Proteomic and Transcriptomic Approaches to Abiotic Stress Tolerance in Rice (Oryza sativaL.)

Dissertação para a obtenção de grau de doutor em Biologia pelo Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa

Potential use of Piper nigrum ethanol extract against pyrethroid-resistant Aedes aegypti larvae

Fractionation of Piper nigrum ethanol extract, biomonitored by assays on pyrethroid-resistant Aedes aegypti larvae yielded isolation of the larvicidal amides piperolein-A and piperine. Comparing LC50 values, the ethanol extract (0. 98 ppm) was the most toxic, followed by piperolein-A (1. 46ppm) and piperine (1. 53ppm).

Evaluation of drought tolerance in several genotypes of spelt (Triticum aestivum Var. Spelta)

Domestication of plants and plant breeding have dramatically eroded the allelic variations of crop species which led to an increasing susceptibility of crop plants to environmental stresses, diseases and pests. Drought is a major environmental stress factor that affects the growth and development of plants so the selection of tolerant genotypes becomes increasingly important with respect to the predicted effects of global warming. In this study, several genotypes of Spelt (Triticum aestivum var. spelta) were tested under low water supply in soil with the aim of to find Spelt genotypes more resistant than wheat to these conditions, and select them so that in future may be used to improve wheat crops. Morphological analyses were performed and mineral and enzymatic analyses and also dry matter production were calculated. Our results suggests that the genotypes Sp53, Sp96, Sp912, Sp757 and Sp804 are a potential ones to use in breeding programs to improve wheat production. Under drought, these genotypes had growth efficiency of 38%, 45%, 64%, 37%, and 31% respectively and also showed higher biomass than modern wheat and were also mineralogical richer. The genotypes Sp96 and Sp912 showed highest activity of all antioxidants enzymes tested. This work proves that Spelt is a good wheat to continue to study in order to improve wheat crops in dry areas and consequently increase the quality of life and health of the populations living in those areas.

Study of salt stress tolerance in spelt

World population is increasing at an alarming rate while food productivity is decreasing due to the effect of various abiotic stresses. Soil salinity is one of the most important abiotic stress and a limiting factor for worldwide plant production. In addition to its important effects on yield, salt stress affects numerous cellular activities, including cell wall composition, photosynthesis, protein synthesis, ions and organic solutes. Up to 20% of the irrigated arable land in arid and semiarid regions is already salt affected and is still expanding. Improving salt tolerant varieties is of major importance, and efforts should be focused on finding adaptive mechanisms which are involved in salinity tolerance. In this study, several spelt wheat (Triticum aestivum var. Spelta) genotypes and one cultivar of modern bread wheat were used to screen them for salt tolerance. Spelt is an old-European cereal crop currently attracting renewed interest as a food grain because it is said to be harder than wheat and requires less fertilizer. Spelt wheat is also becoming very attractive genetic source by plant breeders due to its wide adaptation ability to various stressful conditions such as soil salinity. In this study morphological parameters (e.g., leaf appearance; shoot elongation), dry matter production, mineral nutrients (especially Na and K), and activity of antioxidative enzymes were measured to select superior genotypes of spelt for salt tolerance. The results showed that Spelt genotype Sp41 is a salt sensitive genotype and genotypes Sp69, Sp96 and Sp912 are good candidates for salt tolerant genotypes.

Reasons and benefits associated with ISO 9001 - Certification for sugar and ethanol companies

With increasing business competitiveness, companies have sought to adapt their processes and / or products to worldwide established quality standards in order to achieve a greater share of consumers having as favorable aspect the quality assurance of the products and/or services provided. It was observed that companies of different sizes have different challenges regarding the certification however, the degree of difficulty is the same for all of them. The objective of this paper is to verify the reasons for the implementation of ISO 9001, the obstacles encountered during the implementation, the benefits arising from the use of the quality management system and the degree of difficulty to implement this standard. This work was developed based on a survey involving companies certified with ISO 9001:2008 from the productive sector of sugar, ethanol and derivatives of sugarcane, located in all Brazilian states. It was observed that companies of different sizes have different challenges regarding the certification however the degree of difficulty is the same for all of them. Thus, we believe that expected results represent a very important contribution to examining the reasons, benefits and difficulties of the ISO 9001 to both, the companies and certification bodies, and to researchers.

