Research fields

ATHERO - Activation of endothelial cells, monocytes and adipocytes in pro-atherogenic conditions

Atherosclerosis is a disease affecting middle and large arteries, with an inflammatory component, although the exact molecular mechanisms underlying the pathology are far of being completely elucidated. In order to better understand the early steps of the pathology, that lead to lesion formation, we have developed several in vitro models of cells, human endothelial cells and human or murine monocytes/macrophage. The dysfunction of both these cell types, plays a central role in atherogenesis and in the progression of the lesions. These cells were exposed in vitro to oxidative and/or atherogenic stresses in the presence of LDL (native and oxidized either with copper or myeloperoxidase), but also of peroxynitrite or LPA, a bioactive lipid produced during LDL oxidation. The cellular responses to these stresses are analyzed at the molecular level, by identifying the signalling cascades and the activated transcription factors (PPAR, Nrf2, NFKappaB, tanscription factors of the unfolded protein response (UPR), ...), but also by investigating the changes in gene expression (via transcriptomic and proteomic approaches), to see to what extent and via what mechanisms, cells defend and adapt themselves and to determine when these stressing conditions become deleterious for the cells.

• running projects : 2
• terminated projects : 5
• terminated PHDs : 5

Proteomic approaches and cellular stress

The URBC has a proteomic platform, dedicated to the running and analysis of 2D-eelctrophoresis gels via the performing 2D-DIGE approach. This technique is used to investigate the changes in protein abundance, in response to various stresses (oxidative stress, hypoxia, unfolded protein response, ...). These proteins are then picked in the gel, using a Spotpicker, and trypsinized for identification by mass spectrometry. With the MALDI-TOF mass spectrometer, protein identification is based on the protein tryptic peptide fingerprint, while the Q-TOF mass spectrometer identifies proteins based on the sequence of several peptides obtained after their trypsinization. To investigate the cellular responses to stress, we favour subproteomic approaches, focused for example on the secreted proteins (secretome), the mitochondrial proteome or the lysosomal proteome (collaboration with the URPhyM). This powrful approach is also used in collaboration with the URBO in the field of eco-toxicology, to study environmental stresses in various freshwater organisms. The laboratory is also developing more recently a 2D-DIGE method focused on oxidized proteins (Redox 2D-DIGE) as well as gel-independent approaches.

• terminated projects : 1

BIOCHIPS - DNA microarrays

A biochip is a modified glass slide, carrying multiple nucleotidic sequences or capture probes, that can hybridize with complementary sequences (targets), present in a DNA or RNA population. A robot, called spotter, is used for the mechanical targeting of the capture probes on discrete areas with a diameter of about 300 µm.Various chips have been developed, for instance for the genomic analysis of bacteria, the screening of bacteria resistant to antibiotics, or for gene expression studies expression profile in breast cancer, expression of detoxifying genes in liver, ...). Chips have also been developed for the detection of GMO or for identifying animal virus types.

• terminated projects : 23

DYSO - Organelle dysfunction and cell responses

Mitochondrial stress and dysfunction are studied in cell lines that display a point mutation in mitochondrial DNA or a total absence of mitochondrial genome. In these conditions and cell lines, we analyze molecular mechanisms responsible for the increase sensitivity to pro-apoptotic molecules such as TNF-alpha, TRAIL, staurosporine and etoposide. We are also interested to delineate how adipocytes dedifferentiate when submitted to mitochondrial uncoupling. In addition, in collaboration with Prof. M. Jadot, we investigate cell responses to lysosomal storage disorders. We used cells or animal models deficient for beta-galactosidase or tripeptidyl peptidase I and try to identify new transcription factors and signaling pathways differentially activated in response to lysosomal dysfunction. We are also interested to look for modifications in mitochondrial population (morphology, abundance, and activity) in these conditions. More recently, we started to study the impact of the UPR (unfolded protein response) on mitochondrial population behavior in cells treated with thapsigargin, tunicamycin or brefeldin A. Finally, we collaborate to the understanding of the effects of OMV (outer membrane vesicles) from Brucella spp on eukaryotic cell response in macrophages and more particularly to spontaneous or induced-inflammatory cell response and UPR.

• terminated projects : 5
• terminated PHDs : 4

Development of new biotechnological tools devoted to transcription factors analysis

The control of gene expression requires regulated binding of transcription factors to consensus sequences located in genes promoters. The developement of colorimetric DNA-binding assays of transcription factors to consensus sequences is an advantageous alternative to regular radioactive « supershifts », allowing to assay DNA-binding capacity of a particular transcription factor of interest. This research has been patented and commercialized (by Active Motif and Eppendorf Array Technology.

