Fish and mammalian glut4 traffic characteristics: an evolutionary perspective on the importance of glut4 protein motifs for trafficking

[eng] Glucose transporters (GLUTs) are extremely important for glucose metabolism. Glucose transporters uptake glucose from blood stream into the cells where it can be metabolized. Among the glucose transporters family, GLUT4, which is solely expressed in muscle and adipose tissues, displays a unique feature as it can change its cellular distribution within minutes in response to insulin to regulate glucose uptake. Therefore, the study of GLUT4 cellular trafficking is fundamental to understand its functioning and to deepen our knowledge on glucose homeostasis. In this work, we utilized a GLUT4 fish variant, brown trout GLUT4, to study GLUT4 trafficking and the role of GLUT4 protein motifs in this process, in 3T3-L1 adipocytes. We observed that, in comparison to mammalian GLUT4 (RatGLUT4), brown trout GLUT4 (BtGLUT4) had a much weaker translocation to the plasma membrane in response to insulin which was in part due to a slower cellular trafficking (exocytosis and endocytosis) and to a poor targeting to the GLUT4 storage vesicles responsible for “holding” GLUT4 inside the cell in the absence of insulin; these vesicles represent the main pool of insulin-responsive GLUT4. In this thesis we also studied the most common GLUT4 endocytic routes. We analyzed the contribution of the clathrin-mediated and the cholesterol-dependent endocytic pathways for RatGLUT4 and BtGLUT4 internalization. We observed that whilst RatGLUT4 internalizes through both the clathrin-mediated and the cholesterol-dependent pathways, BtGLUT4 only utilizes the former. It has been suggested that in adipocytes, the main cholesterol-dependent internalization pathway is the caveolar route. The internalization through this pathway is mediated by plasma membrane structures called caveolae. The formation of these structures is dependent on the caveolin-1 protein. To analyze the role of caveolae in GLUT4 internalization we blocked its formation by knocking down caveolin-1 and observed an increase of RatGLUT4 and BtGLUT4 internalization; however, both GLUT4 isoforms showed less internalization through the clathrin-mediated and cholesterol-dependent pathways in the absence of cavolin-1. Therefore, we suggest that in 3T3-L1 adipocytes caveolin-1 knockdown induces internalization of GLUT4 through alternative pathways. GLUT4 trafficking is regulated by cellular machinery that interacts with GLUT4 protein motifs. To analyze the role of the mammalian N-terminal FQQI8 and C-terminal TELEY502 motifs in GLUT4 trafficking we mutated the corresponding motifs in BtGLUT4 (FQHL8 and TELDY495, respectively) and observed that mutations in the C-terminal had little effect on BtGLUT4 trafficking whereas mutations on the N-terminal (especially FQQL8 mutant) improved BtGLUT4 intracellular retention in the absence of insulin. Furthermore, we verified that FQQL8 mutation increased BtGLUT4 retention in a syntaxin-6-rich compartment, possibly the trans-Golgi network. In addition to studying BtGLUT4 mutants we also analyzed the trafficking of a chimera consisting of a RatGLUT4 backbone with the large cytoplasmic loop of BtGLUT4 (L-GLUT4). We observed that L-GLUT4 possessed higher plasma membrane levels in the absence of insulin and as a result a weaker translocation. Moreover, we observed that this was caused, at least in part, by a reduction in the endocytosis of L-GLUT4 in the absence of insulin. We also analyzed the contribution of the clathrin-mediated and cholesterol-dependent pathways for L-GLUT4 internalization and observed that the loop substitution (L-GLUT4) reduced RatGLUT4 internalization through the cholesterol-dependent route. Moreover, in the absence of insulin and in caveolin-1, L-GLUT4 internalization did not increase as much as that of RatGLUT4. The internalization of L-GLUT4 in the absence of caveolin-1 and insulin occurred through a clathrin-mediated pathway, similarly to BtGLUT4, but it also internalized through a cholesterol-dependent pathway, unlike RatGLUT4 and BtGLUT4. In summary, in this thesis we have contributed to increase the knowledge on GLUT4 trafficking and on the roles of the FQQI8 motif and large cytoplasmic loop in this process, in 3T3-L1 adipocytes.[spa] El transportador de glucosa GLUT4 tiene la capacidad de, en respuesta a insulina, cambiar su localización celular y de esta forma regular el transporte de glucosa. En este trabajo, hemos utilizado una variante de GLUT4 de trucha (BtGLUT4) para estudiar el trafico de GLUT4, así como sus dominios proteicos involucrados en este proceso, en adipocitos 3T3-L1. Hemos observado que en comparación con el GLUT4 de mamíferos (RatGLUT4), el BtGLUT4 tenia una menor capacidad de translocación a la membrana plasmática en respuesta a insulina y que esto se debía a una trafico celular mas lento (exocitosis y endocitosis) y a una peor retención en las vesículas responsables por retener el transportador dentro de la célula en ausencia de insulina. En este trabajo hemos observado que RatGLUT4 ha internalizado por la vía de endocitosis mediada por clatrina y por la vía dependiente de colesterol, mientras que BtGLUT4 solo ha utilizado la primera. Además, hemos inhibido la internalización caveolar, mediante bajada de la expresión de caveolina-1, y hemos observado un aumento de la internalización de RatGLUT4 y BtGLUT4. Con el objetivo de estudiar el papel del dominio FQQI8 (extremo -N) de mamífero en el trafico de GLUT4, hemos mutado la secuencia correspondiente en BtGLUT4 (FQHL8) y hemos observado que mutaciones en este dominio han mejorado la retención intracelular de BtGLUT4 en ausencia de insulina. También hemos estudiado el trafico de una quimera que consiste en la secuencia de RatGLUT4 con el lazo citoplasmático largo de BtGLUT4 (L-GLUT4). Hemos observado que la sustitución del lazo ha aumentado los niveles de RatGLUT4 en superficie en ausencia de insulina y que esto era debido, por lo menos en parte, a una menor endocitosis en ausencia de la hormona. También hemos observado que la sustitución del lazo de RatGLUT4 ha reducido su internalización a través de la vía dependiente de colesterol en ausencia de insulina. Además, en ausencia de caveolina-1 y insulina, la internalización de L-GLUT4 ha aumentado menos que la de RatGLUT4 y ha ocurrido a través de las vías mediada por clatrina y dependiente de colesterol