Candida bracarensis: Evaluation of virulence factors and its tolerance to Amphotericin B and Fluconazole

Candida bracarensis is an uncommon Candida species found during an epidemiological study of candidiasis performed in Braga, Portugal. Initially, it was identified as C. glabrata, but recently detailed analyses pointed out their differences. So, little information is still available about C. bracarensis virulence factors and antifungal susceptibilities. Therefore, the main goal of this work is to evaluate the ability of C. bracarensis to form biofilms, to produce hydrolytic enzymes (proteases, phospholipases and hemolysins), as well as its susceptibility to amphotericin B and fluconazole. It was shown, for the first time, that all C. bracarensis strains were able to form biofilms and display proteinase and hemolytic activities. Moreover, although planktonic cells presented antifungal susceptibility, amphotericin B and fluconazole were unable to inhibit biofilm formation and eradicate pre-formed biofilms. Due to the propensity of C. bracarensis to display antifungal resistance and virulence attributes, the control of these emerging pathogens is recommended.

Tolerance to temperature, pH, ammonia and nitrite in cardinal tetra, Paracheirodon axelrodi, an amazonian ornamental fish

Poor water quality condition has been pointed out as one of the major causes for the high mortality of ornamental fishes exported from the state of Amazonas, Brazil. The purpose of the current study was to define water quality standards for cardinal tetra (Paracheirodon axelrodi), by establishing the lower and higher for lethal temperature (LT50), lethal concentration (LC50) for total ammonia and nitrite and LC50 for acid and alkaline pH. According to the findings, cardinal tetra is rather tolerant to high temperature (33.3 ºC), to a wide pH range (acid pH=2.9 and alkaline pH=8.8) and to high total ammonia concentration (23.7 mg/L). However, temperatures below 19.6 ºC and nitrite concentrations above 1.1 mg/L NO2- may compromise fish survival especially during long shipment abroad.

Optimization of low-cost nutritional supplementation for lignocellulosic ethanol fermentation

[Excerpt] Bioethanol from lignocellulosic materials (LCM), also called second generation bioethanol, is considered a promising alternative to first generation bioethanol. An efficient production process of lignocellulosic bioethanol involves an effective pretreatment of LCM to improve the accessibility of cellulose and thus enhance the enzymatic saccharification. One interesting approach is to use the whole slurry from treatment, since allows economical and industrial benefits: washing steps are avoided, water consumption is lower and the sugars from liquid phase can be used, increasing ethanol concentration [1]. However, during the pretreatment step some compounds (such as furans, phenolic compounds and weak acids) are produced. These compounds have an inhibitory effect on the microorganisms used for hydrolysate fermentation [2]. To overcome this, the use of a robust industrial strain together with agro-industrial by-products as nutritional supplementation was proposed to increase the ethanol productivities and yields. (...)

Mapeo de QTL para Mal de Río Cuarto en maíz. QTL mapping for tolerance to Mal de Río Cuarto disease in maize.

El Mal de Río Cuarto (MRC) es una enfermedad del maíz (Zea mays L.), endémica de ciertas zonas de la Argentina y constituye la patología más importante de este cultivo por la severidad de los daños y por la creciente difusión del área geográfica afectada. El empleo de la resistencia genética bajo un manejo integrado de la enfermedad, constituye la estrategia más económica y ambientalmente sustentable para lograr el incremento y la estabilidad en la producción de los cultivos de maíz, reduciendo el uso nocivo de agroquímicos. La reacción a la enfermedad MRC se encuentra influida por una fuerte interacción genotipo-ambiente que dificulta el mejoramiento genético. En los ensayos multiambientales la inconsistencia de la respuesta y la inestabilidad de los genotipos ensayados hace dificultoso llevar a cabo una buena selección basada en los síntomas de la enfermedad. Para contribuir a aumentar la eficacia de los métodos de mejoramiento es posible implementar programas en los que se incluyan herramientas biotecnológicas como los marcadores moleculares, que permiten reducir el efecto ambiental y contribuyen a soslayar, al menos en parte, los inconvenientes planteados por los efectos de la interacción genotipo-ambiente facilitando la identificación de los genotipos buscados. La selección fenotípica realizada convencionalmente, puede ser complementada con la selección asistida por marcadores (marker-assisted selection MAS) consistente en utilizar la información genética que brindan marcadores moleculares de ADN, tales como los SSR, asociados a loci o segmentos cromosómicos (quantitative trait loci QTL) que confieran resistencia a MRC. Si bien los resultados preliminares obtenidos por nuestro grupo de trabajo señalan la presencia de posibles QTLs e informan que la reacción frente a la enfermedad tiene una moderada heredabilidad y una substancial variación debida a la interacción genotipo-ambiente, es necesario confirmar los resultados relativos a los parámetros poblacionales y obtener una mejor delimitación de las regiones genómicas identificadas. Con la finalidad de aumentar la eficiencia de los programas de mejoramiento en el desarrollo de genotipos tolerantes mediante la selección asistida por marcadores utilizando una población de mapeo F2 con un fondo genético diferente y líneas endocriadas recombinantes evaluadas en ensayos multiambientales, se proponen los siguientes objetivos (i) estudiar la forma de herencia de la reacción frente a MRC, (ii) identificar nuevos QTLs asociados a este carácter y (iii) verificar la consistencia de los QTLs identificados previamente.

Aprendizaje durante la ontogenia temprana: Estudio de la participación del sistema opioide en la modulación de aprendizajes mediados por el etanol. LEARNING AND EARLY ONTOGENY: ANALYSIS OF THE INVOLVEMENT OF OPIOID SYSTEM IN FETAL AND INFANTILE ETHANOL-MEDIATED EXPERIENCIES.

Este plan de trabajos es continuidad de una línea de investigación centrada en evaluar los mecanismos responsables de la adquisición, expresión y persistencia de experiencias con el etanol. A partir de ello, indagar acerca de los efectos de esta experiencia sobre comportamientos de búsqueda y autoadministración de etanol en neonatos e infantes de rata. Se pretende analizar la participación del sistema opiáceo en los mecanismos implicados en una memoria fetal y/o infantil, generada como consecuencia de la exposición etílica. En una primera etapa, nos proponemos establecer de qué manera experiencias prenatales con la droga modulan el patrón de auto-administración de alcohol y otros reforzadores, como sacarosa. En este primer bloque de experimentos realizaremos manipulaciones fetales para determinar con mayor grado de especificidad la posible acción del sistema opiáceo en los mecanismos de adquisición de una memoria etílica prenatal. Se realizarán administraciones de etanol y el antagonista opiáceo, directamente a nivel fetal, y se evaluará esta experiencia en un paradigma de condicionamiento neonatal positivo, mediado por la droga. De acuerdo a la evidencia previa, esperamos que la exposición prenatal con la droga facilite la expresión de conductas de consumo y búsqueda del etanol o hacia las claves que señalizan al psicotrópico, tanto durante la infancia como en el neonato. A su vez, cuando la droga es presentada bajo los efectos de un antagonista opiáceo esperamos que estas conductas muestren un perfil similar a las desplegadas por sujetos controles. El segundo bloque de experimentos ha sido ideado con el objeto de indagar acerca de la posible participación del sistema opiáceo en la modulación de los aspectos reforzantes de la droga, a través de un esquema de auto-administración etílica infantil. Se utilizará un paradigma de condicionamiento instrumental adaptado para ratas infantes que consta de dos instancias, una de adquisición de la conducta instrumental (DPs 14-17) en la cual los animales reciben un pulso de refuerzo, como consecuencia de la ejecución de la conducta operante. En una segunda fase se analiza el patrón de búsqueda del reforzador ya que se registra la respuesta instrumental, sin que ocurra el refuerzo por la misma. Para analizar la participación del sistema opiáceo, durante la fase de adquisición de la conducta operante (DPs 16 y 17) los animales serán re-expuestos a mínimas cantidades del reforzador, bajo los efectos de un antagonista opiáceo, momentos previos al ensayo instrumental correspondiente para cada uno de estos días (Exp. 3). Esperamos que el bloqueo del sistema opiáceo, durante esta re-exposición al etanol, sea suficiente para disminuir el patrón de respuesta instrumental hacia el refuerzo etílico. Un último experimento incorporará un tercer evento de re-exposición al etanol -bajo los efectos del antagonista- previo al ensayo de extinción de la conducta instrumental (DP 18). Este nuevo evento tiene por objeto analizar la participación de este sistema neurobiológico en los mecanismos de búsqueda de etanol. Si el sistema opiáceo participa en la modulación de patrones tanto de búsqueda como consumatorios del reforzamiento por etanol, se espera que la re-exposición a la droga bajo los efectos del antagonista, inhiba estas respuestas tanto durante la sesión de adquisición, como de extinción de la conducta operante. Este proyecto intenta profundizar en el conocimiento de los mecanismos que regulan reconocimiento, aceptación, búsqueda y consumo de etanol, como consecuencia de experiencias tempranas con la droga. A su vez, es importante identificar y estudiar los sistemas neurobiológicos involucrados en estos mecanismos. Es por ello que se intenta determinar el rol que ejerce el sistema opiáceo en la adquisición de estas experiencias etílicas a nivel fetal e infantil, que se conoce promueven la búsqueda y el consumo de la droga. Our work is directed to analyze the involvement of the opioid system in the generation of pre- and early postnatal ethanol-related memories. As a first step, maternal manipulations with ethanol will be done. Infants will be evaluated in a paradigm of infantile self-administration of different reinforcers (ethanol, sucrose or water), employing a model of operant conditioning adapted to infant rats. A second experiment will be conducted in order to analyze if a central administration of ethanol, directly to the fetus, modifies subsequent patterns of neonatal conditioned responses to an artificial nipple, mediated by ethanol reinforcing effects. Fetal presentation of ethanol will be accompanied with the injection of an opioid antagonist in order to analyze the involvement of this system in acquisition processes of a fetal ethanol-mediated memory. A second set of studies will be conducted to analyze appetitive and consummatory behaviors in an infant model of ethanol self-administration. Involvement of opioid system in the acquisition or expression of this experience will be also inquired. Infant rats (PDs14-17) have to display a target behavior (nose-poke) to gain access to 5% sucrose or 3.75% ethanol. On PD18 an extinction session will be included. At PDs16-17, 6-hr before training, pups will be re-exposed to ethanol under opioid antagonism effects (naloxone). In a follow up experiment, a re-exposure trial will be included at PD18. Prior extinction, pups will receive naloxone and will be re-exposed to ethanol. We aim to observe if opioid system is modulating etha¬nol reinforcing effects, in terms of both appetitive and consummatory behaviors.

