Differential binding to HLA-C of p50-activating and p58-inhibitory natural killer cell receptors

Natural killer (NK) cell cytotoxicity is regulated in large part by the expression of NK cell receptors able to bind class I major histocompatibility complex glycoproteins. The receptors associated with recognition of HLA-C allospecificities are the two-domain Ig-like molecules, p50 and p58 proteins, with highly homologous extracellular domains but differing in that they have either an activating or inhibitory function, respectively, depending on the transmembrane domain and cytoplasmic tails that they possess. We have compared the binding to HLA-Cw7 of an inhibitory p58 molecule, NKAT2, the highly homologous activating p50 molecule, clone 49, and a second activating p50 molecule, clone 39, which has homologies to both NKAT1 and NKAT2. NKAT2 binds to HLA-Cw7 with very rapid association and dissociation rates. However, the p50 receptors bind only very weakly, if at all, to HLA-C. The molecular basis of this difference is analyzed, and the functional significance of these observations is discussed.

BiP and Immunoglobulin Light Chain Cooperate to Control the Folding of Heavy Chain and Ensure the Fidelity of Immunoglobulin Assembly

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP–heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

Structural interactions of fibroblast growth factor receptor with its ligands

Fibroblast growth factors (FGFs) effect cellular responses by binding to FGF receptors (FGFRs). FGF bound to extracellular domains on the FGFR in the presence of heparin activates the cytoplasmic receptor tyrosine kinase through autophosphorylation. We have crystallized a complex between human FGF1 and a two-domain extracellular fragment of human FGFR2. The crystal structure, determined by multiwavelength anomalous diffraction analysis of the selenomethionyl protein, is a dimeric assemblage of 1:1 ligand:receptor complexes. FGF is bound at the junction between the two domains of one FGFR, and two such units are associated through receptor:receptor and secondary ligand:receptor interfaces. Sulfate ion positions appear to mark the course of heparin binding between FGF molecules through a basic region on receptor D2 domains. This dimeric assemblage provides a structural mechanism for FGF signal transduction.

Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: A model for viral DNA binding

Insolubility of full-length HIV-1 integrase (IN) limited previous structure analyses to individual domains. By introducing five point mutations, we engineered a more soluble IN that allowed us to generate multidomain HIV-1 IN crystals. The first multidomain HIV-1 IN structure is reported. It incorporates the catalytic core and C-terminal domains (residues 52–288). The structure resolved to 2.8 Å is a Y-shaped dimer. Within the dimer, the catalytic core domains form the only dimer interface, and the C-terminal domains are located 55 Å apart. A 26-aa α-helix, α6, links the C-terminal domain to the catalytic core. A kink in one of the two α6 helices occurs near a known proteolytic site, suggesting that it may act as a flexible elbow to reorient the domains during the integration process. Two proteins that bind DNA in a sequence-independent manner are structurally homologous to the HIV-1 IN C-terminal domain, suggesting a similar protein–DNA interaction in which the IN C-terminal domain may serve to bind, bend, and orient viral DNA during integration. A strip of positively charged amino acids contributed by both monomers emerges from each active site of the dimer, suggesting a minimally dimeric platform for binding each viral DNA end. The crystal structure of the isolated catalytic core domain (residues 52–210), independently determined at 1.6-Å resolution, is identical to the core domain within the two-domain 52–288 structure.

Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail

Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a prime target for drugs and herbicides. We have identified the interaction of EPSP synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography. The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain cleft. Glyphosate appears to occupy the binding site of the second substrate of EPSP synthase (phosphoenol pyruvate), mimicking an intermediate state of the ternary enzyme⋅substrates complex. The elucidation of the active site of EPSP synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs.

Phosphorylation-dependent conformational changes in OmpR, an osmoregulatory DNA-binding protein of Escherichia coli.

Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC. We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two-domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the alpha subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.

Conception d’un mécanisme déployable à grand ratio d’expansion et de son système d’actionnement par roues d’inertie pour applications spatiales

