Добірка наукової літератури з теми "Motifs de surface"

Protein surface motifs, which can be defined as commonly appearing patterns of shape and physical properties in protein molecular surfaces, can be considered "possible active sites". We have developed a system for mining surface motifs: SUMOMO which consists of two phases: surface motif extraction and surface motif filtering. In the extraction phase, a given set of protein molecular surface data is divided into small surfaces called unit surfaces. After extracting several common unit surfaces as candidate motifs, they are repetitively merged into surface motifs. However, a large amount of surface motifs is extracted in this phase, making it difficult to distinguish whether the extracted motifs are significant to be considered active sites. Since active sites from proteins with a particular function have similar shape and physical properties, proteins can be classified based on similarity among local surfaces. Thus, in the filtering phase, local surfaces extracted from proteins of the same group are considered significant motifs, and the rest are filtered out. The proposed method was applied to discover surface motifs from 15 proteins belonging to four function groups. Motifs corresponding to all 4 known functional sites were recognised.

The functional behaviour of manufactured surfaces is influenced by the surface topography. Encouraged by the results obtained in the implementation of two-dimensional motifs for practical profiles, a few attempts have been made to arrive at areal motifs for characterization of the surfaces. In this paper, an approach for establishing three-dimensional motifs based on triangular motif is presented. The triangular motif is formed with three peaks and a deep valley between them. To represent the surface by significant motifs, initial motifs are combined using four rules that are extended from the rules defined in ISO 12085 for combining two-dimensional motifs. Application of this technique to practical surfaces shows interesting results for characterizing the manufactured surfaces.

Minimal surfaces arise as energy minimizers for fluid membranes and are thus found in a variety of biological systems. The tight lamellar structures of the endoplasmic reticulum and plant thylakoids are comprised of such minimal surfaces in which right- and left-handed helical motifs are embedded in stoichiometry suggesting global pitch balance. So far, the analytical treatment of helical motifs in minimal surfaces was limited to the small-slope approximation where motifs are represented by the graph of harmonic functions. However, in most biologically and physically relevant regimes the inter-motif separation is comparable with its pitch, and thus this approximation fails. Here, we present a recipe for constructing exact minimal surfaces with an arbitrary distribution of helical motifs, showing that any harmonic graph can be deformed into a minimal surface by exploiting lateral displacements only. We analyse in detail pairs of motifs of the similar and of opposite handedness and also an infinite chain of identical motifs with similar or alternating handedness. Last, we study the second variation of the area functional for collections of helical motifs with asymptotic helicoidal structure and show that in this subclass of minimal surfaces stability requires that the collection of motifs is pitch balanced.

Abstract The receptor for the human granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-R) is a heterodimeric complex consisting of two subunits, GM-R alpha and GM-R beta. Structural analyses have shown a number of highly conserved amino acid motifs present in both GM-R alpha and GM-R beta. These motifs include QYFLY, CXW, XW, and WSXWS motifs in the extracellular domain; a conserved cysteine in the transmembrane domain; and the entire cytoplasmic domain, including the LXVLX box in the carboxy terminal region of the cytoplasmic domain. We have investigated the role of these motifs in GM-R alpha by examining the effects of specific motif mutations on ligand binding and surface expression. Transient expression of these mutant GM-R alpha subunits in COS cells shows that these extracellular motis are essential for ligand binding. Alterations of the cytoplasmic region of GM-R alpha do not alter GM-CSF binding or the reconstitution of high-affinity receptors when coexpressed with GM-R beta. Permeabilization and immunostaining of cells transfected with mutant GM-R alpha subunits yields data suggesting that each of the mutant subunits is present in the cytoplasm. Immunostaining of both intact and permeabilized COS cells transiently transfected with wild-type or mutant GM-R alpha s showed that extracellular domain mutants accumulated in the cytoplasm and were not efficiently transported to the cell surface.

The receptor for the human granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-R) is a heterodimeric complex consisting of two subunits, GM-R alpha and GM-R beta. Structural analyses have shown a number of highly conserved amino acid motifs present in both GM-R alpha and GM-R beta. These motifs include QYFLY, CXW, XW, and WSXWS motifs in the extracellular domain; a conserved cysteine in the transmembrane domain; and the entire cytoplasmic domain, including the LXVLX box in the carboxy terminal region of the cytoplasmic domain. We have investigated the role of these motifs in GM-R alpha by examining the effects of specific motif mutations on ligand binding and surface expression. Transient expression of these mutant GM-R alpha subunits in COS cells shows that these extracellular motis are essential for ligand binding. Alterations of the cytoplasmic region of GM-R alpha do not alter GM-CSF binding or the reconstitution of high-affinity receptors when coexpressed with GM-R beta. Permeabilization and immunostaining of cells transfected with mutant GM-R alpha subunits yields data suggesting that each of the mutant subunits is present in the cytoplasm. Immunostaining of both intact and permeabilized COS cells transiently transfected with wild-type or mutant GM-R alpha s showed that extracellular domain mutants accumulated in the cytoplasm and were not efficiently transported to the cell surface.

From the total time of making traditional Batik, the batik stage is the most time consuming because there are a lot of mistakes due to lack of carefulness when making batik motifs on cloth. To overcome the problem of making Batik motifs, this research utilizes the strengths and capabilities of Marker-Based Augmented Reality technology. The development of Marker-Based Augmented Reality applications is done by replacing digital objects with Batik motif images and markers attached to the fabric. Development of Marker-Based Augmented Reality applications using vertex markers to reproduce Batik motif objects in order to overcome the limitations of the reach of hands when making Batik motifs on fabric Finally, by using Marker-Based Augmented Reality applications batik makers do not need to draw Batik motifs in pencil on fabric because of Batik motifs directly displayed on the fabric surface by the application, so that it is expected to reduce the process time of making Batik motifs compared to making Batik motifs using traditional methods.

