Dynamic analysis of payloads and structures with intermediate modal density

La necesidad de desarrollar técnicas para predecir la respuesta vibroacústica de estructuras espaciales lia ido ganando importancia en los últimos años. Las técnicas numéricas existentes en la actualidad son capaces de predecir de forma fiable el comportamiento vibroacústico de sistemas con altas o bajas densidades modales. Sin embargo, ambos rangos no siempre solapan lo que hace que sea necesario el desarrollo de métodos específicos para este rango, conocido como densidad modal media. Es en este rango, conocido también como media frecuencia, donde se centra la presente Tesis doctoral, debido a la carencia de métodos específicos para el cálculo de la respuesta vibroacústica. Para las estructuras estudiadas en este trabajo, los mencionados rangos de baja y alta densidad modal se corresponden, en general, con los rangos de baja y alta frecuencia, respectivamente. Los métodos numéricos que permiten obtener la respuesta vibroacústica para estos rangos de frecuencia están bien especificados. Para el rango de baja frecuencia se emplean técnicas deterministas, como el método de los Elementos Finitos, mientras que, para el rango de alta frecuencia las técnicas estadísticas son más utilizadas, como el Análisis Estadístico de la Energía. En el rango de medias frecuencias ninguno de estos métodos numéricos puede ser usado con suficiente precisión y, como consecuencia -a falta de propuestas más específicas- se han desarrollado métodos híbridos que combinan el uso de métodos de baja y alta frecuencia, intentando que cada uno supla las deficiencias del otro en este rango medio. Este trabajo propone dos soluciones diferentes para resolver el problema de la media frecuencia. El primero de ellos, denominado SHFL (del inglés Subsystem based High Frequency Limit procedure), propone un procedimiento multihíbrido en el cuál cada subestructura del sistema completo se modela empleando una técnica numérica diferente, dependiendo del rango de frecuencias de estudio. Con este propósito se introduce el concepto de límite de alta frecuencia de una subestructura, que marca el límite a partir del cual dicha subestructura tiene una densidad modal lo suficientemente alta como para ser modelada utilizando Análisis Estadístico de la Energía. Si la frecuencia de análisis es menor que el límite de alta frecuencia de la subestructura, ésta se modela utilizando Elementos Finitos. Mediante este método, el rango de media frecuencia se puede definir de una forma precisa, estando comprendido entre el menor y el mayor de los límites de alta frecuencia de las subestructuras que componen el sistema completo. Los resultados obtenidos mediante la aplicación de este método evidencian una mejora en la continuidad de la respuesta vibroacústica, mostrando una transición suave entre los rangos de baja y alta frecuencia. El segundo método propuesto se denomina HS-CMS (del inglés Hybrid Substructuring method based on Component Mode Synthesis). Este método se basa en la clasificación de la base modal de las subestructuras en conjuntos de modos globales (que afectan a todo o a varias partes del sistema) o locales (que afectan a una única subestructura), utilizando un método de Síntesis Modal de Componentes. De este modo es posible situar espacialmente los modos del sistema completo y estudiar el comportamiento del mismo desde el punto de vista de las subestructuras. De nuevo se emplea el concepto de límite de alta frecuencia de una subestructura para realizar la clasificación global/local de los modos en la misma. Mediante dicha clasificación se derivan las ecuaciones globales del movimiento, gobernadas por los modos globales, y en las que la influencia del conjunto de modos locales se introduce mediante modificaciones en las mismas (en su matriz dinámica de rigidez y en el vector de fuerzas). Las ecuaciones locales se resuelven empleando Análisis Estadístico de Energías. Sin embargo, este último será un modelo híbrido, en el cual se introduce la potencia adicional aportada por la presencia de los modos globales. El método ha sido probado para el cálculo de la respuesta de estructuras sometidas tanto a cargas estructurales como acústicas. Ambos métodos han sido probados inicialmente en estructuras sencillas para establecer las bases e hipótesis de aplicación. Posteriormente, se han aplicado a estructuras espaciales, como satélites y reflectores de antenas, mostrando buenos resultados, como se concluye de la comparación de las simulaciones y los datos experimentales medidos en ensayos, tanto estructurales como acústicos. Este trabajo abre un amplio campo de investigación a partir del cual es posible obtener metodologías precisas y eficientes para reproducir el comportamiento vibroacústico de sistemas en el rango de la media frecuencia. ABSTRACT Over the last years an increasing need of novel prediction techniques for vibroacoustic analysis of space structures has arisen. Current numerical techniques arc able to predict with enough accuracy the vibro-acoustic behaviour of systems with low and high modal densities. However, space structures are, in general, very complex and they present a range of frequencies in which a mixed behaviour exist. In such cases, the full system is composed of some sub-structures which has low modal density, while others present high modal density. This frequency range is known as the mid-frequency range and to develop methods for accurately describe the vibro-acoustic response in this frequency range is the scope of this dissertation. For the structures under study, the aforementioned low and high modal densities correspond with the low and high frequency ranges, respectively. For the low frequency range, deterministic techniques as the Finite Element Method (FEM) are used while, for the high frequency range statistical techniques, as the Statistical Energy Analysis (SEA), arc considered as more appropriate. In the mid-frequency range, where a mixed vibro-acoustic behaviour is expected, any of these numerical method can not be used with enough confidence level. As a consequence, it is usual to obtain an undetermined gap between low and high frequencies in the vibro-acoustic response function. This dissertation proposes two different solutions to the mid-frequency range problem. The first one, named as The Subsystem based High Frequency Limit (SHFL) procedure, proposes a multi-hybrid procedure in which each sub-structure of the full system is modelled with the appropriate modelling technique, depending on the frequency of study. With this purpose, the concept of high frequency limit of a sub-structure is introduced, marking out the limit above which a substructure has enough modal density to be modelled by SEA. For a certain analysis frequency, if it is lower than the high frequency limit of the sub-structure, the sub-structure is modelled through FEM and, if the frequency of analysis is higher than the high frequency limit, the sub-structure is modelled by SEA. The procedure leads to a number of hybrid models required to cover the medium frequency range, which is defined as the frequency range between the lowest substructure high frequency limit and the highest one. Using this procedure, the mid-frequency range can be define specifically so that, as a consequence, an improvement in the continuity of the vibro-acoustic response function is achieved, closing the undetermined gap between the low and high frequency ranges. The second proposed mid-frequency solution is the Hybrid Sub-structuring method based on Component Mode Synthesis (HS-CMS). The method adopts a partition scheme based on classifying the system modal basis into global and local sets of modes. This classification is performed by using a Component Mode Synthesis, in particular a Craig-Bampton transformation, in order to express the system modal base into the modal bases associated with each sub-structure. Then, each sub-structure modal base is classified into global and local set, fist ones associated with the long wavelength motion and second ones with the short wavelength motion. The high frequency limit of each sub-structure is used as frequency frontier between both sets of modes. From this classification, the equations of motion associated with global modes are derived, which include the interaction of local modes by means of corrections in the dynamic stiffness matrix and the force vector of the global problem. The local equations of motion are solved through SEA, where again interactions with global modes arc included through the inclusion of an additional input power into the SEA model. The method has been tested for the calculation of the response function of structures subjected to structural and acoustic loads. Both methods have been firstly tested in simple structures to establish their basis and main characteristics. Methods are also verified in space structures, as satellites and antenna reflectors, providing good results as it is concluded from the comparison with experimental results obtained in both, acoustic and structural load tests. This dissertation opens a wide field of research through which further studies could be performed to obtain efficient and accurate methodologies to appropriately reproduce the vibro-acoustic behaviour of complex systems in the mid-frequency range.

