This project aims at understanding the molecular and cellular mechanisms underlying the supramolecular organization and localization of Golgi-GTs implicated in the biosynthesis of Sda et sLex antigens. These carbohydrate determinants are found in the normal and cancerous gastrointestinal (GI) tract respectively: Sda antigen is highly expressed in normal GI whereas sLex is almost not present. In cancer GI, the opposite is observed: the sLex antigen appears in multiple tumor types at the expense of the Sda antigen, which disappears. Although intensively studied, the mechanisms of regulation leading to the biosynthesis of one or another of these antigens, which involves several alpha2,3-sialyltransferases, alpha1,3-fucosyltransferases and one beta1,4-N-acetylgalactosaminyltransferase (beta4GalNAcT-II) are not yet elucidated. Very recent advances made in the field have shown that the regulation of the beta4GalNAcT-II involved the biosynthesis of the Sda antigen is a key regulation point for this balance and that deregulation of its expression leads to the biosynthesis of the sLex antigen observed in cancer cells. Transcription of the unique B4GALNT2 gene leads to the biosynthesis of two transcripts differing in their 5’-untranslated region. It seems that epigenetic regulation through DNA methylation of the promoter region of the B4GALNT2 gene is not sufficient to explain the down regulated expression of this GT in cancer GI tissues, which up to date has never been studied. Using a classical molecular biology approach, we will determine if the expression of the B4GALNT2 gene relies on one or two promoter regions and we will identify the transcription factors implicated in this regulation, in particular in the diminished expression of the beta4GalNAcT-II observed in cancer GI tract. Interestingly, translation of the two transcripts leads to the potential synthesis of two protein isoforms differing only in their cytoplasmic tail. One shows a regular 6 AA cytoplasmic tail, whereas this other shows an unusually long 66 AA cytoplasmic tail. These differences in the length of the cytoplasmic tails could lead to different molecular interactions between the two beta4GalNAcT-II protein isoforms with cytosolic proteins implicated in the subcellular localization and traffic of GTs in the Golgi apparatus. In particular, recent studies of CDG (Congenital Disorders of Glycosylation) patients has shown that deficiencies in the proteins implicated in vesicular Golgi trafficking could affect subcellular localization of GTs and lead to the biosynthesis of abnormal glycans. Our second objective is thus to study the subcellular localization of the two beta4GalNAcT-II protein isoforms and their potential differential implication in GTs complexes by virtue of their different cytoplasmic tail. Finally, we will assess the implication of vesicular Golgi trafficking proteins, in particular those of the COG complex, in their subcellular localization. To achieve this, we will develop a cell biology approach and observation by fluorescent microscopy (confocal and biophotonique) with FRET techniques to characterize molecular interactions between GT and potentially other proteins. Our third objective is to determine whether these regulations only take place in the human GI tissue, using an integrative biology and a phylogenomic approach to trace back the evolutionary history of the B4GALNT2 gene in vertebrates and to set up a whole organism model for the study of these regulations in vivo, the zebrafish D. rerio.

The comprehension of the cell cycle control in cells is a fundamental task of biology. In bacteria, several model organisms have been studied in order to understand this complex regulation; among them, Caulobacter crescentus is definitely one of the most interesting cases. In this organism, the cell cycle progression is mainly controlled by two-component system proteins that, in bacteria, are the main players of the signal transduction processes. The essential master regulator of cell cycle in C. crescentus is the response regulator CtrA that is activated by the phosphorylation of the cascade CckA-ChpT. However, the activity of CtrA is repressed by the two-component system DivJ-DivK, probably by dynamic delocalization from cell pole. Also another system is inhibiting CtrA and is composed by the protease ClpPX and other factors, such as the response regulator CpdR. Both DivK-inhibition and proteolysis are controlled by CtrA, which activates transcription of divK itself and several genes of the proteolytic degradation, creating redundant negative feedbacks. The conservation across organisms of this circuit has been studied in alpha-proteobacteria, where CtrA is present, using bioinformatic tools; this analysis revealed that the logic of the circuit is conserved across Caulobacter closely related bacteria but the architecture in each organism can significantly vary. The experimental analysis of the variability of cell cycle architecture in different organisms, together with a mechanistic molecular analysis of cell cycle regulation in C. crescentus and close bacteria, are still aspects that need a deeper investigation. In this project I’m proposing to systematically analyze the core components of the regulatory network controlling bacterial cell cycle in Sinorhizobium meliloti, which is another model organism, belonging to alpha-proteobacteria and sharing the logic of cell cycle regulation with C. crescentus. The discoveries, generated by a multidisciplinary approach, will be experimentally compared with C. crescentus. In order to fill a gap of molecular knowledge of cell cycle machinery, the project will investigate factors and complexes that drive cell cycle progression using biochemical and structural biology tools. More in details, the analysis of the principal cell cycle factors in S. meliloti will be carried out by the creation of mutants, fluorescence tagging of each gene and studying the biochemistry of each encoded protein. Moreover, the goal is to genetically compare the functionality of each single factor cross-complementing the deletion and each over-expression of each gene of S. meliloti with the orthologs taken from C. crescentus. This approach will give an indication about the conservation of factors across organisms and how their activity is regulated in different systems. In order to decipher mechanistic insights of the cell cycle in alpha-proteobacteria, the second goal will be to extend the knowledge of the biology of cell cycle factors in C. crescentus and S. meliloti; in particular, elucidating the structural biology of the core factors controlling cell cycle in C. crescentus and S. meliloti.

