Structure of intracellular mature vaccinia virus observed by cryoelectron microscopy.

Intracellular mature vaccinia virus, also called intracellular naked virus, and its core envelope have been observed in their native, unfixed, unstained, hydrated states by cryoelectron microscopy of vitrified samples. The virion appears as a smooth rounded rectangle of ca. 350 by 270 nm. The core seems homogeneous and is surrounded by a 30-nm-thick surface domain delimited by membranes. We show that surface tubules and most likely also the characteristic dumbbell-shaped core with the lateral bodies which are generally observed in negatively stained or conventionally embedded samples are preparation artifacts.

Unicoloniality, recognition and genetic differentiation in a native Formica ant.

Some ants have an extraordinary form of social organization, called unicoloniality, whereby individuals mix freely among physically separated nests. This mode of social organization has been primarily studied in introduced and invasive ant species, so that the recognition ability and genetic structure of ants forming unicolonial populations in their native range remain poorly known. We investigated the pattern of aggression and the genetic structure of six unicolonial populations of the ant Formica paralugubris at four hierarchical levels: within nests, among nests within the same population, among nests of populations within the Alps or Jura Mountains and among nests of the two mountain ranges. Ants within populations showed no aggressive behaviour, but recognized nonnestmates as shown by longer antennation bouts. Overall, the level of aggression increased with geographic and genetic distance but was always considerably lower than between species. No distinct behavioural supercolony boundaries were found. Our study provides evidence that unicoloniality can be maintained in noninvasive ants despite significant genetic differentiation and the ability to discriminate between nestmates and nonnestmates.

Microbial communities in Cerrado soils under native vegetation subjected to prescribed fire and under pasture

The objective of this work was to evaluate the effects of fire regimes and vegetation cover on the structure and dynamics of soil microbial communities, through phospholipid fatty acid (PLFA) analysis. Comparisons were made between native areas with different woody covers ("cerrado stricto sensu" and "campo sujo"), under different fire regimes, and a 20-year-old active palisadegrass pasture in the Central Plateau of Brazil. Microbial biomass was higher in the native plots than in the pasture, and the highest monthly values were observed during the rainy season in the native plots. No significant differences were observed between fire regimes or between communities from the two native vegetation types. However, the principal component (PC) analysis separated the microbial communities by vegetation cover (native x pasture) and season (wet x dry), accounting for 45.8% (PC1 and PC3) and 25.6% (PC2 and PC3), respectively, of the total PLFA variability. Changes in land cover and seasonal rainfall in Cerrado ecosystems have significant effects on the total density of soil microorganisms and on the abundance of microbial groups, especially Gram-negative and Gram-positive bacteria.

Genetic dissimilarity among jabuticaba trees native to southwestern Paraná, Brazil

Knowledge on the genetic diversity within and between genotype groups is of great importance for breeding programs. The purpose of this study was to estimate the genetic dissimilarity among 36 native jabuticaba trees (Plinia cauliflora) from five sites in the southwestern region of Paraná, Brazil. Sixteen fruit traits were analyzed, based on multivariate techniques (canonical variables, Tocher and UPGMA), using Mahalanobis' distance as dissimilarity measure. By the techniques of clustering and graphic dispersion, together with the comparison of means, the genetic diversity among native jabuticaba trees was efficiently identified, indicating a high potential of these genotypes for breeding programs. The traits of greatest importance for dissimilarity were percentage of pulp and of skin, which are easily measured. The clustering structure is related to the collection sites and for breeding programs, genotypes from different sites should be crossed to generate progenies to be tested. Genotypes 'CV5' and 'VT3' should be conserved in genebanks, due to its important agronomic traits.