Design of a Semi-analytical Method to Describe Plasma-Wall Interactions

Electric probes are objects immersed in the plasma with sharp boundaries which collect of emit charged particles. Consequently, the nearby plasma evolves under abrupt imposed and/or naturally emerging conditions. There could be localized currents, different time scales for plasma species evolution, charge separation and absorbing-emitting walls. The traditional numerical schemes based on differences often transform these disparate boundary conditions into computational singularities. This is the case of models using advection-diffusion differential equations with source-sink terms (also called Fokker-Planck equations). These equations are used in both, fluid and kinetic descriptions, to obtain the distribution functions or the density for each plasma species close to the boundaries. We present a resolution method grounded on an integral advancing scheme by using approximate Green's functions, also called short-time propagators. All the integrals, as a path integration process, are numerically calculated, what states a robust grid-free computational integral method, which is unconditionally stable for any time step. Hence, the sharp boundary conditions, as the current emission from a wall, can be treated during the short-time regime providing solutions that works as if they were known for each time step analytically. The form of the propagator (typically a multivariate Gaussian) is not unique and it can be adjusted during the advancing scheme to preserve the conserved quantities of the problem. The effects of the electric or magnetic fields can be incorporated into the iterative algorithm. The method allows smooth transitions of the evolving solutions even when abrupt discontinuities are present. In this work it is proposed a procedure to incorporate, for the very first time, the boundary conditions in the numerical integral scheme. This numerical scheme is applied to model the plasma bulk interaction with a charge-emitting electrode, dealing with fluid diffusion equations combined with Poisson equation self-consistently. It has been checked the stability of this computational method under any number of iterations, even for advancing in time electrons and ions having different time scales. This work establishes the basis to deal in future work with problems related to plasma thrusters or emissive probes in electromagnetic fields.

Estudio de los factores que condicionan, a escala local, la presencia de Epidendrum rhopalostele (Orchidaceae): Implicaciones para su conservación

