Improved walnut mass micropropagation through the combined use of phloroglucinol and FeEDDHA

Despite the socioeconomic importance of walnut trees, poor rooting and recalcitrance to in vitro culture have hampered the establishment of high-yield clonal plantations. To improve walnut micropropagation, we introduced several modifications to current methods and evaluated the effects on microshoot performance and acclimatization. Nine selected genotypes (13-year-old trees) of the commercial hybrid Juglans major 209 x J. regia were cultured in vitro on DKW-C medium supplemented with 4.4 µM BA and 50 µM IBA. A protocol was developed that relies on the use of 0.40 mM phloroglucinol during shoot multiplication, 0.20 mM previous root induction, and 6.81 mg/L Fe3+ (FeEDDHA). Moreover, the addition of 83.2 µM glucose during the root expression phase significantly improved plant survival during acclimatization. Phloroglucinol promoted microshoot elongation but inhibited rooting, especially at concentrations above 0.40 mM. Replacing FeEDTA by FeEDDHA diminished chlorotic symptoms and improved rooting, with up to 90% microshoots developing viable roots. Likewise, glucose was more efficient than sucrose or fructose in promoting plant survival. At the proposed working concentrations, neither glucose nor FeEDDHA caused any noticeable deleterious effect on walnut micropropagation. Microscopic analysis revealed the physical continuity between adventitious roots and stem pericycles. Analysis of leaf genomic DNA with eight polymorphic microsatellite markers was supportive of the clonal fidelity and genetic stability of the micropropagated material. Successful clonal plantations (over 5,800 ramets) have been established by applying this protocol.

Conservación de plantas regeneradas in vitro y análisis de la variación somaclonal de Cinchona officinalis, Linneo

