Aprender numa relação dialógica sustentada pela ação

Relatório de Estágio apresentado à Escola Superior de Educação de Paula Frassinetti para obtenção de grau Mestre em Educação Pré-Escolar e Ensino 1º ciclo do Ensino Básico.

Microfluidic platforms for the characterization of in meso membrane protein crystallization

Membrane proteins, which reside in the membranes of cells, play a critical role in many important biological processes including cellular signaling, immune response, and material and energy transduction. Because of their key role in maintaining the environment within cells and facilitating intercellular interactions, understanding the function of these proteins is of tremendous medical and biochemical significance. Indeed, the malfunction of membrane proteins has been linked to numerous diseases including diabetes, cirrhosis of the liver, cystic fibrosis, cancer, Alzheimer's disease, hypertension, epilepsy, cataracts, tubulopathy, leukodystrophy, Leigh syndrome, anemia, sensorineural deafness, and hypertrophic cardiomyopathy.1-3 However, the structure of many of these proteins and the changes in their structure that lead to disease-related malfunctions are not well understood. Additionally, at least 60% of the pharmaceuticals currently available are thought to target membrane proteins, despite the fact that their exact mode of operation is not known.4-6 Developing a detailed understanding of the function of a protein is achieved by coupling biochemical experiments with knowledge of the structure of the protein. Currently the most common method for obtaining three-dimensional structure information is X-ray crystallography. However, no a priori methods are currently available to predict crystallization conditions for a given protein.7-14 This limitation is currently overcome by screening a large number of possible combinations of precipitants, buffer, salt, and pH conditions to identify conditions that are conducive to crystal nucleation and growth.7,9,11,15-24 Unfortunately, these screening efforts are often limited by difficulties associated with quantity and purity of available protein samples. While the two most significant bottlenecks for protein structure determination in general are the (i) obtaining sufficient quantities of high quality protein samples and (ii) growing high quality protein crystals that are suitable for X-ray structure determination,7,20,21,23,25-47 membrane proteins present additional challenges. For crystallization it is necessary to extract the membrane proteins from the cellular membrane. However, this process often leads to denaturation. In fact, membrane proteins have proven to be so difficult to crystallize that of the more than 66,000 structures deposited in the Protein Data Bank,48 less than 1% are for membrane proteins, with even fewer present at high resolution (< 2Å)4,6,49 and only a handful are human membrane proteins.49 A variety of strategies including detergent solubilization50-53 and the use of artificial membrane-like environments have been developed to circumvent this challenge.43,53-55 In recent years, the use of a lipidic mesophase as a medium for crystallizing membrane proteins has been demonstrated to increase success for a wide range of membrane proteins, including human receptor proteins.54,56-62 This in meso method for membrane protein crystallization, however, is still by no means routine due to challenges related to sample preparation at sub-microliter volumes and to crystal harvesting and X-ray data collection. This dissertation presents various aspects of the development of a microfluidic platform to enable high throughput in meso membrane protein crystallization at a level beyond the capabilities of current technologies. Microfluidic platforms for protein crystallization and other lab-on-a-chip applications have been well demonstrated.9,63-66 These integrated chips provide fine control over transport phenomena and the ability to perform high throughput analyses via highly integrated fluid networks. However, the development of microfluidic platforms for in meso protein crystallization required the development of strategies to cope with extremely viscous and non-Newtonian fluids. A theoretical treatment of highly viscous fluids in microfluidic devices is presented in Chapter 3, followed by the application of these strategies for the development of a microfluidic mixer capable of preparing a mesophase sample for in meso crystallization at a scale of less than 20 nL in Chapter 4. This approach was validated with the successful on chip in meso crystallization of the membrane protein bacteriorhodopsin. In summary, this is the first report of a microfluidic platform capable of performing in meso crystallization on-chip, representing a 1000x reduction in the scale at which mesophase trials can be prepared. Once protein crystals have formed, they are typically harvested from the droplet they were grown in and mounted for crystallographic analysis. Despite the high throughput automation present in nearly all other aspects of protein structure determination, the harvesting and mounting of crystals is still largely a manual process. Furthermore, during mounting the fragile protein crystals can potentially be damaged, both from physical and environmental shock. To circumvent these challenges an X-ray transparent microfluidic device architecture was developed to couple the benefits of scale, integration, and precise fluid control with the ability to perform in situ X-ray analysis (Chapter 5). This approach was validated successfully by crystallization and subsequent on-chip analysis of the soluble proteins lysozyme, thaumatin, and ribonuclease A and will be extended to microfluidic platforms for in meso membrane protein crystallization. The ability to perform in situ X-ray analysis was shown to provide extremely high quality diffraction data, in part as a result of not being affected by damage due to physical handling of the crystals. As part of the work described in this thesis, a variety of data collection strategies for in situ data analysis were also tested, including merging of small slices of data from a large number of crystals grown on a single chip, to allow for diffraction analysis at biologically relevant temperatures. While such strategies have been applied previously,57,59,61,67 they are potentially challenging when applied via traditional methods due to the need to grow and then mount a large number of crystals with minimal crystal-to-crystal variability. The integrated nature of microfluidic platforms easily enables the generation of a large number of reproducible crystallization trials. This, coupled with in situ analysis capabilities has the potential of being able to acquire high resolution structural data of proteins at biologically relevant conditions for which only small crystals, or crystals which are adversely affected by standard cryocooling techniques, could be obtained (Chapters 5 and 6). While the main focus of protein crystallography is to obtain three-dimensional protein structures, the results of typical experiments provide only a static picture of the protein. The use of polychromatic or Laue X-ray diffraction methods enables the collection of time resolved structural information. These experiments are very sensitive to crystal quality, however, and often suffer from severe radiation damage due to the intense polychromatic X-ray beams. Here, as before, the ability to perform in situ X-ray analysis on many small protein crystals within a microfluidic crystallization platform has the potential to overcome these challenges. An automated method for collecting a "single-shot" of data from a large number of crystals was developed in collaboration with the BioCARS team at the Advanced Photon Source at Argonne National Laboratory (Chapter 6). The work described in this thesis shows that, even more so than for traditional structure determination efforts, the ability to grow and analyze a large number of high quality crystals is critical to enable time resolved structural studies of novel proteins. In addition to enabling X-ray crystallography experiments, the development of X-ray transparent microfluidic platforms also has tremendous potential to answer other scientific questions, such as unraveling the mechanism of in meso crystallization. For instance, the lipidic mesophases utilized during in meso membrane protein crystallization can be characterized by small angle X-ray diffraction analysis. Coupling in situ analysis with microfluidic platforms capable of preparing these difficult mesophase samples at very small volumes has tremendous potential to enable the high throughput analysis of these systems on a scale that is not reasonably achievable using conventional sample preparation strategies (Chapter 7). In collaboration with the LS-CAT team at the Advanced Photon Source, an experimental station for small angle X-ray analysis coupled with the high quality visualization capabilities needed to target specific microfluidic samples on a highly integrated chip is under development. Characterizing the phase behavior of these mesophase systems and the effects of various additives present in crystallization trials is key for developing an understanding of how in meso crystallization occurs. A long term goal of these studies is to enable the rational design of in meso crystallization experiments so as to avoid or limit the need for high throughput screening efforts. In summary, this thesis describes the development of microfluidic platforms for protein crystallization with in situ analysis capabilities. Coupling the ability to perform in situ analysis with the small scale, fine control, and the high throughput nature of microfluidic platforms has tremendous potential to enable a new generation of crystallographic studies and facilitate the structure determination of important biological targets. The development of platforms for in meso membrane protein crystallization is particularly significant because they enable the preparation of highly viscous mixtures at a previously unachievable scale. Work in these areas is ongoing and has tremendous potential to improve not only current the methods of protein crystallization and crystallography, but also to enhance our knowledge of the structure and function of proteins which could have a significant scientific and medical impact on society as a whole. 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Clinical outcome after first and recurrent hemorrhage in patients with untreated brain arteriovenous malformation

