Production of active recombinant eIF5A: reconstitution in E.coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine- or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.

Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The influence of the region between residues 220 and 344 and beyond in Phrixotrix railroad worm luciferases green and red bioluminescence

To find the regions having a major influence on the bioluminescence spectra of railroad worm luciferases, we constructed new chimeric luciferases switching the fragments from residues 1-219 and from 220-545 between Phrixotrix viviani (PxvGR; λmax = 548 nm) green light-emitting luciferase and Phrixothrix hirtus (PxhRE; λmax = 623 nm) red light-emitting luciferases. The emission spectrum (λmax = 571 nm) and KM for luciferin in the chimera PxRE220GR (1-219, PxhRE; 220-545, PxvGR) suggested that the region above residue 220 of PxvGR had a major effect on the active site. However, switching the sequence between the residues 226-344 from PxvGR luciferase into PxhRE (PxREGRRE) luciferase resulted in red light emission (λmax = 603 nm), indicating that the region 220-344 by itself does not determine the emission spectrum. Furthermore, the sequence before residue 220 of the green-emitting luciferase is incompatible for light emission with the sequence above residue 220 of PxhRE. These results suggest that the fragments before and after residue 220, which correspond to distinct subdomains, may fold differently in the green- and red-emitting luciferases, affecting the active site conformation.

Detecção e localização de danos em materiais compósitos aplicado em aeronaves utilizando redes neurais artificiais

Pós-graduação em Engenharia Mecânica - FEIS

Production of active recombinant eIF5A: reconstitution in E. coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine- or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.

Interpretação racional do ensaio DPL

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A new method for decision making in multi-objective optimization problems

Many engineering sectors are challenged by multi-objective optimization problems. Even if the idea behind these problems is simple and well established, the implementation of any procedure to solve them is not a trivial task. The use of evolutionary algorithms to find candidate solutions is widespread. Usually they supply a discrete picture of the non-dominated solutions, a Pareto set. Although it is very interesting to know the non-dominated solutions, an additional criterion is needed to select one solution to be deployed. To better support the design process, this paper presents a new method of solving non-linear multi-objective optimization problems by adding a control function that will guide the optimization process over the Pareto set that does not need to be found explicitly. The proposed methodology differs from the classical methods that combine the objective functions in a single scale, and is based on a unique run of non-linear single-objective optimizers.

Development of parallelizable flood inundation models for large scale analysis

Flood disasters are a major cause of fatalities and economic losses, and several studies indicate that global flood risk is currently increasing. In order to reduce and mitigate the impact of river flood disasters, the current trend is to integrate existing structural defences with non structural measures. This calls for a wider application of advanced hydraulic models for flood hazard and risk mapping, engineering design, and flood forecasting systems. Within this framework, two different hydraulic models for large scale analysis of flood events have been developed. The two models, named CA2D and IFD-GGA, adopt an integrated approach based on the diffusive shallow water equations and a simplified finite volume scheme. The models are also designed for massive code parallelization, which has a key importance in reducing run times in large scale and high-detail applications. The two models were first applied to several numerical cases, to test the reliability and accuracy of different model versions. Then, the most effective versions were applied to different real flood events and flood scenarios. The IFD-GGA model showed serious problems that prevented further applications. On the contrary, the CA2D model proved to be fast and robust, and able to reproduce 1D and 2D flow processes in terms of water depth and velocity. In most applications the accuracy of model results was good and adequate to large scale analysis. Where complex flow processes occurred local errors were observed, due to the model approximations. However, they did not compromise the correct representation of overall flow processes. In conclusion, the CA model can be a valuable tool for the simulation of a wide range of flood event types, including lowland and flash flood events.

Increasing Utilization of US Electric Grids via Smart Technologies: Integration of Load Management, Real Time Pricing, Renewables, EVs and Smart Grid Sensors

Electric power grids throughout the world suffer from serious inefficiencies associated with under-utilization due to demand patterns, engineering design and load following approaches in use today. These grids consume much of the world’s energy and represent a large carbon footprint. From material utilization perspectives significant hardware is manufactured and installed for this infrastructure often to be used at less than 20-40% of its operational capacity for most of its lifetime. These inefficiencies lead engineers to require additional grid support and conventional generation capacity additions when renewable technologies (such as solar and wind) and electric vehicles are to be added to the utility demand/supply mix. Using actual data from the PJM [PJM 2009] the work shows that consumer load management, real time price signals, sensors and intelligent demand/supply control offer a compelling path forward to increase the efficient utilization and carbon footprint reduction of the world’s grids. Underutilization factors from many distribution companies indicate that distribution feeders are often operated at only 70-80% of their peak capacity for a few hours per year, and on average are loaded to less than 30-40% of their capability. By creating strong societal connections between consumers and energy providers technology can radically change this situation. Intelligent deployment of smart sensors, smart electric vehicles, consumer-based load management technology very high saturations of intermittent renewable energy supplies can be effectively controlled and dispatched to increase the levels of utilization of existing utility distribution, substation, transmission, and generation equipment. The strengthening of these technology, society and consumer relationships requires rapid dissemination of knowledge (real time prices, costs & benefit sharing, demand response requirements) in order to incentivize behaviors that can increase the effective use of technological equipment that represents one of the largest capital assets modern society has created.