The Effect of Sleep Deprivation on Cardiac Function and Tolerance to Ischemia-Reperfusion Injury in Male Rats

Abstract Background: Sleep deprivation (SD) is strongly associated with elevated risk for cardiovascular disease. Objective: To determine the effect of SD on basal hemodynamic functions and tolerance to myocardial ischemia-reperfusion (IR) injury in male rats. Method: SD was induced by using the flowerpot method for 4 days. Isolated hearts were perfused with Langendorff setup, and the following parameters were measured at baseline and after IR: left ventricular developed pressure (LVDP); heart rate (HR); and the maximum rate of increase and decrease of left ventricular pressure (±dp/dt). Heart NOx level, infarct size and coronary flow CK-MB and LDH were measured after IR. Systolic blood pressure (SBP) was measured at start and end of study. Results: In the SD group, the baseline levels of LVDP (19%), +dp/dt (18%), and -dp/dt (21%) were significantly (p < 0.05) lower, and HR (32%) was significantly higher compared to the controls. After ischemia, hearts from SD group displayed a significant increase in HR together with a low hemodynamic function recovery compared to the controls. In the SD group, NOx level in heart, coronary flow CK-MB and LDH and infarct size significantly increased after IR; also SD rats had higher SBP after 4 days. Conclusion: Hearts from SD rats had lower basal cardiac function and less tolerance to IR injury, which may be linked to an increase in NO production following IR.

Tolerance to salinities shocks of the invasive mussel Limnoperma fortunei under experimental conditions

The golden mussel, Limnoperna fortunei (Dunker, 1857), has been found in the estuarine regions of South America, including the Patos Lagoon (Brazil), a huge choked lagoon with an estuarine region that is highly unstable chemically. Limnoperna fortunei space-temporal variability in the lagoon's estuarine region demonstrated the need to evaluate this species' ability to survive under salinity shocks. A set of experiments was conducted under controlled laboratory conditions. Specimens were tested under salinities of 2, 4, 6, 8 and 12 ppt, and were exposed for periods of 24, 48, 72, 96 and 240 hours. The mussel can survive (90%) up to a salinity shock of 2 ppt for periods of at least 10 days. Considering the influence of climatic and stochastic events and the chemical instability of the Patos Lagoon estuarine region, it's unlikely that populations could survive for longer periods (more than a year) in this area.

Cross-suppression of specific immune responses after oral tolerance

Adult normal inbred mice rendered tolerant to OVA by previous oral exposure do not respond to intraperitonela immunization with DNP-OVA in adjuvant. These tolerant mice also form less DNP-specific antibodies to DNP-KLH when immunized with mixtures of DNP-KLH and DNP-OVA, or less HGG-specific antibodies when immunized with cross-linked conjugates of OVA and HGG. These same procedures increased DNP-specific or HGG-specific responses in non-tolerant control mice. The cross-supperssion was ineffective, however, to inhibit already ongoing antibody responses.

T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.