scientific report - Université catholique de Louvain

Institut des sciences de la vie
Institute of Life Sciences
SCIENTIFIC REPORT
2012
Cover-rapportISV2013-A4.indd All Pages
23/10/13 16:14
1 Table of Contents General information ................................................................................................................................ 4 Acknowledgments ................................................................................................................................... 5 Introduction ............................................................................................................................................. 6 President’s foreword ............................................................................................................................... 6 Group of Cellular, Nutritional & Toxicological Biochemistry (BCNT) ...................................................... 7 Yves‐Jacques Schneider ....................................................................................................................... 7 Group of Biochemistry and Molecular Genetics of Bacteria (BGMB) ................................................... 10 Bernard Hallet ................................................................................................................................... 10 Pascal Hols ......................................................................................................................................... 12 Patrice Soumillion .............................................................................................................................. 14 Group of Nutrition Biology and Environmental Toxicology (BNTE) ...................................................... 17 Cathy Debier ...................................................................................................................................... 17 Yvan Larondelle ................................................................................................................................. 19 Marc Meurens ................................................................................................................................... 22 Jean‐François Rees ............................................................................................................................ 23 Group of Cell Biology (CELL) .................................................................................................................. 24 Patrick Dumont .................................................................................................................................. 24 Françoise Gofflot ............................................................................................................................... 26 Bernard Knoops ................................................................................................................................. 28 Group of « Embryologie moléculaire et cellulaire animale » (EMCA) ................................................... 30 Isabelle Donnay ................................................................................................................................. 30 René Rezsohazy ................................................................................................................................. 32 Group of Molecular Physiology (FYMO) ................................................................................................ 34 Henri Batoko ...................................................................................................................................... 34 Marc Boutry ....................................................................................................................................... 36 François Chaumont ............................................................................................................................ 38 Michel Ghislain .................................................................................................................................. 40 Pierre Morsomme ............................................................................................................................. 41 Scientific platforms ................................................................................................................................ 43 ISV postdoctoral fellowships ................................................................................................................. 44 PhD Students’Day Program ................................................................................................................... 45 ISV Scientific Day ................................................................................................................................... 47 2 Seminars ................................................................................................................................................ 48 PhD dissertations ................................................................................................................................... 50 3 General information Management President: Vice‐presidents: Prof. Bernard KNOOPS Prof. Isabelle DONNAY Prof. François CHAUMONT Administrative Staff Administrative Manager: Administrative Assistant: Finance Manager: Logistic Support: Mrs. Isabelle MAGNOLI Mrs. Véronique LEBRUN Mrs. Michèle ROCHAT Mr. Frank MOREAU Research groups BCNT Group of Cellular, Nutritional & Toxicological Biochemistry Prof. Yves‐Jacques SCHNEIDER (Spokesperson) BGMB Group of Biochemistry and Molecular Genetics of Bacteria Prof. Bernard HALLET Prof. Pascal HOLS Prof. Patrice SOUMILLION (Spokesperson) BNTE Group of Nutrition Biology and Environmental Toxicology Prof. Cathy DEBIER (Spokesperson) Prof. Yvan LARONDELLE Prof. Jean‐François REES CELL Group of Cell Biology Prof. Patrick DUMONT Prof. Françoise GOFFLOT (Spokesperson) Prof. Bernard KNOOPS EMCA Group of Embryologie Moléculaire et Cellulaire Animale Prof. Isabelle DONNAY (Spokesperson) Prof. André MOENS Prof. René REZSOHAZY FYMO Group of Molecular Physiology Prof. Henri BATOKO Prof. Marc BOUTRY Prof. François CHAUMONT (Spokesperson) Prof. Michel GHISLAIN Prof. Pierre MORSOMME 4 Affiliated members Prof. Spiros AGATHOS (ELI) Prof. Pierre BERTIN (ELI) Prof. Xavier DRAYE (ELI) Prof. Yves DUFRENE (IMCN) Prof. Christine DUPONT (IMCN) Prof. Benjamin ELIAS (IMCN) Prof. Marc FRANCAUX (IONS) Prof. Jacques MAHILLON (ELI) Prof. Jacqueline MARCHAND (IMCN) Acknowledgments Here, I would like to thank our Colleagues of the Institute who contributed to this second annual scientific report. I also would like to thank once again our Emeritus Colleagues Claude REMACLE, Jacques FASTREZ and Philippe VAN DEN BOSCH DE AGUILAR who accepted to chair PhD theses juries. Their excellent work has been recognized by the juries as well as by PhD candidates. I would like to thank them this year again for the quality of their work, the time they spent to chair the juries and to examine the manuscripts. Patrice SOUMILLON, Françoise GOFFLOT, Bernard HALLET, Patrick DUMONT, Xavier DE BOLLE are acknowledged for the organization of ISV seminars, the scientific day of the Institute and the PhD students’ day. Of course, I have to thank also our collaborators who played a major role in the organization of these events: Véronique LEBRUN, Philippe BOMBAERTS, Daniel JAL and all the people among technicians, administrative staff and PhD students. Thanks to the generous gift from a private donor combined to the support of the Fondation Louvain that made possible the ISV post‐doctoral fellowship. This is indeed a very nice opportunity to attract talented post‐doctoral fellows. I thank Marc BOUTRY for his commitment and his role as president of the selecting committee. Last but not least, my thanks and gratitude go to Mrs. Isabelle MAGNOLI who collected data and reports to finalize this second annual scientific report of our Institute. 5 Introduction Founded in 1998, the Institute of Life Sciences ‐ Institut des Sciences de la Vie (ISV) ‐ is an interfaculty research Institute of the Université catholique de Louvain. At the end of the 90’s, it appeared that at the UCL in Louvain‐la‐Neuve, research activities in the fields of biochemistry, molecular biology, cell biology, cell physiology and microbiology were dispersed in too many different departments. This fragmentation was clearly not appropriate to create efficient and competitive research groups. Moreover, it was also quite clear that regrouping these groups in an Institute of Life Sciences would be beneficial for interactions between scientists, would favor collaborations between groups, would improve training of young scientists, would allow sharing equipment and would permit the setup of technological platforms. In 2012, the ISV brings together six research groups among which nineteen principal investigators (academics or FNRS researchers) and about 140 people including post‐docs, PhD students, technicians and administrative staff. The main objective of our Institute is still the development of an environment favorable to high profile research activities. Our aims are to promote high quality basic research in life sciences, to improve the scientific training of young researchers, to attract young talented researchers and help them develop a successful team, to favor the setup of interdisciplinary projects, to favor the transfer of know‐how, and finally to promote applied projects that can emerge from basic research. President’s foreword This second annual scientific report provides an overview of scientific activities of the Institute for the period of January 2012 to December 2012 but additional information can be also found on the website of the ISV (http://www.uclouvain.be/en‐isv.html). Overall, this second report must serve to document the activities of the Institute and the achievements for the year 2012. All the best, Bernard Knoops President of the Institute of Life Sciences (2010‐2013) Louvain‐la‐Neuve, October 2013 6 Group of Cellular, Nutritional & Toxicological Biochemistry (BCNT) Yves‐JacquesSchneider
