Proteolytic processing of the nonstructural proteins of dengue 2 virus

The genomes of many positive stranded RNA viruses and of all retroviruses are translated as large polyproteins which are proteolytically processed by cellular and viral proteases. Viral proteases are structurally related to two families of cellular proteases, the pepsin-like and trypsin-like proteases. This thesis describes the proteolytic processing of several nonstructural proteins of dengue 2 virus, a representative member of the Flaviviridae, and describes methods for transcribing full-length genomic RNA of dengue 2 virus. Chapter 1 describes the in vitro processing of the nonstructural proteins NS2A, NS2B and NS3. Chapter 2 describes a system that allows identification of residues within the protease that are directly or indirectly involved with substrate recognition. Chapter 3 describes methods to produce genome length dengue 2 RNA from cDNA templates.

The nonstructural protein NS3 is structurally related to viral trypsinlike proteases from the alpha-, picorna-, poty-, and pestiviruses. The hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins was tested using an efficient in vitro expression system and antisera specific for the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed using T7 RNA polymerase and the RNA translated in reticulocyte lysates. Proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain necessary and sufficient for correct cleavage to the first 184 amino acids of NS3. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.

Biochemical and genetic experiments using viral proteinases have defined the sequence requirements for cleavage site recognition, but have not identified residues within proteinases that interact with substrates. A biochemical assay was developed that could identify residues which were important for substrate recognition. Chimeric proteases between yellow fever and dengue 2 were constructed that allowed mapping of regions involved in substrate recognition, and site directed mutagenesis was used to modulate processing efficiency.

Expression in vitro revealed that the dengue protease domain efficiently processes the yellow fever polyprotein between NS2A and NS2B and between NS2B and NS3, but that the reciprocal construct is inactive. The dengue protease processes yellow fever cleavage sites more efficiently than dengue cleavage sites, suggesting that suboptimal cleavage efficiency may be used to increase levels of processing intermediates in vivo. By mutagenizing the putative substrate binding pocket it was possible to change the substrate specificity of the yellow fever protease; changing a minimum of three amino acids in the yellow fever protease enabled it to recognize dengue cleavage sites. This system allows identification of residues which are directly or indirectly involved with enzyme-substrate interaction, does not require a crystal structure, and can define the substrate preferences of individual members of a viral proteinase family.

Full-length cDNA clones, from which infectious RNA can be transcribed, have been developed for a number of positive strand RNA viruses, including the flavivirus type virus, yellow fever. The technology necessary to transcribe genomic RNA of dengue 2 virus was developed in order to better understand the molecular biology of the dengue subgroup. A 5' structural region clone was engineered to transcribe authentic dengue RNA that contains an additional 1 or 2 residues at the 5' end. A 3' nonstructural region clone was engineered to allow production of run off transcripts, and to allow directional ligation with the 5' structural region clone. In vitro ligation and transcription produces full-length genomic RNA which is noninfectious when transfected into mammalian tissue culture cells. Alternative methods for constructing cDNA clones and recovering live dengue virus are discussed.

Palladium-Catalyzed Decarboxylative and Decarbonylative Transformations in the Synthesis of Fine and Commodity Chemicals

Decarboxylation and decarbonylation are important reactions in synthetic organic chemistry, transforming readily available carboxylic acids and their derivatives into various products through loss of carbon dioxide or carbon monoxide. In the past few decades, palladium-catalyzed decarboxylative and decarbonylative reactions experienced tremendous growth due to the excellent catalytic activity of palladium. Development of new reactions in this category for fine and commodity chemical synthesis continues to draw attention from the chemistry community.

The Stoltz laboratory has established a palladium-catalyzed enantioselective decarboxylative allylic alkylation of β-keto esters for the synthesis of α-quaternary ketones since 2005. Recently, we extended this chemistry to lactams due to the ubiquity and importance of nitrogen-containing heterocycles. A wide variety of α-quaternary and tetrasubstituted α-tertiary lactams were obtained in excellent yields and exceptional enantioselectivities using our palladium-catalyzed decarboxylative allylic alkylation chemistry. Enantioenriched α-quaternary carbonyl compounds are versatile building blocks that can be further elaborated to intercept synthetic intermediates en route to many classical natural products. Thus our chemistry enables catalytic asymmetric formal synthesis of these complex molecules.