Rural water system sustainability : a case study of community-managed water systems in Saramaka communities

Worldwide, rural populations are far less likely to have access to clean drinking water than are urban ones. In many developing countries, the current approach to rural water supply uses a model of demand-driven, community-managed water systems. In Suriname, South America rural populations have limited access to improved water supplies; community-managed water supply systems have been installed in several rural communities by nongovernmental organizations as part of the solution. To date, there has been no review of the performance of these water supply systems. This report presents the results of an investigation of three rural water supply systems constructed in Saramaka villages in the interior of Suriname. The investigation used a combination of qualitative and quantitative methods, coupled with ethnographic information, to construct a comprehensive overview of these water systems. This overview includes the water use of the communities, the current status of the water supply systems, histories and sustainability of the water supply projects, technical reviews, and community perceptions. From this overview, factors important to the sustainability of these water systems were identified. Community water supply systems are engineered solutions that operate through social cooperation. The results from this investigation show that technical adequacy is the first and most critical factor for long-term sustainability of a water system. It also shows that technical adequacy is dependent on the appropriateness of the engineering design for the social, cultural, and natural setting in which it takes place. The complex relationships between technical adequacy, community support, and the involvement of women play important roles in the success of water supply projects. Addressing these factors during the project process and taking advantage of alternative water resources may increase the supply of improved drinking water to rural communities.

Study on the response of IFMIF fission chambers to mixed neutron-gamma fields: PH-2 experimental tests

The engineering design of fissionchambers as on-line radiation detectors for IFMIF is being performed in the framework of the IFMIF-EVEDA works. In this paper the results of the experiments performed in the BR2 reactor during the phase-2 of the foreseen validation activities are addressed. Two detectors have been tested in a mixedneutron-gamma field with high neutron fluence and gamma absorbed dose rates, comparable with the expected values in the HFTM in IFMIF. Since the neutron spectra in all BR2 channels are dominated by the thermal neutron component, the detectors have been surrounded by a cylindrical gadolinium screen to cut the thermal neutron component, in order to get a more representative test for IFMIF conditions. The integrated gamma absorbed dose was about 4 × 1010 Gy and the fast neutron fluence (E > 0.1 MeV) 4 × 1020 n/cm2. The fissionchambers were calibrated in three BR2 channels with different neutron-to-gamma ratio, and the long-term evolution of the signals was studied and compared with theoretical calculations

Influence of termal fluctuations in radiation damage cascade production and defect dynamics in tungsten

The detailed study of the deterioration suffered by the materials of the components of a nuclear facility, in particular those forming part of the reactor core, is a topic of great interest which importance derives in large technological and economic implications. Since changes in the atomic-structural properties of relevant components pose a risk to the smooth operation with clear consequences for security and life of the plant, controlling these factors is essential in any development of engineering design and implementation. In recent times, tungsten has been proposed as a structural material based on its good resistance to radiation, but still needs to be done an extensive study on the influence of temperature on the behavior of this material under radiation damage. This work aims to contribute in this regard. Molecular Dynamics (MD) simulations were carried out to determine the influence of temperature fluctuations on radiation damage production and evolution in Tungsten. We have particularly focused our study in the dynamics of defect creation, recombination, and diffusion properties. PKA energies were sampled in a range from 5 to 50 KeV. Three different temperature scenarios were analyzed, from very low temperatures (0-200K), up to high temperature conditions (300-500 K). We studied the creation of defects, vacancies and interstitials, recombination rates, diffusion properties, cluster formation, their size and evolution. Simulations were performed using Lammps and the Zhou EAM potential for W

Synchrotron strain scanning for residual stress measurement in cold-drawn steel rods

Cold-drawn steel rods and wires retain significant residual stresses as a consequence of the manufacturing process. These residual stresses are known to be detrimental for the mechanical properties of the wires and their durability in aggressive environments. Steel makers are aware of the problem and have developed post-drawing processes to try and reduce the residual stresses on the wires. The present authors have studied this problem for a number of years and have performed a detailed characterization of the residual stress state inside cold-drawn rods, including both experimental and numerical techniques. High-energy synchrotron sources have been particularly useful for this research. The results have shown how residual stresses evolve as a consequence of cold-drawing and how they change with subsequent post-drawing treatments. The authors have been able to measure for the first time a complete residual strain profile along the diameter in both phases (ferrite and cementite) of a cold-drawn steel rod.