Postdoctoral fellow: Alina Martirosyan & Géraldine Nollevaux Scientists: Isabelle Dupont, Aurélie Joly & Thérèse Sergent PhD students: Pauline Beguin, Sophie Bourez, Anne Brasseur, Anouk Kaulmann, Madeleine Polet, Youlia Serikova, Céline Thiry & Julie Winand Graduate students: Bormans Mathieu, Dubois Anne‐Catherine, Hespel Theana, Iweins Michael, Meyer Amandine, Nguyen Thi Le Thuy, Pinto Mateus Valbom Ines Maria, Polet Madeleine, Regnier Pauline, Serikova Youlia. Technicians: Nicole Devilez Research activities 2012: Phenolic compounds are plant secondary metabolites, well known for their antioxidant properties. They appear beneficial for human health, protecting against oxidative stress and related diseases, such as cancers, inflammatory disorders, obesity, cardiovascular and neurodegenerative diseases. In this context, our WALEXTRACT project aims at developing new natural food preservatives as plant extracts produced from agricultural and forestry by‐products. During this year, our research work has mainly focused on the properties of a promising vegetal extract by: (i) the evaluation of the cytotoxicity (effect on cell proliferation and mortality, effect on CYP1A1 activity) and of its intracellular antioxidant and anti‐inflammatory properties using the human intestinal Caco‐2 cells, (ii) the establishment of an in vitro model of the human epidermis based on the HaCaT keratinocytes culture and the assessment of the antioxidant and anti‐inflammatory effects of the extract in this skin model UVB‐stressed, with the view of potential applications. In the frame of another project (CAPPLE), apple extracts from different varieties or variety mixtures have been investigated for their potential health benefits in the prevention of obesity: (i) they have been assayed as inhibitors of intestinal glucose absorption and for their anti‐inflammatory effects in our in vitro model of the inflamed intestinal barrier and (ii) as inhibitors of preadipocytes differentiation, lipid loading and differentiation in coculture of adipocytes and macrophages (CHAIRE DELHAIZE). Silver nanoparticles (AgNPs) are already commonly used in a lot of consumer products e.g. food storage products, socks, toothpaste, ... because of their antibacterial properties. As a result, they can be ingested and found in the gastro‐intestinal tract. However, it seems that their use can lead to adverse effects on human health. AgNPs are cytotoxic from a concentration of 30µg/mL with a decrease of the metabolic activity and the cellular viability. AgNPs also impair the intestinal barrier integrity with an increase of epithelial permeability and a delocalization of the occludin and ZO‐1, two tight junctions proteins. Surprisingly, this effect was not correlated to an intestinal inflammation, as AgNPs decrease the inflammatory chemokine IL‐8 production. The induction of an oxidative stress by AgNPs and toxic silver ions released from them seems to be the main mechanism explaining these 7 observations. Polyphenols, known for their anti‐oxidant properties, reverse largely these effects except from the anti‐inflammatory property. In vitro digestion of AgNPs seems to affect their agglomeration state of the AgNPs, as observed with SEM and their toxicity, as observed through a decrease of the cellular viability and metabolic activity. Synthetic peptides or peptidomimetics could mimic the extracellular matrix in culture. Anchorage dependent cells in culture require an appropriate extracellular matrix for their survival, migration, proliferation, phenotyping and/or differentiation. This environment is usually achieved by extracellular matrix constituents, i.e. collagens, fibronectin, laminin, artificial lamina propria, deposition, adsorption or grafting. However, such animal proteins used in cell culture may induce pro‐inflammatory stress, be unstable against proteolysis or loose activity after adsorption. Synthetic microenvironments could be more suitable for clinical purposes: (i) improved control of physicochemical and mechanical properties, (ii) limited risks of immunogenicity, (iii) increased biosafety (animal free) and (iv) facilitated scale‐up. Moreover, self‐assembling peptide hydrogels could mimic the chemical and mechanical aspects of the natural extracellular matrix by undergoing large deformations, as in mammalian tissues. They also can be functionalized with various biologically active ligands constituting good candidates to a new range of smart biomaterials. The range of biomimetic peptides that direct cell adhesion and are recognized by integrins is large. Recognition sequences derived from different extracellular matrix proteins include RGD, which are specific to different cell lines. In this context, studies were carried out on chemically defined and industrially scalable coatings for animal cell culture, as an alternative to collagen, fibronectin or Matrigel® for laboratory and industrial large scale applications. These are based on self‐assembling short peptides bearing adhesion bioactive sequences like RGD‐derived or other adhesion sequences developed by Peptisyntha sa to coat polystyrene or polyethylene terephthalate surfaces. First studies focused on study of cytocompatibility, cytotoxicity and effect of self‐assembling bioactive peptide coatings on cell adhesion and spreading of Caco‐2 and hADSC cells. Moreover, peptide‐coated surfaces characterisation was performed with AFM and XPS analyses. (DOCTIRIS PhD grant from the Brussels Region (Innoviris)). Research papers: Anti‐inflammatory effect and modulation of cytochrome P450 activities by Artemisia annua tea infusions in human intestinal Caco‐2 cells. Melillo de Magalhães P, Dupont I, Hendrickx A, Joly A, Raas T, Dessy S, Sergent T, Schneider YJ. Food Chem. 2012 Sep 15;134(2):864‐71. Accumulation capacity of primary cultures of adipocytes for PCB‐126: influence of cell differentiation stage and triglyceride levels. Bourez S, Joly A, Covaci A, Remacle C, Larondelle Y, Schneider YJ, Debier C. Toxicol Lett. 2012 Nov 15;214(3):243‐50. Drug delivery to inflamed colon by nanoparticles: comparison of different strategies. Coco R, Plapied L, Pourcelle V, Jérôme C, Brayden DJ, Schneider YJ, Préat V. Int J Pharm. 2013 Jan 2;440(1):3‐12. Anti‐inflammatory effects of pomegranate (Punica granatum L.) husk ellagitannins in Caco‐2 cells, an in vitro model of human intestine. Hollebeeck S, Winand J, Hérent MF, During A, Leclercq J, Larondelle Y, Schneider YJ. Food Funct. 2012 Aug;3(8):875‐85. Uptake of conjugated linolenic acids and conversion to cis‐9, trans‐11‐or trans‐9, trans‐11‐conjugated linoleic acids in Caco‐2 cells. Schneider AC, Mignolet E, Schneider YJ, Larondelle Y. Br J Nutr. 2013 Jan 14;109(1):57‐64. Conversion of t11t13 CLA into c9t11 CLA in Caco‐2 cells and inhibition by sterculic oil. 8 Schneider AC, Beguin P, Bourez S, Perfield JW 2nd, Mignolet E, Debier C, Schneider YJ, Larondelle Y. PLoS One. 2012;7(3) Accumulation of polychlorinated biphenyls in adipocytes: selective targeting to lipid droplets and role of caveolin‐1. Bourez S, Le Lay S, Van den Daelen C, Louis C, Larondelle Y, Thomé JP, Schneider YJ, Dugail I, Debier C. PLoS One. 2012;7(2) Insect cells as factories for biomanufacturing. Drugmand JC, Schneider YJ, Agathos SN. Biotechnol Adv. 2012 Sep‐Oct;30(5):1140‐57. Experimental mycotoxic nephropathy in pigs provoked by a mouldy diet containing ochratoxin A and fumonisin B1. Stoev SD, Gundasheva D, Zarkov I, Mircheva T, Zapryanova D, Denev S, Mitev Y, Daskalov H, Dutton M, Mwanza M, Schneider YJ. Exp Toxicol Pathol. 2012 Nov;64(7‐8):733‐41. PhD theses: 


Pauline Beguin. Metabolism of dietary polyunsaturated fatty acids and their effects on tight junctions in in vitro models of healthy and inflamed intestinal epithelium. Sophie Bourez .Intracellular accumulation of polychlorinated biphenyls in various in vitro models of adipocytes. Céline Thiry. Influence of selenium speciation on its bioavailability from diet and food supplements. Master theses: 









Bormans Mathieu. Evaluation de la toxicité de NP de silice sur un modèle in vitro de barrière intestinale. Dubois Anne‐Catherine. Etude de l’effect d’extraits végétaux de robinier faux‐acacia sur modèle in vitro d’inflamation intestinale (cellules Caco‐2). Hespel Theana. Type 2 diabetes mellitus prevention by phenolic compounds : effects on TNF‐α and NO production in adipose tissue. Iweins Michael. Les effets de composés phénoliques présents dans des extraits de pommes en présence de lactate de calcium sur l’absorption intestinal du glucose et des acides gras : importance dans la prévention de l’obésité. Meyer Amandine. Effets de l’extrait d’écorce de grenade et ses constituants phénoliques principaux sur la différenciation adipocytaire. Nguyen Thi Le Thuy. Bioaccessibilité et biodisponibilité de la curcumine native dans un modèle in vitro de la barrière intestinale humaine. Pinto Mateus Valbom Ines Maria. The benefits of phenolic compounds in the prevention of obesity: Effect on nitric oxide production of macrophages. Polet Madeleine. Evaluation de la toxicité de nanoparticules d’argent sur un modèle in vitro de barrière intestinale. Regnier Pauline. La protéine TRB3 est‐elle impliquée dans l’inhibition du message insulinique induit par le palmitate dans les cellules myogéniques C2C12. Serikova Youlia. Composés phénoliques : effet sur l’absorption intestinale des acides gras. 9 Group of Biochemistry and Molecular Genetics of Bacteria (BGMB) Molecular mechanisms of protein‐DNA transactions BernardHallet
PhD students: ‐ David Blandine (2006‐ 2013, UCL assistant, Co‐sup. P. Hols) ‐ Nauny Philippe (2010‐2012, Co‐sup. P. Soumillion) ‐ Oger Cédric (2011‐ , FRIA) ‐ Duchêne Marie‐Clémence (2011‐, FRIA, Co‐sup. P. Hols) ‐ Goossens Gérôme (2012‐ UCL assistant) Technician: ‐ Nguyen Nathan (UCL, 50%) Research activities 2012: The bacterial transposon Tn4430 as a paradigm for the mechanisms of replicative DNA transposition (C. Oger and G. Goossens, PhD theses; N. Nguyen) Tn4430 is a transposon of the Tn3 family, a widespread family of replicative transposons in bacteria. Transposons of this family were among the very first transposable element to be identified and are often presented as paradigms of replicative transposition in textbooks. Paradoxally, very little is known regarding the exact molecular mechanism of transposition of these elements. In particular, the transposase TnpA of Tn3‐family transposons is an unusually big protein (~1,000 aa) showing little sequence similarity with other proteins. This protein not only catalyse the DNA cut and re‐joining reactions required for transposition, it is also responsible for an intriguing process termed ‘target immunity’ whereby transposons avoid multiple insertions in a same target molecule. During the year, we have shown that target immunity is specific to each transposon of the Tn3 family, with no cross‐immunity between them. This, together with the identification of TnpA mutants that are selectively affected in target immunity was published in Lambin et al.(2012). Biochemical characterization of these mutants showed they form higher‐order nucleoprotein complexes containing one transposase monomer bound to two transposon ends. This paired‐end complex (PEC) corresponds to an activated form of the transposition complex assembled by wild type TnpA. The mutant proteins also exhibit promiscuous catalytic activity, cleaving both DNA strands at the transposon ends. We conclude that transposition is normally regulated at the level of transpososome assembly and that mutations affecting immunity have ‘unlocked’ the transposase making it less demanding with respect to the required activation signal. Based on both in vivo and in vitro data, we have recently proposed a new model for replicative transposition (termed the ‘replisome hi‐jacking’ model) in which transposons specifically target on‐
going replication forks as a mechanism to recruit the host replication machinery during the transposition process. We further documented this model by showing that Tn4430 integrates at a highly specific position of the replication fork, immediately downstream of the 3’OH in the leading strand. Current work aims at further documenting the model in vivo and at characterizing the interaction between TnpA and the replisome. Coordinating bacterial cell wall processing with the cell cycle Distribution of functions among Penicillin Binding Proteins (PBPs) in shaping Lactococcus lactis cells (B. David, end of PhD theses –finished in March 2013.‐, co‐sup. P. Hols) 10 Penicillin Binding Proteins (PBPs) are the key enzymes that are responsible for the biosynthesis of peptidoglycan, the major constituent of the cell wall of Gram‐positive bacteria. Spatial and temporal coordination of these enzymes is crucial for conferring a specific shape to the cells while preserving their integrity during growth. How is this coordination achieved during morphogenesis of ovoid bacterial cells remains poorly documented. Using the ovoid bacterium as a model system, we have provided direct evidences that peripheral and septal growth of ovococci are mediated by functionally separate peptidoglycan (PG) synthetic machineries under the control of the specific class B penicillin binding proteins PBP2b and PBP2x, respectively. Time lapse and sub‐cellular localization analyses revealed that both machineries exhibit specific dynamics, defining separate phases during the cell cycle. PBP2b starts to work early to mediate cell elongation. PBP2x then becomes activated and both machineries appear to work in concert for cell division. Both phases of growth correlate with structural rearrangements of the cytoskeleton protein FtsZ, consistent with the view that FtsZ supports both cell elongation and cell division in ovococci. Although both machineries can be blocked by specific inhibitors, they appear to be functionally interconnected to ensure proper transitions during the cell cycle.The other PBPs of L. lactis appear to play redundant functions, preferentially acting in one or the other biosynthetic pathways. Role of peptidoglycan hydrolases (PGH) in the dynamics of Lactobacillus plantarum cell wall (M.‐C. Duchêne, PhD theses, co‐sup. P. Hols) Peptidoglycan hydrolases play a key role in the dynamics of the bacterial cell wall during growth as they ensure the turn‐over of polymerized material while preserving cell shape and integrity. The mechanisms that regulate these enzymes during the cell cycle remain poorly understood. The aim of M.‐C Duchêne’s theses is to investigate the role of specific PG hydrolases (in the endopeptidase family) from the gram‐positive bacterium L. plantarum whose inactivation was previously found to be lethal or to cause severe morphological defects (T. Rolain theses, July 2013). Efforts have been made to purify certain members of this family (LytA) for biochemical characterization. A conditional mutant of LytA has been constructed and its characterization is under progress. The replication mechanism of pGILO1 prophage from Bacillus thuringiensis: Toward the development of new tools for the molecular evolution of proteins (P. Nauny, PhD theses, co‐sup. P. Soumillion) Unfortunately, P. Nauny failed at his theses progress examination (10/10/2012) and the project could not be continued. Research papers: Dual role for the O‐acetyltransferase OatA in peptidoglycan modification and control of cell septation in Lactobacillus plantarum. Bernard E, Rolain T, David B, André G, Dupres V, Dufrêne YF, Hallet B, Chapot‐Chartier MP, Hols P. (2012) PLoS One. 7(10):e47893. doi: 10.1371/journal.pone.0047893. The histidine kinase PdhS controls cell cycle progression of the pathogenic alphaproteobacterium Brucella abortus. Van der Henst C, Beaufay F, Mignolet J, Didembourg C, Colinet J, Hallet B, Letesson JJ, De Bolle X.(2012) J Bacteriol. 194(19):5305‐14. doi: 10.1128/JB.00699‐12. Separate structural and functional domains of Tn4430 transposase contribute to target immunity Lambin M., Nicolas E., Oger CA, Ngyen N., Prozzi D. and Hallet B. (2012), Mol. Micriobiol., 83: 805‐
820, doi 10.1111/j.1365‐2958.2012.07967.x 11 Group of Biochemistry and Molecular Genetics of Bacteria (BGMB) Molecular Genetics and Genomics of Lactic Acid Bacteria PascalHols
Postdoctoral fellows: Laetitia Fontaine, Astrid Wahl, Wendy Glenisson, Damien Dandoy, Geneviève Deschuyteneer PhD students: Céline Boutry, Blandine David, Thomas Rolain, Elvis Bernard, Benoît Desguin, Marie‐
Clémence Duchêne, Laurie Haustenne (from September 2012) Graduate students: Florence Servais, Sabah El Barkouki Technicians: Brigitte Delplace (BGMB), Marie Henry de Frahan, Benjamin Michiels, André Clippe, Ing. (50%); Research Assistant: Deborah Prozzi; secretary: Véronique Lebrun (BGMB), Liliane Demuylder (BGMB‐CELL) Research activities 2012: The research activities of the team are focused on a specific group of Gram‐positive bacteria, generically referred to as “lactic acid bacteria” (LAB), which are of major importance for food fermentation and for their ability to colonize the gastro‐intestinal tract of mammals. Three model species (Lactobacillus plantarum, Lactococcus lactis, and Streptococcus thermophilus) are studied at the genetic and physiological levels by a multidisciplinary range of genetic, (post‐)genomics, biochemical, and biophysical approaches. More specifically, research areas are: cell‐wall biosynthesis, carbon metabolism, metabolic adaptation to environmental parameters and gene regulation. DNA recombinant technologies are also used to engineer LAB strains as delivery systems for the production of specific compounds for agro‐food and pharmaceutical industry. The main achievements of the team in 2012 are as follows: (i)
Biosynthesis and degradation of the cell envelope The peptidoglycan (PG) is the major constituent of the cell envelope of Gram‐positive bacteria and is a polymer of the disaccharide N‐acetylmuramic acid (MurNAc)‐( ‐1,4)‐N‐acetylglucosamine (GlcNAc) associated with a peptidic stem linked to MurNAc. In L. plantarum, we previously showed that PG glycan strands are highly post‐modified by O‐acetylation on both MurNAc and GlcNAc. In addition to the role of O‐acetylation in the control of the activity of PG hydrolases, we reported that the OatA enzyme responsible of MurNAc O‐acetylation plays a key role in the timing of cell division. Various experiments suggest that OatA could be a novel member of the division machinery (Bernard et al). We also explored the functional role of the predicted peptidoglycan hydrolase (PGH) complement encoded in the genome of L. plantarum by systematic gene deletion. From twelve predicted PGH‐
encoding genes, nine could be individually inactivated and their corresponding mutant strains were characterized regarding their cell morphology, growth, and autolysis under various conditions. From this analysis, we identified two PGHs, the predicted N‐acetylglucosaminidase Acm2 and NplC/P60 D,L‐endopeptidase LytA, as key determinants in the morphology of L. plantarum. Acm2 was demonstrated to be required for the ultimate step of cell separation of daughter cells, whereas LytA appeared to be required for cell shape maintenance and cell‐wall integrity. We showed by autolysis experiments that Acm2 is the major autolysin of L. plantarum. (Rolain et al). In addition, we 12 contributed to the characterization of the two major PGHs of Lactobacillus casei and Lactobacillus rhamnosus (Regulski et al and Claes et al). Our recent work performed in collaboration with Y. Dufrêne on the analysis of the cell wall of lactic acid bacteria by atomic force microscopy was summarized in a review in Micron (Tripathi et al). (ii)
Metabolic adaptation to environmental parameters and gene regulation Recently, we showed that S. thermophilus is naturally competent for DNA transformation under specific conditions. We demonstrated that competence development in this species is controlled at the transcriptional level by a peptide‐mediated quorum‐sensing system (ComRS). This system activates the expression of comX which codes for an alternative sigma factor ( X) that reprogram the RNA polymerase to transcribe late competence genes involved in DNA uptake, protection and chromosomal integration. Since competence is a highly controlled mechanism, we addressed the question of an additional layer of regulation that could take place on the abundance of X. In silico analysis performed on the S. thermophilus genome revealed the presence of a homolog of mecA, which codes for the adaptor protein that is involved in the degradation of the central transcriptional regulator of competence (ComK) by the proteolytic complex ClpCP in Bacillus subtilis. Using reporter strains and microarray experiments, we showed that MecASt and ClpCSt repress the expression of late competence genes and that the abundance of X is higher in the absence of MecA or ClpC. Finally, results of bacterial two‐hybrid assays strongly suggested that MecA interacts with both ComX and ClpC. Based on these results, we concluded that ClpC and MecA act together in the same regulatory circuit to control the abundance of X in S. thermophilus (Boutry et al). Research papers: ‐Bernard E., Rolain T., David B., Andre G., Dupres V., Dufrene Y.F., Hallet B., Chapot‐Chartier M.P. and Hols P. (2012) Dual role for the O‐acetyltransferase OatA in peptidoglycan modification and control of cell septation in Lactobacillus plantarum, PLoS One, 7, e47893 ‐Rolain T., Bernard E., Courtin P., Bron P.A., Kleerebezem M., Chapot‐Chartier M.P. and Hols P. (2012) Identification of key peptidoglycan hydrolases for morphogenesis, autolysis, and peptidoglycan composition of Lactobacillus plantarum WCFS1, Microb Cell Fact, 11, 137 ‐Claes I.J., Schoofs G., Regulski K., Courtin P., Chapot‐Chartier M.P., Rolain T., Hols P., von O., I, Reunanen J., de Vos W.M., Palva A., Vanderleyden J., De Keersmaecker S.C. and Lebeer S. (2012) Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG, PLoS One, 7, e31588 ‐Regulski K., Courtin P., Meyrand M., Claes I.J., Lebeer S., Vanderleyden J., Hols P., Guillot A. and Chapot‐Chartier M.P. (2012) Analysis of the peptidoglycan hydrolase complement of Lactobacillus casei and characterization of the major gamma‐D‐glutamyl‐L‐lysyl‐endopeptidase, PLoS One, 7, e32301 ‐Tripathi P., Beaussart A., Andre G., Rolain T., Lebeer S., Vanderleyden J., Hols P. and Dufrene Y.F. (2012) Towards a nanoscale view of lactic acid bacteria, Micron, 43, 1323‐1330 ‐Boutry C., Wahl A., Delplace B., Clippe A., Fontaine L. and Hols P. (2012) Adaptor protein MecA is a negative regulator of the expression of late competence genes in Streptococcus thermophilus, J Bacteriol, 194, 1777‐1788 PhD theses : ‐Elvis Bernard: Etude du rôle fonctionnel de l’O‐acétylation et de l’amidation du peptidoglycane chez les lactobacilles 13 Group of Biochemistry and Molecular Genetics of Bacteria (BGMB) Proteins and peptides engineering PatriceSoumillion
Postdoctoral fellow: Nadia El Bakkali Taheri PhD students: Gilles Joachim, Pierre Galka, Mary Doumit, Anastassia Vorobieva, Stéphanie Garcia, Heykel Trabelsi, Bruno Baudoux, Gabrielle Woronoff, Julia Wessel, Olivier Box (copromotion with P. Hols), Benoit Desguin (copromotion with P. Hols), Samira Boarbi (copromotion with D. Fretin at the CERVA‐Bxl). Graduate students: Gabriela Gil Alvaradejo, Thomas Delcourt. Technician: Laurence Bausiers; secretary: Véronique Lebrun (50%) Research activities 2012: 1. Directed evolution of enzymes With the general aim of a better understanding of structure‐function relationships in enzymes and their mechanisms of evolution, several projects are dedicated to the engineering of new functions such as specificity, regulation or catalysis and to the exploration of molecular evolutive scenarios. ‐
We optimized a new high throughput screening assays for evolving the specificity of penicillin acylase in microfluidic‐based microdroplets reactors (G. Woronoff). The project was closed in 2012. A manuscript entitled “Activity Fed Translation Assay “AFT”, a new in vitro detection strategy for enzymes in droplets” is under revision. This work was performed in collaboration with A.D. Griffiths (ISIS, Strasbourg). ‐
We have evolved a DD‐peptidase (PBP‐A) into phenotypically active beta‐lactamases (H. Trabelsi). Using a so‐called neutralization drift, PBP‐A mutants that have lost their natural activity but are still covalently acylated by penicillin were generated. Random mutations in these neutralized mutants allowed the emergence of beta‐lactamases but at the expense of fitness costs (T. Delcourt). These results highlight a counterintuitive pathway in enzyme evolution: fitness restrictions are preventing the evolution of PBP‐A into a beta‐lactamase unless PBP‐A looses its natural activity. A project with the aim of evolving PBP‐A fully in vitro in order to overcome fitness problems has been started (J. Wessel). A fluorogenic assay compatible with droplet compartimentalisation was designed and optimized. In parallel, an in vivo study of fitness problems associated with the evolution of beta‐lactamase was initiated (N. El Bakkali Taheri). A library of TEM‐1 beta‐lactamases was created by error prone library and two opposite selection approaches were applied in order to select, on one hand, the enzymes featuring the highest fitness in the absence of any penicillins (with a darwinian selection process) and, on the other hand, the most toxic enzymes that provoke bacterial lysis (with an anti‐darwinian selection process). Clones characterization is underway. 14 ‐
Using the phage display technology, beta‐lactamase insertants libraries constructed from a stabilized starting enzyme have been used for the selection of allosterically regulated enzymes (B. Baudoux). Because of fitness problems associated with the expression level of the enzymes from phagemid libraries, phage libraries were constructed. Enzymes regulated by metallic ions were successfully selected from these libraries. A manuscript entitled “Protein stability promotes tolerance to insertional mutagenesis” is under revision. Beta‐
lactamases recognizing an antibody against an intracellular pathogen (Coxiella), were also selected (S. Boarbi). ‐
The isocitrate dehydrogenase (IDH), an enzyme from the Krebs cycle, as been developed as a new dimeric platform for enzyme evolution (A. Vorobieva). This homodimeric enzyme has its active site at the interface between the subunits. The coexpression of a his‐tagged and a strep‐tagged version of IDH generates two homodimers and one heterodimer. A protocol for purifying and separating the three proteins was optimized. A statistical repartition of the subunits should lead to 50% of heterodimer but only 20 % was observed. This suggests a cotranslational assembly of the dimers. Unfolding studies were also performed and we discovered that isocitrate and magnesium ions greatly enhance the stability of the dimers. Libraries of his‐tagged and strep‐tagged enzymes were built by error prone PCR and directed evolution experiments are underway. A complementary project of IDH evolution but with the genes in a chromosomic context was started, taking advantage of the natural competence of Streptococcus thermophilus (O. Box). ‐
A gene encoding a beta‐lactamase from S. lavendulae reveals a quasi perfect heptapeptide duplication in the essential omega loop that is suspected to be at the ancestral origin of the beta‐lactmase activity. The cloning of the enzyme was performed with the aim of performing a retro‐evolution experiment (G. Joachim). However, no beta‐lactamase was detected (activity and western blot) and many attempts to change the secretion signal failed, suggesting an unexpected toxicity of the enzyme. In vitro expression and cloning into a low copy and tightly regulated vector is underway. 2. Selection of bioactive cyclic peptides We have recently demonstrated the biosynthesis of combinatorial libraries of backbone cyclic peptides in the cytoplasm and periplasm of E. coli. Several projects aim at identifying new bioactive compounds within these libraries. (1) Using an anti‐darwinian selection strategy based on the plasmid recovery after lysis of the interesting clones, we have isolated a clone featuring an increased sensitivity towards oxacillin, a substrate of the AcrAB‐TolC efflux pump (M. Doumit). However the selected peptide is intrinsically toxic and the pump inactivation is most probably indirectly due to an alteration of the electrochemical gradient. The results suggest that there is no peptide inhibitor of the pump in our libraries. (2) In another project, a Darwinian selection strategy is applied for searching cyclic peptide inhibitors of human aquaglyceroporins (AGPs). These inhibitors should allow E. coli to grow in the presence of antimonite, a toxic substrate of AGPs (S. Garcia). Unfortunately, the functional expression of human AGPs in E. coli failed so the project was redirected towards the E. coli porin GlpF. Using an appropriate GlpF expression vector and E. coli strain, culture conditions affording a GlpF‐dependant sensitivity to antimonite were found. Cyclic peptide libraries were transferred in the strain and the selections are underway. (3) In a last project, cyclic peptide that can disrupt the HoxA1‐Pbx interaction in yeast will be searched (P. Galka). A fluorescent screening assay was designed and required the 15 construction of a reporter system integrated in the yeast chromosomes using homologous recombination of in vitro assembled DNA fragments. PCR assemblies were successfully performed but the first attempts to transform the yeast failed. 3. Other research activities – biochemistry of natural proteins One project concerns the biochemical characterization of the lactate racemase of Lactobacillus plantarum, a new metallo‐enzyme containing one or more nickel ions in its active site (B. Desguin, copromotion with P. Hols). The tridimensional structure of the enzyme was solved (collaboration with J.P. Declercq) and a mechanism of Nickel loading requiring several protein chaperones was deciphered and partially reconstituted in vitro. Another project concerns the study of an intein found in a bacterial plant pathogen (G. Gil, N. El Bakkali Taheri). Inteins are small protein elements capable of self‐excision from a polypeptide precursor and are generally found in essential intracellular proteins. In Pseudomonas syringae, we identified an extracellular intein that is trapped in its protein host by a disulfide bond. We showed that disulfide bond reduction triggers intein activation. This property has many potential applications in biotechnology. Research papers: Ponsard I, Soumillion P. (2012) New immunoenzymatic strategy for rapid and selective growth of Salmonella. J Clin Microbiol., 50, 4138‐40. PhD theses: 
