Développement d'un biosenseur BRET permettant le criblage de drogues qui causent l'activation de canaux Kir3 via les récepteurs couplés aux protéines G

Les récepteurs couplés aux protéines G forment des complexes multimériques comprenant protéines G et effecteurs. Nous cherchons à caractériser de tels complexes comprenant les récepteurs opioïdes delta (DOR) et les canaux Kir3, qui nous sont d’intérêt vu leur implication dans l’analgésie des opioïdes. Des expériences d’immunopurification, de BRET et de liaison GTPgS ont été réalisées à l’intérieur de cellules HEK293 transfectées. Les canaux Kir3 ont été co-immunopurifiés avec les DOR, suggérant une interaction spontanée entre récepteur et effecteur. Des essais BRET ont corroboré que l’interaction était présente dans des cellules vivantes et nous ont permis d’identifier une interaction spontanée et spécifique entre DOR/Gg et Gg/Kir3, indiquant leur coexistence en un même complexe. Puisque l’activation du récepteur implique la présence de changements conformationnels à l’intérieur de celui-ci, nous étions intéressés à vérifier si l’information conformationnelle circule à partir du récepteur lié au ligand jusqu’à l’effecteur en aval. Ainsi, nous avons déterminé l’effet de différents ligands sur le signal BRET généré par les paires suivantes : DOR/Gbg, DOR/Kir3 et Kir3/Gbg. Nous avons constaté une modulation de l’interaction DOR/Gbg et Gbg/Kir3 suivant l’ordre d’efficacité des ligands à stimuler la protéine G, ce que nous n’avons pas observé entre DOR et Kir3. Donc, nous concluons que l’information conformationnelle circule du récepteur au canal Kir3 via la protéine Gbg. Ces résultats nous ont permis de développer un biosenseur BRET (EYFP-Gg2/Kir3.1-Rluc) qui pourrait être utilisé dans le criblage à haut débit afin de détecter de nouvelles molécules ayant une grande efficacité à activer les canaux Kir3.

Les littératies multiples et le devenir plurilingue (Keynote)

Cette présentation met en avant la Théorie des Littératies Multiples (TLM) et l’importance de lire, lire le monde et se lire, dans le but de se transformer en contexte plurilingue. La première partie de cette communication sera consacrée à la présentation des principes fondamentaux de la TLM. Il est vrai que la littératie valorisée par l’école est souvent celle qui est la plus prisée dans la recherche et l’enseignement. La TLM enlève la littératie scolaire de sa place privilégiée et l’insère dans un agencement de littératies au foyer, à l’école et dans la communauté. Les littératies, en tant que construit renvoient aux mots, aux gestes, aux attitudes, ou plus exactement, aux façons de parler, de lire, d’écrire et de valoriser les réalités de la vie. Elles sont une façon de devenir avec le monde. Les littératies constituent des textes au sens large (comme par exemple, la musique, l’art, la physique et les athématiques) qui peuvent être visuels,oraux, écrits, tactiles,olfactifs ou numériques. Elles se fusionnent aux contextes sociopolitiques, culturels, économiques, genrés et racialisés, qui de par leur caractère mobile et fluide transforment les littératies génératrices de locuteurs, de scripteurs, d’artistes, d’ava tars et de communautés. Les littératies prennent leur sens en contexte, dans le temps et l’espace où on se trouve De ce fait, leur actualisation n’est pas prédéterminée et est imprévisible. La TLM s’intéresse aux rôles joués par les littératies. Lire, lire le monde et se lire a pour fonction importante entre autres de transformer une vie, une communauté et une société. La deuxième partie de cette communication sera consacrée à un projet de recherche ayant pour objectif d’explorer la façon dont des enfants acquièrent simultanément deux ou plusieurs systèmes d’écriture. Des enfants âgés de 5 à 8 ans ont participé à des activités filmées en salle de classe, au foyer et dans leur quartier. Puis des entretiens ont été menés avec les enfants, leurs parents et leurs enseignants. Ce projet nous permet de mieux saisir ce que signifient en contexte plurilingue les littératies en tant que processus. Le projet s’intéresse à ce qu’implique lire, lire le monde et se lire à l’école, au foyer et dans la communauté. Dans une société pluraliste, nous sommes plus que jamais ensibilisés aux contextes particuliers dans lesquels lire, lire le monde et se lire s’actualisent, qu’il s’agisse d’un nouvel arrivant ou d’une personne vivant en milieu minoritaire.