Diabetes epidemic poses a major socio-economic burden over worldwide. The disease manifests when islets beta-cells from endocrine pancreas fail to release sufficient insulin to compensate for insulin resistance in target tissues. Impairment of beta-cell activity includes loss of glucose-induced insulin secretion and reduction in beta-cell mass. Beside genetic factor, prolonged exposure of beta-cells to an excess of environmental demands such as glucose, unsaturated fatty acids, pro-inflammatory cytokines and oxidized LDL accounts for a large part for development of beta-cell failure in diabetes. The adverse effects of all the stressors rely on activation of the JNK pathway, which is subsequent of the loss of expression of the islet brain 1 (IB1), a JNK scaffold protein that regulates insulin contents, nutrients-induced insulin secretion and cell survival. While IB1 tightly controls the insulin levels and cell survival by inhibiting the JNK pathway, regulation of insulin secretion achieved by the scaffold protein rule out the implication of the pathway. Our findings assume the ATF3, a transcription factor elicited by endoplasmic reticulum stress, as the potential repressor responsible of the diminution in the IB1. Furthermore we highlight the dual leucine zipper kinase (DLK) and annexin2 (anxA2) as keys whereby the scaffold protein exerts its regulatory activity on JNK activity and insulin secretion, respectively. Understanding the mechanism whereby the expression of IB1 decreases in response to diabetogenic factors is crucial for combating the beta cell failure in diabetes. Furthermore, we do believe that elucidating the mechanism through which IB1 regulates the JNK pathway, and more generally, beta cell function, could help to refine existing therapies and/or to explore innovative therapeutic to counteract beta cell defects.

The French « solid state chemistry community » is in a critical position concerning its lowering contribution to the prospection for new inorganic compounds, at the basis of novel properties. This aspect has been largely neglected against more “profitable” studies on dedicated materials : -for energy, -for electronics, for nuclear etc…, which unfortunately essentially consist in optimizing competitive materials, and therefore do not provide new structural architectures, without preconceived ideas about their properties. This national situation is problematic at a multidisciplinary level since chemists, physicists, theoreticians and even industrial partners are penalized in terms of national perspectives. In this context, our project proposes, via an original predictive approach, to elaborate novel inorganic compounds. Concretely, we aim to design new compounds with 1D, 2D and 3D structures using original building units, consisting in oxo-centered OM4 polyhedrons assembled into a structuring framework, the empty spaces being filled by groups of various natures, for which the structural specificities will be fully rationalized. In that sense, our approach is innovative and placed in the frame of the renewal of inorganic chemistry since the majority of structures predicted up to now were obtained through variable stacking of 2D building blocks. The meticulous study of numerous new compounds in the Bi2O3-X2O5-MxOy (M= P, V, As…, X= Li, Na, Cu, Co, Ni, Mg, Cd…) chemical system already enriched our experience in this field and opens great perspectives. From a structural point of view, these phases are deduced from one another by the reorganization of secondary building units (based on O(Bi,M)4 tetrahedrons). We evidenced a particular dependence between the nature of the units and the inter-layers space, leading to empirical rules at the basis of the real prediction of new complex structures. Nevertheless, our ambitions concern the extension of this predictive approach to various chemical systems and the “Design” of structures with various dimensionalities (1D to 3D), leading to different expected properties (dielectric, magnetic, optical…). The work consists in the diversification of the building units’ sizes and topologies, in order to enlarge the self-assembly possibilities. This work involves a systematic rationalization of the preexisting phases to evidence the structural analogies and the chemical parameters controlling the final structure. This tedious work is already under progress in the framework of a review article elaboration (ref 21 in the document). The involved steps (Design/validation/elaboration/structure/properties) integrate the complete solid state chemists’ tools and include the use of structural data banks, DFT based ab initio calculations, diversification of the synthesis methods, diffraction methods and several collaborations for the crystal growth and the characterizations/validations of physico-chemical properties. Here, the originality of our recent electronic microscopy works deserves to be highlighted. Indeed, we have validated the possibility to determine complex structures of the lattice formed by secondary building units O(Bi,M)4 using simple observation/decoding of high resolution imaging. It appears therefore necessary to deepen these studies of high methodological impact. Finally, by analogy with the various spectacular properties (optical, dielectric, magnetic, ionic conductivity…) of the compounds already initiated in the above cited systems, we pretend to predict and elaborate certain inter-atomic topologies at the basis of targeted physico-chemical.