Gabrielle Woronoff : Développement de méthodes pour l’évolution dirigée de la pénicilline G acylase par compartimentalisation in vitro. Mars 2012. Master theses (work in 2011, defense in 2012):  Olivier Box : Vers le couplage entre un liposome et un ARN messager encodant une protéine membranaire. Janvier 2012.  Pierre‐Yves Laruelle : Etude du coût de fitness associé à l’expression de différents variants de la beta‐lactamase TEM‐1. Juin 2012.  Antoine Deschamps (copromotion avec P. Morsomme) : Peptides amphiphiles en brins beta comme alternative aux détergents pour la solubilisation de protéines membranaires. Septembre 2012.  Lucrezia Rinaldi (copromotion avec R. Rezsohazy) : Vers l’identification de peptides cycliques disrupteurs de l’interaction HoxA1‐Pbx. Septembre 2012. 16 Group of Nutrition Biology and Environmental Toxicology (BNTE) CathyDebier
Postdoctoral fellows: Chloé Bonnineau (shared with JF Rees); Graduate students: Sophie Bourez, Marie Vanden Berghe, Caroline Louis; Noa Simon Delso; Undergraduate students: Marie Stas. Technicians: Eric Mignolet (shared with Yvan Larondelle and JF Rees), Christine Turu and Catherine Romanowska (shared with Yvan Larondelle and JF Rees). Secretary: Franck Moreau (shared with Yvan Larondelle and JF Rees). Account manager: Maria Ruiz (shared with Yvan Larondelle and JF Rees) Research activities 2012  Biochemical mechanisms involved in the uptake, storage and mobilization of persistent organic pollutants in the adipose tissue of mammals. In 2012, we have studied the accumulation and intracellular location of several PCB congeners in adipocytes (in vitro model of primary cultures of rat adipocytes). We have also investigated the toxicokinetics of those PCB congeners during adipocyte lipolysis.  Toxicokinetics of persistent organic pollutants during key‐periods of the life cycle of free‐ranging phocid seals. In 2012, we studied the transfer of PCBs, DDTs and PBDEs as well as their metabolites (HO‐PCBs and HO‐PBDEs) from mother to pup during lactation in grey seals. We also studied the mobilization of those organic pollutants from the adipose tissue to the blood circulation in northern elephant seals during their post‐weaning fast (in collaboration with university of Antwerp UA, university of California, Santa Cruz UCSC, Sonoma State university, USA, and University of St Andrews, Scotland).  Relationships between persistent organic pollutants (POPs) and biomarkers of exposure and effects in terrestrial and marine animals (vitamin A and E metabolism, immune function, thyroid hormones). : In 2012, we studied the impact of PCBs and PBDEs on vitamin A metabolism in grey seals (lactating mothers and their pups) (in collaboration with UA and University of St Andrews).  Metabolism of fat‐soluble vitamins and fatty acids in phocid seals during lactation and post‐
weaning fast. In 2012, we focused on the metabolism of the adipose tissue during the post‐
weaning fast of northern elephant seals. We investigated the mobilization of fatty acids from the adipose tissue into the serum and the expression of proteins involved in the hydrolysis of triglycerides as well as in the transport of fatty acids in the cells (in collaboration with UCSC).  Combined effects of nutrition (lipid composition of diet) and chemical stress on the biology of fish: use of in vivo and in vitro models (in collaboration with JF Rees and Y Larondelle, IAP project with UGent, UA, UNamur, KULeuven). This new project started at the end of 2012.  Relationships between the foraging ecology (foraging location and depth, trophic level) of marine mammals (seals and sea lions) and their POP contamination burden. In 2012, we have studied the relationships between the foraging ecology (foraging location and depth, trophic level) of northern elephant seals and their POP contamination burden (in collaboration with UCSC). 17 Research papers 
Debier C, Crocker DE, Houser DS, Vanden Berghe M, Fowler M, Mignolet E, de Tillesse T, Rees, Jean‐François, Thomé, Jean‐Pierre, & Larondelle, Yvan. 2012. Differential changes of fat‐soluble vitamins and pollutants during lactation in northern elephant seal mother‐pup pairs. Comparative biochemistry and physiology. Part A, Molecular & integrative physiology, 162, 323‐30  Vanden Berghe M, Weijs L, Habran S, Das K, Bugli C, Rees JF, Pomeroy PP, Covaci A, Debier C. 2012. Selective transfer of persistent organic pollutants and their metabolites in grey seals during lactation. Environment International, 46, 6‐15  Bourez S, Joly A, Covaci A, Remacle C, Larondelle Y, Schneider YJ, Debier C. 2012. Accumulation capacity of primary cultures of adipocytes for PCB‐126: Influence of cell differentiation stage and triglyceride levels. Toxicology Letters, 214, 243‐250.  Bourez S, Le Lay S, Van Den Daelen C, Louis C, Larondelle Y, Thomé JP, Schneider YJ, Dugail I, Debier C. 2012. Accumulation of polychlorinated biphenyls in adipocytes: selective targeting to lipid droplets and role of caveolin‐1. PLoS One, 7(2), e31834  Schneider AC, Beguin P, Bourez S, Perfield JW, Mignolet E, Debier C, Schneider YJ, Larondelle Y. 2012. Conversion of t11t13 CLA into c9t11 CLA in Caco‐2 Cells and Inhibition by Sterculic Oil. PLoS One, 7(3), e32824  Lemaire B, Debier C, Buc Calderon P, Thomé JP, Stegeman J, Mork J, Rees JF. 2012. Precision‐
cut liver slices to investigate responsiveness of deep‐sea fish to contaminants at high pressure. Environmental Science and Technology, 46, 10310‐10316  Habran S, Crocker DE, Debier C, Das K. 2012. How are trace elements mobilized during the postweaning fast in Northern elephant seals?. Environmental Toxicology and Chemistry, 31, 2354‐2365 Short papers (3 pages – Organohalogen compounds database – Dioxin 2012) 
Louis C, Habran S, Stas M, Tift M, Das K, Dirtu A, Crocker DE, Covaci A, Debier C. 2012. Blubber mobilization of pbdes in northern elephant seals (mirounga angustirostris) during the post‐weaning fasting duration – comparison with PCBs‐ Organohalogen compounds 74 : 907‐910 PhD theses 

Sophie Bourez Intracellular accumulation of polychlorinated biphenyls in various in vitro models of adipocytes (co‐supervision with Prof YJ Schneider, completed in 2012). Marie Vanden Berghe PBDEs, chlorinated POPs and their metabolites in mother and pup seals: influence of the lactation period, relationships with vitamin A status and potential impact on the immune system (completed in 2012) Master theses Marie Stas (BBMC, UCL): Dynamique de mobilisation des PBDEs à partir du tissue adipeux pendant le jeûne post‐sevrage d’éléphants de mer septentrionaux. 18 Group of Nutrition Biology and Environmental Toxicology (BNTE)
Biochemistry of nutrition YvanLarondelle
Postdoctoral fellows Tarik Abboudi (12 months); Christelle André (12 months); Quynh Chau Dang Van (12 months); Laurence Ribonnet (12 months) PhD students: Pauline Beguin (50%); Sylvie Hollebeeck (50%, 1 month); Lai Thi Ngoc Ha; Julie Mellery (8 months); André Nivyobizi (8 months); Claudine Passo Tsamo; Darly Pompeu; Anne‐Catherine Schneider (3 months) Research assistants: Carissa Bichara (12 months); Jonathan Brel (50%, 9 months); Liliana Bejarano (6 months); David Deswysen (50%); Cédric De Taeye (50%, 9 months); Mathieu Haerinck; Julie Mellery (4 months); Stéphanie Meurée (6 months); Jérôme Milet (50%); Tatiana Sokolova (90%; 5 months) Graduate students: Cassandra Castellanos Rodriguez (HE Charlemagne); Géraldine Compère (BIRA); Aline Ferain (BIRA); Elyssa Kay (BIRA); Caroline Nain (ALIT2MC); Anne‐Sophie Pitsaer (BIRC); Sylvestre Valohery Rakotozanany (ALIT2MC); Djilla Seriba (ALIT2MC); Andry Lalasoa Tsiferana (ALIT2MC); Florine Verbrigghe (BIRC) Technicians: Cécile Gardin (9 months); Marc Michotte (6 months); Eric Mignolet (80%); Jérémie Ragmey (4 months); Catherine Romanowska; Christine Turu (40%) Secretary: Franck Moreau (85%) Account officer: Maria Ruiz (about 50%) Research activities 2012 (only the research areas are mentioned – contact Yvan Larondelle for details): Basic research: ‐ Metabolism of different fatty acids in various biological systems (human cells in culture, laboratory animals, ruminants) ‐ Physiological effects of plant‐derived bioactive compounds (phenolic compounds, triterpenes, glucosinolates, unfamiliar fatty acids) in different animal and human models. ‐ Physiological and toxicological consequences of the ingestion of xenobiotics, especially in terms of interactions with other food‐borne compounds. Applied research: ‐ Impact of feeding strategies on the nutritional quality of cow’s milk fat: trans fatty acids, conjugated linoleic acids (CLA), essential fatty acids ‐ Modulation of fermentation processes in the rumen in order to lower the environmental impact of dairy production (methane, nitrogen excretion) ‐ Determination of the dietary requirements of aquaculture fish and impact of feeding strategies on the nutritional quality of their flesh ‐ Development of biological means to lower the toxicity of dietary products contaminated with mycotoxins, pesticides and environmental pollutants 19 ‐ Upgrading of under‐exploited foods (Amazonian and Asian fruits, Andean roots and tubers, agricultural by‐products, regional products), with a specific richness in bioactive compounds (polyphenols, triterpenes, carotenoids, glucosinolates, unfamiliar fatty acids) in the frame of functional food development Research papers: 











Bodin, N., Delfosse, G., Nang Thu T.T., Leboulenge, E., Larondelle, Y., Rollin, X. (2012) Effects of fish size and diet adaptation on growth performances and nitrogen utilization of rainbow trout (Oncorhynchus mykiss W.) juveniles given diets based on free and/or protein‐bound amino acids. Aquaculture, 356/357, 105‐115 Bourez, S., Le Lay, S., Van Den Daelen, C., Louis, C., Larondelle, Y., Thome, J‐P., Schneider, Y‐