Cinchona officinalis (Rubiaceae), especie endémica del Valle de Loja, ubicado en la región sur del Ecuador, es un recurso forestal de importancia medicinal y ecológica, además la especie ha sido catalogada como planta nacional y es un ícono de la región sur por su aporte a la farmacopea mundial. Esta especie, entre los siglos XVII-XIX sufrió una gran presión en sus poblaciones debido a la extracción masiva de la corteza para la cura del paludismo. Aunque la actividad extractiva generó grandes ingresos a la Corona Española y a la región Sur del Ecuador, ésta fue poco o nada sustentable ecológicamente, provocando la desaparición de la especie en muchos sitios de la provincia, pues, en su momento, no se consideraron alternativas de recuperación de las poblaciones naturales. Actualmente la extracción y consumo de la corteza en la zona de origen es baja o nula, sin embargo esta zona enfrenta nuevas amenazas. La deforestación a causa de proyectos de desarrollo en infraestructuras, la práctica de actividades agrícolas y de ganadería, y los efectos del cambio climático han ocasionado, en estos últimos años, la fragmentación de los ecosistemas. La mayoría de los bosques del sur del Ecuador se han convertido en parches aislados (los bosques en los que se distribuye C. officinalis no son la excepción) siendo esta la principal causa para que la especie se encuentre en estado de amenaza. Los individuos de la especie tienen una alta capacidad de rebrote y producen semillas durante todo el año; sin embargo la capacidad germinativa y la tasa de sobrevivencia son bajas, además de estas dificultades la especie requiere de la asociación con otras especies vegetales para su desarrollo, lo cual ha limitado su distribución en pequeños parches aislados. Con esta problemática, la recuperación natural de las poblaciones es una necesidad evidente. Varios trabajos y esfuerzos previos se han realizado a nivel local: i. Identificación de la distribución actual y potencial; ii. Determinación de la fenología y fructificación iii. Programas de educación ambiental, iv. Análisis moleculares para determinar la diversidad genética. v. Ensayos de propagación vegetativa; y otras acciones de tipo cultural. No obstante, el estado de conservación y manejo de las poblaciones naturales no ha mejorado significativamente, siendo necesaria la aplicación de estrategias integradas de conservación in situ y ex situ, que permitan la recuperación y permanencia de las poblaciones naturales a largo plazo. El presente trabajo tiene como fin dar alternativas para el cultivo de tejidos in vitro de Cinchona officinalis centrados en la propagación masiva a partir de semillas, análisis de la fidelidad genética y alternativas de conservación de tejidos. Los objetivos específicos que se plantean son: i. Analizar el proceso de germinación y proliferación in vitro. ii. Evaluar la estabilidad genética en explantes cultivados in vitro, mediante marcadores ISSR. iii. Establecer protocolos de conservación in vitro mediante limitación del crecimiento y criopreservación de segmentos nodales y yemas. Los resultados más significativos de esta investigación fueron: i. El desarrollo de protocolos eficientes para mejorar los porcentajes de germinación y la proliferación de brotes en explantos cultivados in vitro. Para evaluar el efecto de los fenoles sobre la germinación, se determinó el contenido total de fenoles y el porcentaje de germinación en semillas de C. officinalis comparados con una especie de control, C. pubescens. Para inducir a proliferación, se utilizaron segmentos nodales de plántulas germinadas in vitro en medio Gamborg (1968) suplementado con diferentes combinaciones de reguladores de crecimiento (auxinas y citoquininas). Los resultados obtenidos sugieren que el contenido de compuestos fenólicos es alto en las semillas de C. officinalis en comparación con las semillas de C. pubescens. Estos fenoles pueden eliminarse con peróxido de hidrógeno o con lavados de agua para estimular la germinación. La formación de nuevos brotes y callos en la mayoría de las combinaciones de reguladores de crecimiento se observó en un período de 45 días. El mayor porcentaje de proliferación de brotes, formación de callos y presencia de brotes adventicios se obtuvo en medio Gamborg (B5) suplementado con 5.0 mg/l 6-bencil-aminopurina y 3.0 mg/l de ácido indol-3-butírico. ii. La evaluación de la fidelidad genética de los explantes obtenidos con distintas combinaciones de reguladores de crecimiento vegetal y diversos subcultivos. Se realizó el seguimiento a los explantes obtenidos de la fase anterior, determinando el índice de multiplicación y analizando la fidelidad genética de los tejidos obtenidos por las dos vías regenerativas: brotación directa y regeneración de brotes a partir de callos. Este análisis se realizó por amplificación mediante PCR de las secuencias ubicadas entre microsatélites-ISSR (Inter simple sequence repeat). El medio Gamborg (B5) con 3.0 mg/l de AIB y 5.0 mg/l de BAP usado como medio de inducción en la primera etapa de cultivo generó el mayor índice de proliferación (11.5). Un total de 13 marcadores ISSR fueron analizados, 6 de éstos fueron polimórficos. El mayor porcentaje de variación somaclonal fue inducido en presencia de 1.0 mg/l 2,4-D combinado con 0.2 mg/l Kin con un 1.8% en el segundo sub-cultivo de regeneración, la cual incrementó a 3.6% en el tercer sub-cultivo. Todas las combinaciones con presencia de 2,4-D produjeron la formación de callos y presentaron variación genética. Por su parte la fidelidad genética se mantuvo en los sistemas de propagación directa a través de la formación de brotes a partir de meristemos preformados. iii. El establecimiento de protocolos de conservación in vitro y crioconservación de segmentos nodales y yemas. Para la conservación limitando el crecimiento, se cultivaron segmentos nodales en los medios MS y B5 en tres concentraciones de sus componentes (25, 50 y 100%); y en medio B5 más agentes osmóticos como el manitol, sorbitol y sacarosa en diferentes concentraciones (2, 4 y 8%); los cultivos se mantuvieron por 12 meses sin subcultivos. Para el establecimiento de protocolos para la crioconservación (paralización del metabolismo) se usaron yemas axilares y apicales a las cuales se les aplicaron los métodos de encapsulación-deshidratación y vitrificación. La efectividad de los protocolos usados se determinó en función de la sobrevivencia, reducción del crecimiento y regeneración. Los resultados obtenidos en este apartado reflejan que un crecimiento limitado puede mantener tejidos durante 12 meses de almacenamiento, usando medio B5 más manitol entre 2 y 8%. En los protocolos de crioconservación, se obtuvo el mayor porcentaje