In addition to fine chemicals, we became interested in commodity chemical synthesis using renewable feedstocks. In collaboration with the Grubbs group, we developed a palladium-catalyzed decarbonylative dehydration reaction that converts abundant and inexpensive fatty acids into value-added linear alpha olefins. The chemistry proceeds under relatively mild conditions, requires very low catalyst loading, tolerates a variety of functional groups, and is easily performed on a large scale. An additional advantage of this chemistry is that it provides access to expensive odd-numbered alpha olefins.

Finally, combining features of both projects, we applied a small-scale decarbonylative dehydration reaction to the synthesis of α-vinyl carbonyl compounds. Direct α-vinylation is challenging, and asymmetric vinylations are rare. Taking advantage of our decarbonylative dehydration chemistry, we were able to transform enantioenriched δ-oxocarboxylic acids into quaternary α-vinyl carbonyl compounds in good yields with complete retention of stereochemistry. Our explorations culminated in the catalytic enantioselective total synthesis of (–)-aspewentin B, a terpenoid natural product featuring a quaternary α-vinyl ketone. Both decarboxylative and decarbonylative chemistries found application in the late stage of the total synthesis.

Effect of different rice brans on the growth of Thai silver barb (Puntius gonionotus Bleeker) in seasonal ponds

An experiment was conducted for a period of 110 days to study the effect of different rice brans on the growth of Thai silver barb (Puntius gonionotus, Bleeker) in rainfed seasonal ponds (30 m² each). Each of the ponds was stocked with 150 fish with the mean initial body weight of 2.03 ± 0.03 g. There were three treatments namely R1, R2 and R3 each having four replicates. Three types of rice bran namely coarse, auto (fine) and red were applied to the treatments R1, R2 and R3 respectively. Fish received different types of rice bran at a rate of 5% of body weight daily. The water quality parameters were found within the productive range. The results showed that treatment R1 produced significantly (p<0.05) highest growth and treatment R2 produced the lowest growth. The survival rates varied between 77 and 84% with treatment R3 producing the highest survival. However, the overall best production (1530 kg/ha) and economic return for the culture period was obtained in treatment R1 receiving coarse rice bran. The results of the present study demonstrated that the coarse rice bran resulted in better growth and production of P. gonionotus.

Single domain YBCO/Ag bulk superconductors fabricated by seeded infiltration and growth

We have applied the seeded infiltration and growth (IG) technique to the processing of samples containing Ag in an attempt to fabricate Ag-doped Y-Ba-Cu-O (YBCO) bulk superconductors with enhanced mechanical properties. The IG technique has been used successfully to grow bulk Ag-doped YBCO superconductors of up to 25 mm in diameter in the form of single grains. The distribution of Ag in the parent Y-123 matrix fabricated by the IG technique is observed to be at least as uniform as that in samples grown by conventional top seeded melt growth (TSMG). Fine Y-211 particles were observed to be embedded within the Y-123 matrix for the IG processed samples, leading to a high critical current density, Jc, of over 70 kA/cm2 at 77.3 K in self-field. The distribution of Y-211 in the IG sample microstructure, however, is inhomogeneous, which leads to a variation in the spatial distribution of Jc throughout the bulk matrix. A maximum-trapped field of around 0.43 T at 1.2 mm above the sample surface (i.e. including 0.7 mm for the sensor mould thickness) is observed at liquid nitrogen temperature, despite the relatively small grain size of the sample (20 mm diameter × 7 mm thickness). © 2008 IOP Publishing Ltd.

Advanced thermal processing of semiconductor materials in the msec-range

This paper reviews the advances that flash lamp annealing brings to the processing of the most frequently used semiconductor materials, namely silicon and silicon carbide, thus enabling the fabrication of novel microelectronic structures and materials. The paper describes how such developments can translate into important practical applications leading to a wide range of technological benefits. Opportunities in ultra-shallow junction formation, heteroepitaxial growth of thin films of cubic silicon carbide on silicon, and crystallization of amorphous silicon films, along with the technical reasons for using flash lamp annealing are discussed in the context of state-of-the-art materials processing. © 2005 IEEE.