Diversity and genetic structure in natural populations of Hancornia speciosa var. speciosa Gomes in northeastern Brazil

Hancornia speciosa Gomes is a fruit tree native from Brazil that belongs to Apocinaceae family, and is popularly known as Mangabeira. Its fruits are widely consumed raw or processed as fruit jam, juices and ice creams, which have made it a target of intense exploitation. The extractive activities and intense human activity on the environment of natural occurrence of H. speciosa has caused genetic erosion in the species and little is known about the ecology or genetic structure of natural populations. The objective of this research was the evaluation of the genetic diversity and genetic structure of H. speciosa var. speciosa. The genetic variability was assessed using 11 allozyme loci with a sample of 164 individuals distributed in six natural populations located in the States of Pernambuco and Alagoas, Northeastern Brazil. The results showed a high level of genetic diversity within the species (e= 0.36) seeing that the most of the genetic variability of H. speciosa var. speciosa is within its natural populations with low difference among populations ( or = 0.081). The inbreeding values within ( = -0.555) and among populations ( =-0.428) were low showing lacking of endogamy and a surplus of heterozygotes. The estimated gene flow ( m ) was high, ranging from 2.20 to 13.18, indicating to be enough to prevent the effects of genetic drift and genetic differentiation among populations. The multivariate analyses indicated that there is a relationship between genetic and geographical distances, which was confirmed by a spatial pattern analysis using Mantel test (r = 0.3598; p = 0.0920) with 1000 random permutations. The high genetic diversity index in these populations indicates potential for in situ genetic conservation.

Structure-Based, Rational Design of T Cell Receptors.

Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce T cell receptor (TCR) modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction with peptides (p) bound to major histocompatibility complexes (MHC). Using the well-characterized 2C TCR/SIYR/H-2K(b) structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157-165 cancer-testis epitope. Fifty-four percent of the designed sequence replacements exhibited improved pMHC binding as compared to the native TCR, with up to 150-fold increase in affinity, while preserving specificity. Genetically engineered CD8(+) T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity, K D = ∼1 - 5 μM. Beyond the affinity threshold at K D < 1 μM we observed an attenuation in cellular function, in line with the "half-life" model of T cell activation. Our computer-aided protein-engineering approach requires the 3D-structure of the TCR-pMHC complex of interest, which can be obtained from X-ray crystallography. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes when experimental data is not available. Since the accuracy of the models depends on the prediction of the TCR orientation over pMHC, we have complemented the approach with a simplified rigid method to predict this orientation and successfully assessed it using all non-redundant TCR-pMHC crystal structures available. These methods potentially extend the use of our TCR engineering method to entire TCR repertoires for which no X-ray structure is available. We have also performed a steered molecular dynamics study of the unbinding of the TCR-pMHC complex to get a better understanding of how TCRs interact with pMHCs. This entire rational TCR design pipeline is now being used to produce rationally optimized TCRs for adoptive cell therapies of stage IV melanoma.

Experimental assessment of the effects of a Neotropical nocturnal piscivore on juvenile native and invasive fishes

We experimentally examined the predator-prey relationships between juvenile spotted sorubim Pseudoplastystoma corruscans and young-of-the-year invasive and native fish species of the Paraná River basin, Brazil. Three invasive (peacock bass Cichla piquiti, Nile tilapia Oreochromis niloticus, and channel catfish Ictalurus punctatus) and two native (yellowtail tetra Astyanax altiparanae and streaked prochilod Prochilodus lineatus) fish species were offered as prey to P. corruscans in 300 L aquaria with three habitat complexity treatments (0%, 50% and 100% structure-covered). Prey survival was variable through time and among species (C. piquiti < O. niloticus < A. altiparanae < P. lineatus < I. punctatus), depending largely on species-specific prey behavior but also on prey size and morphological defenses. Habitat complexity did not directly affect P. corruscans piscivory but some prey species changed their microhabitat use and shoaling behavior among habitat treatments in predator’s presence. Pseudoplatystoma corruscans preyed preferentially on smaller individuals of those invasive species with weak morphological defensive features that persisted in a non-shoaling behavior. Overall, our results contrast with those in a companion experiment using a diurnal predator, suggesting that nocturnal piscivores preferentially prey on different (rather diurnal) fish species and are less affected by habitat complexity. Our findings suggest that recovering the native populations of P. corruscans might help controling some fish species introduced to the Paraná River basin, particularly C. piquiti and O. niloticus, whose parental care is expected to be weak or null at night

