Assembly and function of fibroblast-derived extracellular matrices

For a tumor to develop and spread, the growth-repressive environment of the host tissue must undergo significant changes. These changes include dramatic modifications in the molecular composition and architecture of the extracellular matrix (ECM). Importantly, different tumors have distinct ECM components, depending on their anatomical site. Accordingly, differences can exist between the ECM of primary tumors and metastatic lesions. The ECM can impact treatment, including the efficacy of resection and accessibility of solid tumors to therapeutic antibodies and small molecules. Conversely, treatment can impact the ECM (e.g. radiotherapy, platinium-based drugs) by promoting the deposition of a dense fibrotic stroma. My laboratory seeks to unravel the cell-dependent mechanisms that drive matrix assembly, and to improve our understanding of the functional interplay between tumor cells and their matrix microenvironment. The tumor ECM is largely synthesized and remodeled by stromal fibroblasts. I will discuss our characterization of matrices produced by head and neck tumor-associated fibroblasts and discuss how these fibrillar networks enriched in so called “oncofetal” matrix proteins convey specific biological signals to the cells they encounter

Raf-1 Activation Prevents Caspase 9 Processing Downstream of Apoptosome Formation

In many cell types, growth factor removal induces the release of cytochrome-c from mitochondria that leads to activation of caspase-9 in the apoptosome complex. Here, we show that sustained stimulation of the Raf-1/MAPK1,3 pathway prevents caspase-9 activation induced by serum depletion in CCL39/ΔRaf-1:ER fibroblasts. The protective effect mediated by Raf-1 is sensitive to MEK inhibition that is sufficient to induce caspase-9 cleavage in exponentially growing cells. Raf-1 activation does not inhibit the release of cytochrome-c from mitochondria while preventing caspase-9 activation. Gel filtration chromatography analysis of apoptosome formation in cells shows that Raf-1/MAPK1,3 activation does not interfere with APAF-1 oligomerization and recruitment of caspase 9. Raf-1-mediated caspase-9 inhibition is sensitive to emetine, indicating that the protective mechanism requires protein synthesis. However, the Raf/MAPK1,3 pathway does not regulate XIAP. Taken together, these results indicate that the Raf-1/MAPK1,3 pathway controls an apoptosis regulator that prevents caspase-9 activation in the apoptosome complex

Improving 3D MA-TIRF Reconstruction with Deconvolution and Background Estimation

International audienceTotal internal reflection fluorescence microscopy (TIRF) produces 2D images of the fluorescent activity integrated over a very thin layer adjacent to the glass coverslip. By varying the illumination angle (multi-angle TIRF), a stack of 2D images is acquired from which it is possible to estimate the axial position of the observed biological structures. Due to its unique optical sectioning capability, this technique is ideal to observe and study biological processes at the vicinity of the cell membrane. In this paper, we propose an efficient reconstruction algorithm for multi-angle TIRF microscopy which accounts for both the PSF of the acquisition system (diffraction) and the background signal (e.g., autofluorescence). It jointly performs volume reconstruction, deconvolution, and background estimation. This algorithm, based on the simultaneous-direction method of mul-tipliers (SDMM), relies on a suitable splitting of the optimization problem which allows to obtain closed form solutions at each step of the algorithm. Finally, numerical experiments reveal the importance of considering the background signal into the reconstruction process, which reinforces the relevance of the proposed approach

Optimal Transport vs Many-to-many assignment for Graph Matching

National audienceGraph matching for shape comparison or network analysis is a challenging issue in machine learning and computer vision. Gener-ally, this problem is formulated as an assignment task, where we seek the optimal matching between the vertices that minimizes the differencebetween the graphs. We compare a standard approach to perform graph matching, to a slightly-adapted version of regularized optimal transport,initially conceived to obtain the Gromov-Wassersein distance between structured objects (e.g. graphs) with probability masses associated to thenodes. We adapt the latter formulation to undirected and unlabeled graphs of different dimensions, by adding dummy vertices to cast the probleminto an assignment framework. The experiments are performed on randomly generated graphs onto which different spatial transformations areapplied. The results are compared with respect to the matching cost and execution time, showcasing the different limitations and/or advantagesof using these techniques for the comparison of graph networks

cDNA cloning and expression of a hamster α-thrombin receptor coupled to Ca2+ mobilization