RIO Country Report Portugal 2014

The report offers an analysis of the R&I system in Portugal for 2014, including relevant policies and funding, with particular focus on topics critical for two EU policies: the European Research Area and the Innovation Union. The report was prepared according to a set of guidelines for collecting and analysing a range of materials, including policy documents, statistics, evaluation reports, websites etc. The report identifies the structural challenges of the Portuguese research and innovation system and assesses the match between the national priorities and those challenges, highlighting the latest policy developments, their dynamics and impact in the overall national context.JRC.J.6-Innovation Systems Analysi

Étude de la relation structure-fonction du segment S6 du canal potassique K��3.1

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Scanning Electron Microscopy of the Human Thyroid Gland and its Disorders

The characteristic scanning electron microscopic features of the normal thyroid gland, benign thyroid lesions such as nodular (adenomatous) and colloid goitre, adenomas and thyroiditis, and malignant tumors such as papillary carcinoma, follicular carcinoma, anaplastic carcinoma and medullary carcinoma are described. One or more cilia are present in the center of the follicular surface of almost every epithelial cell in the normal thyroid gland as well as in most goitres. Their number is reduced in adenomas and differentiated carcinomas. Medullary carcinomas and anaplastic carcinomas usually lack cilia. Variation in distribution and appearance of microvilli seems to be related to functional differences in the normal thyroid and goitres. In neoplastic conditions the abundance of microvilli steadily decreases from ordinary papillary carcinomas to follicular variants of papillary carcinoma and to follicular carcinoma. Most of the cells in medullary carcinoma and anaplastic carcinoma have few or no microvilli. Benign and neoplastic Hürthle cells have a very characteristic appearance. Distinct, smooth-surfaced cells are interspersed among cells rich in microvilli. The literature is reviewed. Our own experience from examinations of 264 thyroid specimens is included