Background and Purpose: The morbidity from spontaneous hemorrhage of untreated brain arteriovenous malformations (AVM) is not well described. Methods: The 241 consecutive AVM patients (mean age 3716 years, 52% women) from the prospective Columbia AVM Databank initially presenting with hemorrhage were evaluated using the Rankin Scale (RS) and the National Institute of Health Stroke Scale (NIHSS). From the 241 AVM patients, 29 (12%) had subsequent intracranial hemorrhage during follow-up. For further comparisons, 84 non-AVM patients with intracerebral hemorrhage from the Northern Manhattan Study (NOMAS) served as a control group. Results: In 241 AVM patients presenting with hemorrhage the median RS was 2 and the median NIHSS was 1 (49% RS 0 to 1, 61% NIHSS 2). The median time between hemorrhage and clinical evaluation was 11 days (mean 219 days). Recurrent AVM hemorrhage during follow-up resulted in no significant increase in morbidity (median RS 2, P0.004; median NIHSS 3, P0.322; time between hemorrhage and study evaluation: median 55 days, mean 657 days). Among AVM-hemorrhage subtypes, parenchymatous AVM hemorrhage was associated with higher stroke morbidity (odds ratio, 2.9; 95% CI, 1.5 to 5.8 for NIHSS 2) than nonparenchymatous hemorrhages. Parenchymatous AVM hemorrhage had a significantly better outcome (median NIHSS 1) than non-AVM related hemorrhage (median NIHSS 12; P0.0001). Conclusions: Hemorrhage, either at initial presentation or during follow-up of untreated AVM patients appears to carry

