Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-03T14:55:33Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação final_bbc.pdf: 741010 bytes, checksum: 2a9032403833869b2c43a2062da8fcd6 (MD5) / Made available in DSpace on 2017-04-03T14:55:33Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação final_bbc.pdf: 741010 bytes, checksum: 2a9032403833869b2c43a2062da8fcd6 (MD5) Previous issue date: 2015-09-02 / FACEPE / A Influenza é uma doença infecciosa aguda, causada por um vírus pertencente à família Orthomyxoviridae. As drogas antivirais e a vacinação são importantes no controle da disseminação da doença, porém alguns vírus adquirem resistência a certas drogas, alertando a necessidade de novas drogas. Triagem de antivirais através de ensaios biológicos são laboriosos e demorados. Com intuito de facilitar a triagem de drogas foram desenvolvidas duas linhagens celulares repórteres distintas. A linhagem Vero-Gluc-NS-Neo é específica para o vírus da influenza, expressa o gene Gaussia luciferase na presença do vírus, e foi desenvolvida através da tranfecção de células Vero com o plasmídeo pGluc-NSNeo. A segunda linhagem, denominada de A549-ISRE-Luc-Hygro, foi desenvolvida a partir da transfecção de células A549 com o plasmídeo pISRE-Luc-Hygro, o qual expressa o gene repórter Firefly luciferase na presença do interferon do tipo (IFN-I). Seguida da transfecção, ambas linhagens foram selecionadas e submetidas a uma clonagem biológica por diluição limitante e os clones selecionados foram então caracterizados quanto à sua especificidade e sensibilidade no ensaio. Resultados importantes e promissores foram obtidos com a linhagem A549-ISRE-Luc-Hygro, a qual se mostrou eficiente para a triagem de antivirais para influenza e drogas indutoras do IFN-I. Em relação à linhagem Vero-Gluc-NS-Neo, apesar do plasmídeo construído se mostrar funcional e específico, não foi possível observar a expressão do gene repórter após a infecção viral, trazendo à tona questionamentos e mostrando ser necessária a realização de ensaios complementares / Influenza is an acute infectious disease caused by viruses belonging to the Orthomyxoviridae family. Antiviral drugs are vital in controlling the spread of the disease, but some viruses become resistant to certain drugs, prompting the need for new drugs. Antiviral screening through biological tests are laborious and time consuming. In order to facilitate the screening of drugs, it was developed two distinct cell lineages reporters. The Vero-Gluc-Neo-NS cells line is specific for the influenza virus expresses the Gaussia luciferase gene in the presence of the virus, and it was developed by transfection of Vero cells with pGluc-NS-Neo plasmid. The second cell line, A549-called ISRE-Luc-Hygro, was developed from the transfection of A549 cells with pISRE-Hygro-Luc plasmid, which expresses the Firefly luciferase reporter gene in the presence of type one interferon (IFN-I). Followed by transfection, both cell lines were selected and subjected to a biological cloning by limiting dilution and selected clones were then characterized for specificity and sensitivity in the assay. Important and promising results were obtained with A549-Hygro-ISRE-Luc cells, which proved to be efficient for screening of antiviral drugs for influenza and IFN-I inducing drugs. Regarding the Vero-Gluc-NS-Neo cell line, despite the plasmid constructed to show functional and specific, it was not possible to observe the reporter gene expression after viral infection, bringing up questions and shown to be necessary to carry out further testing.

The radiation-induced bystander effect is an important phenomenon in the field of radiation biology. It has been shown that cells, after exposure to radiation, can communicate with surrounding cells and affect their physiology. Otherwise-healthy recipient cells can be influenced to undergo cellular senescence or apoptosis through this process. This has potential utilizations for radiation oncology and as well as our understanding of radiation safety. The radiation-induced bystander effect has been extensively investigated since the 1990s, but the scientific community struggles to come to a unanimous decision on how strongly these signals impact the survival of bystander cells. Results show various degrees of impact on cell survival whereas certain studies refute the existence of a radiation-induced bystander effect. This may be due to the fact that there is a great deal of study heterogeneity within the radiation-induced bystander effect community. Most experiments follow a similar general bystander protocol but often use different donor and reporter cell lines that vary in sex, organ of origin, and p53 status. The type of radiation and dose rate also typically differ between experimental designs. In this analysis, 67 in vitro, medium-transfer, radiation-induced, bystander effect studies were retrospectively graphed and analyzed to determine which intrinsic and external factors contributed significantly to the overall survival percentage change observed in reporter cells. A Two-Way ANOVA was conducted on each variable and showed that the reporter cell line, p53 status, and radiation type had a statistically significant effect on survival percentage change. These findings may explain the variation in results seen in past experiments and may help standardize future research allowing for more direct comparisons. / Thesis / Master of Science (MSc)