Gene regulation was up to recently believed to be exerted mainly by proteins. However it has become obvious that small non coding RNA may play an important role in the control of gene expression. The complete sequencing of many genomes from bacteria to mammals, led to the discovery of numerous RNAs, ranging from 20 to 500 nucleotides in length. For instance microRNAs (miRNAs) have recently emerged as a new class of post-transcriptional regulators of gene expression. In bacteria another class of small non coding RNAs (sRNA) has been identified. Like the eukaryotic miRNAs, sRNAs, expressed in response to a stress, are able to regulate selectively the expression of one or several genes by controlling the translation or the stability of the corresponding mRNAs. During the past few years, most of bacterial sRNAs known to date have been identified in the non-pathogenic bacteria Escherichia coli using both computational and experimental methods. Recently many new sRNA have been identified in pathogenic bacteria, such as the two gram positive bacteria Listeria monocytogenes and Staphylococcus aureus. To establish successful infections, bacterial pathogens may use sRNA to control precisely expression of their virulence genes in response to host signals. Strikingly, no regulatory small RNAs have yet been described in the gram-negative, microaerophilic, spiral-shaped bacterium named Helicobacter pylori that colonizes the gastric mucosa of humans. The aim of our project is to identify small non coding RNA that would be involved in the control of virulent gene expression in H. pylori. This bacterium is the aetiological agent of gastritis and peptic ulcers. The development of gastric carcinoma and MALT lymphoma is also strongly associated with long-term chronic H. pylori infection. About 50% of the world population is infected by H. pylori and 9,000 new cases of gastric cancer appear every year in France. The inflammation of the stomach, which is the natural reservoir in which H. pylori persists and grows, leads to lesions in the mucosa and genotoxic events in the gastric epithelial cells. Despite an efficient antibiotic treatment, H. pylori remains one of the most important human bacterial disease. Although several colonization and pathogenesis factors have been identified so far in H. pylori, the molecular mechanisms of the regulation of gene expression in this bacterium are largely unknown. How gene expression is modified to cope with changes in the environment or to facilitate chronic infections in the stomach is poorly defined. The aim of our project is to identify the function of novel sRNAs discovered in H. pylori and to study these sRNA in respect to the regulation of virulence factor gene expression. By the use of an experimental approach (RNomics), we expect to identify sRNA that are specifically induced or repressed during the colonization of the stomach (acidic stress) or in contact with the host epithelial gastric cells. Moreover in the second part of this project, we wish to identify which partners (messenger RNA, proteins or other cellular components) these sRNA are targeting. For this second objective we propose to use several combined approaches: comparative proteomics, bioinformatic searches for complementarities with messenger RNA, and sRNA-associated protein purifications.The discovery of cellular functions of sRNA in a pathogenic bacteria such as H. pylori should improve our global understanding of their role in RNA-mediated virulence gene regulation.

The present project is a in line with continuous effort of the partners to provide better insights into the roles and mechanisms of action of CXCL4/CXCL4L1 chemokines and to develop novel tools for the pharmacological inhibition of angiogenesis. The interaction of the Bikfalvi (partner 1) and the Wu (partner 2) laboratory within this project is critical since the Bikfalvi laboratory is able to carry out all the functional studies while the Wu laboratory is an expert in the analysis of protein structure and of interaction studies. This provides an essential add value and synegism for both laboratories for this project. - The project is devided in 3 major aims that include functional studies as well as structural biology studies related to CXCL4/CXCL4L1 chemokines and of pharmacological inhibitors derived from snake venoms. - ...

Accurate behavioral adaptation to stimuli predicting threatening outcome is critical to survival. An insufficient fear reaction may lead the animal to overlook future signs of danger, whereas overreacting may lead the animal to failure to explore and miss opportunities for feeding or mating. A growing body of evidence indicate that the medial prefrontal cortex (mPFC) plays a key role in the control of fear behavior and that distinct prefrontal sub-regions differentially regulate the expression and inhibition of fear responses. Indeed, whereas lesions or inactivations of the mPFC infralimbic (IL) area promote fear expression, the same manipulations applied to the mPFC prelimbic area (PL) promote fear inhibition. Moreover, it has recently been shown that PL and IL receive segregated inputs from anatomically and functionally distinct amygdala neuronal circuits specifically activated during high and low fear states. These data suggest that a key function of mPFC neuronal circuits might be to integrate inputs from the amygdala and other structures involved in fear behavior to ultimately gate fear expression via projections to specific neuronal circuits._x000D_ However, little is known about the underlying PL/IL neuronal circuits. Is the rapid switch between expression/suppression of fear behaviors mediated by the same circuits or does the mPFC contain distinct neuronal circuits dedicated to the control of opposite behaviors? Which synapses are modified during expression/inhibition of fear behavior? Is there an anatomical organization in terms of afferents and efferent at the level of prefrontal single cells or entire circuits? Are there dedicated prefrontal neurons and circuits projecting to specific targets? What could be the role of such circuits for specific aspects of fear behavior? _x000D_ To address these questions we will use an innovative cross-level approach combining in vivo electrophysiological recording techniques, selective optogenetic manipulations and behavioral approaches to elucidate the anatomical and physiological properties of prefrontal excitatory/inhibitory circuits and to address their functional role in the control of fear behavior. In a first step we will examine the activation and connectivity of excitatory and inhibitory PL/IL neuronal circuits controlling fear expression using in vivo single unit extracellular recordings and extracellular stimulations. In a second step, we will selectively manipulate these circuits during behavior using light-activated proteins to test whether the rapid and reversible activation/silencing of neuronal activity causes behavioral alterations. Finally using in vivo intracellular recordings we will study the plasticity and anatomical properties of prefrontal neuronal circuits controlling fear behavior._x000D_ The expected results will provide a detailed knowledge of the cellular basis of fear behavior and of behavioral control in general. Moreover, elucidating the neuronal circuits controlling fear behavior should also lead to novel therapeutic strategies for psychiatric disorders involving dysregulation of emotional responses such as post-traumatic stress disorder and related psychiatric conditions._x000D_

This proposal is foccused at developing a Low Molecular Weight Gel (LMWG) technology to serve as a smart scaffold for stem cell culture and in situ delivery systems. GELCELLS is based on the complementary chemistry/biology expertises of two partners. The methodology utilized in the proposed studies will marry the expertise of organic chemistry, physico-chemistry, cellular biology, stem cell cultures and a range of techniques to accomplish a comprehensive description of the systems developed. To achieve this ambitious goal, the following tasks have been defined and are listed below. All the tasks can be achieved within the time frame of this project (see figure and table below for the link between the different workpackages of this program). 1. Coordination (INSERM U869, U. Bordeaux) 2. Synthesis of GNLs (INSERM U869, U. Bordeaux) 3. Physico-chemical studies (INSERM U869, U. Bordeaux). Identification of the key physical and structural characteristics of the LMWGs 4. Cells/hydrogel interaction studies in 2D and 3D systems (INSERM U577) 5. Study of nucleic acid entrapment and release (INSERM U869, U. Bordeaux) 6. Transfection assays with matricial systems (INSERM U577, INSERM U869, U. Bordeaux) The following results will be used for patents and/or publications: • Library of glycosylated GNLs • A control of the molecular, supramolecular and physicochemical properties of the LMWG gel matrix and their influence on the cell fate • Successful stem cell culture in LMWG systems • Controlled entrapment and delivery of nucleic acids by the LMWGs matrices • Successful in situ transfection in 2D and 3D matrix