Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-01-18T14:03:34Z No. of bitstreams: 3 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_FINAL-A4.pdf: 4196135 bytes, checksum: 547188bb7660475ceab2a2f39962a9f5 (MD5) Tese_FINAL-A4.pdf: 4196135 bytes, checksum: 547188bb7660475ceab2a2f39962a9f5 (MD5) / Made available in DSpace on 2016-01-18T14:03:34Z (GMT). No. of bitstreams: 3 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_FINAL-A4.pdf: 4196135 bytes, checksum: 547188bb7660475ceab2a2f39962a9f5 (MD5) Tese_FINAL-A4.pdf: 4196135 bytes, checksum: 547188bb7660475ceab2a2f39962a9f5 (MD5) Previous issue date: 2014-03-24 / Glioblastoma (grau IV) é o tumor mais agressivo e infiltrante do sistema nervoso central (SNC), que mostra uma série de mutações, bem como alto grau de vascularidade, polimorfismo e atipia celular nuclear. Infelizmente, diagnóstico precoce de tumores cerebrais é difícil, uma vez que ferramentas de imagem não são eficientes para o diagnóstico correto desses tipos de tumores, levando a falhas no tratamento. Aqui nós descrevemos a síntese, caracterização e conjugação de pontos quânticos ou quantum dots (QDs) de telureto de cádmio (CdTe) recobertos com tiol e com o anticorpo contra a proteína ácida fibrilar glial (anti-GFAP), bem como, a preparação de lipossomas. O método de congelamento e descongelamento foi utilizado para encapsular os QDs em diferentes tipos de lipossomas e liberá-los em células tronco. Os lipossomas vazios e contendo CdTe QDs foram caracterizados por microscopia de fluorescência, microscopia eletrônica de transmissão, tamanho e potencial zeta. Os QDs CdTe-anti-GFAP foram utilizados para um novo direcionamento in vivo e método de imagem para detecção do tipo de tumor glioblastoma U87 xenotransplantado em cérebro de camundongos suíços machos. CdTe QDs não conjugados e CdTe QDs conjugados foram utilizados para marcar U87 linha de células de tumor in vitro e astrócitos saudáveis. A citotoxicidade dos CdTe QDs com fluorescência no verde (530 nm) e no vermelho (644 nm), foi avaliada utilizando MTT nas células U87. O crescimento do tumor foi visualizado no interior do cérebro pela marcação com hematoxilina e eosina e mostrou a entrega com sucesso nas células U87 no parênquima cerebral. CdTe QDs conjugados com anti - GFAP foram injectados na região do tumor e a sua marcação na linhagem celular U87 foi visualizada por microscopia de fluorescência, mostrando dupla marcação com especificidade para vimentina em glioblastomas imunorreactivos. Em comparação com as células tumorais U87, que facilmente foram marcadas com o CdTe QDs conjugados com anti- GFAP, observouse que os astrócitos saudáveis mantidos em culturas primárias tiveram uma maior resistência à sua marcação e foram fracamente marcados. Os resultados descritos aqui direcionam para novas perspectivas na utilização de CdTe QDs na detecção de gliobastoma , sugerindo uma potencial aplicação em cirurgia guiada por imagem. / Glioblastoma (grade IV) is the most aggressive and infiltrating tumor of the central nervous system (CNS), showing a variety of mutations as well as high degree of vascularity, cell polymorphism and nuclear atypia. Unfortunately, early diagnostic of brain tumors is hard, as imaging tools are not efficient for proper diagnosis of these types of tumors, leading to treatment failures. Here we describe the synthesis, characterization and conjugation of thiol-capped CdTe with anti-glial fibrillar acidic protein (anti-GFAP) and preparation of liposomes. The freeze and thaw method was used to encapsulate the QDs in different types of liposomes and deliver them into stem cells. The empty and containing CdTe QDs liposomes were characterized by fluorescence microscopy, transmission electron microscopy, size and zeta potential. A new in vivo targeting and imaging method for U87 glioblastoma tumor type xenotransplanted into male swiss mice brain using aqueous colloidal CdTe quantum dots conjugated to anti-GFAP (CdTeanti- GFAP QDs) was developed. The red emitting CdTe QDs and the conjugated red-emitting CdTe QDs were used to label U87 tumor cell line in vitro and health astrocytes. Toxicity of isolated green (530 nm) and red (644 nm) emitting CdTe QDs, was evaluated using MTT assay applied to U87 cells. The tumor growth was visualized inside the brain by the hematoxylin and eosin staining and showed the successful delivery of the U87 cells into the brain parenchyma. CdTe-anti-GFAP QDs were injected into the tumor region and their uptake by the U87 cell line was visualized by fluorescence microscopy, showing specific double-labeling of vimentinimmunoreactive glioblastoma. Compared to U87 tumor cells, which easily take up anti-GFAP conjugated red-emitting CdTe QDs, healthy astrocytes kept in primary cultures offered more resistance to their incorporation and were weakly labeled. The results reported here provide new perspectives for using CdTe QDs in gliobastoma detection, suggesting their potential application in imaging-guided surgery.