The structures of COPI-coated vesicles reveal alternate coatomer conformations and interactions

Transport between compartments of eukaryotic cells is mediated by coated vesicles. The archetypal protein coats COPI, COPII, and clathrin are conserved from yeast to human. Structural studies of COPII and clathrin coats assembled in vitro without membranes suggest that coat components assemble regular cages with the same set of interactions between components. Detailed three-dimensional structures of coated membrane vesicles have not been obtained. Here, we solved the structures of individual COPI-coated membrane vesicles by cryoelectron tomography and subtomogram averaging of in vitro reconstituted budding reactions. The coat protein complex, coatomer, was observed to adopt alternative conformations to change the number of other coatomers with which it interacts and to form vesicles with variable sizes and shapes. This represents a fundamentally different basis for vesicle coat assembly.

The valuation accuracy of multiples in mergers and acquisitions, and their association with firm misvaluation

This study explores the accuracy and valuation implications of the application of a comprehensive list of equity multiples in the takeover context. Motivating the study is the prevalent use of equity multiples in practice, the observed long-run underperformance of acquirers following takeovers, and the scarcity of multiplesbased research in the merger and acquisition setting. In exploring the application of equity multiples in this context three research questions are addressed: (1) how accurate are equity multiples (RQ1); which equity multiples are more accurate in valuing the firm (RQ2); and which equity multiples are associated with greater misvaluation of the firm (RQ3). Following a comprehensive review of the extant multiples-based literature it is hypothesised that the accuracy of multiples in estimating stock market prices in the takeover context will rank as follows (from best to worst): (1) forecasted earnings multiples, (2) multiples closer to bottom line earnings, (3) multiples based on Net Cash Flow from Operations (NCFO) and trading revenue. The relative inaccuracies in multiples are expected to flow through to equity misvaluation (as measured by the ratio of estimated market capitalisation to residual income value, or P/V). Accordingly, it is hypothesised that greater overvaluation will be exhibited for multiples based on Trading Revenue, NCFO, Book Value (BV) and earnings before interest, tax, depreciation and amortisation (EBITDA) versus multiples based on bottom line earnings; and that multiples based on Intrinsic Value will display the least overvaluation. The hypotheses are tested using a sample of 147 acquirers and 129 targets involved in Australian takeover transactions announced between 1990 and 2005. The results show that first, the majority of computed multiples examined exhibit valuation errors within 30 percent of stock market values. Second, and consistent with expectations, the results provide support for the superiority of multiples based on forecasted earnings in valuing targets and acquirers engaged in takeover transactions. Although a gradual improvement in estimating stock market values is not entirely evident when moving down the Income Statement, historical earnings multiples perform better than multiples based on Trading Revenue or NCFO. Third, while multiples based on forecasted earnings have the highest valuation accuracy they, along with Trading Revenue multiples for targets, produce the most overvalued valuations for acquirers and targets. Consistent with predictions, greater overvaluation is exhibited for multiples based on Trading Revenue for targets, and NCFO and EBITDA for both acquirers and targets. Finally, as expected, multiples based Intrinsic Value (along with BV) are associated with the least overvaluation. Given the widespread usage of valuation multiples in takeover contexts these findings offer a unique insight into their relative effectiveness. Importantly, the findings add to the growing body of valuation accuracy literature, especially within Australia, and should assist market participants to better understand the relative accuracy and misvaluation consequences of various equity multiples used in takeover documentation and assist them in subsequent investment decision making.

Devenir plurilingue : la perspective des littératies multiples

Abstract - English Multiple literacies refers to reading, reading the world and self. This article proposes an understanding of reading that goes beyond its definition in psychology and applied linguistics. This longitudinal project is interested in a conceptualisation of what reading is, how it functions and what it produces in becoming multilingual. Reading is explored through the lens of an empirical study involving five female pupils from senior Kindergarten to Grade 3 observed and interviewed in relation to activities at school and at home. The study took place in Ottawa schools where French is the sole language of instruction. Reading in the context of multiple literacies is conceptualised to disrupt /deterritorialise and to be immanent, offering the potentiality to go beyond what is to what could be. Becoming multilingual is a continuous movement involving networks of rhizomatic connections and reading the world and self. Résumé - Francais Les littératies multiples se réfèrent à la lecture, la lecture du monde et la lecture de soi. Cet article propose une compréhension de la lecture qui dépasse sa définition usuelle en psychologie et en linguistique appliquée. Ce projet longitudinal porte sur la conceptualisation de la lecture, son fonctionnement et ce qu’elle produit dans le devenir plurilingue. La lecture est examinée selon l’optique d’une étude empirique durant laquelle cinq écolières du jardin d’enfants à la 3e année étaient observées et interviewées par rapport à des activités à l’école et à la maison. L’étude a eu lieu dans des écoles d’Ottawa dont la seule langue d’enseignement est le français. Dans le contexte des littératies multiples, la lecture est conceptualisée comme étant perturbatrice/déterritorialisante et immanente. Elle offre la potentialité d’aller au-delà de ce qui est vers ce qui pourrait être. Devenir plurilingue est un mouvement continu faisant appel à des réseaux de connexions rhizomatiques et à la lecture du monde et de soi.

