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This paper describes a teaching experience aimed at providing interactivity to the technique of field diary by using a virtual learning environment. The educational proposal derives from the Federal University of Rio Grande do Sul (UFRGS)'s Teaching Training Program of the Stricto Sensu Post-Graduation Program in Nursing, in which the author, a Master's degree candidate, oriented by her advisor, proposed forming virtual discussion groups in order to write the field diary for an undergraduate discipline in Nursing, with the aim of providing an opportunity for a joint discussion of academic experiences in the realm of practice. The instructors of the discipline in which the proposal was developed also participated in the activities. The virtual technology gave new dynamism to the technique of field diary, making possible an exchange of experiences among the students, the instructor and the author, as well as moments of reflection and discussion regarding the themes faced in the Nursing practice.  相似文献   
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One hundred patients were entered into a double-blind, double-dummy comparison of tripotassium dicitrate bismuthate (TDB) versus ranitidine, to evaluate short-term healing rates, and successfully healed patients were then entered into a follow-up phase to observe relapse rates. At 4 weeks 84% of patients treated with TDB and 68% of those treated with ranitidine had healed. At 8 weeks these figures had risen to 96% and 90%, respectively (p = NS). After a year's follow-up study 84% of patients healed initially with ranitidine had relapsed, whereas in the case of patients healed initially with TDB the relapse rate was 67% (p less than 0.05). The results confirm that in the short term, TDB is as effective as ranitidine, whereas the significantly better protection against relapse offered by TDB compared with ranitidine underlines the importance of restoring mucosal defence, an approach that to date has been somewhat overlooked.  相似文献   
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Since the development of infectious cDNA clones of viral RNA genomes and the means of delivery of the in vitro-synthesized RNA into cells, alphaviruses have become an attractive system for expression of heterologous genetic information. Alphaviruses replicate exclusively in the cytoplasm, and their genetic material cannot recombine with cellular DNA. Alphavirus genome-based, self-replicating RNAs (replicons) are widely used vectors for expression of heterologous proteins. Their current design relies on replacement of structural genes, encoded by subgenomic RNAs (SG RNA), with heterologous sequences of interest. The SG RNA is transcribed from a promoter located in the alphavirus-specific RNA replication intermediate and is not further amplified. In this study, we have applied the accumulated knowledge of the mechanism of alphavirus replication and promoter structures, in particular, to increase the expression level of heterologous proteins from Venezuelan equine encephalitis virus (VEEV)-based replicons. During VEEV infection, replication enzymes are produced in excess to RNA replication intermediates, and a large fraction of them are not involved in RNA synthesis. The newly designed constructs encode SG RNAs, which are not only transcribed from the SG promoter, but are additionally amplified by the previously underused VEEV replication enzymes. These replicons produce SG RNAs and encoded proteins of interest 10- to 50-fold more efficiently than those using a traditional design. A modified replicon encoding West Nile virus (WNV) premembrane and envelope proteins efficiently produced subviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.Alphaviruses are a group of enveloped viruses with a positive-strand RNA genome that replicate in most commonly used cell lines to titers exceeding 1010 infectious units (inf.u)/mL (1, 2). Upon infection, the genomic RNA serves as a template for translation of viral nonstructural proteins that form replication complexes (3). Within a few hours postinfection, these complexes synthesize large amounts of viral genomic and subgenomic (SG) RNA (3). The SG RNA is transcribed from the SG promoter and serves as a template for translation of viral structural proteins: capsid, E2 and E1, which ultimately assemble with genomic RNA into infectious viral particles. This highly efficient virus-specific RNA and protein synthesis, coupled with the availability of infectious cDNA clones, have made alphaviruses an attractive system for designing self-replicating vectors for delivery and expression of heterologous genetic information. The most widely used alphavirus-based expression systems are based on replacement of viral structural genes by a gene(s) of interest (4). These modified viral genomes, termed replicons, can be synthesized in vitro and delivered into cells either by transfection or in infectious viral particles, which deliver essentially every packaged RNA molecule into the cells both in vivo and in vitro.In recent years, significant progress has been made in our understanding of the mechanism of alphavirus replication. Detailed studies have elucidated the structure and function of the RNA promoters, critical aspects of virus–host cell interactions, and the composition of the replication complexes (512). These mechanistic studies of alphavirus replication raised the question of whether we are using their entire expression potential, and whether the traditional replicon design can be further improved to achieve higher levels of heterologous protein production. In this project, we sought to apply the latest advances in understanding of alphavirus RNA replication to design a new generation of Venezuelan equine encephalitis virus (VEEV) genome-based expression systems. The distinguishing feature of these constructs is the modification of the SG RNAs. These SG RNAs have been engineered to contain the cis-acting promoter elements, which are normally present at the 5′ end of the viral genome and mediate genomic RNA replication (8, 13, 14). Thus, in these newly designed VEEV replicons (VEErep), the SG RNAs were not only transcribed from the SG promoter, but were capable of replication/amplification by the VEEV replication complexes. As a result, the heterologous gene expression was more efficient than that of the existing constructs, which use replicons with the standard SG RNAs. The expression level of heterologous protein encoded by the improved replicons was also found to be dependent on coexpression of VEEV capsid protein. The VEEV replicons, which use both amplification of the SG RNA and express capsid protein, provide a platform for development of a variety of more efficient expression systems and have numerous applications. To illustrate this, we have generated a VEEV replicon encoding the premembrane and envelope (prM/E) proteins of West Nile virus (WNV). Particles containing the newly designed replicons induced high levels of WNV E protein expression in vitro and elicited robust protective immunity in mice.  相似文献   
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