Genetic diversity of irrigated barley based on molecular and quantitative data and on malting quality

The objective of this work was to quantify the genetic diversity of elite genotypes of irrigated barley in the Brazilian savanna. Thirty elite barley genotypes from Embrapa Cerrados' collection were evaluated using 160 RAPD markers, 12 agronomic traits related to yield components, and 10 malting quality parameters. The genetic dissimilarity matrices based on molecular markers, quantitative traits, and malting quality characters were calculated and a cluster analysis was performed using the unweighted pair-group method with arithmetic mean (UPGMA) as grouping criterion. High genetic diversity among accessions were observed. The estimated genetic dissimilarities were weakly correlated, showing the complementarity of the different character groups. Selection indices and graphical dispersion analysis allowed the selection of promising genotypes and the indication of suitable crosses for maximizing the heterotic effects in breeding programs for irrigated barley in the Brazilian savanna.

Genetic variability of hull-less barley accessions based on molecular and quantitative data

The objective of this work was to characterize and quantify the genetic, molecular, and agronomic variability of hull-less barley genotypes, for the selection of parents and identification of genotypes adapted to the irrigated production system in the Brazilian Cerrado. Eighteen hull-less barley accessions were evaluated, and three covered barley accessions served as reference. The characterization was based on 157 RAPD molecular markers and ten agronomic traits. Genetic distance matrices were obtained based on molecular markers and quantitative traits. Graphic grouping and dispersion analyses were performed. Genetic, molecular, and agronomic variability was high among genotypes. Ethiopian accessions were genetically more similar, and the Brazilian ones were genetically more distant. For agronomic traits, two more consistent groupings were obtained, one with the most two-rowed materials, and the other with six-rowed materials. The more diverging materials were the two-rowed CI 13453, CN Cerrado 5, CN Cerrado 1, and CN Cerrado 2. The PI 356466, CN Cerrado 1, PI 370799, and CI 13453 genotypes show agronomic traits of interest and, as genetically different genotypes, they are indicated for crossing, in breeding programs.

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.

The maximum voltage drop in an on-chip power distribution network: analysis of square, triangular and hexagonal power pad arrangements

A mathematical model of the voltage drop which arises in on-chip power distribution networks is used to compare the maximum voltage drop in the case of different geometric arrangements of the pads supplying power to the chip. These include the square or Manhattan power pad arrangement, which currently predominates, as well as equilateral triangular and hexagonal arrangements. In agreement with the findings in the literature and with physical and SPICE models, the equilateral triangular power pad arrangement is found to minimize the maximum voltage drop. This headline finding is a consequence of relatively simple formulas for the voltage drop, with explicit error bounds, which are established using complex analysis techniques, and elliptic functions in particular.

Phylogenetic study of the Grimmia Hedw. (Grimmiales, Bryopsida) based on a combination of morphological and molecular characters