Labour segregation and immigrant and native-born wage distributions in Spain: an analysis using matched employer-employee data

This article carries out an empirical examination of the origin of the differences between immigrant and native-born wage structures in the Spanish labour market. Especial attention is given in the analysis to the role played by occupational and workplace segregation of immigrants. Legal immigrants from developing countries exhibit lower mean wages and a more compressed wage structure than native-born workers. By contrast, immigrants from developed countries display higher mean wages and a more dispersed wage structure. The main empirical finding is that the disparities in the wage distributions for the native-born and both groups of immigrants are largely explained by their different observed characteristics, with a particularly important influence in this context of workplace and, particularly, occupational segregation.

Gap junctions in cells of the immune system: structure, regulation and possible functional roles

Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family. According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps of the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response of the immune system.

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60% of activity is retained at 80ºC for 20 min). N-Acylation increased its ordered structure by 4% and decreased its ß-T1 structure by 2%. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures.

Orientation of Non-Native 2-Monosubstituted and 2,3-Disubstituted 1,4-Naphthoquinones in the A1 binding site of PSI and the effect on the rate of electron transfer : an electron paramagnetic resonance spectroscopy study

The proce-ss ofoxygenic photosynthesis is vital to life on Earth. the central event in photosynthesis is light induced electron transfer that converts light into energy for growth. Ofparticular significance is the membrane bound multisubunit protein known as Photosystem I (PSI). PSI is a reaction centre that is responsible for the transfer of electrons across the membrane to reduce NADP+ to NADPH. The recent publication ofa high resolution X-ray structure of PSI has shown new information about the structure, in particular the electron transfer cofactors, which allows us to study it in more detail. In PSI, the secondary acceptor is crucial for forward electron transfer. In this thesis, the effect of removing the native acceptor phylloquinone and replacing it with a series of structurally related quinones was investigated via transient electron paramagnetic resonance (EPR) experiments. The orientation of non native quinones in the binding site and their ability to function in the electron transfer process was determined. It was found that PSI will readily accept alkyl naphthoquinones and anthraquinone. Q band EPR experiments revealed that the non-native quinones are incorporated into the binding site with the same orientation of the headgroup as in the native system. X band EPR spectra and deuteration experiments indicate that monosubstituted naphthoquinones are bound to the Al site with their side group in the position occupied by the methyl group in native PSI (meta to the hydrogen bonded carbonyl oxygen). X band EPR experiments show that 2, 3- disubstituted methyl naphthoquinones are also incorporated into the Al site in the same orientation as phylloquinone, even with the presence of a halogen- or sulfur-containing side chain in the position normally occupied by the phytyl tail ofphylloquinone. The exception to this is 2-bromo-3-methyl --.- _. -. - -- - - 4 _._ _ _ - _ _ naphthoquinone which has a poorly resolved spectrum, making determination of the orientation difficuh. All of the non-native quinones studied act as efficient electron acceptors. However, forward electron transfer past the quinone could only be demonstrated for anthraquinone, which has a more negative midpoint potential than phylloquinone. In the case of anthraquinone, an increased rate of forward electron transfer compared to native PSI was found. From these results we can conclude that the rate ofelectron transfer from Al to Fx in native PSI lies in the normal region ofthe Marcus Curve.