Relatório de estágio para obtenção do grau de mestre em Educação Pré-Escolar e Ensino do 1º Ciclo do Ensino Básico

O presente relatório comporta uma coletânea de descrições da praxis, contempladas por introspeções à luz das teorias estudadas ao longo da minha formação académica. Por outro lado, apresenta como principal propósito refletir acerca da minha intervenção pedagógica e dar a conhecer os pressupostos teóricos e metodológicos que orientaram a minha atuação em contexto Pré-Escolar, no Infantário “O Girassol”, e 1.º Ciclo do Ensino Básico, na Escola Básica do 1.º Ciclo com Pré-Escolar da Ladeira. A articulação entre a teoria e prática decorre da profissão docente solicitar uma formação contínua, uma busca constante na procura de respostas que viabilizem uma melhoria na sua atuação e adequação face aos interesses e necessidades apresentadas pelo grupo de crianças/alunos com que interagem. Desta forma, o profissional, como investigador e reflexivo, deve considerar a metodologia de investigação-ação como basilar ao longo de todo o desenvolvimento do estágio pedagógico. Através da observação participante e do contato com os alunos emergiu a necessidade de promover estratégias de intervenção que contribuíssem para a disciplina, que compilou na seguinte questão: “Como desenvolver comportamentos adequados neste grupo de alunos do 4.º ano de escolaridade?”. Quanto à valência de Pré-Escolar, com base nas mesmas técnicas de recolha de dados, surgiu uma problemática semelhante, com vista na melhoria de comportamentos sociomorais, porém, articulada com os princípios da pedagogia de projeto, desenvolvendo-se uma pergunta de investigação em torno dessa metodologia: “Como a pedagogia de projeto contribui para o maior envolvimento das crianças da sala dos 4 Anos no seu processo de aprendizagem, ao mesmo tempo que fomenta comportamentos adequados?”. Para responder a essas perguntas, no decurso do estágio pedagógico foram utilizadas diversos princípios metodológicos, nomeadamente o trabalho cooperativo, introdução de jogos pedagógicos, o trabalho de projeto, acompanhadas pelo reforço positivo, quer a nível de expressões verbais positivas, quer a nível de recompensas.

Carbon gold rush and carbon cowboys: a new chapter in green mythology?

Individual and collective efforts to mitigate climate change in the form of carbon offsetting and emissions trading schemes have recently become the focus of much media attention. In this paper we explore a subset of the UK national press coverage centered on such schemes. The articles, selected from general as well as specialized business and finance newspapers, make use of gold rush, Wild West and cowboy imagery which is rooted in deeply entrenched myths and metaphors and allows readers to make sense of very complex environmental, political, ethical, and financial issues associated with carbon mitigation. They make what appears complicated and unfamiliar, namely carbon trading and offsetting, seem less complex and more familiar. A critical discussion of this type of imagery is necessary in order to uncover and question tacit assumptions and connotations which are built into it and which might otherwise go unnoticed and unchallenged in environmental communication.

La irrealidad en la cuentística de Quiroga

Contempla el análisis de tres cuentos del escritor Horacio Quiroga: Las moscas, La llama y El conductor del rápido en el aspecto de la realidad-irrealidad. Se afirma que Quiroga no posee un naturalismo orientado a fines científicos, sino hacia fines poéticos o literarios.

A History of Little Havana

This flyer promotes a book presentation by Guillermo J. Grenier and Corinna J. Moebius called "A History of Little Havana". The authors trace the history and growth of Miami's Latin cultural epicenter, Little Havana. The presentation was held at Books&Books in Coral Gables, FL on October 23, 2015.