de recuperación tras la congelación en NL en el tratamiento control seguido por el método crioprotector de encapsulación-deshidratación. Este trabajo brinda alternativas para la propagación de C. officinalis bajo condiciones in vitro, partiendo de material vegetal con alta diversidad genética. El material propagado puede ser fuente de germoplasma para la recuperación y reforzamiento de las poblaciones naturales así como una alternativa de producción para las comunidades locales debido a la demanda actual de corteza de la zona de origen para la elaboración de agua tónica. ABSTRACT Cinchona officinalis (Rubiaceae) is endemic to the Loja Valley, located in the southern area of Ecuador. The importance of this plant as medical and ecological resource is so great that it has been designated as the national flower and is an icon of the southern region for its contribution to the world pharmacopoeia. Between XVII-XIX centuries its population suffered great reduction due to massive harvesting of the bark to cure malaria. Although extraction activity generated large revenues to the Spanish Crown and the southern region of Ecuador, this was not ecologically sustainable, causing the disappearance of the species in many areas of the province, because during that time alternatives to prevent extinction and recover natural populations were not taken in account. Currently the extraction and consumption of bark in the area of origin is almost absent, but this species faces new threats. Deforestation due to infrastructure development, the practice of farming and ranching, and the effects of climate change had led to the fragmentation of ecosystems during the recent years. Most of the forests of southern Ecuador have become isolated patches, including those where C. officinalis is diffused. The lack of suitable habitat is today the main threat for the species. The species has a high capacity for regeneration and produces seeds throughout the year, but the germination rate is low and the growth is slow. In addition, the species requires the association with other plant species to develop. All these factors had limited its distribution to small isolated patches. The natural recovery of populations is essential to face this problem. Several studies and previous efforts had been made at local level: i. Identification of current and potential distribution; ii. Phenology determination. iii. Environmental education programs, iv. Molecular analisis to determine the genetic diversity. v. Testing of vegetative propagation; and other actions of cultural nature. Despite these efforts, the state of conservation and management of natural populations has not improved significantly. Implementation of integrated in situ and ex situ conservation strategies for the recovery and permanence of long-term natural populations is still needed. This work aims to provide alternatives for in vitro culture of tissue of Cinchona officinalis focused on mass propagation from seeds, genetic fidelity analysis and tissue conservation alternatives. The specific aims are: i. Analyze the process of germination and proliferation in vitro. ii. To evaluate the genetic stability of the explants cultured in vitro by ISSR markers. iii. Establish protocols for in vitro conservation by limiting growth and cryopreservation of nodal segments and buds. The most significant results of this research were: i. The development of efficient protocols to improve germination rates and proliferation of buds in explants cultured in vitro. To study the effect of phenols on germination, the total phenolic content and percentage germination was measured in C. officinalis and in a control species, C. pubescens, for comparison. The content of phenolic compounds in C. officinalis seeds is higher than in C. pubescens. These phenols can be removed with hydrogen peroxide or water washes to stimulate germination. To analyze the regeneration, we used nodal explants from seedlings germinated in vitro on Gamborg medium (1968) supplemented with different combinations of growth regulators (auxins and cytokinins) to induce proliferation. The formation of new shoots and calluses was observed within a period of 45 days in most combinations of growth regulators. The highest percentage of shoot proliferation, callus formation and adventitious buds were obtained in B5 medium supplemented with 5.0 mg/l 6-benzyl-aminopurine and 3.0 mg/l indole-3-butyric acid. ii. Evaluating genetic fidelity explants obtained with various combinations of plant growth regulators and different subcultures. The genetic fidelity was analyzed in tissues obtained by the two regenerative pathways: direct sprouting and shoot regeneration from callus. This analysis was performed by PCR amplification of the sequences located between microsatellite-ISSR (Inter Simple Sequence Repeat). Among a total of 13 ISSR markers analyzed, 6 were polymorphic. The highest percentage of somaclonal variation was induced in the presence of 1.0 mg/l 2,4-D combined with 0.2 mg/l Kin with 1.8% in the second round of regeneration, and increased to 3.6% in the third round. The presence of 2,4-D induced genetic variation in all the combinations of growth regulators. Meanwhile genetic fidelity remained systems propagation through direct shoot formation from meristems preformed. iii. Establishing conservation protocols in vitro and cryoconservation of nodal segments and buds. For medium-term conservation (limited growth) nodal segments were cultured in MS and B5 media at three concentrations (25, 50 and 100%); we tested B5 medium with different concentrations of osmotic agents such as mannitol, sorbitol and sucrose (2, 4 and 8%); cultures were maintained for 12 months with regular subculturing. To establish protocols for cryoconservation (cessation of metabolism) different methods of encapsulation-dehydration and vitrification were applied to axillary and apical buds. The effectiveness of the used protocols is determined based on the survival, growth and regeneration success. The results show that these tissues can be maintained in storage for 12 months, using B5 medium plus mannitol between 2 and 8%. The cryoconservation protocol with highest percentage of recovery was obtained by contral treatment, followed by freezing in NL with encapsulation-dehydration method. This work provides alternatives for the propagation in vitro of C. officinalis, starting from plant material with high genetic diversity. The obtained material represents a source of germplasm to support the recovery and strengthening of natural populations as well as a creation of alternative sources for local communities due to the current demand of bark for the preparation of tonic water.