Polyglutamine monomer structure and its implications for molecular self-assembly

Polyglutamine is a naturally occurring peptide found within several proteins in neuronal cells of the brain, and its aggregation has been implicated in several neurodegenerative diseases, including Huntington's disease. The resulting aggregates have been demonstrated to possess ~-sheet structure, and aggregation has been shown to start with a single misfolded peptide. The current project sought to computationally examine the structural tendencies of three mutant poly glutamine peptides that were studied experimentally, and found to aggregate with varying efficiencies. Low-energy structures were generated for each peptide by simulated annealing, and were analyzed quantitatively by various geometry- and energy-based methods. According to the results, the experimentally-observed inhibition of aggregation appears to be due to localized conformational restraint placed on the peptide backbone by inserted prolines, which in tum confines the peptide to native coil structure, discouraging transition towards the ~sheet structure required for aggregation. Such knowledge could prove quite useful to the design of future treatments for Huntington's and other related diseases.

Inventing interventions : strategies of reappropriation in Native American and First Nations literatures

Ma thèse de doctorat, intitulée Inventing Interventions: Strategies of Reappropriation in Native and First Nations Literatures traite du sujet de la réappropriation de la langue anglaise et de la langue française dans les littératures autochtones du Canada et des États-Unis, en tant que stratégie d’intervention de re-narration et de récupération. De fait, mon projet fait abstraction, autant que possible, des frontières nationales et linguistiques, vu que celles-ci sont essentiellement des constructions culturelles et coloniales. Ainsi, l’acte de réappropriation de la langue coloniale implique non seulement la maîtrise de base de cette dernière à des fins de communication, cela devient un moyen envers une fin : au lieu d’être possédés par la langue, les auteurs sur lesquels je me penche ici possèdent à présent cette dernière, et n’y sont plus soumis. Les tensions qui résultent d’un tel processus sont le produit d’une transition violente imposée et expérimentale d’une réalité culturelle à une autre, qui, pour plusieurs, n’a pas réussie et s’est, au contraire, effritée sur elle-même. Je soutiens donc que les auteurs autochtones ont créé un moyen à travers l’expression artistique et politique de répondre (dans le sens de « write back ») à l’oppression et l’injustice. À travers l’analyse d’oeuvres contemporaines écrites en anglais ou en français, que ce soit de la fiction, de l’autobiographie, de la poésie, du théâtre, de l’histoire ou du politique, ma recherche se structure autour de quatre concepts spécifiques : la langue, la résistance, la mémoire, et le lieu. J’examine comment ces concepts sont mis en voix, et comment ils sont interdépendants et s’affectent à l’intérieur du discours particulier issu des littératures autochtones et des différentes stratégies d’intervention (telles la redéfinition ou l’invention) et du mélange de différentes formules littéraires.

Genetic diversity and structure of natural and managed populations of Cedrus atlantica (Pinaceae) assessed using random amplified polymorphic DNA

Cedrus atlantica (Pinaceae) is a large and exceptionally long-lived conifer native to the Rif and Atlas Mountains of North Africa. To assess levels and patterns of genetic diversity of this species. samples were obtained throughout the natural range in Morocco and from a forest plantation in Arbucies, Girona (Spain) and analyzed using RAPD markers. Within-population genetic diversity was high and comparable to that revealed by isozymes. Managed populations harbored levels of genetic variation similar to those found in their natural counterparts. Genotypic analyses Of Molecular variance (AMOVA) found that most variation was within populations. but significant differentiation was also found between populations. particularly in Morocco. Bayesian estimates of F,, corroborated the AMOVA partitioning and provided evidence for Population differentiation in C. atlantica. Both distance- and Bayesian-based Clustering methods revealed that Moroccan populations comprise two genetically distinct groups. Within each group, estimates of population differentiation were close to those previously reported in other gymnosperms. These results are interpreted in the context of the postglacial history of the species and human impact. The high degree of among-group differentiation recorded here highlights the need for additional conservation measures for some Moroccan Populations of C. atlantica.

Structure and function analysis of the poliovirus cis-acting replication element (CRE)

The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5' noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CD(pro) complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.