Insulin stimulates glucose transport in muscle and adipose tissue by triggering the movement of the glucose transporter GLUT-4 from an intracellular compartment to the cell surface. Fundamental to this process is the intracellular sequestration of GLUT-4 in nonstimulated cells. Two distinct targeting motifs in the amino and carboxy termini of GLUT-4 have been previously identified by expressing chimeras comprised of portions of GLUT-4 and GLUT-1, a transporter isoform that is constitutively targeted to the cell surface, in heterologous cells. These motifs-FQQI in the NH2 terminus and LL in the COOH terminus-resemble endocytic signals that have been described in other proteins. In the present study we have investigated the roles of these motifs in GLUT-4 targeting in insulin-sensitive cells. Epitope-tagged GLUT-4 constructs engineered to differentiate between endogenous and transfected GLUT-4 were stably expressed in 3T3-L1 adipocytes. Targeting was assessed in cells incubated in the presence or absence of insulin by subcellular fractionation. The targeting of epitope-tagged GLUT-4 was indistinguishable from endogenous GLUT-4. Mutation of the FQQI motif (F5 to A5) caused GLUT-4 to constitutively accumulate at the cell surface regardless of expression level. Mutation of the dileucine motif (L489L490 to A489A490) caused an increase in cell surface distribution only at higher levels of expression, but the overall cells surface distribution of this mutant was less than that of the amino-terminal mutants. Both NH2- and COOH-terminal mutants retained insulin-dependent movement from an intracellular to a cell surface locale, suggesting that neither of these motifs is involved in the insulin-dependent redistribution of GLUT-4. We conclude that the phenylalanine-based NH2-terminal and the dileucine-based COOH-terminal motifs play important and distinct roles in GLUT-4 targeting in 3T3-L1 adipocytes.

Surface display technology can be defined as that phenotype (protein or peptide) which is linked to a genotype (DNA or RNA) through an appropriate anchoring motif. A bacterial surface display system is based on expressing recombinant proteins fused to sorting signals (anchoring motifs) that direct their incorporation on the cell surface.

Herein we discuss the formation of 1-D polymeric motifs for the silver–flavin complex achieved via rare bidentate coordination for the modified flavin moiety. Further studies revealed facile transfer of the polymeric motif onto the surface.

In spite of their high abundance and importance, aqueous systems are enigmatic on the microscopic scale. In order to obtain information about their geometrical and electronic structure, simple aqueous solutions have been studied experimentally by photo- and Auger electron spectroscopy using the novel liquid micro-jet technique in conjunction with synchrotron radiation. The thesis is thematically divided into three parts. In the first part we utilize the surface sensitivity of photoelectron spectroscopy to probe the distributions of solutes near the water surface. In agreement with recent theoretical predictions we find that large polarizable anions, such as I- and ClO4-, display enhanced surface propensities compared to smaller rigid ions. Surface effects arising from ion-ion interactions at higher electrolyte concentrations and as function of pH are investigated. Studies of linear mono-carboxylic acids and benzoic acid show that the neutral molecular forms of such weak acids are better stabilized at the water surface than their respective conjugate base forms. The second part examines what type of information core-electron spectra can yield about the chemical state and hydration structure of small organic molecules in water. We demonstrate that the method is sensitive to the protonation state of titratable functional groups and that core-level lineshapes are dependent on local water hydration configurations. Using a combination of photoelectron and X-ray absorption spectroscopy we also show that the electronic re-arrangement upon hydrolysis of aldehydes yields characteristic fingerprints in core-level spectra. In the last part of this thesis we study ultrafast charge delocalization dynamics in aqueous solutions using resonant and off-resonant Auger spectroscopy. Intermolecular Coulombic decay (ICD) is found to occur in a number of core-excited solutions where excess energy is transferred between the solvent and the solute. The rate of ultrafast electron delocalization between hydrogen bonded water molecules upon oxygen 1s resonant core-excitation is found to decrease upon solvation of inorganic ions. The presented work is illustrative of how core-level photoelectron spectroscopy can be valuable in the study of fundamental phenomena in aqueous solutions.

La Réalité Augmentée (RA) vise à afficher des informations numériques virtuelles sur des images réelles. Le recalage est important, puisqu’il permet d'aligner correctement les objets virtuels dans le monde réel. Contrairement au tracking qui recale en utilisant les informations de l’image précédente, la localisation à grande échelle (wide baseline localization) calcule la solution en utilisant uniquement les informations présentes dans l’image courante. Il permet ainsi de trouver des solutions initiales au problème de recalage (initialisation) et, n’est pas sujet aux problèmes de « perte de tracking ». Le problème du recalage en RA est relativement bien étudié dans la littérature, mais les méthodes existantes fonctionnent principalement lorsque la scène augmentée présente des textures. Pourtant, pour le recalage avec les objets peu ou pas texturés, il est possible d’utiliser leurs informations géométriques qui représentent des caractéristiques plus stables que les textures. Cette thèse s’attache au problème de recalage basé sur des informations géométriques, et plus précisément sur les points. Nous proposons deux nouvelles méthodes de recalage de points (RRDM et LGC) robustes et rapides. LGC est une amélioration de la méthode RRDM et peut mettre en correspondance des ensembles de motifs de points 2D ou 3D subissant une transformation dont le type est connu. LGC présente un comportement linéaire en fonction du nombre de points, ce qui permet un tracking en temps-réel. La pertinence de LGC a été illustrée en développant une application de calibration de système projecteur-caméra dont les résultats sont comparables avec l’état de l’art tout en présentant des avantages pour l’utilisateur en termes de taille de mire de calibration
Registration is a very important task in Augmented Reality (AR). It provides the spatial alignment between the real environment and virtual objects. Unlike tracking (which relies on previous frame information), wide baseline localization finds the correct solution from a wide search space, so as to overcome the initialization or tracking failure problems. Nowadays, various wide baseline localization methods have been applied successfully. But for objects with no or little texture, there is still no promising method. One possible solution is to rely on the geometric information, which sometimes does not vary as much as texture or color. This dissertation focuses on new wide baseline localization methods entirely based on geometric information, and more specifically on points. I propose two novel point pattern matching algorithms, RRDM and LGC. Especially, LGC registers 2D or 3D point patterns under any known transformation type and supports multipattern recognitions. It has a linear behavior with respect to the number of points, which allows for real-time tracking. It is applied to multi targets tracking and augmentation, as well as to 3D model registration. A practical method for projector-camera system calibration based on LGC is also proposed. It can be useful for large scale Spatial Augmented Reality (SAR). Besides, I also developed a method to estimate the rotation axis of surface of revolution quickly and precisely on 3D data. It is integrated in a novel framework to reconstruct the surface of revolution on dense SLAM in real-time

