Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin

The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates; this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD -> GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity. (C) 2001 Elsevier Science B.V. All rights reserved.

Engineering the structures of nanoporous clays with micelles of alkyl polyether surfactants

Composite clay nanostructures (CCNs) were observed in intercalating Laponite clay with alumina in the presence of alkyl polyether surfactants which contain hydrophobic alkyl chains and ether groups. Such nanostructured clays are highly porous solids consisting of randomly orientated clay platelets intercalated with alumina nanoparticles. The pores in the product solids are larger than the dimension of the surfactant molecules, ranging from 2 to 10 nm. This suggests that the micelles of the surfactant molecules, rather than the molecules, act as templates in the synthesis. Interestingly, it is found that the size of the framework pores was directly proportional to the amount of the surfactants in terms of moles, but shows no evident dependence on the size of the surfactant molecules. Broad pore size distributions were observed for the product CCNs. This study demonstrates that introducing surfactants in the pillaring process of clays is a powerful strategy for tailoring the pore structures of nanoporous clays. With this new technique, it is possible to design and engineer such composite clay nanostructures with desired pore and surface properties by the proper choice of surfactant amounts and preparation conditions.

Hydrophobicity of Templated Silica Xerogels for Molecular Sieving Applications

Silica xerogels were prepared by a sol-gel process catalyzed by acid with tetraethylorthosilicate, and using an organic covalent ligand template (methyltriethoxysilane) or a noncovalent template C6 surfactant (triethylhexylammonium bromide). The influence of hydrotreatment on the structure of templated xerogels is examined in terms of surface area, micropore volume, average pore size, and pore size distribution, and compared against a blank xerogel (nontemplated). The role of surface functional groups was evaluated using Si-29 nuclear magnetic resonance. The structural integrity of the xerogel was maintained to a large extent in samples that had a high contribution of Q(4) species (siloxane groups). Xerogel matrix densification occurred when there was a large concentration of Q(3) and Q(2) species (silanol groups), which also were responsible for increased hydrophilicity. The templated xerogels resulted in up to a 25% concentration of methyl functional groups (T-3 and T-2 species), leading to hydrophobic xerogels. The best results in terms of structural integrity and hydrophobicity were obtained with templated xerogels prepared with the C6 surfactant. The results in this study suggest that surfactant-enhanced condensation reactions lead to structures with a high contribution of Q(4) groups, which are not susceptible to water attack, but are strong enough to oppose matrix densification during rehydration.

Characterisation of templated xerogels for molecular sieve application

This paper presents the results of the characterisation of templated silica xerogels as precursor material for molecular sieve silica membranes for gas separation. The template agent integrated in the xerogel matrix is a methyl ligand covalently bended to the siloxane network in the form of methyltriethoxysilane (MTES). Several surface and microstructural characterisation techniques such as TGA, FTIR, NMR, and nitrogen adsorption have been employed to obtain information on the reaction mechanisms involved in the sol-gel processing of such molecular sieves. The characterisation results show the effects of processing parameters such as heat treatment temperature, and the concentration of the covalently bonded template on the development of the pore structure. It was found that calcination temperature significantly enhanced the condensation reactions thus resulted in more Si-O-Si groups being formed. This was also confirmed with the data of FTIR characterisation showing enhanced silicon bands at higher heat treatment temperatures. As a result of the promoted densification and shrinkable pore network the micropore volume also reduced with increasing methyl ligand molar ratio. However, the mean pore diameter does not change significantly with calcination temperature. While the contribution of the templates towards controlling pore size is less precise, increasing the methyl ligand molar ratio results in the broadening of the pore size distribution and lower pore volume. Higher template concentration induces the collapse of the xerogel matrix due to capillary stress promoting dense xerogels with low pore volume (C) 2001 Elsevier Science B.V. All rights reserved.

2,6,,9-trioxabicyclo[3.3.1]nona-3,7-dienes and 2,4,6,8-tetraoxaadamantanes: Novel chiral spacer units in macrocyclic polyethers