Cette thèse propose de développer des mécanismes déployables pour applications spatiales ainsi que des modes d’actionnement permettant leur déploiement et le contrôle de l’orientation en orbite de l’engin spatial les supportant. L’objectif étant de permettre le déploiement de surfaces larges pour des panneaux solaires, coupoles de télécommunication ou sections de station spatiale, une géométrie plane simple en triangle est retenue afin de pouvoir être assemblée en différents types de surfaces. Les configurations à membrures rigides proposées dans la littérature pour le déploiement de solides symétriques sont optimisées et adaptées à l’expansion d’une géométrie ouverte, telle une coupole. L’optimisation permet d’atteindre un ratio d’expansion plan pour une seule unité de plus de 5, mais présente des instabilités lors de l’actionnement d’un prototype. Le principe de transmission du mouvement d’un étage à l’autre du mécanisme est revu afin de diminuer la sensibilité des performances du mécanisme à la géométrie de ses membrures internes. Le nouveau modèle, basé sur des courroies crantées, permet d’atteindre des ratios d’expansion plans supérieurs à 20 dans certaines configurations. L’effet des principaux facteurs géométriques de conception est étudié afin d’obtenir une relation simple d’optimisation du mécanisme plan pour adapter ce dernier à différents contextes d’applications. La géométrie identique des faces triangulaires de chaque surface déployée permet aussi l’empilement de ces faces pour augmenter la compacité du mécanisme. Une articulation spécialisée est conçue afin de permettre le dépliage des faces puis leur déploiement successivement. Le déploiement de grandes surfaces ne se fait pas sans influencer lourdement l’orientation et potentiellement la trajectoire de l’engin spatial, aussi, différentes stratégies de contrôle de l’orientation novatrices sont proposées. Afin de tirer profit d’une grande surface, l’actionnement par masses ponctuelles en périphérie du mécanisme est présentée, ses équations dynamiques sont dérivées et simulées pour en observer les performances. Celles-ci démontrent le potentiel de cette stratégie de réorientation, sans obstruction de l’espace central du satellite de base, mais les performances restent en deçà de l’effet d’une roue d’inertie de masse équivalente. Une stratégie d’actionnement redondant par roue d’inertie est alors présentée pour différents niveaux de complexité de mécanismes dont toutes les articulations sont passives, c’est-à-dire non actionnées. Un mécanisme à quatre barres plan est simulé en boucle fermée avec un contrôleur simple pour valider le contrôle d’un mécanisme ciseau commun. Ces résultats sont étendus à la dérivation des équations dynamiques d’un mécanisme sphérique à quatre barres, qui démontre le potentiel de l’actionnement par roue d’inertie pour le contrôle de la configuration et de l’orientation spatiale d’un tel mécanisme. Un prototype à deux corps ayant chacun une roue d’inertie et une seule articulation passive les reliant est réalisé et contrôlé grâce à un suivi par caméra des modules. Le banc d’essai est détaillé, ainsi que les défis que l’élimination des forces externes ont représenté dans sa conception. Les résultats montrent que le système est contrôlable en orientation et en configuration. La thèse se termine par une étude de cas pour l’application des principaux systèmes développés dans cette recherche. La collecte de débris orbitaux de petite et moyenne taille est présentée comme un problème n’ayant pas encore eu de solution adéquate et posant un réel danger aux missions spatiales à venir. L’unité déployable triangulaire entraînée par courroies est dupliquée de manière à former une coupole de plusieurs centaines de mètres de diamètre et est proposée comme solution pour capturer et ralentir ces catégories de débris. Les paramètres d’une mission à cette fin sont détaillés, ainsi que le potentiel de réorientation que les roues d’inertie permettent en plus du contrôle de son déploiement. Près de 2000 débris pourraient être retirés en moins d’un an en orbite basse à 819 km d’altitude.

Deep convolutional neural networks for semantic video object segmentation

In this thesis, we propose to infer pixel-level labelling in video by utilising only object category information, exploiting the intrinsic structure of video data. Our motivation is the observation that image-level labels are much more easily to be acquired than pixel-level labels, and it is natural to find a link between the image level recognition and pixel level classification in video data, which would transfer learned recognition models from one domain to the other one. To this end, this thesis proposes two domain adaptation approaches to adapt the deep convolutional neural network (CNN) image recognition model trained from labelled image data to the target domain exploiting both semantic evidence learned from CNN, and the intrinsic structures of unlabelled video data. Our proposed approaches explicitly model and compensate for the domain adaptation from the source domain to the target domain which in turn underpins a robust semantic object segmentation method for natural videos. We demonstrate the superior performance of our methods by presenting extensive evaluations on challenging datasets comparing with the state-of-the-art methods.

Enriquecimento semântico de modelos ecológicos baseados em raciocínio qualitativo : um estudo de caso sobre a dinâmica de comunidades de vegetação lenhosa no cerrado brasileiro

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Ecologia, 2015.