J., Dugail, I., Debier, C. (2012) Accumulation of polychlorinated biphenyls in adipocytes: selective targeting to lipid droplets and role of caveolin‐1. PLOS ONE, 7, e31834, published on 20 February Schneider, A‐C., Beguin, P., Bourez, S., Perfield, J.W., Mignolet, E., Debier, C., Schneider, Y‐J., Larondelle, Y. (2012) Conversion of t11t13 CLA into c9t11 CLA in Caco‐2 cells and inhibition by sterculic oil. PLOS ONE, 2012, 7, e32824, published on 12 March Debier, C., Crocker, D.E., Houser, D.S., Vanden Berghe, M., Fowler, M., Mignolet, E., De Tillesse, T., Rees, J‐F., Thome, J‐P., Larondelle, Y. (2012) Differential changes of fat‐soluble vitamins and pollutants during lactation in northern elephant seal mother‐pup pairs. Comparative Biochemistry and Physiology (Part A), 162, 323‐330. Zhang, Z.Z., Yan, S.H., Li, D.X., Ling, T.J., Meurens, M, Larondelle, Y. (2012) Chemical synthesis and the stability of theanine. Advanced Materials Research, 396‐398, 1273‐1277 Hollebeeck, S., Winand, J., Herent, M‐F., During, A., Leclercq, J., Larondelle, Y., Schneider, Y‐J. (2012) Anti‐inflammatory effects of pomegranate (Punica granatum L.) husk ellagitannins in Caco‐2 cells, an in vitro model of human intestine. Food & Function, 3, 875‐885 Hollebeeck, S., Borlon, F., Schneider, Y‐J., Larondelle, Y., Rogez, H. (2012) Development of a standardized human in vitro digestion protocol based on macronutrient digestion using response surface methodology. Food Chemistry, 138, 1936‐1944. During, A., Debouche, C., Raas, T., Larondelle, Y. (2012) Among plant lignans, pinoresinol has the strongest anti‐inflammatory properties in human intestinal Caco‐2 cells. The Journal of Nutrition, 142, 1798‐1805. Bourez, S., Joly, A., Covaci, A., Remacle, C., Larondelle, Y., Schneider, Y‐J., Debier, C. (2012) Accumulation capacity of primary cultures of adipocytes for PCB‐126: influence of cell differentiation stage and triglyceride levels. Toxicology Letters, 214, 243‐250 Lai, N‐H., Herent, M‐F., Quetin‐Leclercq, J., Nguyen, B‐T., Rogez, H., Larondelle, Y., Andre, C. (2012) Piceatannol, a potent bioactive stilbene, as major phenolic component in Rhodomyrtus tomentosa. Food Chemistry, 138, 1421‐1430 Silva, E.M., Rogez, H., Silva, I.Q., Larondelle, Y. (2012) Improving the desorption of Inga edulis flavonoids from macroporous resin: towards a new model to concentrate bioactive compounds. Food and Bioproducts Processing, 90, 1060‐1067 Rogez, H., Akwie, S.N.L.T., Moura, F.G., Larondelle, Y. (2012) Kinetic modelling of anthocyanin degradation and microorganism growth during post‐harvest storage of açai fruits (Euterpe oleracea). Journal of Food Science, 77, C1299‐C1305 20 Theses: 


Sylvie Hollebeeck: Pomegranate (Punica granatum L.) juice industry by‐products are potential preventive agents against inflammatory bowel diseases (Co‐promotion with Yves‐
Jacques Schneider from ISV) Anne‐Catherine Schneider: Uptake and metabolism of trans and conjugated fatty acids at the intestinal level: different pathways to produce c9t11 CLA André Nivyobizi: Evaluation de différents modèles mathématiques appliqués à la méthode in sacco dans la caractérisation de la valeur nutritive de graminées tropicales et secondaires tempérées pour l’alimentation des ruminants Master theses: 









Cassandra Castellanos Rodriguez : Digestion intestinale de l'isomaltulose chez la vache tarie (Bachelier en agronomie, HE Charlemagne‐Huy) Géraldine Compère : Mise au point d'une méthode basée sur la spectroscopie moyen infrarouge pour la détection et l'identification des composés phénoliques (Bio‐Ingénieur) Aline Ferain : Impact de fibres végétales micronisées sur la biodisponibilité de la zéaralénone (Bio‐Ingénieur) Elyssa Kay : Etude de la formation de composés d'oxydation primaires et secondaires issus des acides gras de l'huile de lin (Linum usitatissimum) lors de vieillissements accélérés à différentes températures (Bio‐Ingénieur) Caroline Nain : Effects of Ricinodendron heudelotii Kernel oil ongrowth perfrormance, fatty acid profile and production of CLA in rainbouw trout ( oncorhynchus mykiss) (Master ALIT) Anne‐Sophie Pitsaer : Micronized natural vegetal fibers as a novel filter aid to reduce the ochratoxin A content of beer and wine (Bio‐Ingénieur) Sylvestre Valohery Rakotozanany : Sources protéiques alternatives chez l'esturgeon Sibérien (Acipenser baer II) (Master ALIT) Djilla Seriba : Biohydrogénation de différentes huiles végétales riches en isomères d'acide α‐
linolénique: une approche in vitro (Master ALIT) Andry Lalasoa Tsiferana : Etude du comportement des composés phénoliques de Rhodomyrtus Tomentosa (aiton) Hassk dans un modèle in vitro de digestion: le cas du piceatannol (Master ALIT) Florine Verbrigghe : Impact de fibres végétales micronisées sur la biodisponibilité des PCBs de l'alimentation chez la truite arc‐en‐ciel (oncorhynchus mykiss) (Bio‐Ingénieur) 21 Group of Nutrition Biology and Environmental Toxicology (BNTE) Spectrolab MarcMeurens
PhD student: Elie Tchuessa KADJI Research activities 2012: Research with the CRA‐W in the project “QUALIPOMME” for the maturity control of apple by Vis/NIR spectrometry. Master these : ‐ Carlos Courtoy : Contrôle de maturité des pommes par spectrophotométrie visible et infrarouge. 22 Group of Nutrition Biology and Environmental Toxicology (BNTE)
Fish toxicology Jean‐FrançoisRees
Postdoctoral fellow Delphine Scaion, Chloé Bonnineau PhD students: none Graduate students: 2 Research activities 2012: We have studied the impacts of Me‐Hg and Hg2+ on cystein‐based antioxidant enzymes such as the thioredoxin‐based peroxidases (peroxyredoxins, PRX) and glutathione peroxidase in eel brain and liver. Results indicate that these enzymes are more sensitive to Hg2+ than to MeHg. In vivo exposure to Hg2+ of eels induced an increase in PRX activity in the liver. Contamination profiles of experimentally‐exposed eels to MeHg and Hg2+ reveal that , as in wild eels, the brain content in MeHg and Hg2+ differ from the liver and muscle. They also indicate that MeHg might be demethylated and converted into Hg2+ in the brain. Research papers: • Lemaire, Benjamin, et al. "Precision‐cut liver slices to investigate responsiveness of deep‐sea fish to contaminants at high pressure." Environmental Science & Technology 46.18 (2012): 10310‐
10316. • Debier, Cathy, et al. "Differential changes of fat‐soluble vitamins and pollutants during lactation in northern elephant seal mother–pup pairs." Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 162.4 (2012): 323‐330. • Vanden Berghe, Marie, et al. "Effects of polychlorobiphenyls, polybromodiphenylethers, organochlorine pesticides and their metabolites on vitamin A status in lactating grey seals." Environmental research (2012). • Vanden Berghe, Marie, et al. "Selective transfer of persistent organic pollutants and their metabolites in grey seals during lactation." Environment international 46 (2012): 6‐15. Master theses : • David Neukermans : Biodisponibilité et impacts des espèces organiques et inorganiques du mercure dans le cerveau de l’anguille européenne (Anguilla anguilla Linnaeus) • Anne‐Sophie Bauwin : Impact du mercure sur l’activité enzymatique du système péroxyrédoxine‐thiorédoxine dans le cerveau de l’anguille européenne (Anguilla anguilla L.). 23 Group of Cell Biology (CELL) Tumor cell biology PatrickDumont
PhD students: Benjamin Demoulin (from Sept 2010, FRIA), Castrogiovanni Cédric (from Sept 2011, FNRS‐TELEVIE), Ana Sofia Silva Gallardo (from Jan 2010, FRIA) Graduate students: Florence Wagner, Liliana Gutierrez Quintero Secretary: Véronique Guns Research activities 2012: The tumor suppressor p53 has the distinction of being the most frequently mutated gene in human cancer. As a transcription factor, p53 is able to induce the expression of a variety of genes involved in inhibition of cell growth and induction of type I programmed cell death (apoptosis), as well as to repress the expression of pro‐survival genes such as those encoding for MDR1 or BCL‐2. In addition, in the presence of an apoptotic stimulus, p53 translocates to the mitochondria and triggers mitochondrial outer membrane permeabilization (MOMP), leading to release of the mitochondrial effectors of apoptosis into the cytosol. Notably, p53 interacts with BAK, a pro‐apoptotic BCL‐2 family member, allowing its oligomerization at the MOM. Our projects aim to study p53‐dependent apoptosis, to investigate p53’s activity alteration in cancer as well as to determine the mechanisms of action of natural molecules displaying promising chemopreventive or chemotherapeutic activities: 1) Effects of MAGE‐A expression on p53‐dependent apoptosis. Although silent in the vast majority of adult tissues, MAGE‐A (Melanoma Antigen‐A) genes are frequently expressed in different cancer types including breast, colon and lung cancer. Our project aims at characterizing whether an expression of MAGE‐A1, ‐A2, ‐A3, ‐A4, ‐A6 and ‐A12 can alter sensitivity of cancer cells to apoptosis‐
inducing stimuli, including drugs commonly used in cancer chemotherapy. Our data indicate that overexpression of several of these MAGE‐A in breast cancer cells enhances their resistance to DNA damaging agents, such as doxorubicin and camptothecin, as well as the MDM2 inhibitor nutlin‐3a. We are currently investigating the mechanisms involved. Data show that some MAGE‐A interact with the tumor suppressor p53 modulating its activation and stability following a stress. 2) Influence of post‐translational modifications of p53 on its mitochondrial activity. Following different types of stresses (DNA damage, oncogene activation,…), p53 translocates to the MOM where it acts similarly to activating BCL‐2 family members, such as tBID. At the MOM, p53 interacts directly with BAK and triggers its oligomerization, therefore allowing formation of pores and release of the mitochondrial effectors of apoptosis into the cytosol. MOMP is commonly considered has a point of no return in the decision of a cell to commit suicide by apoptosis. Post‐translational modifications of p53 play a major role in regulation of its activity. For example, they allow its stabilization after a stress, regulate its subcellular localization as well as transcriptional activity. Although post‐translational modifications of p53 have been extensively studied in the context of its transcriptional activity, little is known concerning their influence on its mitochondrial function. We have currently identified two post‐translational modifications specifically carried by the tumor suppressor when present at the mitochondria, suggesting that they may influence its mitochondrial 24 translocation in response to an apoptotic stimulus or its ability to trigger MOMP. Study of point mutants at these two sites will allow further characterization of their role. 3) Influence of anti‐apoptotic BCL‐2 family members on the mitochondrial activity of wt p53. Anti‐