Sodom and Gomorrah in Pompeii

O presente estudo analisa o contexto de uma inscrição encontrada em Pompeios e na qual se lêem os nomes das duas cidades bíblicas de Sodoma e Gomorra. Partindo da hipótese de o grafito ter sido escrito antes ou no momento da erupção do Vesúvio, considera-se a possibilidade de o seu autor, eventualmente um judeu, ter tido a intenção de relacionar o terramoto de 62 ou a catástrofe de 79 d. C. com a ira divina que na tradição bíblica teria destruído Sodoma e Gomorra.O presente estudo analisa o contexto de uma inscrição encontrada em Pompeios e na qual se lêem os nomes das duas cidades bíblicas de Sodoma e Gomorra. Partindo da hipótese de o grafito ter sido escrito antes ou no momento da erupção do Vesúvio, considera-se a possibilidade de o seu autor, eventualmente um judeu, ter tido a intenção de relacionar o terramoto de 62 ou a catástrofe de 79 d. C. com a ira divina que na tradição bíblica teria destruído Sodoma e Gomorra.This paper studies the context of an inscription found in Pompeii, which refers to the Biblical cities of Sodom and Gomorrah. If the graffito was written before or during the volcanic eruption, it is possible that its Author, probably a Jew, had connected the earthquake of 62 or the catastrophe of 79 A.D. with the Divine wrath that, according the Biblical tradition, had destroyed Sodom and Gomorrah

METHANOLYSIS AND ETHANOLYSIS OF ANIMAL FATS: A COMPARATIVE STUDY OF THE INFLUENCE OF ALCOHOLS

Biodiesel from animal fats with methanol and ethanol was produced in the presence of sodium methoxide and sodium ethoxide as catalysts. Two samples of pork fats and one natural beef tallow were directly transesterified with a good final product yield: 87.7, 86.7 and 86.3% for methanolysis, and 78.4, 82.6 and 82.7% for ethanolysis, respectively. Methyl ester content was also determined, being higher than 96.5 mass% for all the samples prepared. The presence of natural C17:0 in animal fats makes it necessary to correct the method pro¬posed in the standard EN 14103 (2003). Biodiesel density at 15 C of the samples was between 870 and 876 kg/m3, within the acceptance range of standard EN 14214, and the dynamic viscosity at 40 °C of the produced biodiesels was in the range of 4.5 to 5.16 mm2/s, also fulfilling requirements of EN 14214 standard. The iodine value is much lower than the superior limit established by EN 14214 standard but oxidation stability (OSI) is lower than the required limit, 6 h, of the standard, which can be attributed to the lack of natural antioxidants in tallows

Ship Hull Representation by Non-Uniform Rational B-Spline Surface Patches

The purpose of this work is to propose a new method for representing the ship hull shape with mathematic surfaces so that geometric data can be generated for any point on the hull where required to assist the production process. An extensive survey of previous work is presented covering both the use of parametric curves and surfaces to model the ship hull and also the most relevant software systems developed for that purpose. The main methods and algorithms available for the generation and edition of curves and surfaces are presented and compared taking into consideration the intended application. From the analysis of the formulations available it was concluded that the most adequate one, which however had not yet been extensively used to model ship hulls was the Non-Uniform Rational B-Splines (NURBS), due to the potential of their capability to represent exactly conic curves and surfaces. Therefore these surfaces were selected as the basis of the method developed in this thesis. A procedure is proposed for the representation of a given hull form in a two step approach, creating first a wireframe model over which the surface patches are generated. Both curves and surfaces are based on the NURBS formulation. To create the wireframe model, first a set of longitudinal boundary lines is selected, dividing the surface into areas of similar shape. Then, these lines are fitted by curves and faired to some extent. Next, transverse sections are defined and split by the boundary lines. Surface patches are then generated over the transverse section curves within the limits of each patch. Finally, to obtain the traditional representation of the ship surface by transverse sections, buttocks and waterlines, contour lines are generated for constant values of x, y and z coordinates. A computer system has been developed incorporating an interface that allows the visualization of the curves and surfaces being modeled. The system incorporates several algorithms for generation and edition of curves and surfaces, in addition to the main contribution of this thesis which is the use of NURBS to represent the ship hull surface. The system also incorporates curve and surface analysis tools and some basic fairing algorithms so that during the several steps of the creation of the model, the fairness of the curves and surfaces can be evaluated and improved to some extent. The procedure is tested and compared with an existing commercial system through some application examples, of a complete hull and in more detail in the bow region, showing that good results can be obtained with the system presented here