Desenvolvimento de um controle externo positivo do tipo viral - like - particle para utilização em um teste diagnóstico de arboviroses emergentes

Orientador: Prof. Dr. Marco Aurélio KriegerCoorientadores: Drª. Rita de Cássia Pontello Rampazzo, Profª. Drª. Vanete ThomazSoccolTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa : Curitiba, 22/06/2018Inclui referênciasResumo: Os arbovírus como dengue, Zika e chikungunya têm se proliferado principalmente em regiões tropicais e subtropicais por conta de condições climáticas, ambientais e falta de saneamento. Os sinais e sintomas das arboviroses citadas podem ser bastante semelhantes e representam um problema para o diagnóstico clínico. Assim, o diagnóstico laboratorial é imprescindível para definição das condutas terapêuticas a serem adotadas no tratamento dos pacientes suspeitos. Os testes mais usados no diagnóstico laboratorial são os sorológicos e moleculares, sendo que os testes moleculares apresentam a vantagem de serem mais sensíveis e específicos, enquanto que os sorológicos têm a desvantagem de apresentar riscos de reações cruzadas, uma vez que esses vírus tem genomas bastante semelhantes. O objetivo deste trabalho foi construir um controle externo positivo único contendo sequencias dos arbovírus dengue, Zika e chikungunya e de um transcrito humano específico para validar as reações do kit BIOMOL ZDC. A construção do controle foi feita utilizando técnicas de engenharia genética, foi desenhada uma sequência sintética única contendo fragmentos de todos os alvos do kit, e esta foi sintetizada e clonada em um sistema de expressão conhecido, contendo o promotor T7 e o fago MS2 modificado. Após a expressão e purificação foi feita a extração do RNA das partículas e a amplificação na RT- qPCR. Na análise verificou-se que todos os alvos do kit kit BIOMOL ZDC presentes no controle construído foram amplificados, validando todo o processo desde a extração, a transcrição reversa até a reação de PCR. Portanto, o controle construído atendeu aos objetivos deste projeto e desta forma o kit BIOMOL ZDC que já estava sendo usado para pesquisa no Brasil sem a presença do controle positivo, passou a ser utilizado com o módulo completo pelos mesmos laboratórios que obtiveram resultados semelhantes. Portanto, este produto (kit com o controle), desenvolvido com tecnologia nacional é biosseguro, tendo custo menor e alto valor agregado, e potencial para ser utilizado a fim de atender as necessidades da saúde pública brasileira. Palavras-chave: arbovírus, controle externo, diagnóstico molecular, RTqPCR.Abstract: The arboviruses such as dengue, Zika and chikungunya have proliferated mainly in tropical and subtropical regions due to climatic and environmental conditions and lack of sanitation. The signs and symptoms of the aforementioned arboviruses can be very similar and represent a problem for the clinical diagnosis. Thus, the laboratory diagnosis is essential for defining the therapeutic behaviors to be adopted in the treatment of suspected patients. The most commonly used tests in laboratory diagnosis are serological and molecular tests; molecular tests have the advantage of being more sensitive and specific, whereas serological tests have the disadvantage of presenting cross-reactivity risks, since these viruses have very similar genomes. The objective of this work was to construct a single positive external control containing dengue, Zika and chikungunya arbovirus sequences and a specific human transcript to validate the reactions of the BIOMOL ZDC kit. Construction of the control was done using genetic engineering techniques, a single synthetic sequence containing fragments of all kit targets was drawn and synthesized and cloned into a known expression system containing the T7 promoter and the modified MS2 phage. After the expression and purification, the RNA extraction from the particles and the amplification in the RT- qPCR were made. In the analysis it was verified that all the targets of the BIOMOL ZDC kit present in the constructed control were amplified validating the whole process from the extraction, the reverse transcription until the PCR reaction. Therefore, the constructed control met the objectives of this project and in this way the BIOMOL ZDC kit that was already being used for research in Brazil without the presence of the positive control was used with the complete module by the same laboratories that obtained similar results. Therefore, this product (kit with the control), developed with national technology is biosecure, having a lower cost and high added value, having potential to be used to meet the needs of Brazilian public health. Keywords: arbovirus, external control, molecular diagnosis, RT- qPCR

Chapter 14 - Restoration Processes after Blackouts

This chapter aims at illustrating the power system dynamic behavior during restoration scenarios in transmission networks, with particular reference to the analysis of thermal power plant black-start capabilities. This chapter is structured into three parts. Section 14.2 briefly reviews the requirements and the structure of power system restoration processes. The unbundling and liberalization of electrical power systems justifies the reassessment of power system restoration strategies, also by increasing the number of black-start resources, traditionally only represented by hydro stations and gas turbines (GTs). Hence, Section 14.3 deals with the analysis of the black-start capabilities of thermal stations equipped with both steam turbines (STs) and GTs. Moreover, accurate modeling of the dynamic interaction between the power plant and the restoring electrical power system, throughout the use of modern computer simulation tools, appears to be of considerable help to study the early stage of the restoration plans and to design specific control systems to support the feasibility and reliability of black-start maneuvers. Therefore, Section 14.3 also includes the analysis of the typical transients occurring during load restoration maneuvers and islanding formation for two representative types of power plants, namely a once-through boiler steam group repowered by a gas turbine and a combined combined-cycle unit. The models of the two relevant simulators and their identification by means of experimental data are described in Section 14.4

A Mixed Integer Linear Programming Approach to the Optimal Reconfiguration of Electrical Distribution Networks with Embedded Generators

The paper focuses on the solution of the minimum loss reconfiguration problem of distribution networks, including embedded generation, by means of a mixed integer linear programming (MILP) model. The proposed model takes into account typical operating constraints of distribution networks (radial configuration, branch ampacity limits and bus voltage requirements). The accuracy of the results and the computational performances of the proposed MILP model are evaluated by making reference to test networks already adopted in the literature for the problem of interest

A Synchrophasor Estimation Algorithm for the Monitoring of Active Distribution Networks in Steady State and Transient Conditions

The paper presents a PMU prototype specifically developed for active distribution network applications. The paper describes the method developed to accurately measure the synchrophasors, its implementation into a real-time microcontroller and the relevant characterization in both steady state and transient conditions. Concerning these last points, the paper presents a test procedure suitably conceived to characterize the PMU with both steady state – single tone and distorted – and frequency-varying signals simulating power systems electromechanical transients. The results show that the developed PMU exhibits high accuracy levels that are not modified by the harmonic distortion of the reference signal and by its frequency-varying transient behavior and compatible with their use into power distribution networks

Effects of nearby buildings on lightning induced voltages on overhead power distribution lines

For the case of urban overhead lines, the presence of nearby buildings is expected to affect the overvoltages induced by nearby cloud-to-ground lightning return strokes. So far, this effect has been seldom taken into account in the literature on the subject. The paper presents a 3D FEM model that calculates the lightning electromagnetic pulse (LEMP) taking into account the presence of a building placed in proximity of the LEMP-coupled overhead line. As a first approximation, all the metallic elements are assumed as perfect conductors, as well as the ground plane. The calculated fields are then introduced in the Agrawal et al. coupling model for the calculation of the induced voltages. The results of a sensitivity analysis carried by varying the model and dimensions of the building, its distance to the line and the position of the lightning stroke location are presented and discussed