Pour une réponse immunitaire efficace, une interaction optimale entre les cellules T et les cellules présentatrices d'antigène (APC) est nécessaire. Elle se présente sous la forme d'un contact cellulaire à différentes échelles allant du moléculaire (1-10 nm) au cellulaire (1-10 micromètres). La liaison entre les récepteurs spéciaux des cellules T (TCR) et leurs ligands sur une APC entraîne une réorganisation moléculaire à plus grande échelle menant d'abord à la formation de micro-clusters de TCR, puis à une restructuration à l'échelle cellulaire de la membrane et du cytosquelette. La création d'un substrat artificiel avec des amas de ligands qui induisent à leur tour l'accumulation de TCR est un outil important pour comprendre le lien entre l'organisation du TCR et de son ligand, l'organisation du cytosquelette d'actine et l'impact de ces deux facteurs sur le comportement cellulaire global, notamment l'adhérence et la signalisation. Nous avons développé un nouveau substrat basé sur la nanotechnologie et utilisé une stratégie alternative basée sur l'auto-assemblage colloïdal pour montrer que le TCR est clairement groupé sur des points de 700 nm et non pas sur des points de 400 nm. L'actine est distribuée de manière homogène sous forme de réseau dans la plupart des cellules, mais dans quelques-unes d'entre elles, elle apparaît sous la forme de points co-localisés avec les amas de ligands. Une observation plus fine à l'aide de la microscopie de reconstruction optique aléatoire indique que les points peuvent en fait être des sites où des faisceaux d'actine se croisent pour former des nœuds non visibles à moindre résolution
For an efficient immune response, an optimal interaction between T-cells and antigen presenting cells (APC) is required; it takes the form of a cell-cell contact involving different scales ranging from the molecular (1-10 nm) to the cellular (1-10 micrometer). The ligation of the special T cell receptors (TCR) to its ligands on an APC, leads to larger scale molecular reorganisation leading first to formation of TCR micro-clusters, and later to cell-scale restructuring of both the membrane and the cytoskeleton. Patterning an artificial substrate with ligand-clusters that in turn induce TCR-clustering is an important tool to understand the link between the organisation of TCR and its ligand, the organisation of the actin cytoskeleton and the impact of both on overall cell behavior including adhesion and signaling. We developed a new nanotechnology based substrate (ligand-dot size down to 250 nm) and also used an alternative strategy based on colloidal self-assembly (700 or 400 nm) to show that TCR is clearly clustered on 700 nm dots but not on smaller 400 nm dots. Actin is homogeneously distributed in the form of a network in most cells but in a few of them, it appears as dots that co-localize with the ligand clusters. Finer observation using stochastic optical reconstruction microscopy indicates that the dots may in fact be sites where actin bundles cross each other forming nodes that are not visible at lower resolution. This work confirms a close link between T cell receptor organisation and actin structure

Otar iosseliani, cineaste georgien, ne en 1934 a tbilissi, est l'auteur d'une oeuvre originale et attachante qui ne compte que peu de films (huit longs-metrages de fiction en plus de trente ans). Mais elle constitue assurement une des plus importantes du cinema contemporain. C'est en tout cas ce que tente de demontrer cette these, en trois temps. D'abord, a travers l'analyse d'un processus de creation tout a la fois philosophique, primitif et teleologique, c'est-a-dire, comparable a celui du demiurge, replace dans la lignee du cinema muet et base sur une methode refusant categoriquement le hasard. Ensuite, en dissequant la fonction du regard du cineaste sur le reel. Que ce soit dans ses documentaires (il en a realise cinq), dans ses trois premiers films tournes en georgie, ou en apprivoisant des cultures etrangeres (principalement la france mais aussi l'afrique), le regard qu'il porte sur le monde fait de lui un cineaste universel. Enfin, en tentant de restituer le plus fidelement possible une des caracteristiques esthetiques de son cinema : le rythme, tant sonore que plastique et structural. Tout au long de ces trois parties, il s'agit egalement de definir l'ethique de iosseliani.

Ce travail de thèse a eu pour objectif de développer de nouvelles méthodes de dépôts et de formation de motifs pour mieux contrôler l'organisation de molécules d'intérêt biologique sur des substrats solides.
Dans la première partie du manuscrit, nous présentons une méthode originale, la micro-aspiration, permettant de réaliser des assemblages réversibles de canaux microfluidiques sur un substrat et servir à guider les liquides. Nous avons étudié les propriétés de ces systèmes avec des modèles physiques simples et appliqué ces phénomènes à la micromanipulation de liquides, le dépôt de protéines à diverses concentrations sur un substrat, la fabrication de motifs de polymères, nanoparticules, gels, etc.
Dans la seconde partie, nous avons exploré des nouvelles méthodes de dépôt de films de phospholipides multicouches sur des substrats solides et les avons appliqués à la fabrication de vésicules unilamellaires géantes de taille contrôlée. Tout d'abord, l'adaptation de techniques conventionnelles (micro-contact printing, moulage, etc.) a permis d'obtenir des motifs de phospholipides de taille micrométrique. Les dépôts ont ensuite été réalisés par retrait d'un ménisque en situation d'évaporation (assemblage capillaire). Nous avons identifié deux régimes de dépôt en fonction de l'importance relative des forces visqueuses et de l'évaporation, permettant un contrôle de l'épaisseur du film jusqu'à 200 nm à la bicouche près. L'émergence d'instabilités de mouillage ou le guidage sur micro-structures ont permis en outre de réaliser des motifs variés. En utilisant des substrats comme électrodes, ces différents niveaux d'organisation ont permis d'obtenir par électroformation des vésicules unilamellaires géantes de taille contrôlée. L'ensemble de ces travaux ouvre de nouvelles voies à la réalisation de surfaces et de motifs micrométriques d'intérêt biologique.