Bayesian signal processing

The application of Bayes' Theorem to signal processing provides a consistent framework for proceeding from prior knowledge to a posterior inference conditioned on both the prior knowledge and the observed signal data. The first part of the lecture will illustrate how the Bayesian methodology can be applied to a variety of signal processing problems. The second part of the lecture will introduce the concept of Markov Chain Monte-Carlo (MCMC) methods which is an effective approach to overcoming many of the analytical and computational problems inherent in statistical inference. Such techniques are at the centre of the rapidly developing area of Bayesian signal processing which, with the continual increase in available computational power, is likely to provide the underlying framework for most signal processing applications.

Towards jet flow LES of conceptual nozzles for acoustic predictions

Progress in simulating chevron nozzle jet flows using ILES/RANS-ILES approaches and using the Ffowcs Williams and Hawkings (FW-H) surface integral method to predict the radiated far field sound is presented in this paper. With the focus on the realistic chevron geometries, SMC001 and SMC006, coarse and fine meshes are generated in the range of 3∼13 million mesh cells. Throughout this work, to minimize numerical dissipation introduced by mesh quality issues, the hexahedral cell type is used. Numerical simulations are then carried out with cell-vertex and cell-centered codes. Despite the modest grids, mean velocities and turbulent statistics are found to be in reasonable accord with measurements. Also, far field sound levels predicted by the FW-H post processor are encouraging. Copyright © 2008 by the American Institute of Aeronautics and Astronautics, Inc.

Adoption of recommended quality control practices by fish processing factories

Responses to mailed questionnaires from 31 fish processing factories revealed that for 55 recommended practices in quality control, the percentage of adopters varied from 21.43 to 100.The adoption index was not correlated to any of five variables studied. The mean adoption index did not differ significantly between five other criteria related to fish processing. Rate of rejection or reprocessing at the factory itself was highly correlated with the amount of water used per tonne of processed material.

Unified compression model for venice lagoon natural silts

Over the last 50 years, the city of Venice, Italy, has observed a significant increase in the frequency of flooding. Numerous engineering solutions have been proposed, including the use of movable gates located at the three lagoon inlets. A key element in the prediction of performance is the estimation of settlements of the foundation system of the gates. The soils of Venice Lagoon are characterized by very erratic depositional patterns of clayey silts, resulting in an extremely heterogeneous stratigraphy with discontinuous layering. The soils are also characterized by varying contents of coarse and fine-grained particles. In contrast, the mineralogical composition of these deposits is quite uniform, which allows us to separate the influence of mineralogy from that of grain size distribution. A comprehensive geotechnical testing program was performed to assess the one-dimensional compression of Venice soils and examine the factors affecting the response in the transition from one material type to another. The compressibility of these natural silty clayey soils can be described by a single set of constitutive laws incorporating the relative fraction of granular to cohesive material. © 2007 ASCE.

The release behavior of inorganic nitrogen and phosphorus in sediment during disturbance

Sediments and surface water were sampled in a tide flat in the Huiquan Bay, Qingdao, China in January 2004 to simulate the exchange of NH4-N/NO3-N/PO43- between sediments and surface water. A working system was designed with which samples were shaken at 60, 120 and 150 revolutions per minute (r/min). Experiment results show that NH4-N concentration in water at shaking rate of 60 r/min decreased gradually, while at 120 r/min increased gradually. In resuspension, fine-grained sediments contributed most NH4-N to the seawater, followed by medium-grained and coarse-grained sediments. The NO3-N concentration in water had a negative relation, with the shaking rate; the medium-grained sediments contributed more NO3-N to seawater than the coarse- and fine-grained sediments. The PO43- concentration is positively related with the shaking rate, the fine-grained sediments were the main N and P contributor to the seawater, followed by medium- and coarse-grained sediments.