Reconstructing the event stratigraphy from the complex structural - stratigraphic architecture of an Archaean volcanic – intrusive – sedimentary succession : the Boorara Domain, Eastern Goldfields Superterrane, Western Australia

Two main deformational phases are recognised in the Archaean Boorara Domain of the Kalgoorlie Terrane, Eastern Goldfields Superterrane, Yilgarn Craton, Western Australia, primarily involving southover- north thrust faulting that repeated and thickened the stratigraphy, followed by east northeast – west-southwest shortening that resulted in macroscale folding of the greenstone lithologies. The domain preserves mid-greenschist facies metamorphic grade, with an increase to lower amphibolite metamorphic grade towards the north of the region. As a result of the deformation and metamorphism, individual stratigraphic horizons are difficult to trace continuously throughout the entire domain. Volcanological and sedimentological textures and structures, primary lithological contacts, petrography and geochemistry have been used to correlate lithofacies between faultbounded structural blocks. The correlated stratigraphic sequence for the Boorara Domain comprises quartzo-feldspathic turbidite packages, overlain by high-Mg tholeiitic basalt (lower basalt), coherent and clastic dacite facies, intrusive and extrusive komatiite units, an overlying komatiitic basalt unit (upper basalt), and at the stratigraphic top of the sequence, volcaniclastic quartz-rich turbidites. Reconstruction of the stratigraphy and consideration of emplacement dynamics has allowed reconstruction of the emplacement history and setting of the preserved sequence. This involves a felsic, mafic and ultramafic magmatic system emplaced as high-level intrusions, with localised emergent volcanic centres, into a submarine basin in which active sedimentation was occurring.

School architecture; one-, two-, three- and four-room school buildings.

Mode of access: Internet.

Thermal conductivity of configurable two-dimensional carbon nanotube architecture and strain modulation

We reported the thermal conductivity of the two-dimensional carbon nanotube (CNT)-based architecture, which can be constructed through welding of single-wall CNTs by electron beam. Using large-scale nonequilibrium molecular dynamics simulations, the thermal conductivity is found to vary with different junction types due to their different phonon scatterings at the junction. The strong length and strain dependence of the thermal conductivity suggests an effective avenue to tune the thermal transport properties of the CNT-based architecture, benefiting the design of nanoscale thermal rectifiers or phonon engineering.

Interaction Between Two Residues in the Inter-Domain Interface of Escherichia coli Peptidase N Modulates Catalytic Activity

The role of interaction between Asn259 (catalytic domain) with Gln821 (C-terminal domain) in PeptidaseN was investigated. The k(cat) of PeptidaseN containing Asn259Asp or Gln821Glu is enhanced whereas it is suppressed in Asn259AspGln821Glu. Structural analysis shows this interaction to change the relative disposition of active site residues, which modulates catalytic activity.

Inhibition of Human Two-Pore Domain K+ Channel TREK1 by Local Anesthetic Lidocaine: Negative Cooperativity and Half-of-Sites Saturation Kinetics

TWIK-related K+ channel TREK1, a background leak K+ channel, has been strongly implicated as the target of several general and local anesthetics. Here, using the whole-cell and single-channel patch-clamp technique, we investigated the effect of lidocaine, a local anesthetic, on the human (h) TREK1 channel heterologously expressed in human embryonic kidney 293 cells by an adenoviral-mediated expression system. Lidocaine, at clinical concentrations, produced reversible, concentration-dependent inhibition of hTREK1 current, with IC50 value of 180 mu M, by reducing the single-channel open probability and stabilizing the closed state. We have identified a strategically placed unique aromatic couplet (Tyr352 and Phe355) in the vicinity of the protein kinase A phosphorylation site, Ser348, in the C-terminal domain (CTD) of hTREK1, that is critical for the action of lidocaine. Furthermore, the phosphorylation state of Ser348 was found to have a regulatory role in lidocaine-mediated inhibition of hTREK1. It is interesting that we observed strong intersubunit negative cooperativity (Hill coefficient = 0.49) and half-of-sites saturation binding stoichiometry (half-reaction order) for the binding of lidocaine to hTREK1. Studies with the heterodimer of wild-type (wt)-hTREK1 and Delta 119 C-terminal deletion mutant (hTREK1(wt)-Delta 119) revealed that single CTD of hTREK1 was capable of mediating partial inhibition by lidocaine, but complete inhibition necessitates the cooperative interaction between both the CTDs upon binding of lidocaine. Based on our observations, we propose a model that explains the unique kinetics and provides a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

Using Relationships for Matching Textual Domain Models with Existing Code

We address the task of mapping a given textual domain model (e.g., an industry-standard reference model) for a given domain (e.g., ERP), with the source code of an independently developed application in the same domain. This has applications in improving the understandability of an existing application, migrating it to a more flexible architecture, or integrating it with other related applications. We use the vector-space model to abstractly represent domain model elements as well as source-code artifacts. The key novelty in our approach is to leverage the relationships between source-code artifacts in a principled way to improve the mapping process. We describe experiments wherein we apply our approach to the task of matching two real, open-source applications to corresponding industry-standard domain models. We demonstrate the overall usefulness of our approach, as well as the role of our propagation techniques in improving the precision and recall of the mapping task.