Résumé Les mousses sont la plus ancienne lignée de plantes terrestres et leur longue évolution a été accompagnée par des tendances à la simplification des caractères morphologiques. Ce phénomène a quelque peu compliqué les reconstructions phylogénétiques basées sur la morphologie. Les analyses génétiques ont permis de donner de nouvelles informations dans le cadre des analyses phylogénétiques et une réévaluation de certains caractères morphologiques. La plupart des études combinant les données morphologiques et moléculaires ne concernent que des niveaux systématiques élevés comme l'ordre ou la famille et très peu considèrent le niveau du genre. La présente étude tend à tester les relations phylogénétiques du genre Grimmia à l'aide d'une combinaison de caractères morphologiques et moléculaires. Les 40 espèces de Grimmia utilisées dans la première partie de cette étude représentent la majorité des espèces trouvées en Eurasie, un des centres de diversification du genre. Lors de l'analyse morphologique, 52 caractères morphologiques/anatomiques (33 du gamétophyte et 19 du sporophyte) ont été numérisés. Malgré le peu de support statistique des arbres, la topologie des arbres est stable. Les Grimmia, comme décrit précédemment, sont paraphylétiques. Trois clades, correspondant respectivement aux sous-genres Rhabdogrimmia Limpr, Litoneuron I.Hagen et Gasterogrimmia Schimp. sont présents, tandis que le restant des taxons appartenant aux Grimmia forment un groupe non-résolu et indistinct des autres Grimmiaceae. Les séquences chloroplastiques trnL-trnF et rps4 combinés à la morphologie ont été ensuite utilisés pour reconstruire la phylogénie des Grimmia. Les arbres obtenus soutiennent la monophylie des Grimmiaceae tandis que les Grimmia, sont paraphylétiques. Deux clades principaux correspondant aux "Rhabdogrimmia" et aux "Grimmia" se détachent. Seules les espèces de "Rhabdogrimmia" produisent des gemmules foliaires (reproduction asexuée). Dans une étude considèrant 91 séquences trrIL-trnF les espèces appartenant aux "Rhabdogrimrnia" (reproduction asexuée essentiellement) ont des variabilités intraspécifique très faible et interspécifique relativement élevée tandis que les "Grimmia" possèdent la tendance inverse (plus de reproduction sexuée). Summary The mosses are a very old land plant lineage and their long evolutionary history has been accompanied by a trend of morphological character simplifications. This phenomenon has somewhat complicated morphological based phylogenetic reconstructions. Genetic analyses have provided new insights for phylogenetic studies, and have allowed morphological data to be re¬evaluated. Most of the studies combining morphological and molecular data have concerned the higher systematic levels of order and family and only have few considered the genus. The present study aims to test the phylogenetic relationships of the genus Grimmia using a combination of morphological and molecular characters. The 40 chosen Grimmia species represent the majority of those found in Eurasia, one diversification centers of the genus. For the morphological analysis, 52 morphological/anatomical characters (33 gametophyte and 19 sporophyte characters) were numerized. Although the internal statistical support was relatively low, the tree topologies were stable. Grimmia as currently defined was found to be paraphyletic. Three subclades, corresponding to the subgenera Rhabdogrimmia Limpr., Litoneuron I.Hagen, and Gasterogrimmia Schimp. were observed in the trees, while the reminder of the Grimmia species formed an unresolved group indistinct from other Grimmiaceae. Chloroplast (trnL-trnF and rps4) DNA sequences combined with morphology were used to reconstruct the phylogeny of Grimmia. The resulting trees supported the monophyly of Grimmiaceae and that the genus Grimmia, as currently defined, as paraphyletic. Two main clades were resolved corresponding to "Rhabdogrimmia" and "Grimmia". The species belonging to "Rhabdogrimmia" produce foliar-gemmae (asexual reproduction). In a study using 91 sequences of trnL-trnF,"Rhabdogrimmia" species (mainly asexual reproduction) have very low intraspecific variability and high interspecific variability whereas the "Grimmia" species possess the inverse tendency.

Molecular phylogenetics and temporal diversification in the genus Aeromonas based on the sequences of five housekeeping genes

Several approaches have been developed to estimate both the relative and absolute rates of speciation and extinction within clades based on molecular phylogenetic reconstructions of evolutionary relationships, according to an underlying model of diversification. However, the macroevolutionary models established for eukaryotes have scarcely been used with prokaryotes. We have investigated the rate and pattern of cladogenesis in the genus Aeromonas (γ-Proteobacteria, Proteobacteria, Bacteria) using the sequences of five housekeeping genes and an uncorrelated relaxed-clock approach. To our knowledge, until now this analysis has never been applied to all the species described in a bacterial genus and thus opens up the possibility of establishing models of speciation from sequence data commonly used in phylogenetic studies of prokaryotes. Our results suggest that the genus Aeromonas began to diverge between 248 and 266 million years ago, exhibiting a constant divergence rate through the Phanerozoic, which could be described as a pure birth process.

Comment on 'The Molecular Evolutionary Patterns of the Insulin/FOXO Signaling Pathway'

Letter to the Editor on Wang M, Wang Q, Wang Z, Zhang X, Pan Y. The molecular evolutionary patterns of the insulin/FOXO signaling pathway

CNDOL: A fast and reliable method for the calculation of electronic properties of very large systems. Applications to retinal binding pocket in rhodopsin and gas phase porphine.

Very large molecular systems can be calculated with the so called CNDOL approximate Hamiltonians that have been developed by avoiding oversimplifications and only using a priori parameters and formulas from the simpler NDO methods. A new diagonal monoelectronic term named CNDOL/21 shows great consistency and easier SCF convergence when used together with an appropriate function for charge repulsion energies that is derived from traditional formulas. It is possible to obtain a priori molecular orbitals and electron excitation properties after the configuration interaction of single excited determinants with reliability, maintaining interpretative possibilities even being a simplified Hamiltonian. Tests with some unequivocal gas phase maxima of simple molecules (benzene, furfural, acetaldehyde, hexyl alcohol, methyl amine, 2,5 dimethyl 2,4 hexadiene, and ethyl sulfide) ratify the general quality of this approach in comparison with other methods. The calculation of large systems as porphine in gas phase and a model of the complete retinal binding pocket in rhodopsin with 622 basis functions on 280 atoms at the quantum mechanical level show reliability leading to a resulting first allowed transition in 483 nm, very similar to the known experimental value of 500 nm of "dark state." In this very important case, our model gives a central role in this excitation to a charge transfer from the neighboring Glu(-) counterion to the retinaldehyde polyene chain. Tests with gas phase maxima of some important molecules corroborate the reliability of CNDOL/2 Hamiltonians.