Improving Optical Performance of Concentrator Cells by Means of a Deposited Nanopattern Layer

Multijunction solar cells (MJSC) use anti-reflective coatings (ARC) to minimize Fresnel reflection losses for a family of light incidence angles. These coatings adapt the refractive index of the cell to that of the surrounding medium. Patterns with sizes in the range of the light wavelength can be used to further reduce reflections through diffraction. Transparent nanopatterns with a gradual profile, called moth-eye nanostructures, can adapt the refractive index of the optical interfaces (often with n∼1.5) used to encapsulate concentrator solar cells to that of the air (n air∼1). Here we show the effect of a nanometric moth-eye ARC with a round motif deposited on commercial MJSC that achieves short-circuit current (I SC) gains greater than 2% at normal incidence and even higher in the case of tilted illumination. In this work, MJSC with different moth-eye ARC are characterized under quantum efficiency (QE) as well as under concentrated illumination I-V in order to assess their potential. Simulations based on coupled wave analysis (RCWA) are used to fit the experimental results with successful results.

Bem-estar subjetivo e bem-estar no trabalho em profissionais de educação física

Atualmente, bem-estar cada vez mais ganha espaço como parte importante e integrante da vida das pessoas. Pesquisas vêm buscando destacar fatores positivos que exerçam influencia em sua vida pessoal, e conseqüentemente em seu trabalho. O objetivo deste estudo foi verificar as relações entre bem-estar subjetivo (satisfação geral com a vida, afetos positivos e afetos negativos) e bem-estar no trabalho (satisfação no trabalho, envolvimento com o trabalho e comprometimento organizacional afetivo) em professores de educação física que atuavam em escolas e academias, descrevendo os níveis de bem-estar subjetivo e bem-estar no trabalho, análises de variância e correlação entre os construtos. A amostra foi composta por 124 professores de educação física, 34 atuavam em escolas particulares e 90 atuavam em academias, sendo 81 do sexo masculino e 43 do sexo feminino, solteiros e casados e faixa etária entre 21 e 59 anos, com escolaridade distribuída desde o ensino superior completo até o mestrado concluído. O instrumento de coleta foi um questionário auto-aplicavel composto por cinco escalas que mediram as variáveis do estudo e um questionário de dados complementares. Os resultados deste estudo revelaram que BES e BET guardam relações entre si. Os professores são considerados pessoas relativamente felizes com sua vida pessoal e revelaram uma relação mediana no que se refere a sua vida profissional. Os resultados das correlações relacionadas ao BES sinalizaram que o acúmulo de experiências negativas ao longo da vida poderia reduzir a vivencia de experiências positivas e de avaliações positivas sobre a vida em geral e vice-versa e ao BET, satisfação no trabalho, envolvimento com o trabalho e comprometimento organizacional afetivo são interdependentes. No que se refere às comparações, para BES os professores que atuavam em academias obtiveram médias significativamente maiores de afetos negativos do que os de escolas e por outro lado, a média de satisfação geral com a vida dos professores de escolas foi significativamente superior à dos que atuavam em academias demonstrando que estes vivenciaram mais experiências negativas do que os que trabalhavam em escolas e que os professores de escola eram mais satisfeitos com sua vida do que os de academias. Para BET, o quadro geral se mantém semelhante para professores de educação física que atuavam em escolas e academias. Futuros estudos deveriam aumentar o número de professores bem como, investigar outras áreas de atuação da educação física e compara-las com o presente estudo ampliando os estudos envolvendo bemestar e professores de educação física.(AU)