El análisis de los factores que determinan el establecimiento y supervivencia de orquídeas epífitas, incluyen: a) las condiciones microambientales de los bosques que las mantienen, b) preferencias por las características de los hospederos donde crecen, c) limitación en la dispersión de semillas, d) interacciones planta-planta, y e) asociaciones micorrízicas para la germinación y resultan esenciales para el desarrollo de estrategias para la conservación y manejo de este grupo de plantas. Este trabajo ha evaluado la importancia de estos factores en Epidendrum rhopalostele, orquídea epífita del bosque de niebla montano, a través de los análisis de los patrones espaciales de los árboles que la portan y de la propia orquídea, a escala de población, estudios de asociación y métodos moleculares. Estos últimos han consistido en el uso de marcadores AFLP para el análisis de la estructura genética de la orquídea y en la secuenciación-clonación de la región ITS para la identificación de los hongos micorrízicos asociados. El objetivo de esta tesis es, por tanto, una mejor comprensión de los factores que condicionan la presencia de orquídeas epífitas en los remanentes de bosque de niebla montano y una evaluación de las implicaciones para la conservación y mantenimiento de sus hábitats y la permanencia de sus poblaciones. El estudio fue realizado en un fragmento de bosque de niebla montano de sucesión secundaria situado al este de la Cordillera Real, en los Andes del sur de Ecuador, a 2250 m.s.n.m y caracterizado por una pendiente marcada, temperatura media anual de 20.8°C y precipitación anual de 2193 mm. En este fragmento se mapearon, identificaron y caracterizaron todos los árboles presentes con DBH > 1 cm y todos los individuos de Epidendrum rhopalostele. Así mismo se tomaron muestras de hoja para obtener ADN de todas las orquídeas registradas y muestras de raíces de individuos con flor de E. rhopalostele, uno por cada forófito, para el análisis filogenético de micorrizas. Análisis espaciales de patrones de puntos basados en la K de Ripley y la distancia al vecino más cercano fueron usados para los árboles, forófitos y la población de E. rhopalostele. Se observó que la distribución espacial de árboles y forófitos de E. rhopalostele no es aleatoria, ya que se ajusta a un proceso agregado de Poisson. De ahí se infiere una limitación en la dispersión de las semillas en el fragmento estudiado y en el establecimiento de la orquídea. El patrón de distribución de la población de E. rhopalostele en el fragmento muestra un agrupamiento a pequeña escala sugiriendo una preferencia por micro-sitios para el establecimiento de la orquídea con un kernel de dispersión de las semillas estimado de 0.4 m. Las características preferentes del micro-sitio como tipos de árboles (Clusia alata y árboles muertos), tolerancia a la sombra, corteza rugosa, distribución en los dos primeros metros sugieren una tendencia a distribuirse en el sotobosque. La existencia de una segregación espacial entre adultos y juveniles sugiere una competencia por recursos limitados condicionada por la preferencia de micro-sitio. La estructura genética de la población de E. rhopalostele analizada a través de Structure y PCoA evidencia la presencia de dos grupos genéticos coexistiendo en el fragmento y en los mismos forófitos, posiblemente por eventos de hibridización entre especies de Epidendrum simpátricas. Los resultados del análisis de autocorrelación espacial efectuados en GenAlex confirman una estructura genético-espacial a pequeña escala que es compatible con un mecanismo de dispersión de semillas a corta distancia ocasionada por gravedad o pequeñas escorrentías, frente a la dispersión a larga distancia promovida por el viento generalmente atribuida a las orquídeas. Para la identificación de los micobiontes se amplificó la región ITS1-5.8S-ITS2, y 47 secuencias fueron usadas para el análisis filogenético basado en neighborjoining, análisis bayesiano y máximum-likelihood que determinó que Epidendrum rhopalostele establece asociaciones micorrízicas con al menos dos especies diferentes de Tulasnella. Se registraron plantas que estaban asociadas con los dos clados de hongos encontrados, sugiriendo ausencia de limitación en la distribución del hongo. Con relación a las implicaciones para la conservación in situ resultado de este trabajo se recomienda la preservación de todo el fragmento de bosque así como de las interacciones existentes (polinizadores, micorrizas) a fin de conservar la diversidad genética de esta orquídea epífita. Si fuere necesaria una reintroducción se deben contemplar distancias entre los individuos en cada forófito dentro de un rango de 0.4 m. Para promover el reclutamiento y regeneración de E. rhopalostele, se recomienda que los forófitos correspondan preferentemente a árboles muertos o caídos y a especies, como Clusia alata, que posean además corteza rugosa, sean tolerantes a la sombra, y en el área del sotobosque con menor luminosidad. Además es conveniente que las