Notre objectif est la modelisation de la formation de complexes proteiques par la recherche de zones de complementarite entre surfaces moleculaires. La complementarite geometrique est une caracteristique indispensable dans la reconnaissance des proteines entre elles, en effet les associations proteiques stables sont dues en particulier aux effets hydrophobiques et aux interactions polaires, qui sont des forces agissant a faible distance. Le probleme de la complementarite moleculaire est aborde par la definition de l'enveloppe convexe d'un nuage tridimensionnel de points. A partir de son enveloppe convexe, on peut definir la profondeur et la hauteur de tout point de la surface moleculaire. La profondeur, definie comme la distance entre le point et la face associee de l'enveloppe convexe, permet de localiser les zones creuses (trous) de la surface moleculaire. La hauteur, definie comme la distance du point a sa face associee sur une surface convexe interne, permet de localiser les zones elevees, (bosses) de la surface moleculaire. Profondeur et hauteur sont ici considerees comme deux quantites complementaires pouvant permettre la recherche de zones complementaires. Chacune des deux surfaces moleculaires est representee par une carte de contour d'iso-profondeur ou d'iso-hauteur, profondeur pour le substrat et hauteur pour l'inhibiteur. Par une comparaison des deux cartes, les zones de complementarite susceptibles de constituer le site de liaison sont selectionnees sur la base de differents criteres de dissemblance: coefficient de correlation, erreur moyenne d'ajustement, indice de non-recouvrement. Cette demarche d'analyse a ete appliquee a l'etude du complexe trypsine-inhibiteur et s'est averee efficace pour retrouver le site de formation du complexe