Bem-estar subjetivo e bem-estar no trabalho em profissionais de educação física

Atualmente, bem-estar cada vez mais ganha espaço como parte importante e integrante da vida das pessoas. Pesquisas vêm buscando destacar fatores positivos que exerçam influencia em sua vida pessoal, e conseqüentemente em seu trabalho. O objetivo deste estudo foi verificar as relações entre bem-estar subjetivo (satisfação geral com a vida, afetos positivos e afetos negativos) e bem-estar no trabalho (satisfação no trabalho, envolvimento com o trabalho e comprometimento organizacional afetivo) em professores de educação física que atuavam em escolas e academias, descrevendo os níveis de bem-estar subjetivo e bem-estar no trabalho, análises de variância e correlação entre os construtos. A amostra foi composta por 124 professores de educação física, 34 atuavam em escolas particulares e 90 atuavam em academias, sendo 81 do sexo masculino e 43 do sexo feminino, solteiros e casados e faixa etária entre 21 e 59 anos, com escolaridade distribuída desde o ensino superior completo até o mestrado concluído. O instrumento de coleta foi um questionário auto-aplicavel composto por cinco escalas que mediram as variáveis do estudo e um questionário de dados complementares. Os resultados deste estudo revelaram que BES e BET guardam relações entre si. Os professores são considerados pessoas relativamente felizes com sua vida pessoal e revelaram uma relação mediana no que se refere a sua vida profissional. Os resultados das correlações relacionadas ao BES sinalizaram que o acúmulo de experiências negativas ao longo da vida poderia reduzir a vivencia de experiências positivas e de avaliações positivas sobre a vida em geral e vice-versa e ao BET, satisfação no trabalho, envolvimento com o trabalho e comprometimento organizacional afetivo são interdependentes. No que se refere às comparações, para BES os professores que atuavam em academias obtiveram médias significativamente maiores de afetos negativos do que os de escolas e por outro lado, a média de satisfação geral com a vida dos professores de escolas foi significativamente superior à dos que atuavam em academias demonstrando que estes vivenciaram mais experiências negativas do que os que trabalhavam em escolas e que os professores de escola eram mais satisfeitos com sua vida do que os de academias. Para BET, o quadro geral se mantém semelhante para professores de educação física que atuavam em escolas e academias. Futuros estudos deveriam aumentar o número de professores bem como, investigar outras áreas de atuação da educação física e compara-las com o presente estudo ampliando os estudos envolvendo bemestar e professores de educação física.(AU)

Glutathione synthesis is essential for mouse development but not for cell growth in culture

Glutathione (GSH) is a major source of reducing equivalents in mammalian cells. To examine the role of GSH synthesis in development and cell growth, we generated mice deficient in GSH by a targeted disruption of the heavy subunit of γ-glutamylcysteine synthetase (γGCS-HStm1), an essential enzyme in GSH synthesis. Embryos homozygous for γGCS-HStm1 fail to gastrulate, do not form mesoderm, develop distal apoptosis, and die before day 8.5. Lethality results from apoptotic cell death rather than reduced cell proliferation. We also isolated cell lines from homozygous mutant blastocysts in medium containing GSH. These cells also grow indefinitely in GSH-free medium supplemented with N-acetylcysteine and have undetectable levels of GSH; further, they show no changes in mitochondrial morphology as judged by electron microscopy. These data demonstrate that GSH is required for mammalian development but dispensable in cell culture and that the functions of GSH, not GSH itself, are essential for cell growth.

A repressible female-specific lethal genetic system for making transgenic insect strains suitable for a sterile-release program

We have developed a tetracycline-repressible female-specific lethal genetic system in the vinegar fly Drosophila melanogaster. One component of the system is the tetracycline-controlled transactivator gene under the control of the fat body and female-specific transcription enhancer from the yolk protein 1 gene. The other component consists of the proapoptotic gene hid under the control of a tetracycline-responsive element. Males and females of a strain carrying both components are viable on medium supplemented with tetracycline, but only males survive on normal medium. A strain with such properties would be ideal for a sterile-insect release program, which is most effective when only males are released in the field.

