SYSMET: SYSTEMS BIOLOGY OF MEMBRANE TRAFFICKING

Studies on membrane trafficking have expanded massively over the last 40 years. During this time, research has led to an understanding of the molecular mechanisms underlying membrane trafficking pathways, providing crucial insights into several fundamental events. Although we have gained detailed knowledge about the molecular organization of membrane trafficking machineries there is a lack of a global view of its function, organization and regulation. In addition, many genes of the membrane trafficking machinery have been associated with diseases. In the majority of cases, disease manifestation is tissue-specific despite the ubiquitous expression of the causal gene. Explanations for this phenomenon may be found either in the specific requirements and demands of a cell within a given tissue or in differences in the expression of disease gene interactors. The main aim of this project was to delineate sets of co-expressed membrane trafficking genes and proteins (membrane trafficking modules; MTMs) across tissues. For this purpose we curated a list of 1,261 genes that have been described as part of membrane trafficking machineries in different cellular organelles, around which we have developed a bioinformatics pipeline in order to address two specific questions: a) are membrane trafficking genes organized in MTMs, defined as communities of co-expressed genes, and are they associated with general cellular functions? b) do disease genes have specific membrane-trafficking co-expressed communities in those tissues that are affected by the disease? To address these questions we used data from the Genotype-Tissue Expression (GTEx) project, a catalog of human tissue-specific gene expression patterns obtained from \u201cnon-diseased\u201d tissues sampled from recently deceased human donors. With regards to the first question, we analyzed the expression patterns of the trafficking genes in twenty-five different tissues and used weighted correlation network analysis (WGCNA) to derive highly preserved MTMs. We have analyzed in more detail one that includes genes apparently involved in collagen secretion. Instead for the second question we applied differential co-expression before the WGCNA to generate tissue-specific MTMs to understand how specific membrane trafficking gene modules might be organized in human tissues

A toxicogenomic data space for system-level understanding and prediction of EDC-induced toxicity.

Endocrine disrupting compounds (EDCs) are a persistent threat to humans and wildlife due to their ability to interfere with endocrine signaling pathways. Inspired by previous work to improve chemical hazard identification through the use of toxicogenomics data, we developed a genomic-oriented data space for profiling the molecular activity of EDCs in an in silico manner, and for creating predictive models that identify and prioritize EDCs. Predictive models of EDCs, derived from gene expression data from rats (in vivo and in vitro primary hepatocytes) and humans (in vitro primary hepatocytes and HepG2), achieve testing accuracy greater than 90%. Negative test sets indicate that known safer chemicals are not predicted as EDCs. The rat in vivo-based classifiers achieve accuracy greater than 75% when tested for invitro to in vivoextrapolation. This study reveals key metabolic pathways and genes affected by EDCs together with a set of predictive models that utilize these pathways to prioritize EDCs in dose/time dependent manner and to predict EDCevokedmetabolic diseases

High-Yield Method for Isolation and Culture of Endothelial Cells from Rat Coronary Blood Vessels Suitable for Analysis of Intracellular Calcium and Nitric Oxide Biosynthetic Pathways

We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This methods makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed