Molecular modeling study of the binding and signaling properties of the TCRpMHC complex

The activation of the specific immune response against tumor cells is based on the recognition by the CD8+ Cytotoxic Τ Lymphocytes (CTL), of antigenic peptides (p) presented at the surface of the cell by the class I major histocompatibility complex (MHC). The ability of the so-called T-Cell Receptors (TCR) to discriminate between self and non-self peptides constitutes the most important specific control mechanism against infected cells. The TCR/pMHC interaction has been the subject of much attention in cancer therapy since the design of the adoptive transfer approach, in which Τ lymphocytes presenting an interesting response against tumor cells are extracted from the patient, expanded in vitro, and reinfused after immunodepletion, possibly leading to cancer regression. In the last decade, major progress has been achieved by the introduction of engineered lypmhocytes. In the meantime, the understanding of the molecular aspects of the TCRpMHC interaction has become essential to guide in vitro and in vivo studies. In 1996, the determination of the first structure of a TCRpMHC complex by X-ray crystallography revealed the molecular basis of the interaction. Since then, molecular modeling techniques have taken advantage of crystal structures to study the conformational space of the complex, and understand the specificity of the recognition of the pMHC by the TCR. In the meantime, experimental techniques used to determine the sequences of TCR that bind to a pMHC complex have been used intensively, leading to the collection of large repertoires of TCR sequences that are specific for a given pMHC. There is a growing need for computational approaches capable of predicting the molecular interactions that occur upon TCR/pMHC binding without relying on the time consuming resolution of a crystal structure. This work presents new approaches to analyze the molecular principles that govern the recognition of the pMHC by the TCR and the subsequent activation of the T-cell. We first introduce TCRep 3D, a new method to model and study the structural properties of TCR repertoires, based on homology and ab initio modeling. We discuss the methodology in details, and demonstrate that it outperforms state of the art modeling methods in predicting relevant TCR conformations. Two successful applications of TCRep 3D that supported experimental studies on TCR repertoires are presented. Second, we present a rigid body study of TCRpMHC complexes that gives a fair insight on the TCR approach towards pMHC. We show that the binding mode of the TCR is correctly described by long-distance interactions. Finally, the last section is dedicated to a detailed analysis of an experimental hydrogen exchange study, which suggests that some regions of the constant domain of the TCR are subject to conformational changes upon binding to the pMHC. We propose a hypothesis of the structural signaling of TCR molecules leading to the activation of the T-cell. It is based on the analysis of correlated motions in the TCRpMHC structure. - L'activation de la réponse immunitaire spécifique dirigée contre les cellules tumorales est basée sur la reconnaissance par les Lymphocytes Τ Cytotoxiques (CTL), d'un peptide antigénique (p) présenté à la suface de la cellule par le complexe majeur d'histocompatibilité de classe I (MHC). La capacité des récepteurs des lymphocytes (TCR) à distinguer les peptides endogènes des peptides étrangers constitue le mécanisme de contrôle le plus important dirigé contre les cellules infectées. L'interaction entre le TCR et le pMHC est le sujet de beaucoup d'attention dans la thérapie du cancer, depuis la conception de la méthode de transfer adoptif: les lymphocytes capables d'une réponse importante contre les cellules tumorales sont extraits du patient, amplifiés in vitro, et réintroduits après immunosuppression. Il peut en résulter une régression du cancer. Ces dix dernières années, d'importants progrès ont été réalisés grâce à l'introduction de lymphocytes modifiés par génie génétique. En parallèle, la compréhension du TCRpMHC au niveau moléculaire est donc devenue essentielle pour soutenir les études in vitro et in vivo. En 1996, l'obtention de la première structure du complexe TCRpMHC à l'aide de la cristallographie par rayons X a révélé les bases moléculaires de l'interaction. Depuis lors, les techniques de modélisation moléculaire ont exploité les structures expérimentales pour comprendre la spécificité de la reconnaissance du pMHC par le TCR. Dans le même temps, de nouvelles techniques expérimentales permettant de déterminer la séquence de TCR spécifiques envers un pMHC donné, ont été largement exploitées. Ainsi, d'importants répertoires de TCR sont devenus disponibles, et il est plus que jamais nécessaire de développer des approches informatiques capables de prédire les interactions moléculaires qui ont lieu lors de la liaison du TCR au pMHC, et ce sans dépendre systématiquement de la résolution d'une structure cristalline. Ce mémoire présente une nouvelle approche pour analyser les principes moléculaires régissant la reconnaissance du pMHC par le TCR, et l'activation du lymphocyte qui en résulte. Dans un premier temps, nous présentons TCRep 3D, une nouvelle méthode basée sur les modélisations par homologie et ab initio, pour l'étude de propriétés structurales des répertoires de TCR. Le procédé est discuté en détails et comparé à des approches standard. Nous démontrons ainsi que TCRep 3D est le plus performant pour prédire des conformations pertinentes du TCR. Deux applications à des études expérimentales des répertoires TCR sont ensuite présentées. Dans la seconde partie de ce travail nous présentons une étude de complexes TCRpMHC qui donne un aperçu intéressant du mécanisme d'approche du pMHC par le TCR. Finalement, la dernière section se concentre sur l'analyse détaillée d'une étude expérimentale basée sur les échanges deuterium/hydrogène, dont les résultats révèlent que certaines régions clés du domaine constant du TCR sont sujettes à un changement conformationnel lors de la liaison au pMHC. Nous proposons une hypothèse pour la signalisation structurelle des TCR, menant à l'activation du lymphocyte. Celle-ci est basée sur l'analyse des mouvements corrélés observés dans la structure du TCRpMHC.

Antigen binding properties of purified immunoglobulin A and reconstituted secretory immunoglobulin A antibodies.

The hybridoma cell line ZAC3 expresses Vibrio cholerae lipopolysaccharide (LPS)-specific mouse IgA molecules as a heterogeneous population of monomeric (IgAm), dimeric (IgAd), and polymeric (IgAp) forms. We describe a gentle method combining ultrafiltration, ion-exchange chromatography, and size exclusion chromatography for the simultaneous and qualitative separation of the three molecular forms. Milligram quantities of purified IgA molecules were recovered allowing for direct comparison of the biological properties of the three forms. LPS binding specificity was tested after purification; IgAd and IgAp were found to bind strongly to LPS whereas IgAm did not. Secretory IgA (sIgA) could be reconstituted in vitro by combining recombinant secretory component (rSC) and purified IgAd or IgAp, but not IgAm. Surface plasmon resonance-based binding experiments using LPS monolayers indicated that purified reconstituted sIgA and IgA molecules recognize LPS with identical affinity (KA 1.0 x 10(8)M-1). Thus, this very sensitive assay provides the first evidence that the function of SC in sIgA complex is not to modify the affinity for the antigen. KA falls to 6.6 x 10(5) M-1 when measured by calorimetry using detergent-solubilized LPS and IgA, suggesting that the LPS environment is critical for recognition by the antibody.

Short and long-term effects of tillage systems and nutrient sources on soil physical properties of a Southern Brazilian Hapludox

Soil tillage promotes changes in soil structure. The magnitude of the changes varies with the nature of the soil, tillage system and soil water content and decreases over time after tillage. The objective of this study was to evaluate short-term (one year period) and long-term (nine year period) effects of soil tillage and nutrient sources on some physical properties of a very clayey Hapludox. Five tillage systems were evaluated: no-till (NT), chisel plow + one secondary disking (CP), primary + two (secondary) diskings (CT), CT with burning of crop residues (CTb), and CT with removal of crop residues from the field (CTr), in combination with five nutrient sources: control without nutrient application (C); mineral fertilizers, according to technical recommendations for each crop (MF); 5 Mg ha-1 yr-1 of poultry litter (wetmatter) (PL); 60 m³ ha-1 yr-1 of cattle slurry (CS) and; 40 m³ ha-1 yr-1 of swine slurry (SS). Bulk density (BD), total porosity (TP), and parameters related to the water retention curve (macroporosity, mesoporosity and microporosity) were determined after nine years and at five sampling dates during the tenth year of the experiment. Soil physical properties were tillage and time-dependent. Tilled treatments increased total porosity and macroporosity, and reduced bulk density in the surface layer (0.00-0.05 m), but this effect decreased over time after tillage operations due to natural soil reconsolidation, since no external stress was applied in this period. Changes in pore size distribution were more pronounced in larger and medium pore diameter classes. The bulk density was greatest in intermediate layers in all tillage treatments (0.05-0.10 and 0.12-0.17 m) and decreased down to the deepest layer (0.27-0.32 m), indicating a more compacted layer around 0.05-0.20 m. Nutrient sources did not significantly affect soil physical and hydraulic properties studied.

Surface states in template synthesized tin oxide nanoparticles

Tin-oxide nanoparticles with controlled narrow size distributions are synthesized while physically encapsulated inside silica mesoporous templates. By means of ultraviolet-visible spectroscopy, a redshift of the optical absorbance edge is observed. Photoluminescence measurements corroborate the existence of an optical transition at 3.2 eV. The associated band of states in the semiconductor gap is present even on template-synthesized nanopowders calcined at 800°C, which contrasts with the evolution of the gap states measured on materials obtained by other methods. The gap states are thus considered to be surface localized, disappearing with surface faceting or being hidden by the surface-to-bulk ratio decrease.

Enhancement of the photoelectrochemical properties of Cl-doped ZnO nanowires by tuning their coaxial doping profile

Arrays of vertically aligned ZnO:Cl/ZnO core-shell nanowires were used to demonstrate that the control of the coaxial doping profile in homojunction nanostructures can improve their surface charge carrier transfer while conserving potentially excellent transport properties. It is experimentally shown that the presence of a ZnO shell enhances the photoelectrochemical properties of ZnO:Cl nanowires up to a factor 5. Likewise, the ZnO shell promotes the visible photoluminescence band in highly conducting ZnO:Cl nanowires. These lines of evidence are associated with the increase of the nanowires" surface depletion layer

Influence of premetallization surface treatment on the formation of Schottky Au-nGaN contacts

The influence of premetallization surface preparation on the structural, chemical, and electrical properties of Au-nGaN interfaces has been investigated by x-ray photoemission spectroscopy (XPS), current-voltage measurement (I-V) and cross-section transmission electron microscopy (TEM). XPS analysis showed that the three GaN substrate treatments investigated i.e., ex situ hydrofluoric acid etch, in situ anneal in ultrahigh-vacuum (UHV), and in situ Ga reflux cleaning in UHV result in surfaces increasingly free of oxygen contamination. XPS and TEM characterization of Au-nGaN formed after the three premetallization surface treatments show that HF etching and UHV annealing produce abrupt, well-defined interfaces. Conversely, GaN substrate cleaning in a Ga flux results in Au/GaN intermixing. I-V characterization of Au¿nGaN contacts yields a Schottky barrier height of 1.25 eV with a very low-ideality factor and very good contact uniformity for the premetallization UHV anneal, while the Ga reflux cleaning results in a much lower barrier (0.85 eV), with poor ideality and uniformity. I-V and XPS results suggest a high density of acceptor states at the surface, which is further enhanced by UHV annealing. These results are discussed in the context of current models of Schottky barrier formation.

Aluminum hydroxy-interlayered minerals and chemical properties of a subtropical Brazilian Oxisol under no-tillage and conventional tillage

No tillage systems significantly influence the soil system, but knowledge about the effects on the mineralogy of tropical and subtropical soils is limited. This study evaluated the long-term effects (26 years) of no-tillage (NT) on aluminum hydroxy-interlayered minerals of a subtropical Oxisol in Southern Brazil (Guarapuava, PR), compared to the same soil under conventional tillage (CT). The clay fraction (< 2 µm) in soil samples of the surface horizons of a field experiment under both management systems was analyzed by X-ray diffraction (XRD) to identify and characterize Al hydroxy-interlayered minerals before and after treatment with sodium citrate to remove intra-layer material. Soil liquid (solution) and solid phases were also characterized. The contents of total organic C, exchangeable cations, P, and the values of extractable acidity and cation exchange capacity as well as electrical conductivity and levels of dissolved organic C, basic cations, aluminum, Si, and sulfur in the soil solution were higher in the NT soil. Under both soil management systems, more than 90 % of the total soluble Al was complexed with organic compounds, with similar Al activity. No significant changes were detected by 2:1 clay mineral XRD analyses in terms of extension or intercalation of Al-hydroxy-polymers in the no-tilled in comparison to the conventionally tilled soil. In both soil management systems, Al and Si activities in the soil solution indicated thermodynamic stability of 2:1 clay minerals with partially occupied by hydroxy-Al, suggesting deceleration in the intercalation process and a tendency of transforming clay minerals from extensive into partial intercalation.