Esta investigación se enmarca en una de las principales líneas que en la actualidad movilizan las reflexiones en la didáctica de las ciencias: la influencia del uso de modelos en el desarrollo del conocimiento científico de los estudiantes. En particular, el proceso de construcción y revisión del modelo ser vivo por niños y niñas de segundo curso del primer ciclo en una escuela pública de Barcelona durante la realización de una secuencia didáctica sobre los microorganismos. El objetivo principal consistió en analizar cómo los distintos modos comunicativos y las actividades experimental, lectora y comparativa contribuyen a la construcción del modelo ser vivo por estos alumnos. La implementación en el aula de la unidad didáctica diseñada permitió la obtención de datos de la investigación. Los instrumentos de recogida de datos fueron: cuestionarios KPSI (inicial y final), producciones gráficas, escritas y orales. El análisis de los datos se hizo a partir del análisis del contenido de las producciones de los alumnos, lo que permitió identificar la contribución de cada modo comunicativo y de cada actividad a partir del desempeño alcanzado en la progresión de aprendizaje al construir el modelo ser vivo. Así mismo, desde el marco teórico de la multimodalidad fue posible reconocer la relación de cooperación o de especialización de los distintos modos comunicativos en este proceso. Los resultados analizados revelaron que cada modo comunicativo contribuyó de forma específica. La representación gráfica permitió al alumnado expresar el proceso de evolución gradual de las caries, los elementos que creían esenciales para este proceso, el tamaño y cantidad del bicho, su morfología (siendo la más frecuente la similar a un insecto o humanoide) y su ubicación. La representación textual y oral permitió al alumnado enunciar procesos de interacción entre el alimento, la caries y el ser vivo, así mismo a evidenciar la confusión entre los conceptos caries y bacteria. Analizando los tres modos comunicativos se observa que la relación más frecuente entre ellos es la especialización, en las representaciones individuales el dibujo alcanzó un mayor nivel de desempeño en la progresión de aprendizaje con relación a los demás modos comunicativos. Así mismo, la cooperación entre los modos comunicativos fue encontrada en otros casos, cuando las representaciones del alumnado se encontraban en el mismo nivel de desempeño. En las discusiones entre iguales, constatamos que a través del modo oral se alcanza un nivel mayor de desempeño, en tres de los cuatro grupos, caracterizando por tanto una relación de especialización con el dibujo y el texto y mostrando el potencial del discurso oral en la construcción de conocimiento. A través del análisis del contenido de los elementos argumentativos generados en la discusión entre iguales, se observó que el alumnado dio mucha importancia a aspectos formales del dibujo, siendo el color el más representativo. Los aspectos más fundamentales del modelo se produjeron una vez decididos los aspectos formales. La influencia de las tres actividades propuestas fue diversa. El planteamiento de la actividad experimental – elaboración del yogur – fue demasiado ambicioso y los alumnos no fueron capaces de reconocer la importancia del medio en la vida de las bacterias, aunque si permitió discutir el efecto de la temperatura. La actividad lectora permitió reconocer de manera explícita la relación entre consumo de dulces, reproducción y crecimiento de las bacterias y desarrollo de las caries. Así mismo, la actividad comparativa de la bacteria con otro ser vivo (conejo) permitió a los estudiantes generalizar el modelo ser vivo construido en el curso anterior, aplicándolo a un organismo microscópico. Esta actividad permitió al alumnado progresar en su nivel de desempeño al reconocer la presencia de células en