The regulation of AMP-activated protein kinase by vascular endothelial growth factor

The function of the vascular endothelium is to maintain vascular homeostasis, by providing an anti-thrombotic, anti-inflammatory and vasodilatory interface between circulating blood and the vessel wall, meanwhile facilitating the selective passage of blood components such as signaling molecules and immune cells. Dysfunction of the vascular endothelium is implicated in a number of pathological states including atherosclerosis and hypertension, and is thought to precede atherogenesis by a number of years. Vascular endothelial growth factor A (VEGF) is a crucial mitogenic signaling molecule, not only essential for embryonic development, but also in the adult for regulating both physiological and pathological angiogenesis. Previous studies by our laboratory have demonstrated that VEGF-A activates AMP-activated protein kinase (AMPK), the downstream component of a signaling cascade important in the regulation of whole body and cellular energy status. Furthermore, studies in our laboratory have indicated that AMPK is essential for VEGF-A-stimulated vascular endothelial cell proliferation. AMPK activation typically stimulates anabolic processes and inhibits catabolic processes including cell proliferation, with the ultimate aim of redressing energy imbalance, and as such is an attractive therapeutic target for the treatment of obesity, metabolic syndromes, and type 2 diabetes. Metabolic diseases are associated with adverse cardiovascular outcomes and AMPK activation is reported to have beneficial effects on the vascular endothelium. The mechanism by which VEGF-A stimulates AMPK, and the functional consequences of VEGF-A-stimulated AMPK activation remain uncertain. The present study therefore aimed to identify the specific mechanism(s) by which VEGF-A regulates the activity of AMPK in endothelial cells, and how this might differ from the activation of AMPK by other agents. Furthermore, the role of AMPK in the pro-proliferative actions of VEGF-A was further examined. Human aortic and umbilical vein endothelial cells were therefore used as a model system to characterise the specific effect(s) of VEGF-A stimulation on AMPK activation. The present study reports that AMPK α1 containing AMPK complexes account for the vast majority of both basal and VEGF-A-stimulated AMPK activity. Furthermore, AMPK α1 is localized to the endoplasmic reticulum when sub-confluent, but translocated to the Golgi apparatus when cells are cultured to confluence. AMPK α2 appears to be associated with a structural cellular component, but neither α1 nor α2 complexes appear to translocate in response to VEGF-A stimulation. The present study confirms previous reports that when measured using the MTS cell proliferation assay, AMPK is required for VEGF-A-stimulated endothelial cell proliferation. However, parallel experiments measuring cell proliferation using the Real-Time Cell Analyzer xCELLigence system, do not agree with these previous reports, suggesting that AMPK may in fact be required for an aspect of mitochondrial metabolism which is enhanced by VEGF-A. Studies into the mitochondrial activity of endothelial cells have proved inconclusive at this time, but further studies into this are warranted. During previous studies in our laboratory, it was suggested that VEGF-A-stimulated AMPK activation may be mediated via the diacylglycerol (DAG)-sensitive transient receptor potential cation channel (TRPCs -3, -6 or -7) family of ion channels. The present study can neither confirm, nor exclude the expression of TRPCs in vascular endothelial cells, nor rule out their involvement in VEGF-A-stimulated AMPK activation; more specific investigative tools are required in order to characterise their involvement. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP)-stimulated Ca2+ release from acidic intracellular organelles is not required for AMPK activation by VEGF-A. Despite what is known about the mechanisms by which AMPK is activated, far less is known concerning the downregulation of AMPK activity, as observed in human and animal models of metabolic disease. Phosphorylation of AMPK α1 Ser485 (α2 Ser491) has recently been characterised as a mechanism by which the activity of AMPK is negatively regulated. We report here for the first time that VEGF-A stimulates AMPK α1 Ser485 phosphorylation independently of the previously reported AMPK α1 Ser485 kinases Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2). Furthermore, inhibition of protein kinase C (PKC), the activity of which is reported to be elevated in metabolic disease, attenuates VEGF-A- and phorbol 12-myristate 13-acetate (PMA)-stimulated AMPK α1 Ser485 phosphorylation, and increases basal AMPK activity. In contrast to this, PKC activation reduces AMPK activity in human vascular endothelial cells. Attempts to identify the PKC isoform responsible for inhibiting AMPK activity suggest that it is one (or more) of the Ca2+-regulated DAG-sensitive isoforms of PKC, however cross regulation of PKC isoform expression has limited the present study. Furthermore, AMPK α1 Ser485 phosphorylation was inversely correlated with human muscle insulin sensitivity. As such, enhanced AMPK α1 Ser485 phosphorylation, potentially mediated by increased PKC activation may help explain some of the reduced AMPK activity observed in metabolic disease.

Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine

Background: In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. Results: The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. Conclusion: This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.

Mathematical Programming Models for Influence Maximization on Social Networks

In this dissertation, we apply mathematical programming techniques (i.e., integer programming and polyhedral combinatorics) to develop exact approaches for influence maximization on social networks. We study four combinatorial optimization problems that deal with maximizing influence at minimum cost over a social network. To our knowl- edge, all previous work to date involving influence maximization problems has focused on heuristics and approximation. We start with the following viral marketing problem that has attracted a significant amount of interest from the computer science literature. Given a social network, find a target set of customers to seed with a product. Then, a cascade will be caused by these initial adopters and other people start to adopt this product due to the influence they re- ceive from earlier adopters. The idea is to find the minimum cost that results in the entire network adopting the product. We first study a problem called the Weighted Target Set Selection (WTSS) Prob- lem. In the WTSS problem, the diffusion can take place over as many time periods as needed and a free product is given out to the individuals in the target set. Restricting the number of time periods that the diffusion takes place over to be one, we obtain a problem called the Positive Influence Dominating Set (PIDS) problem. Next, incorporating partial incentives, we consider a problem called the Least Cost Influence Problem (LCIP). The fourth problem studied is the One Time Period Least Cost Influence Problem (1TPLCIP) which is identical to the LCIP except that we restrict the number of time periods that the diffusion takes place over to be one. We apply a common research paradigm to each of these four problems. First, we work on special graphs: trees and cycles. Based on the insights we obtain from special graphs, we develop efficient methods for general graphs. On trees, first, we propose a polynomial time algorithm. More importantly, we present a tight and compact extended formulation. We also project the extended formulation onto the space of the natural vari- ables that gives the polytope on trees. Next, building upon the result for trees---we derive the polytope on cycles for the WTSS problem; as well as a polynomial time algorithm on cycles. This leads to our contribution on general graphs. For the WTSS problem and the LCIP, using the observation that the influence propagation network must be a directed acyclic graph (DAG), the strong formulation for trees can be embedded into a formulation on general graphs. We use this to design and implement a branch-and-cut approach for the WTSS problem and the LCIP. In our computational study, we are able to obtain high quality solutions for random graph instances with up to 10,000 nodes and 20,000 edges (40,000 arcs) within a reasonable amount of time.

Modelo de Medición de la Gestión Estratégica Mediante el Empleo de una Estructura de Cuadro de Mando Integral Dirigido a las Empresas Pertenecientes al Sector Manufacturero de Artículos de Talabartería y Guarnicionería en Venezuela

En éste artículo se analiza las características y dimensiones de los indicadores de las Estrategias Genéricas (Porter, 1980) y de los Cuadro de Mando Integral (Kaplan y Norton, 1992), para posteriormente integrarlas y conjugarlas con el propósito de conformar un “Modelo de Medición de la Gestión Estrategia mediante una Estructura del Cuadro de Mando Integral para el Sector Manufacturero de Talabartería y Guarnicionería de Venezuela” (CMI-EGP). Los datos fueron recolectados con dos cuestionarios basados en dimensiones de éstas dos teorías, relacionadas con la alineación entre el recurso humano y la gestión organizacional. Es decir, donde cada dependencia busca alinear los enfoques estratégicos propios de la organización, para así convertirse en un factor de éxito. La metodología empírica empleada esta basada en la técnica de reducción de datos o análisis factorial y por un análisis confirmatorio mediante la técnica Structural Equation Models (SEM), que es una herramienta integral de modelización multiecuacional que fusiona la econometría con los principios de medición de la psicología y la sociología. Esta técnica estadística de análisis multivariante tiene como objetivos primordiales, el aumentar la capacidad explicativa del investigador y la eficacia estadística. La investigación proporciona una modelización confirmatoria que correlaciona las variables latentes y manifiestas, que determinan el grado de relación y alineación entre las cuatro perspectivas de cuadro de mando integral (procesos internos, financieros, del cliente y aprendizaje y crecimiento) y las estrategias genéricas de Porter. Para el procesamiento se emplea el software LISREL versión más reciente 8.8 del año 2009, que es un programa usado en el análisis de ecuaciones estructurales, que fue desarrollado en los años setenta por Karl Jöreskog y Dag Sörbom, ambos profesores de la Universidad de Upsala, Suecia.