Physical properties of a humic cambisol under tillage and cropping systems after twelve years

Soil is the basis underlying the food production chain and it is fundamental to improve and conserve its productive capacity. Imbalanced exploitation can degrade agricultural areas physical, chemical and biologically. The objective of this study was to evaluate some soil physical properties and their relation with organic carbon contents of a Humic Dystrudept under conventional tillage (CT) and no-tillage (NT), for 12 years in rotation (r) and succession (s) cropping systems. The experiment was carried out in Lages, SC (latitude 27 º 49 ' S and longitude 50 º 20 ' W, 937 m asl), using crop sequences of bean-fallow-maize-fallow-soybean in conventional tillage rotation; maize-fallow in conventional tillage succession; bean-oat-maize-turnip-soybean-vetch in no-tillage rotation; and maize-vetch in no-tillage succession. The experimental design was completely randomized with four replications. The soil samples were collected in the layers 0-2.5, 2.5-5, 5-10, and 10-20 cm. The following properties were analyzed: soil density, porosity, aggregate stability, degree of flocculation, water retention, infiltration, mechanical strength, and total organic carbon. Soil aggregation in the surface layer (0-5 cm) was better in the no-tillage than the conventional system, related to higher microporosity, organic carbon contents and water retention capacity, indicating that a periodical tillage of this soil is unnecessary. Infiltration was highest in no-tillage with crop succession.

Microbial biomass and soil chemical properties under different land use systems in northeastern Pará

The increase in agricultural production in the Brazilian Amazon region is mostly a result of the agricultural frontier expansion, into areas previously influenced by humans or of native vegetation. At the same time, burning is still used to clear areas in small-scale agricultural systems, leading to a loss of the soil productive capacity shortly after, forcing the opening of new areas. This study had the objective of evaluating the effect of soil preparation methods that involve plant residue shredding, left on the surface or incorporated to the soil, with or without chemical fertilization, on the soil chemical and biological properties. The experiment was conducted in 1995, in an experimental field of Yellow Latosol (Oxisol) of the Embrapa Amazônia Oriental, northeastern Pará (Brazil). The experiment was arranged in randomized blocks, in a 2x6 factorial design, with two management systems and six treatments evaluated twice. The management systems consisted of rice (Oriza sativa), followed by cowpea (Vigna unguiculata) with manioc (Manihot esculenta). In the first system the crops were planted in two consecutive cycles, followed by a three-year fallow period (natural regrowth); the second system consisted of one cultivation cycle and was left fallow for three years. The following treatments were applied to the secondary forest vegetation: slash and burn, fertilized with NPK (Q+NPK); slash and burn, without fertilizer NPK (Q-NPK); cutting and shredding, leaving the residues on the soil surface, fertilized with NPK (C+NPK); cutting and shredding, leaving residues on the soil surface, without fertilizer (C-NPK); cutting and shredding, with residue incorporation and fertilized with NPK (I+NPK); cutting and shredding, with residue incorporation and without NPK fertilizer (I-NPK). The soil was sampled in the rainier season (April 2006) and in the drier season (September 2006), in the 0-0.1 m layer. From each plot, 10 simple samples were collected in order to generate a composite sample. In the more intensive management system the contents of microbial C (Cmic) and microbial N (Nmic) were higher, while the C (Corg) level was higher in the less intensive system. The treatments with highest Cmic and Nmic levels were those with cutting, shredding and distribution of biomass on the soil surface. Under both management systems, the chemical characteristics were in ranges that classify the soil as little fertile, although P and K (in the rainy season) were higher in the less intensive management system.

Nuclear pairing: surface or bulk?

We analyze how the spatial localization properties of pairing correlations are changing in a major neutron shell of heavy nuclei. It is shown that the radial distribution of the pairing density depends strongly on whether the chemical potential is close to a low or a high angular momentum level and has little sensitivity to whether the pairing force acts at the surface or in the bulk. The pairing density averaged over one major shell is, however, rather flat, exhibiting little dependence on the pairing force. Hartree-Fock-Bogoliubov calculations for the isotopic chain 100-132Sn are presented for demonstration purposes.