提高勘探成熟区地震资料精度的方法研究

With the Oil field exploration and exploitation, the problem of supervention and enhaning combination gas recovery was faced.then proposing new and higher demands to precision of seismic data. On the basis of studying exploration status,resource potential,and quality of 3D seismic data to internal representative mature Oil field, taking shengli field ken71 zone as study object, this paper takes advantage of high-density 3D seismic technique to solving the complex geologic problem in exploration and development of mature region, deep into researching the acquisition, processing of high-density 3D seismic data. This disseration study the function of routine 3D seismic, high-density 3D seismic, 3D VSP seismic,and multi-wave multi-component seismic to solving the geologic problem in exploration and development of mature region,particular introduce the advantage and shortage of high-density 3D seismic exploration, put forward the integrated study method of giving priority to high-density 3D seismic and combining other seismic data in enhancing exploration accuracy of mature region. On the basis of detailedly studying acquisition method of high-density 3D seismic and 3D VSP seismic,aming at developing physical simulation and numeical simulation to designing and optimizing observation system. Optimizing “four combination” whole acquisition method of acquisition of well with ground seimic and “three synchron”technique, realizing acquisition of combining P-wave with S-wave, acquisition of combining digit geophone with simulation geophone, acquisition of 3D VSP seismic with ground seimic, acquisition of combining interborehole seismic,implementing synchron acceptance of aboveground equipment and downhole instrument, common use and synchron acceptance of 3D VSP and ground shots, synchron acquisition of high-density P-wave and high-density multi-wave, achieve high quality magnanimity seismic data. On the basis of detailedly analysising the simulation geophone data of high-density acquisition ,adopting pertinency processing technique to protecting amplitude,studying the justice matching of S/N and resolution to improving resolution of seismic profile ,using poststack series connection migration,prestack time migration and prestack depth migration to putting up high precision imaging,gained reliable high resolution data.At the same time carrying along high accuracy exploration to high-density digit geophone data, obtaining good improve in its resolution, fidelity, break point clear degree, interbed information, formation characteristics and so on.Comparing processing results ,we may see simulation geophone high-density acquisition and high precision imaging can enhancing resolution, high-density seismic basing on digit geophone can better solve subsurface geology problem. At the same time, fine processing converted wave of synchron acquisition and 3D VSP seismic data,acquiring good result. On the basis of high-density seismic data acquisition and high-density seismic data processing, carry through high precision structure interpretation and inversion, and preliminary interpretation analysis to 3D VSP seismic data and multi-wave multi-component seismic data. High precision interpretation indicates after high resolution processing ,structural diagram obtaining from high-density seismic data better accord with true geoligy situation.