Slabsim: Simulador de dispositivos ópticos integrados

En los diseños y desarrollos de ingeniería, antes de comenzar la construcción e implementación de los objetivos de un proyecto, es necesario realizar una serie de análisis previos y simulaciones que corroboren las expectativas de la hipótesis inicial, con el fin de obtener una referencia empírica que satisfaga las condiciones de trabajo o funcionamiento de los objetivos de dicho proyecto. A menudo, los resultados que satisfacen las características deseadas se obtienen mediante la iteración de métodos de ensayo y error. Generalmente, éstos métodos utilizan el mismo procedimiento de análisis con la variación de una serie de parámetros que permiten adaptar una tecnología a la finalidad deseada. Hoy en día se dispone de computadoras potentes, así como algoritmos de resolución matemática que permiten resolver de forma veloz y eficiente diferentes tipos de problemas de cálculo. Resulta interesante el desarrollo de aplicaciones que permiten la resolución de éstos problemas de forma rápida y precisa en el análisis y síntesis de soluciones de ingeniería, especialmente cuando se tratan expresiones similares con variaciones de constantes, dado que se pueden desarrollar instrucciones de resolución con la capacidad de inserción de parámetros que definan el problema. Además, mediante la implementación de un código de acuerdo a la base teórica de una tecnología, se puede lograr un código válido para el estudio de cualquier problema relacionado con dicha tecnología. El desarrollo del presente proyecto pretende implementar la primera fase del simulador de dispositivos ópticos Slabsim, en cual se puede representar la distribución de la energía de una onda electromagnética en frecuencias ópticas guiada a través de una una guía dieléctrica plana, también conocida como slab. Este simulador esta constituido por una interfaz gráfica generada con el entorno de desarrollo de interfaces gráficas de usuario Matlab GUIDE, propiedad de Mathworks©, de forma que su manejo resulte sencillo e intuitivo para la ejecución de simulaciones con un bajo conocimiento de la base teórica de este tipo de estructuras por parte del usuario. De este modo se logra que el ingeniero requiera menor intervalo de tiempo para encontrar una solución que satisfaga los requisitos de un proyecto relacionado con las guías dieléctricas planas, e incluso utilizarlo para una amplia diversidad de objetivos basados en esta tecnología. Uno de los principales objetivos de este proyecto es la resolución de la base teórica de las guías slab a partir de métodos numéricos computacionales, cuyos procedimientos son extrapolables a otros problemas matemáticos y ofrecen al autor una contundente base conceptual de los mismos. Por este motivo, las resoluciones de las ecuaciones diferenciales y características que constituyen los problemas de este tipo de estructuras se realizan por estos medios de cálculo en el núcleo de la aplicación, dado que en algunos casos, no existe la alternativa de uso de expresiones analíticas útiles. ABSTRACT. The first step in engineering design and development is an analysis and simulation process which will successfully corroborate the initial hypothesis that was made and find solutions for a particular. In this way, it is possible to obtain empirical evidence which suitably substantiate the purposes of the project. Commonly, the characteristics to reach a particular target are found through iterative trial and error methods. These kinds of methods are based on the same theoretical analysis but with a variation of some parameters, with the objective to adapt the results for a particular aim. At present, powerful computers and mathematical algorithms are available to solve different kinds of calculation problems in a fast and efficient way. Computing application development is useful as it gives a high level of accurate results for engineering analysis and synthesis in short periods of time. This is more notable in cases where the mathematical expressions on a theoretical base are similar but with small variations of constant values. This is due to the ease of adaptation of the computer programming code into a parameter request system that defines a particular solution on each execution. Additionally, it is possible to code an application suitable to simulate any issue related to the studied technology. The aim of the present project consists of the construction of the first stage of an optoelectronics simulator named Slabsim. Slabism is capable of representing the energetic distribution of a light wave guided in the volume of a slab waveguide. The mentioned simulator is made through the graphic user interface development environment Matlab GUIDE, property of Mathworks©. It is designed for an easy and intuitive management by the user to execute simulations with a low knowledge of the technology theoretical bases. With this software it is possible to achieve several aims related to the slab waveguides by the user in low interval of time. One of the main purposes of this project is the mathematical solving of theoretical bases of slab structures through computing numerical analysis. This is due to the capability of adapting its criterion to other mathematical issues and provides a strong knowledge of its process. Based on these advantages, numerical solving methods are used in the core of the simulator to obtain differential and characteristic equations results that become represented on it.

Towards measuring the complexity of introducing semantics into a company

The Semantics Difficulty Model (SDM) is a model that measures the difficult of introducing semantics technology into a company. SDM manages three descriptions of stages, which we will refer to as ?snapshots?: a company semantic snapshot, data snapshot and semantic application snapshot. Understanding a priory the complexity of introducing semantics into a company is important because it allows the organization to take early decisions, thus saving time and money, mitigating risks and improving innovation, time to market and productivity. SDM works by measuring the distance between each initial snapshot and its reference models (the company semantic snapshots reference model, data snapshots reference model, and the semantic application snapshots reference model) with Euclidian distances. The difficulty level will be "not at all difficult" when the distance is small, and becomes "extremely difficult" when the the distance is large. SDM has been tested experimentally with 2000 simulated companies with arrangements and several initial stages. The output is measured by five linguistic values: "not at all difficult, slightly difficult, averagely difficult, very difficult and extremely difficult". As the preliminary results of our SDM simulation model indicate, transforming a search application into integrated data from different sources with semantics is a "slightly difficult", in contrast with data and opinion extraction applications for which it is "very difficult".