Beta-Turn Conformations In Crystal-Structures Of Model Peptides Containing Alpha,Alpha-Di-N-Propylglycine And Alpha,Alpha-Di-N-Butylglycine

The crystal state conformations of three peptides containing the alpha, alpha-dialkylated residues, alpha,alpha-di-n-propylglycine (Dpg) and alpha,alpha-di-n-butylglycine (Dbg), have been established by x-ray diffraction. Boc-Ala-Dpg-Ala-OMe (I) and Boc-Ala-Dbg-Ala-OMe (III) adopt distorted type II beta-turn conformations with Ala (1) and Dpg/Dbg (2) as the corner residues. In both peptides the conformational angles at the Dxg residue (I: phi = 66.2 degrees, psi = 19.3 degrees; III: phi = 66.5 degrees, psi = 21.1 degrees) deviate appreciably from ideal values for the i + 2 residue in a type II beta-turn. In both peptides the observed (N...O) distances between the Boc CO and Ala(3) NH groups are far too long (I: 3.44 Angstrom; III: 3.63 Angstrom) for an intramolecular 4 --> 1 hydrogen bond. Boc-Ala-Dpg-Ala-NHMe (II) crystallizes with two independent molecules in the asymmetric unit. Both molecules IIA and IIB adopt consecutive beta-turn (type III-III in IIA and type III-I in IIB) or incipient 3(10)-helical structures, stabilized by two intramolecular 4 --> 1 hydrogen bonds. In all four molecules the bond angle N-C-alpha-C' (tau) at the Dxg residues are greater than or equal to 110 degrees. The observation of conformational angles in the helical region of phi,psi space at these residues is consistent with theoretical predictions.

Solution conformations of penta and heptapeptides containing repetitive α-aminoisobutyryl-Image -alanyl and α -aminoisobutyryl-Image -valyl sequences

The presence of folded solution conformations in the peptides Boc-Ala-(Aib-Ala)2-OMe, Boc-Val-(Aib-Val) 2-OMe, Boc-Ala-(Aib-Ala)3-OMe and Boc-Val-(Aib-Val)3-OMe has been established by 270MHz 1H NMR. Intramolecularly H-bonded NH groups have been identified using temperature and solvent dependence of NH chemical shifts and paramagnetic radical induced broadening of NH resonances. Both pentapeptides adopt 310 helical conformations possessing 3 intramolecular H-bonds in CDCl3 and (CD3)2SO. The heptapeptides favour helical structures with 5 H-bonds in CDCl3. In (CD3)2SO only 4 H-bonds are readily detected.

The helical conformations of 14- and 16-residue fragments of suzukacillin, a membrane channel-forming polypeptide

The suzukacillin fragments, Boc-Ala-Aib-Aib-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-OMe (14), Boc-Ala-Aib-Ala-Aib-Aib-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-OMe (16G) and the completely apolar 16-residue peptide in which the glutamine residue has been replaced by alanine (16A) have been studied by 270 MHz 1H-HMR, in C2HCl3 and (C2H3)2SO solution. Intramolecularly hydrogen-bonded NH groups have been identified by temperature and solvent dependence of chemical shifts. Peptides 14 and 16A adopt folded 310 helical conformations stabilized by 11 and 13 hydrogen bonds, respectively. In peptide 16G there are 12 intramolecular hydrogen bonds, with the glycine NH being solvent-exposed, in contrast to 14 and 16A.