orquídeas en su distribución vertical estén ubicadas en los primeros metros. En conclusión, la limitación en la dispersión, las características del micro-sitio, las interacciones intraespecíficas y con especies congenéricas simpátricas y las preferencias micorrízicas condicionan la presencia de esta orquídea epífita en este tipo de bosque. ABSTRACT The analysis of factors that determine the establishment and survival of epiphytic depends on factors such as a) microenvironmental conditions of forest, b) preference for host characteristics where orchids grow, c) seed dispersal limitation, d) plant-plant interaction, e) priority mycorrhizal associations for germination, are essential for the development of strategies for management and conservation. This work evaluated the importance of these factors in Epidendrum rhopalostele, an epiphytic orchid of montane cloud forest through the analysis of spatial patterns of host trees and the orchid, in a more specific scale, with association studies and molecular methods, including AFLPs for orchid population genetic structure and the sequencing of the ITS region for associated mycorrhizal fungi. The aim of this thesis is to understand the factors that condition the presence of epiphytic orchids in the remnants of montane cloud forest and to assess the implications for the conservation and preservation of their habitats and the persistence of the orchid populations. The study was carried out in a fragment of montane cloud forest of secondary succession on the eastern slope of Cordillera Real in the Andes of southern Ecuador, located at 2250 m a.s.l. characterized by a steep slope, mean annual temperature of 20.8°C and annual precipitation of 2193 mm. All trees with DBH > 1 cm were mapped, characterized and identified. All E. rhopalostele individuals present were counted, marked, characterized and mapped. Leaf samples of all orchid individuals were collected for DNA analysis. Root samples of flowering E. rhopalostele individuals were collected for phylogenetic analysis of mycorrhizae, one per phorophyte. Spatial point pattern analysis based on Ripley`s K function and nearest neighbor function was used for trees, phorophytes and orchid population. We observed that spatial distribution of trees and phorophytes is not random, as it adjusts to a Poisson cluster process. This suggests a limitation for seed dispersal in the study fragment that is affecting orchid establishment. Furthermore, the small-scale spatial pattern of E. rhopalostele evidences a clustering that suggests a microsite preference for orchid establishment with a dispersal kernel of 0.4 m. Microsite features such as types of trees (dead trees or Clusia alata), shade tolerance trees, rough bark, distribution in the first meters suggest a tendency to prefer the understory for their establishment. Regarding plant-plant interaction a spatial segregation between adults and juveniles was present suggesting competition for limited resources conditioned for a microsite preference. Analysis of genetic structure of E. rhopalostele population through Structure and PCoA shows two genetic groups coexisting in this fragment and in the same phorophyte, possibly as a result of hybridization between sympatric species of Epidendrum. Our results of spatial autocorrelation analysis develop in GenAlex confirm a small-scale spatial-genetic structure within the genetic groups that is compatible with a short-distance dispersal mechanism caused by gravity or water run-off, instead of the long-distance seed dispersal promoted by wind generally attributed to orchids. For mycobionts identification ITS1-5.8S-ITS2 rDNA region was amplified. Phylogenetic analysis was performed with neighborjoining, Bayesian likelihood and maximum-likelihood for 47 sequences yielded two Tulasnella clades. This orchid establishes mycorrhizal associations with at least two different Tulasnella species. In some cases both fungi clades were present in same root, suggesting no limitation in fungal distribution. Concerning the implications for in situ conservation resulting from this work, the preservation of all forest fragment and their interactions (pollinators, mycorrhiza) is recommended to conserve the genetic diversity of this species. If a reintroduction were necessary, distances between individuals in each phorophyte within a range of 0.4 m, are recommended. To promote recruitment and regeneration of E. rhopalostele it is recommended that phorophytes correspond to dead or fallen trees or species, such as Clusia alata. Trees that have rough bark and are shade tolerant are also recommended. Furthermore, regarding vertical distribution, it is also convenient that orchids are located in the first meter (in understory, area with less light). In conclusion, limitation on seed dispersal, microsite characteristics, plant-plant interactions or interaction with cogeneric sympatric species and mycorrhizal preferences conditioned the presence of this epiphytic orchid in this fragment forest.