Fyra speciallärares syn på Wendickmodellens användning som en läsutvecklingsmetod : En intervjustudie med speciallärare för grundskolans mellanår.

Syftet med den här undersökningen har varit att undersöka hur fyra speciallärare använder Wendickmodellen i årskurs 4-6 samt att undersöka deras inställning till Wendickmodellen som läsutvecklingsmodell. Undersökningen gjordes genom kvalitativa intervjuer med fyra speciallärare för grundskolans mellanår. Alla dessa har på ett eller annat sätt kommit i kontakt med, och använt sig av, Wendickmodellen, men det är inte alla som använder den i dag. Som teoretisk utgångspunkt har Vygotskijs sociokulturella perspektiv samt Deweys pragmatiska perspektiv använts. Resultatet visar att Wendickmodellens vara eller icke vara inte är vad som avgör om en utlärningssituation är tillfredsställande eller inte, utan snarare att det är det aktiva valet av metod från läraren som avgör. Resultatet visar också att det oftast inte går att använda sig av endast en modell, i alla fyra intervjuer som gjordes blev det tydligt att alla lärare valde att arbeta på olika sätt med sina elever, oavsett om de använde sig av Wendickmodellen eller inte.

Fyra speciallärares syn på Wendickmodellens användning som en läsutvecklingsmetod. : En intervjustudie med speciallärare för grundskolans mellanår.

Syftet med den här undersökningen har varit att undersöka hur fyra speciallärare använder Wendickmodellen i årskurs 4-6 samt att undersöka deras inställning till Wendickmodellen som läsutvecklingsmodell. Undersökningen gjordes genom kvalitativa intervjuer med fyra speciallärare för grundskolans mellanår. Alla dessa har på ett eller annat sätt kommit i kontakt med, och använt sig av, Wendickmodellen, men det är inte alla som använder den i dag. Som teoretisk utgångspunkt har Vygotskijs sociokulturella perspektiv samt Deweys pragmatiska perspektiv använts. Resultatet visar att Wendickmodellens vara eller icke vara inte är vad som avgör om en utlärningssituation är tillfredsställande eller inte, utan snarare att det är det aktiva valet av metod från läraren som avgör. Resultatet visar också att det oftast inte går att använda sig av endast en modell, i alla fyra intervjuer som gjordes blev det tydligt att alla lärare valde att arbeta på olika sätt med sina elever, oavsett om de använde sig av Wendickmodellen eller inte.

Déterminants génétiques de la variabilité des triglycérides plasmatiques en réponse à une supplémentation en acides gras oméga-3 d’origine marine

Il est reconnu que la consommation d’acides gras (AG) oméga-3 (n-3) d’origine marine est bénéfique pour la prévention des maladies cardiovasculaires (MCV), notamment en raison de leurs effets hypotriglycéridémiants. Toutefois, il existe une importante hétérogénéité dans la réponse des triglycérides (TG) plasmatiques à une supplémentation en AG n-3 et ce phénomène est en partie attribuable à des facteurs génétiques. Notre groupe de recherche a récemment réalisé une étude d’association à l’échelle du génome (GWAS) sur les participants de l’étude Fatty Acid Sensor (FAS), qui a permis d’identifier plusieurs loci associés à la réponse des TG suite à une supplémentation de 3g d’AG n-3 par jour. La plupart de ces loci sont localisés dans les gènes IQCJ, NXPH1, PHF17 et MYB. Des effets du génotype ainsi que des interactions gène-diète ont été observés avec plusieurs polymorphismes nucléotidiques simples (SNPs) des quatre gènes candidats. Ces résultats suggèrent que des variations génétiques à l’intérieur de gènes identifiés par GWAS peuvent expliquer en partie la variabilité de la réponse des TG plasmatiques à une supplémentation en AG n-3 d’origine marine.