apoptotic BCL2 family members are often overexpressed in cancer cells. This is for example the case of BCL‐2 itself, BCL‐XL and MCL1. They participate to the different mechanisms that progressively render cancer cells less and less sensitive to apoptotic stimuli. This project aims to analyze whether the level of expression of anti‐apoptotic BCL‐2 family members has an impact on the mitochondrial activity of p53. Specifically, we will determine the interactions between these proteins and the tumor suppressor, test whether their expression inhibits the mitochondrial translocation of p53 during apoptosis and study whether the mitochondrial activity of p53 is itself compromised (BAK oligomerization, release of the mitochondrial effectors of apoptosis). 4) Study of the kinetic of the mitochondrial translocation of p53. Previous studies show that mitochondrial translocation of p53 is a very early event during the apoptotic process. The tumor suppressor is found at the mitochondria only one hour after an apoptotic insult. In addition, our recent data show that p53’s mitochondrial localization is a transient event. Although p53 interacts with mitochondrial caspase‐3, loss of mitochondrial localization is not due to caspase‐mediated degradation of p53. The pro‐apoptotic BCL‐2 family member BAK is the major anchor of p53 at the MOM: the tumor suppressor is always absent from the mitochondrial fraction of stressed BAK‐/‐ mouse embryo fibroblasts, in contrast to BAK wt cells. We are currently investigating whether transience of p53’s mitochondrial localization is linked to BAK oligomerization status. Master theses : 
Denis Martin (Juin 2012): Influences des gènes MAGE‐A sur l’apoptose dépendante du suppresseur de tumeur p53. 25 Group of Cell Biology (CELL) Molecular and integrative neurobiology FrançoiseGofflot
PhD students: Benoit Lizen, Bertrand Hutlet Graduate students: Diane Bissen, Cécile Coste (2011‐2012); Deborah Sauvegarde (2012‐2013) Technicians: Konstantin Doshishti‐Agolli; Marie‐Thérèse Ahn (75% CELL); Secretary: Véronique Guns (CELL) Research activities 2012 The Hox genes code for a family of transcription factors that determine the identity of cells and tissues in the developing embryo. Although their expression and functions within the embryo are well established, very little is known about their distribution at foetal, post‐natal and adult stages. Several studies have reported Hox gene expression in normal adult tissues, opening new questions about their regulation and biological function at adulthood besides their role in development. In addition, inappropriate expression of particular Hox genes is associated with tumour development and malignant progression in adult tissues. Our research project is in keeping with these important aspects of Hox genes, their biological functions at adulthood and their potential involvement in diseases and oncogenesis, with emphasis on the central nervous system (CNS). During the year 2012, our research work has been mainly focused on (i) the production of a comprehensive expression atlas of the all 39 Hox genes in the adult CNS in mouse; (ii) the characterization of the spatio‐temporal regulation of Hoxa5 in the postnatal and adult CNS; (iii) the development of mouse models for conditional inactivation of Hoxa5 genes and (iv) the characterization of Hox gene expression in medulloblastoma cell lines. Our first action to elucidate the late functions of Hox genes in the CNS was to establish a comprehensive, quantitative and qualitative, expression atlas of the 39 Hox genes in the adult CNS in mouse. Using RT‐qPCR we found that 24 Hox genes were still expressed in the adult brain, notably in the forebrain. Using in situ hybridization (ISH) we showed that paralogy groups PG2‐8 Hox gene expression was maintained in the hindbrain at adulthood. The spatial colinearity, which characterizes Hox genes embryonic expression, was still observed with sequential antero‐posterior expression boundaries. Moreover, the main mossy and climbing fibres precerebellar nuclei expressed PG2‐8 Hox genes according to their migration origins. Within the forebrain, we mapped Hoxb1, Hoxb3, Hoxb4, Hoxd3 and Hoxa5 expression in restricted areas of the sensory cortex as well as in specific thalamic nuclei. Our data emphasize the importance of Hox genes far beyond their role of patterning genes, suggesting a functional relevance in the adult brain, potentially in relation to synaptic plasticity. These results are currently being prepared for publication. A second action to investigate Hox late CNS functions was to select specific Hox genes on the basis of their particular distribution as revealed in the first action, and to characterize in more details their spatio‐temporal regulation. In 2012, we mainly worked on Hoxa5. Indeed, our quantitative analysis by RT‐qPCR revealed that Hoxa5 is broadly expressed in the adult mouse brain, notably in anterior regions where there is no expression during development. Using ISH on brain cryosections, we confirmed these results and showed that Hoxa5 is expressed in specific sub‐regions of the brain 26 mapping different somatosensory and motor pathways. In addition, our results suggested that Hoxa5 transcript/protein appear in these original domains of expression after birth, at postnatal stages. Using cellular mapping of Hoxa5 expression by ISH and by immunohistochemistry (IHC), we could identify neurons as the main population expressing Hoxa5, at the opposite of glial cells in which no transcripts or protein were detected. Within the neuronal populations co‐labelling using both IHC and ISH suggested that Hoxa5 is restricted to glutamatergic and GABAergic subpopulations, while no transcripts/proteins were detected in cholinergic, adrenergic and dopaminergic neurons. These results were presented as poster at the COST/ISF Workshop on the Function of Hox and Tale Homeoproteins in Development and Disease (Madrid, November 2012). Building upon these data, our third action was the generation and phenotyping of conditional loss‐
of‐function mutant mice for Hoxa5. A transgenic mouse line with a floxed allele of Hoxa5 was obtained via collaboration with Dr L. Jeannotte (Université de Laval, Canada). This transgenic line is being crossed with an inducible CreERT2 mouse line, which will allow temporal inactivation of Hoxa5 in all tissues, the CMV‐CreERT2 (collaboration with the group of Filippo Rijli, FMI, Basel). Protocols of tamoxifen induction for temporal inactivation at late gestation stages were established and are currently optimized. Finally, to bring this project one step further in the analysis of Hox functions and dysfunctions in the nervous system, the last action focused on Hox gene expression in nervous system tumours, mainly medulloblastoma (MB). Preliminary bioinformatics analysis (collaboration with Dr Dirk Geerts, Erasmus University Medical Center, Rotterdam, Nederland) shows a correlation between the expression of some HOX genes, in particular the HOXD and HOXA genes, and the differential clinical outcome of human MBs. In order to validate the data from the bioinformatics analysis, and to be able to manipulate experimentally HOX genes expression, we next sought a relevant cellular model. We thus obtained 7 MB cell lines (Dr M. Hamdi, Academic Medical Center, Amsterdam, Netherlands) and characterized the expression profile of the 11 HOXA genes and 9 HOXD genes by RT‐qPCR. Three cell lines present interesting profiles: the DAOY (HOXA and HOXD positive); the D341 (HOXA and HOXD negative); and the UW228‐2 (HOXA negative and HOXD positive). These cell lines will thus be further characterized to evaluate their proliferative and invasive characteristics. Master theses: 


Diane Bissen (2011‐2012) Dynamique spatio‐temporelle de l’expression de Hoxa5 et Hoxb5 dans le cervelet fœtal et adulte . Cécile Coste (2011‐2012) Caractérisation cellulaire de l’expression de gènes appartenant au cluster HoxB dans le cerveau post‐natal et adulte de la souris. Deborah Sauvegarde (2012‐2013) Caractérisation neuro‐anatomique et cellulaire de la protéine Hoxa5 dans le cerveau de souris adultes. 27 Group of Cell Biology (CELL) Antioxidant enzymes of animal cells BernardKnoops
Postdoctoral fellow:Julie Goemaere PhD students: Valérie Van der Eecken, Oksana Kuznetsova, Geoffroy Walbrecq, Marc Pirson, Typhanie Maurer Graduate students: Stéphanie Annet, Typhanie Maurer Technicians: André Clippe, Ing. (50%), Daniel Jal (CELL), Coralie Piget (CELL); secretary: Véronique Guns (CELL), Michèle Rochat (CELL‐FYMO‐ISV), Liliane Demuylder (CELL‐BGMB). Research activities 2012: In 1988, the first eukaryotic member of the peroxiredoxin (Prdx or Prx) superfamily was discovered as a 25 kDa protein in the yeast Saccharomyces cerevisiae. This enzyme, named thiol‐specifc antioxidant protein, was shown to protect against oxidation in a thiol‐containing system. Since then, numerous Prdx members have been identified in many organisms, defining a superfamily which is highly conserved throughout evolution and represented in all kingdoms of life. It appeared that Prdxs are highly expressed in virtually all living species. They are among the most abundant proteins in Escherichia coli and represent 0.1 to 0.8% of total soluble proteins in mammalian cells. The Prdx superfamily includes three isoforms in the bacterium Escherichia coli, five in the yeast Saccharomyces cerevisiae, nine in plant Arabidopsis thaliana and six in mammals. Prdxs show no significant sequence homology with catalase, superoxide dismutases or selenocysteine‐containing glutathione peroxidases. Their catalytic mechanism does not require any redox cofactors such as heme, flavin or metal ions. Prdxs are thiol‐dependent enzymes that reduce hydrogen peroxide, alkyl hydroperoxides and, for some of them, peroxynitrite with high catalytic rates (~107 M‐1s‐1) [4]. They use thioredoxins or glutaredoxins as electron donors. Prdx5 is the last isoform identified and characterized among the six mammalian Prdxs. Prdx5 is also the only so‐called atypical 2‐Cys Prdx in mammals and according to its amino acid sequence, it is the most divergent mammalian isoform. Prdx5 is largely distributed