Operational short term health impact assessment of air pollution modelling system over Europe

The last decade, scientific studies have indicated an association between air pollution to which people are exposed and wide range of adverse health outcomes. We have developed a tool which is based on a model (MM5-CMAQ) running over Europe with 50 km spatial resolution, based on EMEP annual emissions, to produce a short-term forecast of the impact on health. In order to estimate the mortality change (forecasted for the next 24 hours) we have chosen a log-linear (Poisson) regression form to estimate the concentration-response function. The parameters involved in the C-R function have been estimated based on epidemiological studies, which have been published. Finally, we have derived the relationship between concentration change and mortality change from the C-R function which is the final health impact function.

The activation domain of the enhancer binding protein p45NF-E2 interacts with TAFII130 and mediates long-range activation of the α- and β-globin gene loci in an erythroid cell line

We have used the interaction between the erythroid-specific enhancer in hypersensitivity site 2 of the human β-globin locus control region and the globin gene promoters as a paradigm to examine the mechanisms governing promoter/enhancer interactions in this locus. We have demonstrated that enhancer-dependent activation of the globin promoters is dependent on the presence of both a TATA box in the proximal promoter and the binding site for the erythroid-specific heteromeric transcription factor NF-E2 in the enhancer. Mutational analysis of the transcriptionally active component of NF-E2, p45NF-E2, localizes the critical region for this function to a proline-rich transcriptional activation domain in the NH2-terminal 80 amino acids of the protein. In contrast to the wild-type protein, expression of p45 NF-E2 lacking this activation domain in an NF-E2 null cell line fails to support enhancer-dependent transcription in transient assays. More significantly, the mutated protein also fails to reactivate expression of the endogenous β- or α-globin loci in this cell line. Protein-protein interaction studies reveal that this domain of p45 NF-E2 binds specifically to a component of the transcription initiation complex, TATA binding protein associated factor TAFII130. These findings suggest one potential mechanism for direct recruitment of distal regulatory regions of the globin loci to the individual promoters.