In silico investigation on protein domains structure and linear/structural motifs can strongly boost functional analyses and technological design. Protein surface features study is crucial to understanding Protein-Protein Interactions (PPI); in particular, surface and pockets conservation and variation, in terms of hydrophobicity, steric hindrance and electrostatics can act as driving forces in protein evolution and functional specialization. Therefore, molecular modeling and structure comparison techniques play an important role in shedding light on “protein behavior” and this PhD work took advantage from integrating computational approaches based on some known molecular modeling methods, such as e.g. Homology Modeling, Fold Recognition, Ab initio Modeling, PBE (Poisson-Boltzmann Electrostatics), Protein-peptide Docking and Hydropathy Analysis with structure and sequence comparison and scanning tools and, of course, with feedback from wet lab analyses performed by co-workers. Such an integrative approach was followed along investigations on a number of different biological systems: • Surface determinants in H5N1 type A Influenza viruses: Here, an analysis of surface determinants from H5N1 haemagglutinin, involved in host-viral interaction, was completed and then published. Genomic variation is very high in influenza A viruses. However, viral evolution and spreading are strongly influenced by immunogenic features and capacity to bind host cells, depending in turn on the two major capsidic proteins (haemagglutinin and neuraminidase). Current analyses of viral evolution are based on serological and primary sequence comparison; however, comparative structural analysis of capsidic proteins can provide functional insights on surface regions possibly crucial to antigenicity and cell binding. We performed molecular modeling and extensive structural comparison of influenza virus haemagglutinin and of their domains and sub-regions to investigate type- and/or domain specific variation. We found that structural closeness and primary sequence similarity are not always tightly related; moreover, type-specific features could be inferred when comparing surface properties of haemagglutinin subregions, monomers and trimers, in terms of electrostatics and hydropathy. Focusing on H5N1, we found that the variation at the receptor binding domain (RBD) surface intriguingly relates to branching of still circulating clades from those ones that are no longer circulating. Recent evidence on the association between electrostatic fingerprints at the haemagglutinin receptor binding surface and the evolutionary success and spreading of H5N1 avian influenza clades prompted us to perform further integrated phylogenetic and structural bioinformatic analysis in H9N2 viruses. In fact, influenza A virus is a zoonotic agent with a significant impact both on public health and poultry industry and switch to human host has been reported for both H5N1 and H9N2 viruses. We performed the evolutionary analysis of a large and non-redundant viral strain dataset, leading to clustering of H9N2 viruses in five groups. Then and according to recent evidence on H5N1, congruence resulted among phylogenetic data and surface electrostatic fingerprints from structural comparison. In particular, surface feature fingerprints could be inferred that relate group specific variation in electrostatic charges and isocontour to well-known hemagglutinin sites involved in modulation of immune escape and host specificity. Results from this second work strengthen suggestion that when integrating up-to-date phylogenetic analyses with sequence-based and structural investigation of surface features may represent a front-end strategy for inferring trends and relevant mechanisms in influenza virus evolution. • Domain architecture variation in mammalian protein trafficking: Human VAMP7b is the most interesting variant among those produced by alternative splicing of the encoding gene SYBL1. Production of VAMP7b variants is determined by skipping of exon 6 which in turn results in coding sequence frameshift. We found that this event is conserved in other mammalian species. VAMP7b shares with the main isoform the N-terminal, inhibitory longin domain and the first half of the SNARE motif. In mammals, VAMP7b is a truncated protein in which the C-terminal half of the SNARE motif and the transmembrane region are replaced by short and variable peptides. Intriguingly instead, only in human and apes sequence frameshift determined by exon 6 skipping results in the creation of a novel unique domain of unknow function, hence human VAMP7b is not truncated but even 40 residues longer than the main isoform. Since existence of such “long” isoform and of its unique domain at protein level were confirmed by specific antibodies, we embarked on in silico dissection of the novel domain by position specific matrix sequence analysis and by ab initio structural modeling. Moreover, since the N-terminal region of the SNARE motif is conserved and it is known to mediate intramolecular binding to the Longin domain, we investigated both in vivo (by two-hybrid in yeast analysis) and in vitro (by NMR analysis) on conservation of the closed conformation. Furthermore, SCL of both VAMP7b and Ykt6b was investigated using GFP and RFP chimeras. Last but not least, b isoforms of the longin genes were analyzed by qPCR and found to be developmentally regulated. • Binding motif regulating neurite outgrowth and guidance: Fine tuning of PPIs by variation in domain architecture or by changing local motifs by surface features modulation can regulate both extracellular and intracellular signaling pathways. Extracellular PPIs can play a central role in heterologous recognition (e.g. host-pathogen) as well as in homologous signaling among cells from the same organism. Proteins exposed at the plasma membrane (PM) can interact each other and with the extracellular matrix (ECM) to provide positional information and guidance cues. Cell adhesion molecules (CAMs) are PM proteins mediating either attractive or repulsive signals by homo- and heterophilic interactions of their extracellular domains (Eds). CAM EDs are most often composed by Ig-like or Fibronectin type III fold repeats. Current evidence suggests that the four N-terminal Ig 1-4 domains of CAM EDs play a major role in such homo- or heterophilic interactions and in particular an important interaction motif is contributed by repeat Ig2. In our lab, biomimetic peptides have been developed by reproducing the known or predicted interaction motifs from the Ig2 domain of human L1CAM and the single Ig domain of human LINGO1, i.e. two proteins that play a crucial role in neurite outgrowth and guidance and in neuronal differentiation. Based on the somehow surprising structural and sequence conservation of the motif region (even when proteins show very different ED architectures), we started investigating on variation and conservation of the putative motif region by means of homology search, regular expression and finally by structural modeling and comparison. Preliminary results highlighted strong conservation of the central Arg residue in the interaction motif, while in other positions of the motif residue properties rather than specific residues are conserved. Such evidence is in agreement with finding that mutation of such residue in L1CAM is responsible for a severe neurological disorder, while mutations at other residues of the motif, results in less severe phenotype. This suggests the motif is an epitope positionally conserved around the central Arg allowing limited, but significant structural variability in surrounding sequence. In order to check such a hypothesis, a structural superposition of the Ig domains containing the interaction motif was performed, confirming that the peptide motif itself is positionally conserved but the highest positional and structural conservation concerns the central Arg residue. Experiments with peptides mutated in the central Arg showed biological activity of these peptides in terms of neuritogenesis signalling. These works carry out a bioinformatic protocol for the characterization of interaction determinants and their functional modulation, easily transportable to other proteins.