AbstractThe serine protease α-thrombin (thrombin) potently stimulates G-protein-coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G-protein-coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C-coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N-terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N-terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion-binding exosite of thrombin

Classification of the fibronectin variants with curvelets

International audienceThe role of the extracellular matrix (ECM) in the evolution of certain diseases (e.g. fibrosis, cancer) is generally accepted but yet to be completely understood. A numerical model that captures the physical properties of the ECM, could convey certain connections between the topology of its constituents and their associated biological features. This study addresses the analysis and modeling of fibrillar networks containing Fibronectin (FN) networks, a major ECM molecule, from 2D confocal microscopy images. We leveraged the advantages of the fast discrete curvelet transform (FDCT), in order to obtain a multiscale and multidirectional representation of the FN fibrillar networks. This step was validated by performing a classification among the different variants of FN upregulated in disease states with a multi-class classification algorithm, DAG-SVM. Subsequently, we designed a method to ensure the invariance to rotation of the curvelet features. Our results indicate that the curvelets offer an appropriate discriminative model for the FN networks, that is able to characterize the local fiber geometry

Fibronectin Extra Domains tune cellular responses and confer topographically distinct features to fibril networks

International audienceCellular fibronectin (FN; also known as FN1) variants harboring one or two alternatively spliced so-called extra domains (EDB and EDA) play a central bioregulatory role during development, repair processes and fibrosis. Yet, how the extra domains impact fibrillar assembly and function of the molecule remains unclear. Leveraging a unique biological toolset and image analysis pipeline for direct comparison of the variants, we demonstrate that the presence of one or both extra domains impacts FN assembly, function and physical properties of the matrix. When presented to FN-null fibroblasts, extra domain-containing variants differentially regulate pH homeostasis, survival, and TGF- β by tuning the magnitude of cellular responses, rather than triggering independent molecular switches. Numerical analyses of fiber topologies highlight significant differences in variant-specific structural features and provide a first step for the development of a generative model of FN networks to unravel assembly mechanisms and investigate the physical and functional versatility of extracellular matrix landscapes

Los Montes Submarinos de la Plataforma Marginal de Motril, Mar de Alborán: Interacción con las Masas de Agua Profunda

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L'étude de la régulation du couplage excitation-contraction dans le muscle lisse vasculaire : mécanismes cellulaires et moléculaires de l'hypertension pulmonaire

L'hypertrophie vasculaire médiane pulmonaire dans l'hypertension pulmonaire primaire (PPH) est principalement liée à une augmentation de la prolifération et à une diminution de l'apoptose dans les cellules du muscle lisse de l'artère pulmonaire humaine (PASMCs). Le premier objectif de cette étude était d'élucider la pathogenèse PPH. Dans un premier temps, j'ai examiné les effets apoptotiques des protéines morphogénétiques osseuses (BMPs) sur les cellules PASMCs humaines normales et analyse si les effets induits par les BMPs sont altérés dans les cellules PASMCs de patients PPH. Le traitement des cellules normales PASMCs avec BMP-2 et BMP-7 entraîne une augmentation considérable du pourcentage de cellules entrant en apoptose. L'inhibition de l'apoptose induite par les BMPs dans les cellules PASMCs chez les patients PPH pourrait jouer un rôle important dans le développement de l'hypertrophie vasculaire médiane pulmonaire. Dans un second temps, j'ai analysé les mécanismes cellulaires et moléculaires de la vasoconstriction pulmonaire hypoxique. L'hypoxie chronique augmente la prolifération des cellules du muscle lisse de l'artère pulmonaire en surexprimant les gènes AP-1 dépendants et codant pour des facteurs vaso-actifs d'origine endothéliale et des facteurs mitogènes impliqués dans le développement de l'hypertension pulmonaire. L'effet de l'hypoxie chronique a été examiné sur la capacite de AP-1 a fixer l'ADN, l'entrée capacitive du calcium, et l'expression des gènes TRP dans les cellules endothéliales de l'artère pulmonaire humaine (PAECs). Mes travaux suggèrent que l'augmentation de la fixation de AP-1 via le calcium pourrait jouer un rôle important en augmentant l'expression génique dépendante de AP-1, en stimulant la prolifération des cellules vasculaires pulmonaires, et enfin en remodelant les vaisseaux pulmonaires chez les patients avec une hypertension pulmonaire due à l'hypoxie.NICE-BU Sciences (060882101) / SudocSudocFranceF