Soil chemical properties related to acidity under successive pig slurry application

Pig slurry application as soil manure can alter the chemical properties of the soil and affect its acidity, modifying the environment for crop growth and development. The objective of this study was to evaluate the chemical properties related to soil acidity subjected to successive applications of pig slurry. The experiment was conducted in May 2000, in an experimental area of the Federal University of Santa Maria (UFSM) under no-tillage and lasted until January 2008. Nineteen surface applications of 0, 20, 40, and 80 m³ ha-1 of pig slurry were performed, during a period of 100 months and the soil sampled in the end (layers 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-25, 25-30, 30-35, 35-40, 40-50 and 50-60 cm). The application of pig slurry increased soil pH values, an effect that could reach the depth of 8 cm without affecting the potential acidity values. The applications also resulted in accumulation of Ca and Mg exchangeable levels in the surface layers, increasing base saturation and reducing Al saturation. Long-term applications induced an increase in organic matter in the deeper layers. However, the effect of this residue on the potential CEC was less significant and restricted to the surface layers.

Soil uses in the sugarcane fallow period to improve chemical and physical properties of two latosols (oxisols)

Sugarcane production should be integrated with crop diversification with a view to competitive and sustainable results in economic, social and environmental aspects. The purpose of this study was to assess the influence of different soil uses during the sugarcane fallow period on the chemical and physical properties of eutroferric Red Latosol - LVef (Oxisol) and Acric Latosol - LVw (Acric Oxisol), in Jaboticabal, São Paulo State, Brazil (21º14'05'' S, 48º17'09'' W, 600 m asl). A randomized block design was used with five replications and four treatments, consisting of different soil uses (crops) in the sugarcane fallow period: soybean only, soybean/fallow/soybean, soybean/millet/soybean, and soybean/sunn hemp/soybean. After two soybean crops, the LVef chemical properties remained at intermediate to high levels; while those of the LVw, classified as intermediate to high in the beginning, increased to high levels. Thus, the different soil uses during the sugarcane fallow period allowed the maintenance of LVef fertility levels and the improvement of those of the LVw. Two soybean crops increased macroporosity in the 0.0-0.1 m layer of the LVef; reduced soil aggregates in the 0.0-0.1 and 0.1-0.2 m layers of both soils, and reduced aggregate stability in these two layers of the LVw. Planting pearl millet or sunn hemp between the two soybean growing seasons promoted the formation of larger soil aggregates in the surface layer (0.0-0.1 m) of the LVw.

Physical properties and particle-size fractions of soil organic matter in crop-livestock integration

Crop-livestock integration represents an interesting alternative of soil management, especially in regions where the maintenance of cover crops in no-tillage systems is difficult. The objective of this study was to evaluate soil physical and chemical properties, based on the hypothesis that a well-managed crop-livestock integration system improves the soil quality and stabilizes the system. The experiment was set up in a completely randomized design, with five replications. The treatments were arranged in a 6 x 4 factorial design, to assess five crop rotation systems in crop-livestock integration, and native forest as reference of soil undisturbed by agriculture, in four layers (0.0-0.05; 0.05-0.10; 0.10-0.15 and 0.15-0.20 m). The crop rotation systems in crop-livestock integration promoted changes in soil physical and chemical properties and the effects of the different systems were mainly detected in the surface layer. The crops in integrated crop-livestock systems allowed the maintenance of soil carbon at levels equal to those of the native forest, proving the efficiency of these systems in terms of soil conservation. The systems influenced the environmental stability positively; the soil quality indicator mineral-associated organic matter was best related to aggregate stability.

Time-dependent couplings and crossover length scales in nonequilibrium surface roughening

We show that time-dependent couplings may lead to nontrivial scaling properties of the surface fluctuations of the asymptotic regime in nonequilibrium kinetic roughening models. Three typical situations are studied. In the case of a crossover between two different rough regimes, the time-dependent coupling may result in anomalous scaling for scales above the crossover length. In a different setting, for a crossover from a rough to either a flat or damping regime, the time-dependent crossover length may conspire to produce a rough surface, although the most relevant term tends to flatten the surface. In addition, our analysis sheds light into an existing debate in the problem of spontaneous imbibition, where time-dependent couplings naturally arise in theoretical models and experiments.