Protein–protein interactions among the Aux/IAA proteins

The plant hormone indoleacetic acid (IAA) transcriptionally activates early genes in plants. The Aux/IAA family of early genes encodes proteins that are short-lived and nuclear-localized. They also contain a putative prokaryotic βαα DNA binding motif whose formation requires protein dimerization. Here, we show that the pea PS-IAA4 and Arabidopsis IAA1 and IAA2 proteins perform homo- and heterotypic interactions in yeast using the two-hybrid system. Gel-filtration chromatography and chemical cross-linking experiments demonstrate that the PS-IAA4 and IAA1 proteins interact to form homodimers in vitro. Deletion analysis of PS-IAA4 indicates that the βαα containing acidic C terminus of the protein is necessary for homotypic interactions in the yeast two-hybrid system. Screening an Arabidopsis λ-ACT cDNA library using IAA1 as a bait reveals heterotypic interactions of IAA1 with known and newly discovered members of the Arabidopsis Aux/IAA gene family. The new member IAA24 has similarity to ARF1, a transcription factor that binds to an auxin response element. Combinatorial interactions among the various members of the Aux/IAA gene family may regulate a variety of late genes as well as serve as autoregulators of early auxin-regulated gene expression. These interactions provide a molecular basis for the developmental and tissue-specific manner of auxin action.

Specificity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: Interactions of promoter DNA, transcription factor μE3, and Smad proteins

Transforming growth factor β (TGF-β) regulates a broad range of biological processes, including cell growth, development, differentiation, and immunity. TGF-β signals through its cell surface receptor serine kinases that phosphorylate Smad2 or Smad3 proteins. Because Smad3 and its partner Smad4 bind to only 4-bp Smad binding elements (SBEs) in DNA, a central question is how specificity of TGF-β-induced transcription is achieved. We show that Smad3 selectively binds to two of the three SBEs in PE2.1, a TGF-β-inducible fragment of the plasminogen activator inhibitor-1 promoter, to mediate TGF-β-induced transcription; moreover, a precise 3-bp spacer between one SBE and the E-box, a binding site for transcription factor μE3 (TFE3), is essential for TGF-β-induced transcription. Whereas an isolated Smad3 MH1 domain binds to TFE3, TGF-β receptor-mediated phosphorylation of full-length Smad3 enhances its binding to TFE3. Together, these studies elucidate an important mechanism for specificity in TGF-β-induced transcription of the plasminogen activator inhibitor-1 gene.

Long-range disruption of gene expression by a selectable marker cassette

Recent studies have suggested that the retention of selectable marker cassettes (like PGK–Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in “knockout” mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the β-like globin gene cluster. The insertion of PGK–Neo into the granzyme B gene, the most 5′ gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK–Neo cassette into the β-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3′ to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these long distance effects are unknown, the expression of PGK–Neo can be “captured” by the regulatory domain into which it is inserted. These results suggest that the PGK–Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.

Distinct short-term plasticity at two excitatory synapses in the hippocampus

A single mossy fiber input contains several release sites and is located on the proximal portion of the apical dendrite of CA3 neurons. It is, therefore, well suited to exert a strong influence on pyramidal cell excitability. Accordingly, the mossy fiber synapse has been referred to as a detonator or teacher synapse in autoassociative network models of the hippocampus. The very low firing rates of granule cells [Jung, M. W. & McNaughton, B. L. (1993) Hippocampus 3, 165–182], which give rise to the mossy fibers, raise the question of how the mossy fiber synapse temporally integrates synaptic activity. We have therefore addressed the frequency dependence of mossy fiber transmission and compared it to associational/commissural synapses in the CA3 region of the hippocampus. Paired pulse facilitation had a similar time course, but was 2-fold greater for mossy fiber synapses. Frequency facilitation, during which repetitive stimulation causes a reversible growth in synaptic transmission, was markedly different at the two synapses. At associational/commissural synapses facilitation occurred only at frequencies greater than once every 10 s and reached a magnitude of about 125% of control. At mossy fiber synapses, facilitation occurred at frequencies as low as once every 40 s and reached a magnitude of 6-fold. Frequency facilitation was dependent on a rise in intraterminal Ca2+ and activation of Ca2+/calmodulin-dependent kinase II, and was greatly reduced at synapses expressing mossy fiber long-term potentiation. These results indicate that the mossy fiber synapse is able to integrate granule cell spiking activity over a broad range of frequencies, and this dynamic range is substantially reduced by long-term potentiation.