Gli studi in silico aventi per oggetto la struttura di domini proteici e di motif sia strutturali che lineari, sono in grado di fornire un importante apporto in termini di comprensione di funzione e nelle biotecnologie. Lo studio delle caratteristiche a carico della superficie proteica si rivelano essenziali nella comprensione delle Interazioni Proteina-Proteina (PPI); in particolare, la conservazione e variazione della superficie proteica e delle relative cavità in termini di idrofobicità, ingombro sterico e caratteristiche elettrostatiche, possono essere considerate come la forza in grado di guidare l’evoluzione e la specializzazione funzionale delle proteine stesse. Alla luce di quanto sopra esposto, tecniche come la Modellistica Molecolare ed il confronto tra strutture giocano un ruolo importante nel chiarire il modus operandi delle proteine e questo progetto di Dottorato ha proprio sfruttato l’approccio integrato di alcune ben note tecniche di biologia computazionale basate sulla Modellistica Molecolare come, ad esempio, Homology Modeling, Fold Recognition, Ab initio Modeling, PBE (Poisson-Boltzmann Electrostatics), Protein-peptide Docking e Hydropathy Analysis con confronto di sequenze e strutture. Elemento indispensabile e prezioso, ovviamente, il feedback ottenuto dagli esperimenti al banco effettuati dai nostri collaboratori. Questo approccio integrato è stato dunque applicato a differenti sistemi biologici: • Individuazione di determinanti di superficie in virus influenzali di tipo A H5N1: è stata effettuata un’analisi dei determinanti di superficie a carico dell’emoagglutinina proveniente dal virus influenzale H5N1, coinvolta nell’interazione virus-ospite. Questo lavoro ha già condotto ad una pubblicazione. La variazione genomica è elevata nei virus influenzali di tipo A. L’evoluzione e la diffusione dei virus sono molto influenzate dalle caratteristiche immunogeniche e dalla capacità del virus, di interagire con le cellule dell’ospite tramite le due più importanti proteine presenti sul capside virale: l’emoagglutinina e la neuraminidasi. Le analisi oggi a disposizione sono basate sul confronto dell’attività sierologica e di sequenze primarie; alla luce di ciò, l’analisi strutturale di queste proteine capsidiche può essere in grado di svelare delle conoscenze a riguardo di certe regioni presenti sulla superficie proteica che possono essere cruciali per l’antigenicità e per il legame alle cellule dell’ospite. L’emoagglutinina, sezionata nei suoi domini e subdomini, è stata da noi studiata con metodi di Modellistica Molecolare e sottoposta a confronti strutturali fini, per individuare quelle variazioni che potessero risultare tipo/dominio specifiche. Abbiamo evidenziato che la vicinanza strutturale e la similarità di sequenza primaria non sempre sono correlate; in più, caratteristiche tipo-specifiche di sottoregioni dell’emoagglutinina, monomeri e trimeri, possono essere rivelate grazie al confronto delle loro proprietà di superficie, (in termini di elettrostatica ed idrofobicità) appartenenti a sottoregioni dell’emoagglutinina, monomeri e trimeri. In questo lavoro ci siamo focalizzati sul virus H5N1 e abbiamo scoperto che il dominio di legame recettoriale dell’emoagglutinina (RBD) presenta delle variazioni tra clade circolanti e non più circolanti. Le recenti scoperte riguardanti l’associazione tra la disposizione delle cariche al RBD ed il successo in termini evolutivi e di diffusione del virus H5N1 ci hanno spinto ad eseguire analisi integrate di filogenesi e biologia strutturale a carico dei virus H9N2. Infatti, l’influenza A è un agente zoonotico in grado di produrre un grosso impatto sia sulla salute pubblica che sull’industria del pollame, avendo la capacità di effettuare il salto d’ospite, come riportato proprio per H5N1 ed H9N2. Abbiamo effettuato un’analisi evoluzionistica su un grande dataset non ridondante di ceppi virali e questo ci ha consentito di individuare cinque gruppi di virus H9N2. In accordo con le precedenti analisi effettuate per H5N1, abbiamo ottenuto accordo tra i dati filogenetici con quelli ottenuti dalle analisi di confronto strutturale. In particolare, emerge che la variazione della disposizione delle cariche coincide con quella di siti noti dell’emoagglutinina coinvolti nell’evasione al sistema immunitario e nella specificità d’ospite. I risultati ottenuti da questo secondo lavoro pongono l’accento sull’importanza dell’integrazione tra analisi di tipo filogenetiche e di biologia strutturale nella scoperta di nuovi meccanismi evolutivi dei virus dell’influenza. • Variazione dell’architettura di domini in proteine di mammifero coinvolte nel traffico vescicolare: la proteina umana VAMP7b è la più interessante tra quelle prodotte per splicing alternativo del gene SYBL1. La produzione di VAMP7b è causata dal salto dell’esone 6 che si traduce in uno slittamento della sequenza codificante. Abbiamo scoperto che questo evento è conservato in altre specie di mammiferi. VAMP7b condivide con l’isoforma principale il dominio inibitorio longin N-terminale e la prima metà dello SNARE motif. Nei mammiferi, VAMP7b è una proteina tronca in cui al C-terminale metà dello SNARE motif e la regione transmembrana sono sostituite da peptidi corti e variabili. È molto interessante notare come negli uomini e nelle scimmie antropomorfe lo slittamento della regione codificante determinato dal salto dell’esone 6 abbia prodotto un nuovo dominio di funzione sconosciuta: proprio per questo VAMP7b umana non è tronca, ma addirittura 40 residui più lunga rispetto all’isoforma principale. Dal momento che l’esistenza di questa isoforma “lunga” ed il suo nuovo dominio sono stati confermati a livello proteico grazie all’ausilio di specifici anticorpi, abbiamo effettuato una dissezione in silico del nuovo dominio adoperando un’analisi di sequenza di tipo matrice posizione-specifica (PSI-BLAST), seguita da da Modellistica Strutturale di tipo ab initio. In più, dal momento che la regione N-terminale dello SNARE motif è conservata ed è nota nel mediare il legame intramolecolare al dominio Longin, abbiamo appurato la conservazione della conformazione chiusa sia in vivo (saggio del doppio ibrido in lievito) che in vitro (analisi NMR). Inoltre, la localizzazione subcellulare (SCL) di VAMP7b e Ykt6b è stata studiata adoperando chimere contenenti GFP e RFP. Non ultimo, le isoforme b dei geni longin sono stati analizzati tramite qPCR e si è scoperto essere regolate durante lo sviluppo. • Motif di legame con azione regolatoria sulla crescita e l’indirizzamento neuronale: La regolazione fine delle interazioni proteina-proteina che avviene grazie alle variazioni nell’architettura dei domini o dal cambiamento di motif locali indotto dalla modulazione di caratteristiche di superficie, è in grado di regolare i percorsi di segnalazione sia a livello intra- che extracellulare. Le interazioni proteina-proteina extracellulari possono giocare un ruolo fondamentale nel riconoscimento eterologo (es. ospite-patogeno) come nella segnalazione omologa tra cellule appartenenti allo stesso organismo. Le proteine esposte in membrana plasmatica (PM) possono interagire le une con le altre e con la matrice extracellulare (ECM) per consentire informazioni posizionali e segnali di indirizzamento. Le molecole di adesione cellulare (CAMs) sono proteine della membrana plasmatica in grado di mediare segnali sia di natura attrattiva che repulsiva grazie ad interazioni omo- ed eterofiliche a carico dei loro domini extracellulari (EDs). Questi ultimi sono composti per la magior parte da domini ripetuti aventi fold di tipo Ig o Fibronectina di tipo III. Le attuali conoscenze suggeriscono che i 4 domini extracellulari N-terminali di tipo Ig siano importanti nelle interazioni omo- o eterofiliche ed in modo particolare il dominio Ig2 è provvisto di un importante motif di interazione. Nel nostro laboratorio abbiamo sviluppato dei peptidi biomimetici che riproducono i motif di interazione conosciuti o predetti appartenenti al dominio Ig2 di L1CAM umana e al singolo dominio Ig di LINGO1 umana, proteine, queste, che giocano un ruolo fondamentale nella crescita, nell’indirizzamento e nel differenziamento neuronale. Sulla base della conservazione strutturale della regione del motif (anche tra proteine con architetture molto diverse dei loro EDs), abbiamo iniziato a studiarne la variazione di sequenza mediante analisi per omologia e per espressioni regolari, per infine tornare al livello strutturale mediante Modellistica Molecolare. I risultati preliminari indicano una forte conservazione dell’Arginina centrale presente nel motif d’interazione, mentre nelle altre posizioni del motif si osserva la conservazione di proprietà dei residui piuttosto che la presenza di specifici residui. Questa evidenza è in accordo con il dato di fatto che la mutazione dell’Arginina in L1CAM è responsabile di un serio disordine neurologico, mentre mutazioni a carico di altri residui del motif causano un fenotipo meno grave. Questo suggerisce che il motif è un epitopo posizionalmente conservato attorno all’Arginina centrale in grado di consentire una variabilità limitata ma significativa nella sequenza circostante. Per verificare quest’ipotesi è stata effettuata una superimposizione strutturale dei domini Ig contenenti il motif d’interazione: il risultato ha confermato che il peptide contenente il motif è di per sé conservato posizionalmente e che la conservazione maggiore sia a livello posizionale che struturale è a carico del residuo centrale di Arginina. Esperimenti con peptidi mutati nell’Arginina centrale hanno dimostrato un’attività in termini di segnalazione nella neuritogenesi. Questi lavori hanno consentito di sviluppare un protocollo bioinformatico per la caratterizzazione di determinanti d’interazione e della loro modulazione funzionale, facilmente trasportabile su altre proteine.