Nevertheless, this assay requires pre-existing knowledge of the transcription factor of interest, and performant antibodies. The purpose of our new project is to develop a method allowing, without a priori, the identification of all the proteins binding an oligonucleotidic sequence of interest. This method includes the purification of proteins through « DNA-affinity », followed by tryptic digest of proteins, separation of resulting peptides by ion exchange chromatography and identification by nano-LC-MS/MS. More than a hundred of proteins, including transcription factors and coactivators/corepressors, can be identified though interaction with a 300 bp oligonucleotide sequence.

• terminated projects : 3

HYPOXIA - Adaptation of cells to hypoxia : role of the transcriptional factor HIF

We are studying the metabolic modifications and the changes in gene expression induced by hypoxia on cells and tissues. We are investigating the mechanisms leading to HIf-1 activation under hypoxia as well as to the role of HIF-1 in the modulation of apoptosis. Moreover, the study of the effects of hypoxia on mitochondrial respiration has led to the design of new molecules with anti-ischemic properties.Finally, we proposed a role for hypoxia in the appearance of varicose vein based on the response of endothelial cells to hypoxia.

• terminated projects : 3
• terminated PHDs : 4

Design of a serum-free medium for MRC-5 cells

The aim of our work is to formulate a serum free medium for MRC-5 cells (human diploid fibroblasts from fetal lung) which are commonly used in the industrial field for the manufacture of biologicals.

Animal serum (mainly bovine) is essential for cell culture because it is an important source of nutrients, growth and attachment factors, protection agents¿ It is also used to inhibit trypsin in subculture protocol and for cryopreservation. However, it presents several disadvantages especialy for the pharmaceutical industry :

• substantial cost,
• variability between batches,
• serum is an undefined mixture,
• poor reliability of serum supplying,
• last but not least, it is a source of pathogenic contaminants (adventitious agents) such as fungi, bacteria, viruses or agents of the bovine spongiform encephalopathy (BSE - until now, no detectable infectivity was found in serum, but it remains a theoritical risk).

Therefore, the current tendency is to recommend the elimination of serum and even more the elimination of all the animal-derived raw material from the culture media used in the manufacture of biologicals for human therapy or vaccination.

Several serum free media have already been developed for transformed cell lines (e.g. CHO, hybridoma or recombinant myeloma cells) but nothing is available for normal cells. Our project focuses on human diploid MRC-5 cells. Because of their limited life-span, these cells have particular requests on the quality of the culture medium.

We are developing a long term culture protocol (several weeks) in which serum is replaced at the level of the culture medium, trypsin inhibition and the cryopreservation medium. After a preliminar screening to identify the essential factors for the MRC-5 cell proliferation, a prototype medium is now tested in long term cultures and the cell culture conditions are under optimalization. Afterwards, the cells adapted to the serum free medium will be further characterized at the molecular l

• terminated projects : 1

Stress induced premature senescence - SIPS

We proposed that exposures of cells to sublethal stress may trigger the appearance of biomarkers of senescence. This prediction was based on the thermodynamics of open systems and considered stress as fluctuations threatening the stability of biological systems (Toussaint et al., Mech. Ageing Dev., 61, 1991). We demonstrated that stress with tert-butylhydroperoxide (t-BHP) and ethanol at sublethal concentrations, repeated (at each cumulative population doubling or every two days) or not, triggered the appearance of the typical morphology of senescence in human diploid fibroblasts (Toussaint et al., Mech. Ageing Dev., 64, 1992). These findings were confirmed by later studies which were extended to other biomarkers of senescence. Indeed fibroblasts treated with t-BHP or H2O2 display many morphological changes, growth arrest in G1/S phase of the cell cycle, long-term p21waf-1 overexpression and pRb hypophosphorylation, senescence-associated ß galactosidase activity at pH 6.0, senescence-associated changes in gene expression, and the common 4,977 kb deletion in the mitochondrial DNA after t-BHP treatment and in senescent fibroblasts (Toussaint et al., Exp. Gerontol, 30, 1995, Dumont et al., Free Radic. Biol. Med, 28, 2000, Dumont et al., Ann.NY Acad. Sci., 2000). We have also shown that repeated exposures of fibroblasts to cytokines (IL-1a, TNF-a, TGF-ß1) also triggers the appearance of biomarkers of senescence (Dumont et al., J. Anat., in press).

Proteome analysis allowed to identify several tenths of proteins which expression is shared by senescent cells and by cells in SÌPS. However proteins have been found which expression is shared by senescent cells only or by cells in SÌPS caused by t-BHP or ethanol.

These results suggest there is a mechanism whereby sublethal stresses lead cells to a state close to replicative senescence which could be differently regulated since proteins are specific to SÌPS.

• terminated projects : 10
• terminated PHDs : 5