ambos seres vivos. Analizar el proceso de construcción del modelo ser vivo a través de la identificación de niveles de desempeño en la progresión de aprendizaje facilita el reconocimiento de dificultades y puede orientar en el diseño de las secuencias de aprendizaje implementadas en el aula. / This research falls within one of the main lines that currently move the reflections on science education: the influence of the use of models in the development of scientific knowledge of the students. In particular, in the process of construction and revision of the living being model by children in the second year of primary education in a public school in Barcelona during the performance of a learning sequence on microorganisms. The main objective was to analyze to what extent the different communicative modes and experimental, reading and comparative activities contribute to the construction of the living being model of these students. The implementation of the designed learning sequence allowed obtaining research data. Data was collected using KPSI questionnaires (collected at the beginning and at the end of the sequence), drawings of the students as well as their written and oral productions. Data analysis consisted in content analysis of the worksheets of the students, which allowed to identify the contribution of each communicative mode as well as the contribution of each activity from the performance achieved in the learning progression in the construction of the living being model. Likewise, the theoretical framework of multimodality was used to analyze and recognize the relationship between the different communicative modes in this process, which could be cooperation or specialization. The results revealed that each communicative mode contributed in a specific way to the construction of the living being model. The graphic representation (drawings) allowed students to express the gradual process of evolution of dental caries process as well as the components they believed essential to this process: size and quantity of the bug, its morphology (most frequently being similar to an insect or humanoid) and its location. Oral and textual representations allowed students to spell out the processes of interaction between food, dental caries and a living being, it also allowed to highlight the conceptual confusion between dental caries and bacteria. By analyzing the three communicative modes it is observed that the most common relation between them is the specialization: in the individual representations the drawing reached a higher level of performance in learning progression than the other communicative modes. Furthermore, the relation of cooperation between communicative modes was found in other cases, where representations of students reached the same level of performance. In peer discussion activities, it can be noted that through the oral mode a higher level of performance is reached in three of the four groups, therefore characterizing a relationship of specialization between drawing and text, and showing the potential of oral discourse in the construction of knowledge. Through the content analysis of the argumentative elements generated during the peer discussion, it was noted that students gave great importance to formal aspects of the drawing, the color being the more representative. The most fundamental aspects of the model ocurred once the formal features were set. The influence to the construction of the living being model of the three proposed activities was different. The approach of the experimental activity - preparation of yogurt - was too ambitious and the students were not able to recognize the importance of the environment in the life of the bacteria, but it allowed to discuss the effect of temperature. The reading activity allowed to recognize explicitly the relationship between consumption of candies, reproduction and growth of bacteria and the development of dental caries. Furthermore, the comparative activity between bacteria and another living being (a rabbit) allowed students to generalize the living being model (which they had worked in the previous year), applying it to a microscopic organism. This activity allowed the students to progress in their level of performance to recognize the presence of cells in both living beings. Analyzing the process of construction of the living being model by the identification of levels of performance in a learning progression, facilitates the recognition of difficulties and can guide the design of learning sequences to be implemented in the classroom.