boxes of different sizes. The intermittent formation of solute clusters gives a free energy that is proportional to the loss in exposed molecular surface area with a constant of proportionality of 45 ± 6 cal/mol⋅Å2. The molecular surface area is the envelope of the solute cluster that is impenetrable by solvent and is somewhat smaller than the more traditional solvent-accessible surface area, which is the area transcribed by the radius of a solvent molecule rolled over the surface of the cluster. When we apply a factor relating molecular surface area to solvent-accessible surface area, we obtain 24 cal/mol⋅Å2. Ours is the first direct calculation, to our knowledge, of the hydrophobic interaction from molecular dynamics simulations; the excellent qualitative and quantitative agreement with experiment proves that simple van der Waals interactions and atomic point-charge electrostatics account for the most important driving force in biology.

Una política de defensa europea compatible con la Alianza Atlántica

Desde que entrara en vigor del Tratado de Lisboa, la UE ha avanzado de forma considerable, aunque claramente insuficiente, en el desarrollo y aplicación de la Política Común de Seguridad y Defensa (PCSD) durante estos últimos casi seis años. A pesar de que se ha agravado el empeoramiento de los riesgos y amenazas internacionales, los países miembros han disminuido sus presupuestos de defensa, debido a diferentes razones, especialmente a la crisis económica, en estos mismos años. Por ello, la única respuesta posible es mejorar la eficiencia a través de medidas de Pooling and Sharing, es decir, incrementando las respuestas comunes, especialmente a través del desarrollo y profundización de la Política Común de Seguridad y Defensa. Ésta debe empezar por la puesta en marcha de la Cooperación Estructurada Permanente, siendo ésta compatible con la Alianza Atlántica, tal y como establece el Tratado.

Clinical Characterization of Asymptomatic or Minimally Symptomatic Young Patients Showing Signs Compatible With Dry Eye: A Pilot Study

Purpose: To evaluate and characterize the clinical profile of young asymptomatic or minimally symptomatic patients without diagnosis of dry eye but showing signs compatible with dry eye syndrome (DES). Methods: Prospective study including a total of 50 consecutive subjects with ages ranging from 18 to 40 years that were identified as asymptomatic or minimally symptomatic by means of the Ocular Surface Disease Index (OSDI) (score of <22). In all patients, a complete battery of tests for the diagnosis of DES was performed including the evaluation of the tear film break-up time (TFBUT), the level of corneal and conjunctival staining, and the eyelid and Meibomian morphology. Results: The OSDI score was significantly higher in women than in men (median: 12.5 vs. 5.3, P=0.01). Low grades of ocular surface staining, dysfunction of Meibomian gland expression, and alteration of quality of Meibomian secretions were observed in 56%, 58%, and 84% of eyes, respectively. More eyes with some dysfunction of Meibomian gland expressibility had a TFBUT less than 5 sec (P=0.033). A statistically significant difference in the OSDI score was found between patients with and without systemic allergies (P=0.036) and between male and female (P=0.01). Likewise, the OSDI score was significantly higher in those women wearing contact lenses compared with those not wearing them (P=0.012). Conclusions: Asymptomatic or minimally symptomatic young subjects may present low grades of clinical signs compatible with DES, with a trend to more symptomatology in women and allergic patients. These outcomes should be confirmed in future studies with larger samples.

Development of a magnetic resonance compatible wrist device for the analysis of movement encoding in the brain

Tese de mestrado integrado em Engenharia Biomédica e Biofísica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016

About BLS data diskettes : for use with IBM-compatible microcomputers Lotus 1-2-3 or ASCII file format.

Mode of access: Internet.

Real cedula de S.M. y señores del Consejo, por la qual se manda observar en Madrid el Reglamento formado para el establecimiento de escuelas gratuitas en los barrios de él, en que se dé educacion á las niñas, extendiéndose á las capitales, ciudades y villas populosas de estos reinos en lo que sea compatible con la proporción y circunstancias de cada una, y lo demás que se expresa

Certifica la copia Pedro Escolano de Arrieta.

Time of travel and dispersion of solutes in a 36.4-mile reach of the North Platte River downstream from Casper, Wyoming /

Bibliography: p. 17.

A treatise on free agency : maintaining that the immutability of the divine nature is perfectly compatible with the moral freedom of the intellectual world; also, A dissertation on the Mosaic account of the creation; comprising its most remarkable internal evidences respecting the antiquity of the earth, &c. /

From the library of B. George Ulizio.

Essay on the prize-question, whether the use of distilled liquors, or traffic in them, is compatible, at the present time, with making a profession of Christianity? /

A reply to "questions as originally proposed through the agency of the secretary of the American Temperance Society."--P. [3].

A production-compatible microelectronic test pattern for evaluating photomask misalignment /

Includes bibliographical references.