Exhibiting Antonio Canova: Display and the Transformation of Sculptural Theory argues that the display of Canova’s sculptures in the late eighteenth and early nineteenth centuries acted as a catalyst for discourse across a broad range of subjects. By enshrining his marble figures alongside plaster casts of ancient works, bathing them in candlelight, staining and waxing their surfaces, and even setting them in motion on rotating bases, Canova engaged viewers intellectually, physically, and emotionally. These displays inspired discussions on topics as diverse as originality and artistic production, the association between the sculptural surface, flesh, and anatomy, the relationship between painting and sculpture, and the role of public museums. Beholders’ discussions also shaped the legacy of important sculptural theories. They helped usher in their modern definitions and created the lenses through which we experience and interpret works of art, establishing modern attitudes not just towards sculpture, but towards cultural patrimony in general.

This research will focus on three small-scale mock-up reconstructions of several embellishment motifs used by renaissance painter Nikola Bozidarevic from Dubrovnik (Croatia). The reconstructions were carried out as a part of the practical portion of the Technical analyses and historical reconstructions course at the Arts Academy of the University of Split during the academic year 2020/2021. During the reconstruction process a number of questions concerning the painter’s working properties aroused. The attempt was made to answer which methods of transferring the pattern on the gilded surface could have been used. Further questions concerned the subsequent steps of decoration, for example did the pouncing of the surface occurred before or after delineation of the motifs in thick paint. In spite the fact that most of these questions remind ambiguous, the experience gained from the reconstruction process emphasized the importance of understanding the practical aspects of decorative techniques as well as the high level of knowledge about the painter’s materials required for this kind of process.

Vascularization is critical for success of tissue engineering applications. Previous studies by us and others have shown that self-assembling peptide nanoscaffold RAD16-II promotes de novo capillary formation (angiogenesis) in vitro and neovascularization in vivo, and is a promising material for tissue engineering applications [1, 2]. However, the molecular mechanisms for cell interactions with this material are not known. Angiogenesis is mediated via interactions between integrins, which are expressed on the surface of activated endothelial cells (ECs), and extracellular matrix proteins. Among several integrins, αvβ3 is the most abundant and influential receptor regulating angiogenesis [3]. The αvβ3 integrin binds to its ligands via Arg-Gly-Asp (RGD) biding motif. However, there are no RGD motifs on RAD 16-II peptide. Instead, it contains three RAD motifs. Studies have shown that non-specific binding of αvβ3 with RAD can be retained through R and D sides [4]. The objective of this study, therefore, is to elucidate the underlying molecular mechanisms of RAD16-II nanoscaffold interactions with microvascular endothelial cells. We hypothesize that non-specific interactions between RAD16-II peptide nanoscaffold and αvβ3 integrin result in phosphorylations of β3 cytoplasmic domain, which then activate downstream angiogenic signaling pathways and promote angiogenesis.

The objective of this study is to develop a portable hand held diagnostics platform for monitoring pollutants and water quality testing. We are developing a lab-on-chip (LOC) device for in-situ synthesis of gold nano-particles and for using a colorimetric peptide assay for water quality monitoring. The gold nano-particles are synthesized in-situ in our experiments. The gold nano-particles exhibit various optical properties due to their Surface Plasmon Resonance (SPR). These stabilized mono-disperse gold nano-particles are coated with bio-molecular recognition motifs on their surfaces. The stabilization and functionalization with bio-molecular recognition motif provides flexibility for various applications. For example, the gold nano-particles synthesized by this process are tested for their ability to be recognized by a surface coated with anti-Flg antibodies. The LOC consists of micro-wells housing different reagents and samples that feed to a common reaction chamber. The reaction products are delivered to several waste chambers in a pre-defined sequence to enable subsequent reagents/ samples to flow into the reaction chamber. Passive flow actuation is obtained by capillary driven flow (wicking). Dissolvable micro-structures are used as passive micro-valves that actuate at predefined intervals and do not require any external power source for actuation. The microfluidic chip (LOC) and the dissolvable microstructures are fabricated using soft lithography techniques. The passive valves are incorporated into the microfluidics platform by novel micro-fabrication and bonding techniques.

The paper describes Late Roman and Early Byzantine decorated fine ware vessels and fragments found on both sides of the Kerch Strait (city of Kytaia, Džurg-Oba necropolis and fortlet Il’ičevskoe), and in Chersonesos. The finds represent the recently identified group of fine ceramics called Late Roman Pontic Burnished Ware. They are characterized by very re fined, pale pinkish - light brownish clay containing some very fine flakes of mica and single lumps of lime of various sizes. The inside surface of the dishes is carefully burnished, producing a slight lustre, and contains elaborated decoration executed by delicate incising in dried clay after burnishing and before firing (sgraffito-like technique). The decorative compositions contain centrally placed big cross, in two cases flanked by palm-branches or cypresses of similar size, and in one case completed with representation of four birds between arms of the cross. The central compositions are surrounded by two concentric grooved lines accompanied in two cases by a long spiral scratched on their outside. The outer surface of the examined dishes was finished with less care, it has some turning marks and a dull appearance. The jugs with their burnished surfaces bear incised decoration in the upper part of their bodies. In both cases, it contains a friese composed of simple motifs such as palm trees and branches, threeleaf branches and rope patterns

Abstract This paper extends the recent work of Xia and Ge (1999) to develop methods for the exact analysis of the swept surface of a cylindrical surface undergoing two-parameter rational Bézier motions. Instead of the approach of analyzing the point trajectory of an object motion for swept volume analysis, this paper seeks to develop a new approach to swept volume analysis by studying the plane trajectory of a rational motion. It seeks to bring together recent work in swept volume analysis, plane representation of developable surfaces, as well as computer aided synthesis of freeform rational motions. The results have applications in design and approximation of freeform surfaces as well as tool path planning for 5-axis machining of freeform surfaces.

