Proteínas SAG1, SAG2, SAG3 de toxoplasma gondii como perspectivas para o desenvolvimento de protótipo vacinal contra a toxoplasmose

Toxoplasmosis is a zoonosis of worldwide distribution caused by the protozoan Toxoplasma gondii, triggering dangerous complications in immunocompromised patients and pregnant women, as well as having great economic impact for the livestock. So far the control of toxoplasmosis is made primarily by chemotherapy. However, most drugs used routinely have some limitations. In order to control this disease, several research groups, including ours, has been working to develop a medical-veterinary vaccine based on parasite antigens, vectors and protocols of immunization. In this study were implemented and standardized methodologies for amplification and cloning of recombinant immunogens in the system for the development of a prototype vaccine, based on the surface antigens of T. gondii and recombinant adenovirus encoding these antigens. Genes encoding BAG1, GRA2 and SAG1 proteins were amplified. We established a strategy for cloning SAG1, SAG2, SAG3 and TgAMA1- genes in recombinant system. The genes encoding SAG1 and SAG2 were cloned and their sequences showed high similarity with sequences from GenBank. The virtual translation of these proteins showed polymorphisms in the amino acid sequence, which can be correlated with levels of antigenicity. Simultaneously, the adenovirus encoding the SAGs (HAdSAGs) were expanded, purificated and characterizated. Immunization of C57bl/6 mice, using viral supernatant was not enought to elicit immune responses at high levels, being required HAdSAGs titration for future immunizations. Therefore, this study allowed the cloning of the two genes important for the development of a prototype vaccine. Besides, implementations methodologies that permit advancements in the development of a vaccine against toxoplasmosis using adenovirus to express proteins of the parasite

Efeitos da deficiência de vitamina A na função pulmonar de crianças após infecção respiratória viral

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Produção e avaliação de nanoemulsão catiônica como possível vetor não-viral para pIRES2-EGFP

Gene therapy is based on the transfer of exogenous genetic material into cells or tissues in order to correct, supplement or silencing a particular gene. To achieve this goal, efficient vehicles, viral or non-viral, should be developed. The aim of this work was to produce and evaluate a nanoemulsion system as a possible carrier for no-viral gene therapy able to load a plasmid model (pIRES2-EGFP). The nanoemulsion was produced by the sonication method, after been choose in a pseudo-ternary phase diagram build with 5 % of Captex 355®, 1.2 % of Tween 80®, 0.8 % of Span 80®, 0.16% of stearylamine and water (to 100 %). Measurements of droplet size, polydispersity index (PI), zeta potential, pH and conductivity, were performed to characterize the system. Results showed droplets smaller than 200 nm (PI < 0.2) and zeta potential > 30 mV. The formulation pH was near to 7.0 and conductivity was that expected to oil in water systems (70 to 90 μS/s) A scale up study, the stability of the system and the best sterilization method were also evaluated. We found that the system may be scaled up considering the time of sonication according to the volume produced, filtration was the best sterilization process and nanoemulsions were stable by 180 days at 4 ºC. Once developed, the complexation efficiency of the plasmid (pDNA) by the system was tested by agarose gel electrophoresis retardation assay.. The complexation efficiency increases when stearylamine was incorporated into aqueous phase (from 46 to 115 ng/μL); regarding a contact period (nanoemulsion / pDNA) of at least 2 hours in an ice bath, for complete lipoplex formation. The nanoemulsion showed low toxicity in MRC-5 cells at the usual transfection concentration, 81.49 % of survival was found. So, it can be concluded that a nanoemulsion in which a plasmid model was loaded was achieved. However, further studies concerning transfectation efficiency should be performed to confirm the system as non-viral gene carrier

Algoritmo memético com infecção viral: uma aplicação ao problema do caixeiro viajante assimétrico

The Combinatorial Optimization is a basic area to companies who look for competitive advantages in the diverse productive sectors and the Assimetric Travelling Salesman Problem, which one classifies as one of the most important problems of this area, for being a problem of the NP-hard class and for possessing diverse practical applications, has increased interest of researchers in the development of metaheuristics each more efficient to assist in its resolution, as it is the case of Memetic Algorithms, which is a evolutionary algorithms that it is used of the genetic operation in combination with a local search procedure. This work explores the technique of Viral Infection in one Memetic Algorithms where the infection substitutes the mutation operator for obtaining a fast evolution or extinguishing of species (KANOH et al, 1996) providing a form of acceleration and improvement of the solution . For this it developed four variants of Viral Infection applied in the Memetic Algorithms for resolution of the Assimetric Travelling Salesman Problem where the agent and the virus pass for a symbiosis process which favored the attainment of a hybrid evolutionary algorithms and computational viable

Estratégias de Produção in vitro de Bioinseticida Viral: influências do Isolado, da Cinética e do Modo de operação

Societal concerns about environmental sustainability has lead to the development of ecologically-friendly alternatives to chemical insecticides for crop protection. One such alternative is biological pest control. In particular, baculoviruses are well suited as insect biopesticides due to their narrow host specificity and relative ease of propagation. In Brazil, the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is the main biological control agent employed for the soybean pest, Anticarsia gemmatalis. This baculovirus biopesticide is currently produced using caterpillars, but increasing market demand for the product has encouraged the development of an in vitro manufacturing process, which can be scaled up to much higher virus productivities. In this study, three wild-type AgMNPV isolates (AgMNPV-2D, AgMNPV-MP2 and AgMNPV-MP5) and a recombinant form (vAgEGT-LacZ) were characterised in terms of occlusion body (OB) production and infection kinetics, to enable future optimisation of the in vitro production process. These viruses were propagated using a Spodoptera frugiperda (IPLB-SF21) insect cell line grown in shaker-flask batch cultures. Among the virus isolates tested, AgMNPV-MP5 was found to be the best producer, yielding (5.3±0.85)x108 OB/mL after 8 days post infection. The characterisation of vAgEGT-LacZ propagation in suspension cell cultures has not been previously reported in the literature; hence it became the main focus for this thesis. In particular, it was carried out a study on the effect of the multiplicity of infection (MOI) on OB production. Five successive batches were performed getting a final production (8.9±1.42)x1014 occlusion bodies, considering that production is related for a bioreactor with final volume of 10m3. A low MOI associated with a fed-batch process for vAgEGT-LacZ production was found to support a 3-fold higher OB yield when compared to the default batch process (1.8x107 and 5.3x107 OB/mL, respectively). This yield is competitive with regards to the production process.