Requirement of phosphatidylglycerol for photosynthetic function in thylakoid membranes

To investigate the role of phosphatidylglycerol (PG) in photosynthesis, we constructed a mutant defective in the CDP-diacylglycerol synthase gene from a cyanobacterium, Synechocystis sp. PCC6803. The mutant, designated as SNC1, required PG supplementation for growth. Growth was repressed in PG-free medium concomitantly with the decrease in cellular content of PG. These results indicate that PG is essential, and that SNC1 is defective in PG synthesis. Decrease in PG content was accompanied by a reduction in the cellular content of chlorophyll, but with little effect on the contents of phycobilisome pigments, which showed that levels of chlorophyll–protein complexes decreased without alteration of those of phycobilisomes. Regardless of the decrease in the PG content, CO2-dependent photosynthesis by SNC1 was similar to that by the wild type on a chlorophyll basis, but consequently became lower on a cell basis. Simultaneously, the ratio of oxygen evolution of photosystem II (PSII) measured with p-benzoquinone to that of CO2-dependent photosynthesis, which ranged between 1.3 and 1.7 in the wild type. However, it was decreased in SNC1 from 1.3 to 0.4 during the early growth phase where chlorophyll content and CO2-dependent photosynthesis were little affected, and then finally to 0.1, suggesting that PSII first lost its ability to reduce p-benzoquinone and then decreased in its level and actual activity. These results indicate that PG contributes to the accumulation of chlorophyll–protein complexes in thylakoid membranes, and also to normal functioning of PSII.

petit1, a Conditional Growth Mutant of Arabidopsis Defective in Sucrose-Dependent Elongation Growth1

The hypocotyl of Arabidopsis is well suited for the analysis of cell elongation because it elongates without cell division. We have isolated a new class of recessive mutants, petit1 (pet1), which are defective in aspects of hypocotyl elongation. The short-hypocotyl phenotype of pet1 is caused by shortened cells. The cells of the elongation zone of the hypocotyl are often deformed. pet1 also shows defects in elongation of the roots, flower stalk, leaves, petals, pedicels, and siliques, and these defects cannot be repaired by the application of auxin, gibberellin, brassinolide, or an inhibitor of ethylene biosynthesis. The short-hypocotyl phenotype of pet1 is pronounced only in growth medium supplemented with sucrose, which has promotive effects on hypocotyl elongation. In pet1 this effect is much reduced, causing the sucrose-dependent short-hypocotyl phenotype of pet1. pet1 accumulates more soluble sugars than the wild type and also shows more intensive iodo-starch staining in the cotyledon and hypocotyl. These results indicate that PETIT1 is involved in a sugar-dependent elongation process that may include correct assembly of expanding cell wall architecture.

The Boron Requirement and Cell Wall Properties of Growing and Stationary Suspension-Cultured Chenopodium album L. Cells1

Suspension-cultured Chenopodium album L. cells are capable of continuous, long-term growth on a boron-deficient medium. Compared with cultures grown with boron, these cultures contained more enlarged and detached cells, had increased turbidity due to the rupture of a small number of cells, and contained cells with an increased cell wall pore size. These characteristics were reversed by the addition of boric acid (≥7 μm) to the boron-deficient cells. C. album cells grown in the presence of 100 μm boric acid entered the stationary phase when they were not subcultured, and remained viable for at least 3 weeks. The transition from the growth phase to the stationary phase was accompanied by a decrease in the wall pore size. Cells grown without boric acid or with 7 μm boric acid were not able to reduce their wall pore size at the transition to the stationary phase. These cells could not be kept viable in the stationary phase, because they continued to expand and died as a result of wall rupture. The addition of 100 μm boric acid prevented wall rupture and the wall pore size was reduced to normal values. We conclude that boron is required to maintain the normal pore structure of the wall matrix and to mechanically stabilize the wall at growth termination.

Mutations affecting conserved cysteine residues within the extracellular domain of Neu promote receptor dimerization and activation.

Overexpression of the Neu/ErbB-2 receptor tyrosine kinase has been implicated in the genesis of human breast cancer. Indeed, expression of either activated or wild-type neu in the mammary epithelium of transgenic mice results in the induction of mammary tumors. Previously, we have shown that many of the mammary tumors arising in transgenic mice expressing wild-type neu occur through somatic activating mutations within the neu transgene itself. Here we demonstrate that these mutations promote dimerization of the Neu receptor through the formation of disulfide bonds, resulting in its constitutive activation. To explore the role of conserved cysteine residues within the region deleted in these altered Neu proteins, we examined the transforming potential of a series of Neu receptors in which the individual cysteine residues were mutated. These analyses indicated that mutation of certain cysteine residues resulted in the oncogenic activation of Neu. The increased transforming activity displayed by the altered receptors correlated with constitutive dimerization that occurred in a disulfide bond-dependent manner. We further demonstrate that addition of 2-mercaptoethanol to the culture medium interfered with the specific transforming activity of the mutant Neu receptors. These observations suggest that oncogenic activation of Neu results from constitutive disulfide bond-dependent dimerization.

Ascorbic acid is essential for the release of insulin from scorbutic guinea pig pancreatic islets.

Pancreatic islets from young normal and scorbutic male guinea pigs were examined for their ability to release insulin when stimulated with elevated D-glucose. Islets from normal guinea pigs released insulin in a D-glucose-dependent manner showing a rapid initial secretion phase and three secondary secretion waves during a 120-min period. Islets from scorbutic guinea pigs failed to release insulin during the immediate period, and only delayed and decreased responses were observed over the 40-60 min after D-glucose elevation. Insulin release from scorbutic islets was greatly elevated if 5 mM L-ascorbic acid 2-phosphate was supplemented in the perifusion medium during the last 60 min of perifusion. When 5 mM L-ascorbic acid 2-phosphate was added to the perifusion medium concurrently with elevation of medium D-glucose, islets from scorbutic guinea pigs released insulin as rapidly as control guinea pig islets and to a somewhat greater extent. L-Ascorbic acid 2-phosphate without elevated D-glucose had no effect on insulin release by islets from normal or scorbutic guinea pigs. The pancreas from scorbutic guinea pigs contained 2.4 times more insulin than that from control guinea pigs, suggesting that the decreased insulin release from the scorbutic islets was not due to decreased insulin synthesis but due to abnormal insulin secretion.

Rapid acquisition of dendritic spines by visual thalamic neurons after blockade of N-methyl-D-aspartate receptors.

N-Methyl-D-aspartate (NMDA) receptors play an important role in the development of retinal axon arbors in the mammalian lateral geniculate nucleus (LGN). We investigated whether blockade of NMDA receptors in vivo or in vitro affects the dendritic development of LGN neurons during the period that retinogeniculate axons segregate into on-center and off-center sublaminae. Osmotic minipumps containing either the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV) or saline were implanted in ferret kits at postnatal day 14. After 1 week, LGN neurons were intracellularly injected with Lucifer yellow. Infusion of D-APV in vivo led to an increase in the number of branch points and in the density of dendritic spines compared with age-matched normal or saline-treated animals. To examine the time course of spine formation, crystals of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate were placed in the LGN in brain slices from 14- to 18-day-old ferrets. Labeled LGN cell dendrites were imaged on-line in living slices by confocal microscopy, with slices maintained either in normal perfusion medium or with the addition of D-APV or NMDA to the medium. Addition of D-APV in vitro at doses specific for blocking NMDA receptors led to a > 6-fold net increase in spine density compared with control or NMDA-treated slices. Spines appeared within a few hours of NMDA receptor blockade, indicating a rapid local response by LGN cells in the absence of NMDA receptor activation. Thus, activity-dependent structural changes in postsynaptic cells act together with changes in presynaptic arbors to shape projection patterns and specific retinogeniculate connections.

Fisiologia e formação de partículas lipídicas durante o crescimento da levedura Yarrowia lipolytica IMUFRJ 50682.