Molecular mechanism of a green-shifted, pH-dependent red fluorescent protein mKate variant.

Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm) and emits green fluorescence (525 nm). At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies.

Modeling and manufacturability assessment of bistable quantum-dot cells

We have investigated the behavior of bistable cells made up of four quantum dots and occupied by two electrons, in the presence of realistic confinement potentials produced by depletion gates on top of a GaAs/AlGaAs heterostructure. Such a cell represents the basic building block for logic architectures based on the concept of quantum cellular automata (QCA) and of ground state computation, which have been proposed as an alternative to traditional transistor-based logic circuits. We have focused on the robustness of the operation of such cells with respect to asymmetries derived from fabrication tolerances. We have developed a two-dimensional model for the calculation of the electron density in a driven cell in response to the polarization state of a driver cell. Our method is based on the one-shot configuration-interaction technique, adapted from molecular chemistry. From the results of our simulations, we conclude that an implementation of QCA logic based on simple ¿hole arrays¿ is not feasible, because of the extreme sensitivity to fabrication tolerances. As an alternative, we propose cells defined by multiple gates, where geometrical asymmetries can be compensated for by adjusting the bias voltages. Even though not immediately applicable to the implementation of logic gates and not suitable for large scale integration, the proposed cell layout should allow an experimental demonstration of a chain of QCA cells.

Electronic properties of a quantum wire with arbitrary bending angle

The electron transmission and bound state properties of a quantum wire with a sharp bend at arbitrary angle are studied, extending results on the right angle sharp bend (the L¿shaped wire). These new results are compared to those of a similar structure, the circular bend wire. The possibility of using a bent wire to perform transistor action is also discussed.

Impact of T cell receptor affinity on the molecular mechanisms modulating CD8 T cell signalling and function against NY-ESO-1 tumour antigen