The unusual chiral heterocyclic systems, trioxabicyclo[3.3.1]nona-3,7-dienes (bridged bisdioxines), are incorporated as novel spacer molecules into macrocyclic polyether ring systems of various sizes (8, 9 as well as 11-15) by cyclocondensation reaction of the! bisacid chloride 4b or bisesters 6,7 and 10, with several ethylene glycols. The 2:2 macrocycles 12-14 are obtained in approximately 50:50 mixtures of diastereomers. These conclusions are mainly based on HPLC data presented in Table I as well as X-ray analyses of (1R,5R)-8c (space group Pbca, a = 10.163(3) Angstrom, b = 18.999(4) Angstrom, c = 36.187(10) Angstrom, V = 6987(3) Angstrom(3), Z = 8, d(calc) = 1.218 g cm(-3), 6974 reflections, R = 0.0553.), mesolrac-11 (space group P (1) over bar, a = 10.472(5) Angstrom, b = 16.390(5) Angstrom, c = 17.211(5) Angstrom, alpha = 98.69(2)degrees, beta = 93.04(2)degrees, gamma = 98.52(2)degrees, V = 2879.3(18) Angstrom(3), Z = 2, d(calc) = 1.173 g cm(-3), 11,162 reflections, R = 0.0945) and meso-12 (space group P2(1)/c, a = 9.927(2), b = 18.166(3), c = 17.820(3) Angstrom, beta = 96.590(10)degrees, V = 3192.3(10)Angstrom(3), Z = 4, D-c = 1.109 g cm(-3), 3490 reflections, R = 0.0646). The 1:1 macrocycles 8b,c are also formed by intramolecular transesterification of the open-chain bisesters 7b,c and their formation is favored by the use of metal ions as templates. The bridged bisdioxine moieties in 8b and 12 are converted into the corresponding chiral tetra-oxaadamantane spacers to afford macrocycles 16 and 17. Preliminary metal ion complexation studies with selected species (8c, 11-14) were also performed.

A framework for table driven testing of Java classes

With the advent of object-oriented languages and the portability of Java, the development and use of class libraries has become widespread. Effective class reuse depends on class reliability which in turn depends on thorough testing. This paper describes a class testing approach based on modeling each test case with a tuple and then generating large numbers of tuples to thoroughly cover an input space with many interesting combinations of values. The testing approach is supported by the Roast framework for the testing of Java classes. Roast provides automated tuple generation based on boundary values, unit operations that support driver standardization, and test case templates used for code generation. Roast produces thorough, compact test drivers with low development and maintenance cost. The framework and tool support are illustrated on a number of non-trivial classes, including a graphical user interface policy manager. Quantitative results are presented to substantiate the practicality and effectiveness of the approach. Copyright (C) 2002 John Wiley Sons, Ltd.

Towards synthetic vaccines built on carbohydrate cores

Lipophilic polyfunctional carbohydrate core/templates have been designed and developed for drug/vaccine delivery. Three carbohydrate-based templates containing four protected N-terminal arms were synthesised from glucose and galactose. Methyl alpha-D-glucopyranoside was converted to two derivatives bearing a carboxylic acid handle for attachment to solid supports, spacer arms of differing hydrophilicity, and phthaloyl-protected amino groups suitable for peptide chain extension. beta-D-Galactopyranosyl azide was converted to a template bearing a carboxylic acid handle and four BOC-protected amines. All the templates were found to be suitable for attachment to solid supports and subsequent cleavage from resins, using either BOC- or FMOC-methodologies.

Conformationally constrained macrocycles that mimic tripeptide beta-strands in water and aprotic solvents

The beta-strand conformation is unknown for short peptides in aqueous solution, yet it is a fundamental building block in proteins and the crucial recognition motif for proteolytic enzymes that enable formation and turnover of all proteins. To create a generalized scaffold as a peptidomimetic that is preorganized in a beta-strand, we individually synthesized a series of 15-22-membered macrocyclic analogues of tripeptides and analyzed their structures. Each cycle is highly constrained by two trans amide bonds and a planar aromatic ring with a short nonpeptidic linker between them. A measure of this ring strain is the restricted rotation of the component tyrosinyl aromatic ring (DeltaG(rot) 76.7 kJ mol(-1) (16-membered ring), 46.1 kJ mol(-1) (17-membered ring)) evidenced by variable temperature proton NMR spectra (DMF-d(7), 200-400 K). Unusually large amide coupling constants ((3)J(NH-CHalpha) 9-10 Hz) corresponding to large dihedral angles were detected in both protic and aprotic solvents for these macrocycles, consistent with a high degree of structure in solution. The temperature dependence of all amide NH chemical shifts (Deltadelta/T7-12 ppb/deg) precluded the presence of transannular hydrogen bonds that define alternative turn structures. Whereas similar sized conventional cyclic peptides usually exist in solution as an equilibrium mixture of multiple conformers, these macrocycles adopt a well-defined beta-strand structure even in water as revealed by 2-D NMR spectral data and by a structure calculation for the smallest (15-membered) and most constrained macrocycle. Macrocycles that are sufficiently constrained to exclusively adopt a beta-strand-mimicking structure in water may be useful pre-organized and generic templates for the design of compounds that interfere with beta-strand recognition in biology.