Conformations Of Peptides Containing 1-Aminocyclohexane-Carboxylic Acid (Acc6) - Crystal-Structures Of 2 Model Peptides

The crystal structures of two peptides containing 1-aminocyclohexanecarboxylic acid (Acc6) are described. Boc-Aib-Acc6-NHMe · H2O adopts a β-turn conformation in the solid state, stabilized by an intramolecular 4 → 1 hydrogen bond between the Boc CO and methylamide NH groups. The backbone conformational angles (φAib = – 50.3°, ψAib = – 45.8°; φAcc6 = – 68.4°, ψAcc6 = – 15°) lie in between the values expected for ideal Type I or III β-turns. In Boc-Aib-Acc6-OMe, the Aib residue adopts a partially extended conformation (φAib = – 62.2°, ψAib = 143°) while the Acc6residue maintains a helical conformation (φAcc6 = 48°, ψAcc6= 42.6°). 1H n.m.r. studies in CDCl3 and (CD3)2SO suggest that Boc-Aib-Acc6-NHMe maintains the β-turn conformation in solution.

Stereochemistry of Peptides Containing 1-AminocyclopentanecarboxylicA cid ( Am5): Solution and Solid-state Conformations of Boc-Acc '-Acc'-NHMe

The conformational analysis of a protected homodipeptide of 1-aminocyclopentanecarboxylic acid (Acc5) has been carried out. 1H-nmr studies establish a -turn conformation for Boc-Acc5-Acc5-NHMe in chloroform and dimethylsulfoxide solutions involving the methylamide NH in an intramolecular hydrogen bond. Supportive evidence for the formation of an intramolecular hydrogen bond is obtained from ir studies. X-ray diffraction studies reveal a type III -turn conformation in the solid state stabilized by a 4 1 hydrogen bond between the Boc CO and methylamide NH groups. The , values for both Acc5 residues are close to those expected for an ideal 310-helical conformation ( ± 60°, ±30°).

Conformations of the amino aterminal tetrapeptide of emerimicins and antiamoebins in solution and in the solid state

The conformations of Boc-l-Phe-(AiB)3-OH (1) and Boc-l-Phe-(Aib)3-OMe (2) which correspond to the amino terminal sequence of the emerimicins and antiamoebins have been studied in solution using 270 MHz 1H n.m.r. In dimethyl sulphoxide solution both peptides show the presence of two strongly solvent shielded Aib NH groups, consistent with a consecutive β-turn conformation, involving the Aib(3) and Aib(4) NH groups in intramolecular 4 → I hydrogen bonds. This folded conformation is maintained for 2 in chloroform solution. Nuclear Overhauser effect studies provide evidence for a Type II Phe-Aib β-turn. An X-ray diffraction study of Boc-(d,l)-Phe-(Aib)3-OH establishes a single type III(III′) β-turn conformation with Aib(2)-Aib(3) as the corner residues. A single intramolecular 4 → I hydrogen bond between Phe(I) CO and Aib(4) NH groups is observed in the crystal. The solution conformation may incorporate a consecutive type II-III′ structure for the Phe(1)-Aib(2)-Aib(3) segment, with the initial type II β-turn being destabilized by intermolecular interactions in the solid state.

Proline ring conformations corresponding to a bistable jump model from 13C spin-lattice relaxation times

A formalism for extracting the conformations of a proline ring based on the bistable jump model of R. E. London [(1978) J. Am. Chem. Soc. 100, 2678-2685] from 13C spin-lattice relaxation times (T1) is given. The method is such that the relaxation data are only partially used to generate the conformations; these conformations are constrained to satisfy the rest of the relaxation data and to yield acceptable ring geometry. An alternate equation for T1 of 13C nuclei to that of London is given. The formalism is illustrated through an example.

Helical conformations of three crystalline pentapeptide fragments of suzukacillin, a membrane channel forming polypeptide

The crystal structures of three pentapeptide fragments of suzukacillin-A have been determined. Boc-Aib-Pro-Val-Aib-Val-OMe (peptide 1–5) adopts a distorted helical conformation, stabilized by three intramolecular hydrogen bonds (two 5→1, one 4→1). Boc-Ala-Aib-Ala-Aib-Aib-OMe (peptide 6–10) and Boc-Leu-Aib-Pro-Val-Aib-OMe (peptide 16–20) adopt 310 helical structures stabilized by three and two 4→1 intramolecular hydrogen bonds, respectively. These structures provide substantial support for a largely helical conformation for the suzukacillin membrane channel.

Theoretical studies on the conformations of higher gangliosides

The possible conformations of higher gangliosides (GD3, GT1a. GT1b, GQ1b) have been determined by computing their potential energy using semi-empirical potential functions. The favoured conformation of the disialic acid fragment in these gangliosides is independent of its position (internal or terminal). The favoured conformations of these gangliosides have also been correlated to their biological activity. The results suggest that tetanus toxin and sendai virus may have a large binding site which can accommodate at least four sugar residues.