花岗岩风化土微观结构及其工程地质特性研究

Saprolite is the residual soil resulted from completely weathered or highly weathered granite and with corestones of parent rock. It is widely distributed in Hong Kong. Slope instability usually happens in this layer of residual soil and thus it is very important to study the engineering geological properties of Saprolite. Due to the relic granitic texture, the deformation and strength characteristics of Saprolite are very different from normal residual soils. In order to investigate the effects of the special microstructure on soil deformation and strength, a series of physical, chemical and mechanical tests were conducted on Saprolite at Kowloon, Hong Kong. The tests include chemical analysis, particle size analysis, mineral composition analysis, mercury injection, consolidation test, direct shear test, triaxial shear test, optical analysis, SEM & TEM analysis, and triaxial shear tests under real-time CT monitoring.Based on the testing results, intensity and degree of weathering were classified, factors affecting and controlling the deformation and strength of Saprolite were identified, and the interaction between those factors were analyzed.The major parameters describing soil microstructure were introduced mainly based on optical thin section analysis results. These parameters are of importance and physical meaning to describe particle shape, particle size distribution (PSD), and for numerical modeling of soil microstructure. A few parameters to depict particle geometry were proposed or improved. These parameters can be used to regenerate the particle shape and its distribution. Fractal dimension of particle shape was proposed to describe irregularity of particle shapes and capacity of space filling quantitatively. And the effect of fractal dimension of particle shape on soil strength was analyzed. At the same time, structural coefficient - a combined parameter which can quantify the overall microstructure of rock or soil was introduced to study Saprolite and the results are very positive. The study emphasized on the fractal characteristics of PSD and pore structure by applying fractal theory and method. With the results from thin section analysis and mercury injection, it was shown that at least two fractal dimensions Dfl(DB) and Df2 (Dw), exist for both PSD and pore structure. The reasons and physical meanings behind multi-fractal dimensions were analyzed. The fractal dimensions were used to calculate the formation depth and weathering rate of granite at Kowloon. As practical applications, correlations and mathematical models for fractal dimensions and engineering properties of soil were established. The correlation between fractal dimensions and mechanical properties of soil shows that the internal friction angle is mainly governed by Dfl 9 corresponding to coarse grain components, while the cohesion depends on Df2 , corresponding to fine grain components. The correlations between the fractal dimension, friction angle and cohesion are positive linear.Fractal models of PSD and pore size distribution were derived theoretically. Fragmentation mechanism of grains was also analyzed from the viewpoint of fractal. A simple function was derived to define the theoretical relationship between the water characteristic curve (WCC) and fractal dimension, based on a number of classical WCC models. This relationship provides a new analytical tool and research method for hydraulic properties in porous media and solute transportation. It also endues fractal dimensions with new physical meanings and facilitates applications of fractal dimensions in water retention characteristics, ground water movement, and environmental engineering.Based on the conclusions from the fractal characteristics of Saprolite, size effect on strength was expressed by fractal dimension. This function is in complete agreement with classical Weibull model and a simple function was derived to represent the relationship between them.In this thesis, the phenomenon of multi-fractal dimensions was theoretically analyzed and verified with WCC and saprolite PSD results, it was then concluded that multi-fractal can describe the characteristics of one object more accurately, compared to single fractal dimension. The multi-fractal of saprolite reflects its structural heterogeneity and changeable stress environment during the evolution history.

Using Speculation to Reduce Server Load and Service Time on the WWW

Speculative service implies that a client's request for a document is serviced by sending, in addition to the document requested, a number of other documents (or pointers thereto) that the server speculates will be requested by the client in the near future. This speculation is based on statistical information that the server maintains for each document it serves. The notion of speculative service is analogous to prefetching, which is used to improve cache performance in distributed/parallel shared memory systems, with the exception that servers (not clients) control when and what to prefetch. Using trace simulations based on the logs of our departmental HTTP server http://cs-www.bu.edu, we show that both server load and service time could be reduced considerably, if speculative service is used. This is above and beyond what is currently achievable using client-side caching [3] and server-side dissemination [2]. We identify a number of parameters that could be used to fine-tune the level of speculation performed by the server.

From Parallel Sequence Representations to Calligraphic Control: A Conspiracy of Neural Circuits

Calligraphic writing presents a rich set of challenges to the human movement control system. These challenges include: initial learning, and recall from memory, of prescribed stroke sequences; critical timing of stroke onsets and durations; fine control of grip and contact forces; and letter-form invariance under voluntary size scaling, which entails fine control of stroke direction and amplitude during recruitment and derecruitment of musculoskeletal degrees of freedom. Experimental and computational studies in behavioral neuroscience have made rapid progress toward explaining the learning, planning and contTOl exercised in tasks that share features with calligraphic writing and drawing. This article summarizes computational neuroscience models and related neurobiological data that reveal critical operations spanning from parallel sequence representations to fine force control. Part one addresses stroke sequencing. It treats competitive queuing (CQ) models of sequence representation, performance, learning, and recall. Part two addresses letter size scaling and motor equivalence. It treats cursive handwriting models together with models in which sensory-motor tmnsformations are performed by circuits that learn inverse differential kinematic mappings. Part three addresses fine-grained control of timing and transient forces, by treating circuit models that learn to solve inverse dynamics problems.

Second language processing: an analysis of theory, problems and possible solutions

Second Language Processing examines the problems facing learners in the second language classroom from the theoretical perspectives of Processing Instruction (structured input) and Enhanced Input. These two theories are brought to bear on a variety of processing problems, such as the difficulty of connecting second language grammatical forms encoding tense and mood as well as noun-adjective agreement with their meaning. Empirical studies examine a range of languages including Japanese, Italian and Spanish, through which the authors suggest practical solutions to these processing problems.