It is well established that cytotoxic T lymphocytes play a pivotal role in the protection against intracellular pathogens and tumour cells. Such protective immune responses rely on the specific T cell receptor (TCR)-mediated recognition by CD8 T cells of small antigenic peptides presented in the context of class-I Major Histocompatibility Complex molecules (pMHCs) on the surface of infected or malignant cells. The strength (affinity/avidity) of this interaction is a major correlate of protection. Although tumour-reactive CD8 T cells can be observed in cancer patients, anti-tumour immune responses are often ineffective in controlling or eradicating the disease due to the relative low TCR affinity of these cells. To overcome this limitation, tumour-specific CD8 T cells can be genetically modified to express TCRs of improved binding strength against a defined tumour antigen before adoptive cell transfer into cancer patients. We previously generated a panel of TCRs specific for the cancer-testis antigen NY-ESO-l,57.165 with progressively increased affinities for the pMHC complex, thus providing us with a unique tool to investigate the causal link between the surface expression of such TCRs and T cell activation and function. We recently demonstrated that anti-tumour CD8 T cell reactivity could only be improved within physiological affinity limits, beyond which drastic functional declines were observed, suggesting the presence of multiple regulatory mechanisms limiting T cell activation and function in a TCR affinity-dependent manner. The overarching goal of this thesis was (i) to assess the precise impact of TCR affinity on T cell activation and signalling at the molecular level and (ii) to gain further insights on the mechanisms that regulate and delimitate maximal/optimized CD8 T cell activation and signalling. Specifically, by combining several technical approaches we characterized the activation status of proximal (i.e. CD3Ç, Lek, and ZAP-70) and distal (i.e. ERK1/2) signalling molecules along the TCR affinity gradient. Moreover, we assessed the extent of TCR downmodulation, a critical step for initial T cell activation. CD8 T cells engineered with the optimal TCR affinity variants showed increased activation levels of both proximal and distal signalling molecules when compared to the wild-type T cells. Our analyses also highlighted the "paradoxical" status of tumour-reactive CD8 T cells bearing very high TCR affinities, which retained strong proximal signalling capacity and TCR downmodulation, but were unable to propagate signalling distally (i.e. pERKl/2), resulting in impaired cell-mediated functions. Importantly, these very high affinity T cells displayed maximal levels of SHP-1 and SHP-2 phosphatases, two negative regulatory molecules, and this correlated with a partial pERKl/2 signalling recovery upon pharmacological SHP-l/SHP-2 inhibition. These findings revealed the putative presence of inhibitory regulators of the TCR signalling cascade acting very rapidly following tumour-specific stimulation. Moreover, the very high affinity T cells were only able to transiently express enhanced proximal signalling molecules, suggesting the presence of an additional level of regulation that operates through the activation of negative feedback loops over time, limiting the duration of the TCR-mediated signalling. Overall, the determination of TCR-pMHC binding parameters eliciting optimal CD8 T cell activation, signalling, and effector function while guaranteeing high antigen specificity, together with the identification of critical regulatory mechanisms acting proximally in the TCR signalling cascade, will directly contribute to optimize and support the development of future TCR-based adoptive T cell strategies for the treatment of malignant diseases. -- Les lymphocytes T CD8 cytotoxiques jouent un rôle prédominant dans la protection contre les pathogènes intracellulaires et les cellules tumorales. Ces réponses immunitaires dépendent de la spécificité avec laquelle les récepteurs T (TCR) des lymphocytes CD8 reconnaissent les peptides antigéniques présentés par les molécules du complexe Majeur de Histocompatibilité de classe I (pCMH) à la surface des cellules infectées ou malignes. La force (ou affinité/avidité) de l'interaction du TCR-pCMH est un corrélat majeur de protection. Les réponses immunitaires sont cependant souvent inefficaces et ne permettent pas de contrôler ou d'éliminer les cellules tumorales chez les patients atteint du cancer, et ce à cause de la relative faible reconnaissance des TCRs exprimés par les lymphocytes T CD8 envers les antigènes tumoraux. Afin de surmonter cette limitation, les cellules T anti-tumorales peuvent être génétiquement modifiées en les dotant de TCRs préalablement optimisés afin d'augmenter leur reconnaissance ou affinité contre les antigènes tumoraux, avant leur ré¬infusion dans le patient. Nous avons récemment généré des cellules T CD8 exprimant un panel de TCRs spécifiques pour l'antigène tumoral NY-ESO-l157.16J avec des affinités croissantes, permettant ainsi d'investiguer la causalité directe entre l'affinité du TCR-pCMH et la fonction des cellules T CD8. Nous avons démontré que la réactivité anti-tumorale pouvait être améliorée en augmentant l'affinité du TCR dans une intervalle physiologique, mais au delà duquel nous observons un important déclin fonctionnel. Ces résultats suggèrent la présence de mécanismes de régulation limitant l'activation des cellules T de manière dépendante de l'affinité du TCR. Le but de cette thèse a été (i) de définir l'impact précis de l'affinité du TCR sur l'activation et la signalisation des cellules T CD8 au niveau moléculaire et (ii) d'acquérir de nouvelles connaissances sur les mécanismes qui régulent et délimitent l'activation et la signalisation maximale des cellules T CD8 optimisées. Spécifiquement, en combinant plusieurs approches technologiques, nous avons caractérisé l'état d'activation de différentes protéines de la voie de signalisation proximale (CD3Ç, Lek et ZAP-70) et distale (ERK1/2) le long du gradient d'affinité du TCR, ainsi que l'internalisation du TCR, une étape clef dans l'activation initiale des cellules T. Les lymphocytes T CD8 exprimant des TCRs d'affinité optimale ont montré des niveaux d'activation augmentés des molécules proximales et distales par rapport aux cellules de type sauvage (wild-type). Nos analyses ont également mis en évidence un paradoxe chez les cellules T CD8 équipées avec des TCRs de très haute affinité. En effet, ces cellules anti-tumorales sont capables d'activer leurs circuits biochimiques au niveau proximal et d'internaliser efficacement leur TCR, mais ne parviennent pas à propager les signaux biochimiques dépendants du TCR jusqu'au niveau distal (via phospho-ERKl/2), avec pour conséquence une limitation de leur capacité fonctionnelle. Finalement, nous avons démontré que SHP-1 et SHP-2, deux phosphatases avec des propriétés régulatrices négatives, étaient majoritairement exprimées dans les cellules T CD8 de très hautes affinités. Une récupération partielle des niveaux d'activation de ERK1/2 a pu être observée après l'inhibition pharmacologique de ces phosphatases. Ces découvertes révèlent la présence de régulateurs moléculaires qui inhibent le complexe de signalisation du TCR très rapidement après la stimulation anti-tumorale. De plus, les cellules T de très hautes affinités ne sont capables d'activer les molécules de la cascade de signalisation proximale que de manière transitoire, suggérant ainsi un second niveau de régulation via l'activation de mécanismes de rétroaction prenant place progressivement au cours du temps et limitant la durée de la signalisation dépendante du TCR. En résumé, la détermination des paramètres impliqués dans l'interaction du TCR-pCMH permettant l'activation de voies de signalisation et des fonctions effectrices optimales ainsi que l'identification des mécanismes de régulation au niveau proximal de la cascade de signalisation du TCR contribuent directement à l'optimisation et au développement de stratégies anti-tumorales basées sur l'ingénierie des TCRs pour le traitement des maladies malignes.