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.

Osteoarthritis is a joint disease characterized by the breakdown of articular cartilage. The field of tissue engineering is interested in developing methods to produce biological alternatives to current orthopedic procedures. Lubricin is a molecule which is important in the proper lubrication of articular cartilage. It is a challenge in the field of tissue engineering to produce cartilage with sufficient lubricin expression. Developing a reporter cell for lubricin allowed for a more efficient investigation of the conditions wh­­­ich may influence its expression. By comparing "optimized" and traditional media solutions, it was determined that the use of a previously reported type II collagen optimized media would negatively affect the expression of lubricin. This information indicates the need to further evaluate the conditions which are conducive to producing cartilage with both sufficient types of type II collagen and lubricin.

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)

Le mépliement et l'agrégation des protéines sont à l'origine de nombreuses maladies neurodégénératives, dont la maladie de Huntington (HD) et la maladie de Parkinson (PD). Même si l’agrégation de différentes protéines liées à des maladies est bien documentée, on en sait peu sur l'interaction entre les protéines mal repliées et les cellules neuronales, qui leur permettent de se propager et affecter différentes régions du cerveau. L'objectif de ma thèse était de générer des modèles cellulaires rapporteurs de la huntingtine et l’α-synucléine, protéines dont le mauvais repliement et l'agrégation sont à l'origine de HD et PD respectivement, et utiliser ces modèles cellulaires pour étudier les interactions entre les agrégats et des lignées cellulaires de mammifères. Notre but c’était de documenter les propriétés de liaison et d’absorption de ces agrégats par les cellules rapporteuses, et les conséquences de leur internalisation pour les cellules. Deux modèles cellulaires de neuroblastome (SH-SY5Y et Neuro2A) et un modèle de cellules d’ostéoblastome (U2OS) exprimant la protéine fluorescente ChFP ont été générés pour HD. Pour simuler ce qui se passe au sein de neurones réels, des cellules de neuroblastome ont été induites à se différencier. Des différences de fixation, internalisation, nucléation de la protéine endogène et localisation finale des agrégats de polyglutamine internalisés ont été observées entre les cellules différenciées et non différenciées. Des cellules rapporteuses U2OS ont été utilisées pour déterminer les différences d’infectiosité entre des fibres de HttExon1 assemblés en présence ou en l’absence de la protéine de choc thermique constitutivement exprimée chez l'Homme Hsc70. Hsc70 a un effet protecteur car il rend les fibres moins infectieuses pour les cellules de mammifères en culture. Enfin, un modèle cellulaire de neuroblastome (Neuro2A) rapporteur pour PD exprimant l’α-synucléine fusionnée à la protéine ChFP a été utilisé pour déterminer des différences de liaison, pénétration, absorption, nucléation de la protéine endogène et persistance entre deux polymorphismes d’α-synucléine générés par notre équipe. L'hétérogénéité observée dans différents patients souffrant de synucléinopaties pourrait s'expliquer par différents polymorphes d’assemblages protéiques d’α-synucléine présents dans les cerveaux des malades, ce qui doit être pris en compte pour les développements thérapeutiques futurs.Ces modèles cellulaires rapporteurs pour différentes maladies sont un système valable pour l'étude de différents processus cellulaires liés à l'interaction entre les protéines agrégées exogènes et des cellules de mammifères en culture. Nos résultats indiquent un mécanisme commun par lequel les différentes protéines agrégées peuvent interagir avec des cellules en culture: les protéines mal repliées exogènes sont capables de se lier à des membranes cellulaires, les pénétrer, entrer dans l'espace intracellulaire et recruter des protéines endogènes solubles. Même si cela semble être un mécanisme générique pour des protéines infectieuses telles que la α-synucléine ou la huntingtine, des lignées cellulaires avec différents phénotypes montrent différences de vulnérabilité à la présence de protéines agrégées. Ceci suggère la présence de récepteurs spécifiques à la surface de la cellule capables de reconnaître des structures de type amyloïde. D'autres études sont nécessaires pour déterminer la nature de ces récepteurs et si sa modulation pourrait être utile pour contrôler la propagation des ces maladies dans le cerveau. / Protein misfolding and aggregation are at the origin of many neurodegenerative diseases, including Huntington’s disease (HD) and Parkinson’s disease (PD). Even if the aggregation of different disease-related proteins is well documented, little is known about the interaction between those misfolded proteins and neuronal cells that allow them to spread and affect several regions of the brain. The objective of my thesis was to generate reporter cellular models of huntingtin and α-synuclein, proteins whose misfolding and aggregation are at the origin of HD and PD respectively, and use these cell models for studying the interactions between misfolded protein aggregates and mammalian cell lines. We aimed to document the binding and uptake properties of those aggregates by reporter cells and the consequences of their internalization for the cells. Two neuroblastoma cell models (SH-SY5Y and Neuro2A) and an osteoblastoma cell model (U2OS) expressing the fluorescent protein ChFP were generated as mammalian reporter cell lines for HD. To mimic what happens in real neurons, neuroblastoma reporter cells were induced to differentiate. Differences in binding, internalization, nucleation of the endogenous protein and final localization of the internalized polyglutamine aggregates were observed between differentiated and undifferentiated cells. U2OS reporter cells were used for determining differences in the infectivity of HttExon1 fibrils assembled in the presence or in the absence of the constitutively expressed heat shock protein Hsc70, suggesting a protective effect of Hsc70, since it renders the fibrils less infectious to mammalian cells. Finally, a neuroblastoma reporter cell model (Neuro2A) of PD expressing α-synuclein fused to the fluorescent and reporter protein ChFP was used to determine the different binding, penetration, uptake, nucleation of the endogenous protein and persistence properties of two α-synuclein polymorphs generated by our team. The heterogeneity observed in different patients suffering from synucleinopathies could be explained due to different α-synuclein assemblies present in diseased brains, what needs to be taken into account for future therapeutic developments. These reporter cellular models for different diseases are a valid system for the study of different cellular processes related with the interaction between exogenous aggregated proteins and mammalian cells in culture. Our results indicate a common mechanism by which different aggregated proteins can interact with cells in culture: exogenous misfolded proteins are able to bind cell membranes, penetrate them, enter the intracellular space and recruit endogenous soluble proteins. Even if this seems to be a generic mechanism for infectious proteins such as α-synuclein or huntingtin, different cell lines or cell phenotypes show distinct vulnerability to the presence of aggregated proteins. This strongly suggests the presence of specific receptors at the surface of the cell able to recognize amyloid-like structures. Further investigations are needed to determine the nature of these receptors and whether their modulation might be helpful for controlling the spread of these diseases within the brain.