A levedura Yarrowia lipolytica tem sido muito investigada, especialmente por ser um microrganismo oleaginoso, ou seja, capaz de acumular grandes quantidades de lipídios, o que ocorre majoritariamente em organelas denominadas partículas lipídicas. Estes lipídios apresentam várias potenciais aplicações biotecnológicas, como por exemplo na produção de óleo microbiano (single cell oil) e na produção de biodiesel. Durante este projeto de mestrado, objetivou-se estudar a fisiologia de duas linhagens da levedura Y. lipolytica, sendo uma tradicionalmente estudada pela comunidade científica internacional (linhagem w29) e outra isolada da Baía da Guanabara, no Rio de Janeiro (linhagem IMUFRJ 50682). Foram realizados cultivos em frascos agitados tipo Erlenmeyer com defletores tampados com algodão (volume total 500 mL, volume de meio 100 mL, 28 oC e 200 rotações por minuto), durante os quais foi possível: 1) escolher um meio de cultivo de composição totalmente definida, com tiamina como único fator de crescimento, adequado a estudos de fisiologia quantitativa com esta levedura; 2) verificar que Y. lipolytica não é capaz de crescer com sacarose ou xilose como única fonte de carbono; 3) verificar que Y. lipolytica cresce com velocidade específica de crescimento máxima (Máx) de 0,49 h-1 num meio complexo contendo glicose, extrato de levedura e peptona (meio YPD), 0,31 h-1 em meio definido com glicose como única fonte de carbono e 0,35 h-1 no mesmo meio, mas com glicerol como única fonte de carbono, sem excreção de metabólitos para o meio de cultivo; 4) verificar que ocorreu limitação por oxigênio nos cultivos em frasco agitado, sendo este o motivo pelo qual as células deixaram de crescer exponencialmente; 5) verificar que o uso de ureia, em vez de sulfato de amônio, como fonte de nitrogênio, contribui para uma variação menor do pH durante os cultivos, sem prejuízo ao crescimento da levedura; 6) observar que, ao se restringir a oferta de nitrogênio à levedura (aumento da relação C/N inicial no meio de 12,6 para 126), as células têm sua morfologia alterada e apresentam maior quantidade de partículas lipídicas; 7) determinar uma composição elementar para a biomassa de Y. lipolytica (CH1,98O0,58N0,13), em que os átomos de carbono encontram-se em média mais reduzidos do que na biomassa de leveduras como Saccharomyces cerevisiae e Dekkera bruxellensis. Foram também realizados cultivos em biorreator em batelada (1 L de volume útil, 28 oC, aerobiose plena e pH controlado em 5,0), durante os quais foi possível: a) estabelecer um protocolo de cultivo para Y. lipolytica em biorreator (que envolvem agitação mecânica, aeração e uso de anti-espumante, entre outras diferenças em relação aos cultivos em frasco); b) confirmar os valores dos principais parâmetros fisiológicos apresentados por esta levedura, anteriormente obtidos a partir de cultivos em frasco; c) confirmar que o fator de conversão de substrato a células (Yx/s) é maior para cultivos realizados com glicerol como fonte única de carbono (0,53 g/g para a linhagem IMUFRJ 50682), do que com glicose (0,48 g/g para a mesma linhagem). Finalmente, cultivos realizados em quimiostato com glicerol como fonte de carbono e energia, limitados por amônio (fonte de nitrogênio, relação C/N 126), às vazões específicas de 0,25 h-1 e 0,15 h-1, permitiram observar que o número de partículas lipídicas por célula de Y. lipolytica permaneceu em torno de 2 em ambas as situações e houve uma diminuição no teor de nitrogênio nas células quando a velocidade específica de crescimento diminuiu de 0,25 para 0,15 h-1.

Propostes d’aula per a un enfocament motivador de la literatura

Els textos literaris, a banda de ser un terreny molt òptim per a desenvolupar la sensibilitat i ensenyar a sentir, són un recurs útil també per a millorar la competència lingüística de l’alumnat de manera motivadora i divertida. Aquests textos, junt a altres, formen part del treball diari a l’aula del Portafolis de Valencià on l’alumnat reflexiona sobre com millorar el seu nivell en aquesta llengua. La nostra proposta presenta seqüències didàctiques d’activitats on es desenvolupen tant les habilitats productives com receptives a traves del treball de textos literaris com l’endevinalla, les nadaletes, els textos narratius amb imatge (curt, fotonovel·la, còmic), el rap, els contes, la poesia...