Large amplitude vibration of thermo-electro-mechanically stressed FGM laminated plates

This paper presents a large amplitude vibration analysis of pre-stressed functionally graded material (FGM) laminated plates that are composed of a shear deformable functionally graded layer and two surface-mounted piezoelectric actuator layers. Nonlinear governing equations of motion are derived within the context of Reddy's higher-order shear deformation plate theory to account for transverse shear strain and rotary inertia. Due to the bending and stretching coupling effect, a nonlinear static problem is solved first to determine the initial stress state and pre-vibration deformations of the plate that is subjected to uniform temperature change, in-plane forces and applied actuator voltage. By adding an incremental dynamic state to the pre-vibration state, the differential equations that govern the nonlinear vibration behavior of pre-stressed FGM laminated plates are derived. A semi-analytical method that is based on one-dimensional differential quadrature and Galerkin technique is proposed to predict the large amplitude vibration behavior of the laminated rectangular plates with two opposite clamped edges. Linear vibration frequencies and nonlinear normalized frequencies are presented in both tabular and graphical forms, showing that the normalized frequency of the FGM laminated plate is very sensitive to vibration amplitude, out-of-plane boundary support, temperature change, in-plane compression and the side-to-thickness ratio. The CSCF and CFCF plates even change the inherent hard-spring characteristic to soft-spring behavior at large vibration amplitudes. (C) 2003 Elsevier B.V. All rights reserved.

Postbuckling of piezoelectric FGM plates subject to thermo-electro-mechanical loading

In this paper, we examine the postbuckling behavior of functionally graded material FGM rectangular plates that are integrated with surface-bonded piezoelectric actuators and are subjected to the combined action of uniform temperature change, in-plane forces, and constant applied actuator voltage. A Galerkin-differential quadrature iteration algorithm is proposed for solution of the non-linear partial differential governing equations. To account for the transverse shear strains, the Reddy higher-order shear deformation plate theory is employed. The bifurcation-type thermo-mechanical buckling of fully clamped plates, and the postbuckling behavior of plates with more general boundary conditions subject to various thermo-electro-mechanical loads, are discussed in detail. Parametric studies are also undertaken, and show the effects of applied actuator voltage, in-plane forces, volume fraction exponents, temperature change, and the character of boundary conditions on the buckling and postbuckling characteristics of the plates. (C) 2003 Elsevier Science Ltd. All rights reserved.

Surge damping analysis in pipe systems: modelling and experiments

The current study focuses on the analysis of pressure surge damping in single pipeline systems generated by a fast change of flow, conditions. A dimensionless form of pressurised transient flow equations was developed. presenting the main advantage of being independent of the system characteristics. In lack of flow velocity profiles. the unsteady friction in turbulent regimes is analysed based on two new empirical corrective-coefficients associated with local and convective acceleration terms. A new, surge damping approach is also presented taking into account the pressure peak time variation. The observed attenuation effect in the pressure wave for high deformable pipe materials can be described by a combination of the non-elastic behaviour of the pipe-wall with steady and unsteady friction effects. Several simulations and experimental tests have been carried out. in order to analyse the dynamic response of single pipelines with different characteristics, such as pipe materials. diameters. thickness. lengths and transient conditions.

Marine cyanobacteria compounds with anticancer properties: a review on the implication of apoptosis

Marine cyanobacteria have been considered a rich source of secondary metabolites with potential biotechnological applications, namely in the pharmacological field. Chemically diverse compounds were found to induce cytoxicity, anti-inflammatory and antibacterial activities. The potential of marine cyanobacteria as anticancer agents has however been the most explored and, besides cytotoxicity in tumor cell lines, several compounds have emerged as templates for the development of new anticancer drugs. The mechanisms implicated in the cytotoxicity of marine cyanobacteria compounds in tumor cell lines are still largely overlooked but several studies point to an implication in apoptosis. This association has been related to several apoptotic indicators such as cell cycle arrest, mitochondrial dysfunctions and oxidative damage, alterations in caspase cascade, alterations in specific proteins levels and alterations in the membrane sodium dynamics. In the present paper a compilation of the described marine cyanobacterial compounds with potential anticancer properties is presented and a review on the implication of apoptosis as the mechanism of cell death is discussed.

Geração de simuladores de filas para diferentes contextos com estudo de casos para redes de computadores

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Analysis of tongue shape and motion in speech production using statistical modeling

The mechanisms of speech production are complex and have been raising attention from researchers of both medical and computer vision fields. In the speech production mechanism, the articulator’s study is a complex issue, since they have a high level of freedom along this process, namely the tongue, which instigates a problem in its control and observation. In this work it is automatically characterized the tongues shape during the articulation of the oral vowels of Portuguese European by using statistical modeling on MR-images. A point distribution model is built from a set of images collected during artificially sustained articulations of Portuguese European sounds, which can extract the main characteristics of the motion of the tongue. The model built in this work allows under standing more clearly the dynamic speech events involved during sustained articulations. The tongue shape model built can also be useful for speech rehabilitation purposes, specifically to recognize the compensatory movements of the articulators during speech production.

e-Recruitment: descrição e inferência sobre oferta e procura

Trabalho de Projeto para obtenção do grau de Mestre em Engenharia Informática e de Computadores