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2015-03-27T14:27:58Z No. of bitstreams: 2 Dissertação - Laís Cristina da Silva - 2014.pdf: 4002977 bytes, checksum: c54a0aa96c3be46a95b8df8e9a407c1d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-27T15:49:49Z (GMT) No. of bitstreams: 2 Dissertação - Laís Cristina da Silva - 2014.pdf: 4002977 bytes, checksum: c54a0aa96c3be46a95b8df8e9a407c1d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-27T15:49:49Z (GMT). No. of bitstreams: 2 Dissertação - Laís Cristina da Silva - 2014.pdf: 4002977 bytes, checksum: c54a0aa96c3be46a95b8df8e9a407c1d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-09-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The main predictive models of absorption of potential new drugs in preclinical stage are focused on the gastrointestinal mucosa, given the predominance of this pathway in drug administration. Often, the fraction absorbed (Fa) can be predicted in ex vivo models (p.e. Ussing chambers), in vitro (p.e. Caco-2 cells monolayers), intestinal perfusion studies in situ and in vivo absorption. In the present study, from an adaptation of Snapwell ™ inserts, a new ex vivo model to evaluate the permeability of substances passively absorbed is proposed. High permeable drugs (metoprolol, caffeine and theophylline) and low permeable drugs (atenolol, ranitidine and cimetidine) were maintained in an incubator at 37 ° C under constant stirring (60 rpm) and carbogenic atmosphere (5% CO2). The viability of the jejunal membrane (52 Ω.cm2 ± 8.0) was observed remaining above 20 Ω.cm2 for 120 min incubation, under all conditions evaluated, including the addition of co-solvents (1% DMSO and 1% EtOH). Values of apparent permeability coefficients obtained (Papp) were characteristic of ex vivo permeation studies (3.8 to 12.6 x10-6 cm / s). Strong correlation was observed between the data obtained here versus data intestinal perfusion in vivo (r = 0.89), as well as the fraction absorbed in humans (r = 0.85), reported in the literature. Additionally, the model features high sensitivity and accuracy compared to other commonly used models in classification permeability of substances. In line, we can infer that the MTSSNAPWELL model demonstrates, yet, potential application in studies of screening for selection of low molecular weight, such as potential phytochemicals, as well as their synthetic analogues evaluated with low amount of sample (ca 10 mg). / Os principais modelos preditivos da absorção de potenciais novos fármacos na etapa pré-clinica são focados na mucosa gastrointestinal, haja vista a predominância desta via na administração medicamentosa. Frequentemente, a fração absorvida (Fa) pode ser predita em modelos ex vivo em câmaras de Ussing, in vitro em monocamadas de células Caco-2, perfusão intestinal in situ e estudos de absorção in vivo. No presente estudo, a partir de uma adaptação do aparato Snapwell™, um novo modelo ex vivo de avaliação da permeabilidade para substâncias absorvidas por difusão passiva é proposto. Substâncias de alta (metoprolol, cafeína e teofilina) e baixa (atenolol, ranitidina e cimetidina) permeabilidade, foram mantidos em incubadora à 37OC, sob agitação constante (60 rpm) e atmosfera carbogênica (5% CO2). A viabilidade da membrana jejunal (52 ± 8,0 Ω.cm2) foi observada mantendose acima de 20 Ω.cm2 por até 120 min de incubação, sob todas condições avaliadas incluindo a adição de co-solventes (DMSO 1% e EtOH 1%). Os valores de coeficientes de permeabilidade aparente obtidos (Papp) mostraram-se característicos de estudos ex vivo de permeação (3,8 – 12,6 x10-6 cm/s). Forte correlação foi observada entre os dados aqui obtidos versus dados de perfusão intestinal in vivo (r = 0,89), assim como da fração absorvida em humanos (r = 0,85), relatados na literatura. Adicionalmente, o modelo apresenta elevada sensibilidade e precisão frente aos demais modelos comumente utilizados na classificação da permeabilidade de substâncias. Em consonância, pode-se inferir que o modelo MTSSNAPWELL demonstra, até o momento, potencial aplicação em estudos de screening para seleção de moléculas de baixo peso molecular, tais como potenciais fitofármacos, assim como seus análogos sintéticos avaliados com baixa quantidade de amostra (c.a. 10 mg).