Relationships of dehydroepiandrosterone sulfate in the elderly with functional, psychological, and mental status, and short-term mortality: A French community-based study

In human beings of both sexes, dehydroepiandrosterone sulfate (DHEAS) circulating in blood is mostly an adrenally secreted steroid whose serum concentration (in the micromolar range and 30–50% higher in men than in women) decreases with age, toward ≈20–10% of its value in young adults during the 8th and 9th decades. The mechanism of action of DHEA and DHEAS is poorly known and may include partial transformation into sex steroids, increase of bioavailable insulin-like growth factor I, and effects on neurotransmitter receptors. Whether there is a cause-to-effect relationship between the decreasing levels of DHEAS with age and physiological and pathological manifestations of aging is still undecided, but this is of obvious theoretical and practical interest in view of the easy restoration by DHEA administration. Here we report on 622 subjects over 65 years of age, studied for the 4 years since DHEAS baseline values had been obtained, in the frame of the PAQUID program, analyzing the functional, psychological, and mental status of a community-based population in the south-west of France. We confirm the continuing decrease of DHEAS serum concentration with age, more in men than in women, even if men retain higher levels. Significantly lower values of baseline DHEAS were recorded in women in cases of functional limitation (Instrumental Activities of Daily Living), confinement, dyspnea, depressive symptomatology, poor subjective perception of health and life satisfaction, and usage of various medications. In men, there was a trend for the same correlations, even though not statistically significant in most categories. No differences in DHEAS levels were found in cases of incident dementia in the following 4 years. In men (but not in women), lower DHEAS was significantly associated with increased short-term mortality at 2 and 4 years after baseline measurement. These results, statistically established by taking into account corrections for age, sex, and health indicators, suggest the need for further careful trials of the administration of replacement doses of DHEA in aging humans. Indeed, the first noted results of such “treatment” are consistent with correlations observed here between functional and psychological status and endogenous steroid serum concentrations.

An in vitro study of the dynamic features of the major histocompatibility complex class I complex relevant to its role as a versatile peptide-receptive molecule

The major histocompatibility complex class I complex consists of a heavy chain and a light chain (β2-microglobulin, β2m), which assemble with a short endogenously derived peptide in the endoplasmic reticulum. The class I peptide can be directly exchanged, either at the cell surface or, as recently described, in vesicles of the endocytic compartments, thus allowing exogenous peptides to enter the class I presentation pathway. To probe the interactions between the components of the class I molecule, we analyzed the exchange of peptide and β2m by using purified, recombinant H2-Kb/peptide complexes in a cell-free in vitro system. The exchange of competitor peptide was primarily dependent on the off-rate of the original peptide in the class I binding groove. Peptide exchange was not enhanced by the presence of exogenous β2m, as exchange occurred to the same extent in its absence. Thus, the exchange of peptide and β2m are independent events. The exchange rate of β2m also was not affected by the dissociation rates of the original peptides. Furthermore, peptides could substantially exchange into class I molecules over a pH range of 5.5 to 7.5, conditions prevalent in certain endocytic compartments. We conclude that the dynamic properties of the components of class I molecules explain its function as a highly peptide-receptive molecule. The major histocompatibility complex class I can readily receive peptides independent of the presence of exogenous β2m, even at a low pH. Such properties are relevant to class I peptide acquisition, which can occur at the cell surface, as well as in specialized endosomes.