Estado nutricional y etapas de cambio comportamental frente a la actividad física niños y adolescentes de Bogotá, Colombia: Estudio FUPRECOL

Objetivo: El propósito del estudio fue relacionar la etapa en el cambio en el comportamiento frente a la actividad física y el estado nutricional en escolares entre 9 y 17 años de Bogotá- Colombia, pertenecientes al estudio FUPRECOL. Método: Se trata de un estudio transversal, en 6.606 niños y adolescentes entre 9 y 17 años, pertenecientes a 24 instituciones educativas, de Bogotá-Colombia. Se aplicó de manera auto-diligenciada el cuestionario de cambio de comportamiento en función a la intensión de realizar actividad física (CCC-Fuprecol) y se midió el peso y la estatura para determinar el estado nutricional con el índice de masa corporal (IMC). Resultados: El porcentaje de respuesta fue 94% y se consideraron válidos 6,606 registros, siendo 58.3 % (n=3.850) niñas con un promedio de edad de 12,7±2,3 años. En la población general, el 5,3 % de los escolares se encontraban en etapa de pre-contemplación, 31,8 % en contemplación, el 26,7 % en acción y el 36,2 % en etapa de mantenimiento. Al comparar la etapa de cambio con el estado nutricional por IMC, los escolares clasificados en obesidad mostraron mayor frecuencia de respuesta en la etapa de pre-contemplación, mientras que los escolares con peso saludable acusaron mayores porcentajes en la etapa de mantenimiento. Conclusión: En escolares de Bogotá, Colombia, se encontró una relación estadísticamente significativa entre a la intensión de realizar actividad con el estado nutricional medido con el IMC. Fomentar la promoción de la actividad física y monitorear el estado nutricional, deberá ser una prioridad en las agendas y políticas públicas dentro del ámbito escolar.

Metabolic syndrome and associated factors in a population-based sample of schoolchildren in Colombia: The FUPRECOL Study

In contrast to the definition of metabolic syndrome (MetS) in adults, there is no standard definition of MetS in pediatric populations. We aimed to assess the differences in the prevalence of MetS in children and adolescents aged 9–17 years in the city of Bogota (Colombia) using four different operational definitions for these age groups and to examine the associated variables. A total of 673 children and 1,247 adolescents attending public schools in Bogota (54.4% girls; age range 9–17.9 years) were included. The prevalence of MetS was determined by the definitions provided by the International Diabetes Federation (IDF) and three published studies by Cook et al., de Ferranti et al., and Ford et al. The prevalence of MetS was 0.3%, 6.3%, 7.8%, and 11.0% according to the IDF, Cook et al., Ford et al., and de Ferranti et al. definitions, respectively. The most prevalent components were low high-density lipoprotein cholesterol and high triglyceride levels, whereas the least prevalent components were abdominal obesity and hyperglycemia. Overall, the prevalence of MetS was higher in obese than in non-obese schoolchildren. In conclusion, MetS diagnoses in schoolchildren strongly depend on the definition chosen. These findings may be relevant to health promotion efforts for Colombian youth to develop prospective studies and to define which cut-offs are the best indicators of future morbidity.

Factores asociados al desplazamiento activo en bicicleta al colegio en el Distrito Bogotá, Colombia. Estudio FUPRECOL

Introducción: El transporte activo (TA) puede ser una oportunidad para incrementar los niveles de actividad física diarios de los niños y adolescentes, además de destacarse como una estrategia práctica, accesible y sostenible a largo plazo. Objetivos: El objetivo del presente estudio es doble: Analizar los patrones de desplazamiento activo en bicicleta al y desde el centro educativo, y b) Identificar los factores asociados al uso de la bicicleta como TA; en una muestra de niños y jóvenes pertenecientes a escuelas oficiales de Bogotá, Colombia. Material y métodos: Se trata de un sub-análisis del estudio FUPRECOL en 8060 niños y adolescentes entre los 9-17 años de edad). El modo de desplazamiento del escolar fue determinado a través de la pregunta: “¿Durante los últimos 7 días, usaste bicicleta para ir al colegio/escuela y volver a la casa?. Dicha respuesta se categorizó en activos “Si” (si se desplazan en bicicleta) y pasivos “No” (si se desplazan en vehículo motorizado). Se midieron parámetros antropométricos de peso, talla y perímetro de cintura. El máximo nivel de estudios alcanzados por la madre/padre (no reporta, primaria o secundaria/técnico o tecnólogo/universitario o postgrado) y la composición del hogar (vive con padre/vive con madre/con ambos padres/con abuelos/otros familiares) se auto-reportó por los padres. Las relaciones entre el TA y los factores anteriormente descritos se analizaron mediante regresión logística binaria. Resultados: El 21,9% del total de la muestra reporta usar la bicicleta como medio de transporte y el 7,9% acumula más de 120 minutos al día. Se observó una mayor probabilidad de usar la bicicleta como medio de desplazamiento activo a la escuela en los varones, en los jóvenes entre 9 y 12 años, y en aquellos cuyo padre/madre reportaron mayor grado académico, es decir, “universitario/postgrado”. 3 Conclusión: Los hallazgos del presente estudio sugieren que es necesario promover el TA desde la niñez, poniendo mayor énfasis en el paso a la adolescencia y en las jóvenes, para así aumentar los niveles diarios de AF de estos.