Abstract This paper brings together line geometry, kinematic geometry of line-symmetric motions, and computer aided geometric design to develop a method for geometric design of rational Bézier line-symmetric motions. By taking advantage of the kinematic geometry of a line-symmetric motion, the problem of synthesizing a rational Bézier line-symmetric motion is reduced to that of designing a rational Bézier ruled surface. In this way, a recently developed de Casteljau algorithm for line-geometric design of ruled surfaces can be applied. An example is presented in which the Bennet motion is represented as a rational Bézier line-symmetric motion whose basic surface is a hyperboloid.

The numerical prediction of chaos and quasi-periodic motion on the homoclinic surface of a 2-DOF nonlinear Hamiltonian system is presented through the energy spectrum method. For weak interactions, the analytical conditions for chaotic motion in such a Hamiltonian system are presented through the energy incremental energy approach. The Poincare mapping surfaces of chaotic motions for such nonlinear Hamiltonian systems are illustrated. The chaotic and quasiperiodic motions on the phase planes, displacement subspace (or potential domains), and the velocity subspace (or kinetic energy domains) are illustrated for a better understanding of motion behaviors on the homoclinic surface. Through this investigation, it is observed that the chaotic and quasi-periodic motions almost fill on the homoclinic surface of the 2-DOF nonlinear Hamiltonian systems. The resonant-periodic motions are theoretically countable but numerically inaccessible. Such conclusions are similar to the ones in the KAM theorem even though the KAM theorem is based on the small perturbation.

We have applied molecular dynamics method and semi-empirical potential [1] to obtain the realistic picture of Si surface layers relaxation.The starting configuration was taken as a parallelepiped containing 864 atoms. There were 12 layers with 72 atoms in each one. Periodic boundary conditions were used in two dimensions. At first all atoms were in normal lattice positions. The relaxation of Si surface, which corresponds to (001) plane was investigated. MD method was applied in its standard form i.e. the equations of motion were solved by using of the central difference scheme. The time-step was 10-14s.

The goal of the one-year feasibility study was to identify specific cytotoxic T-lymphocyte (CTL) epitopes to Neosporacaninum in the natural bovine host in order to make progress toward developing an effective peptide-based vaccine against bovine neosporosis. We tested the hypothesis that: N. caninum SRS2 peptides contain immunogenicCTLepitope clusters cross-presented by multiple bovine MHC-I and MHC-IIhaplotypes. The specific objectives were: (1) Map bovine CTLepitopes of N. caninum NcSRS-2 and identify consensus MHC-I and class-II binding motifs; and (2) Determine if subunit immunization with peptides containing N. caninum-specificCTLepitopes cross-reactive to multiple bovine MHChaplotypes induces a CTL response in cattle with disparate MHChaplotypes. Neosporosis is a major cause of infectious abortion and congenital disease in cattle, persisting in cattle herds via vertical transmission.5 N. caninum abortions are reported in Israel; a serological survey of 52 Israeli dairy herds with reported abortions indicated a 31% infection rate in cows and 16% infection rate in aborted fetuses.9,14 Broad economic loss due to bovine neosporosis is estimated at $35,000,000 per year in California, USA, and $100,000,000 (Australian) per year in Australia and New Zealand.13 Per herd losses in a Canadian herd of 50 cattle are estimated more conservatively at $2,305 (Canadian) annually.4 Up to date practical measures to reduce losses from neosporosis in cattle have not been achieved. There is no chemotherapy available and, although progress has been made toward understanding immunity to Neospora infections, no efficacious vaccine is available to limit outbreaks or prevent abortions. Vaccine development to prevent N. caninum abortion and congenital infection remains a high research priority. To this end, our research group has over the past decade: 1) Identified the importance of T-lymphocyte-mediated immunity, particularly IFN-γ responses, as necessary for immune protection to congenital neosporosis in mice,1,2,10,11 and 2) Identified MHC class II restricted CD4+ CTL in Neosporainfected Holstein cattle,16 and 3) Identified NcSRS2 as a highly conserved surface protein associated with immunity to Neospora infections in mice and cattle.7,8,15 In this BARD-funded 12 month feasibility study, we continued our study of Neospora immunity in cattle and successfully completed T-lymphocyte epitope mapping of NcSRS2 surface protein with peptides and bovine immune cells,15 fulfilling objective 1. We also documented the importance of immune responses NcSRS2 by showing that immunization with native NcSRS2 reduces congenital Neospora transmission in mice,7 and that antibodies to NcSRS2 specifically inhibition invasion of placental trophoblasts.8 Most importantly we showed that T-lymphocyte responses similar to parasite infection, namely induction of activated IFN-γ secreting Tlymphocytes, could be induced by subunit immunization with NcSRS2 peptides containing the Neospora-specificCTLepitopes (Baszler et al, In preparation) fulfilling objective 2. Both DNA and peptide-based subunit approaches were tested. Only lipopeptide-based NcSRS2 subunits, modified with N-terminal linked palmitic acid to enhance Toll-like receptors 2 and 1 (TLR2-TLR1), stimulated robust antigen-specific T-lymphocyte proliferation, IFN-γ secretion, and serum antibody production across different MHC-IIhaplotypes. The discovery of MHC-II cross-reactive T-cellinducing parasite peptides capable of inducing a potentially protective immune response following subunit immunization in cattle is of significant practical importance to vaccine development to bovine neosporosis. In addition, our findings are more widely applicable in future investigations of protective T-cell, subunit-based immunity against other infectious diseases in outbred cattle populations.