αβ T cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization

Cellular immunity is mediated by the interaction of an αβ T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have αβ TCRs that can recognize both self- and foreign peptide–MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive αβ T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired αβ heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2Kb-dEV8, H-2Kbm3-dEV8, H-2Kb-SIYR, and H-2Ld-p2Ca spans a range of affinities from Kd = 10−4 to 10−6M for the syngeneic (H-2Kb) and allogeneic (H-2Kbm3, H-2Ld) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the α and β chains. X-ray quality crystals of complexes of 2C with H-2Kb-dEV8, H-2Kbm3-dEV8 and H-2Kb-SIYR have been grown.

Comparing protein–ligand interactions in solution and single crystals by Raman spectroscopy

By using a Raman microscope, we show that it is possible to probe the conformational states in protein crystals and crystal fragments under growth conditions (in hanging drops). The flavin cofactor in the enzyme para-hydroxybenzoate hydroxylase can assume two conformations: buried in the protein matrix (“in”) or essentially solvent-exposed (“out”). By using Raman difference spectroscopy, we previously have identified characteristic flavin marker bands for the in and out conformers in the solution phase. Now we show that the flavin Raman bands can be used to probe these conformational states in crystals, permitting a comparison between solution and crystal environments. The in or out marker bands are similar for the respective conformers in the crystal and in solution; however, significant differences do exist, showing that the environments for the flavin's isoalloxazine ring are not identical in the two phases. Moreover, the Raman-band widths of the flavin modes are narrower for both in and out conformers in the crystals, indicating that the flavin exists in a more limited range of closely related conformational states in the crystal than in solution. In general, the ability to compare detailed Raman data for complexes in crystals and solution provides a means of bridging crystallographic and solution studies.

RNA tertiary interactions in the large ribosomal subunit: The A-minor motif

Analysis of the 2.4-Å resolution crystal structure of the large ribosomal subunit from Haloarcula marismortui reveals the existence of an abundant and ubiquitous structural motif that stabilizes RNA tertiary and quaternary structures. This motif is termed the A-minor motif, because it involves the insertion of the smooth, minor groove edges of adenines into the minor groove of neighboring helices, preferentially at C-G base pairs, where they form hydrogen bonds with one or both of the 2′ OHs of those pairs. A-minor motifs stabilize contacts between RNA helices, interactions between loops and helices, and the conformations of junctions and tight turns. The interactions between the 3′ terminal adenine of tRNAs bound in either the A site or the P site with 23S rRNA are examples of functionally significant A-minor interactions. The A-minor motif is by far the most abundant tertiary structure interaction in the large ribosomal subunit; 186 adenines in 23S and 5S rRNA participate, 68 of which are conserved. It may prove to be the universally most important long-range interaction in large RNA structures.

Template-nucleated alanine-lysine helices are stabilized by position-dependent interactions between the lysine side chain and the helix barrel.

The helicity in water has been determined for several series of alanine-rich peptides that contain single lysine residues and that are N-terminally linked to a helix-inducing and reporting template termed Ac-Hel1. The helix-propagating constant for alanine (sAla value) that best fits the properties of these peptides lies in the range of 1.01-1.02, close to the value reported by Scheraga and coworkers [Wojcik, J., Altmann, K.-H. & Scheraga, H.A. (1990) Biopolymers 30, 121-134], but significantly lower than the value assigned by Baldwin and coworkers [Chakrabartty, A., Kortemme, T. & Baldwin, R.L. (1994) Protein Sci. 3,843-852]. From a study of conjugates Ac-Hel1-Ala(n)-Lys-Ala(m)-NH2 and analogs in which the methylene portion of the lysine side chain is truncated, we find that the unusual helical stability of Ala(n)Lys peptides is controlled primarily by interactions of the lysine side chain with the helix barrel, and only passively by the alanine matrix. Using 1H NMR spectroscopy, we observe nuclear Overhauser effect crosspeaks consistent with proton-proton contacts expected for these interactions.