in mammalian tissues [6,7]. Prdx5 exhibits also a remarkably wide subcellular distribution compared with the other mammalian Prdxs. Indeed, in human and mouse, Prdx5 can be localized in the cytosol, peroxisomes, mitochondria and in the nucleus depending on the cell type. These features, as well as other characteristics such as its high affinity for peroxynitrite and organic peroxides, suggest that Prdx5 may possess specific functions in mammalian cells, acting mainly as a cytoprotective antioxidant enzyme, compared with the five other isoforms. During the year 2012, our research work has been mainly focused on: (i) the generation of Prdx5 knockout mice, (ii) the function of Prdx5 in peroxisomes and (iii) the role of the mitochondrial thioredoxin system in spinal cord development. To generate Prdx5 knockout mice, a plasmid with the gene‐trap (gt) Prdx5 construct was obtained from the EUCOMM consortium and homologous recombination in ES cells was performed. ES cells 28 were microinjected into blastocysts, and chimeras were obtained in collaboration with Profs. Fadel Tissir and Patrick Jacquemin (UCL‐Brussels). Unfortunately, none of the chimeras transmitted mutated alleles. Additional four generated chimeric males were generated and were then intercrossed with C57BL/6N females to generate F1 offsprings. Heterozygotous mice Prdx5+/gt are now crossed in our animal facility to obtain homozygotous mice Prdx5gt/gt. Crossing will be pursued with Flp and Cre mice to obtain Prdx5‐/‐ animals. The successful inactivation of the Prdx5 gene in Prdx5gt/gt and Prdx5‐/‐ will be confirmed by Northern blotting and Western blotting analyses on Prdx5gt/gt and Prdx5‐/‐ mice. In order to assess the role of peroxisomal Prdx5 in redox regulation in peroxisomes and mitochondria, 158N murine oligodendrocyte cells stably transfected and presenting one of the following four different conditions of Prdx5 expression were previously generated: (i) basal expression, (ii) underexpression, (iii) peroxisomal overexpression only and (iv) basal expression and peroxisomal overexpression. In the different clones, we measured basal redox state and redox state responses to metabolic oxidative stress (inhibition of catalase by 3‐amino‐1,2,4‐triazole) or direct oxidative stress (exposure to tert‐butyl hydroperoxide, tBHP), using roGFP2 based probes targeted to peroxisomes or mitochondria. Our data suggest that mitochondria of 158N cells have a more oxidized basal redox state than peroxisomes and that Prdx5 expression levels differing from the basal level can destabilize and/or even increase basal peroxisomal and mitochondrial redox states. Previous studies conducted in our laboratory showed that mitochondrial Prdx5, Prdx3 and thioredoxin‐2 (Trx2) are highly expressed in murine motor neurons during embryogenesis, suggesting a role of the mitochondrial thioredoxin system during the spinal cord development. To study the functional role of Trx2 in chicken spinal cord, in ovo, the cDNA coding for chicken Trx2 was cloned in pCMS‐EGFP vector. Electroporation of vectors to overexpress Trx2 or electroporation of morpholinos to silence Trx2 were performed. Altogether, our data suggest that Trx2 is indeed involved in the control of motor neuron survival during spinal cord development. Research paper: ‐J. Goemaere J. & Knoops B. (2012) Peroxiredoxin distribution in the mouse brain with emphasis on neuronal populations affected in neurodegenerative disorders. J Comp Neurol, 520, 258‐280 PhD theses : ‐Valérie Van der Eecken: Evolution of peroxiredoxin 5 mitochondrial targeting in mammals: molecular mechanisms and functional consequences Master theses : ‐Stéphanie ANNET : Etude de l’expression de la Nucléorédoxine dans la moelle épinière de souris au cours du développement embryonnaire ‐Typhanie MAURER : Expression et suroxydation des peroxyrédoxines dans les astrocytes SOD1G93A exposés au peroxyde d’hydrogène et au peroxynitrite 29 Group of « Embryologie moléculaire et cellulaire animale » (EMCA)
IsabelleDonnay
PhD students Delphine Paul, Wendy Sonnet, Emmanuelle Ghys, Caroline Sauvegarde, Delphine Delvaux, Matthew Dallemagne Graduate students: Matthew Dallemagne (2011‐2012) ; Jean‐Charles de Marneffe (2012‐2013) Technicians and secretary: Philippe Bombaerts, Marie‐Anne Mauclet, Raphaël Chiarelli, Nathan Nguyen Research activities 2012: Hox genes in oocytes and early embryos There are 39 Hox genes in mammals, clustered in four genomic loci (A‐D), and subdivided into 13 paralogy groups according to their sequence similarities and relative position along the clusters. Two projects are conducted under the co‐supervision of R. Rezsohazy aiming to better characterize the expression and roles of Hox genes and their co‐factors from the Three Amino Acid Loop Extension (TALE) family before gastrulation, period that was not investigated before. Two mammalian embryonic models are studied in parallel: the mouse and the bovine. We confirm that those genes are expressed at early stages of development, which raises the possibility that Hox genes could play roles in oogenesis, including oocyte maturation, in the maternal control of early embryo development, in the activation of the embryonic genome, or in the control of the first cell differentiation events during the morula‐blastocyst transition. During the year 2012, our research work has been mainly focused on (i) the role of HOXB9 during oocyte maturation and early embryonic development in the bovine (ii) the expression of the TALE genes during the first differentiation steps (morula to blastocyst transition) in the mouse and the bovine. The profile of expression of HOXB9 gene we previously established in the bovine is compatible with a role of the protein during oocyte maturation and maternal to embryonic transition. siRNA targeting HOXB9 mRNAs were injected in immature oocytes or zygotes. A significant decrease in HOXB9 transcripts was observed 24h post‐injection (up to 70% decrease). However, no impact was observed on oocyte maturation, embryo development or quality. Moreover, the level of transcripts was no more affected after the maternal to embryonic transition, 4 days post‐injection. We then tested the specificity and sensitivity of a commercial antibody directed against the human HOXB9 protein in order to detect the bovine protein by immunofluorescence. The protein was found at all stages, from the oocyte to the blastocyst, mainly located in the nucleus. It was also observed in cumulus and sperm cells. In parallel, the study of the expression of TALE genes during the morula to blastocyst transition was carried on. To reveal to what extent TALE genes expression at these early stages is a conserved feature among mammals, this study was performed in parallel in the bovine and mouse models. We demonstrated the transcription and translation of TALE genes, before gastrulation in the two species. At least one member of Meis, Pbx and Prep subfamilies was found expressed at the RNA and protein 30 levels but different patterns of expression were observed between genes and between species, suggesting specific gene regulations. Differences between male and female embryos in early development Shifts in sex ratio are often observed when producing bovine embryos in vitro. The shift is already observed at the blastocyst stage, well before the differentiation of the gonads. Our study aimed to better understand the mechanisms leading to such shift depending on the culture conditions. We demonstrated that female embryos are more prone to apoptosis than male ones, whatever the culture conditions. The gender difference was more important when embryo culture was performed with serum, a medium leading to a shift in sex ratio in favor of male embryos. During the year 2012 we completed the data on apoptosis using the sexed semen of one bull. A new protocol was then set up to confirm the data on apoptosis using the unsexed semen from another bull. For that purpose, embryos were fixed and stained to detect apoptotic cells, and then sexed by PCR. This approach needs an accurate and sensitive PCR to avoid any bias and is still under investigation. In parallel, we started to study the impact of oxidative stress induced during embryo culture on embryo development and apoptosis depending on the gender. Set up of a cryobank for the preservation of livestock breeds This project, funded by the RW, started in 2005 and aims to preserve the genetic diversity of local breeds of sheep, horse, pig and cattle by the setting up of a cryobank. It is conducted in co‐
promotion with Ph. Baret (ELI). The last season of ovine semen collection was performed in 2012. Around 16.000 semen straws from 80 rams belonging to three endangered ovine local breeds are now stored in liquid nitrogen. A scientific and technical committee was set up to define the strategy of development of the cryobank, as well as the conditions of use of the stored material. Research papers - Sonnet W., Rezsohazy R., Donnay I. Characterization of TALE genes expression during the first lineage segregation in mammalian embryos. Dev Dyn. 2012, 241, 1827‐1839. - Dumasy J.‐F., Daniaux C., Donnay I., Baret P.V. Genetic diversity and networks of exchange : a combined approach to assess intra‐breed diversity. Genetics Selection Evolution, 2012, 44, 17. PhD theses: Wendy Sonnet: Etude de l’expression des genes Hox et TALE lors des premières étapes de différenciation embryonnaire chez les mammifères (juin 2012 ; co‐promoteur R. Rezsohazy) Delphine Paul: Hox genes and early mammalian embryogenesis : from expression profiles to loss‐of‐
function strategies (octobre 2012; co‐promoteur R. Rezsohazy) Master theses: Matthew Dallemagne: Effet du sexe des embryons bovins sur leur développement précoce (janvier 2012) 31 Group of « Embryologie moléculaire et cellulaire animale » (EMCA)
Mode of action of homeodomain transcription factors RenéRezsohazy
PhD students: Isabelle Bergiers (until June 2012), Laure Bridoux, Miloud Nichane (until November 2012), Arnaud Taminiau, Pierre Deprez (until July 2012; co‐supervision with Prof. B. Lengelé, IREC, UCL), Delphine Paul (until October 2012; co‐supervision with Prof. I. Donnay, ISV, UCL), Caroline Sauvegarde (co‐supervision with Prof. I. Donnay, ISV, UCL), Wendy Sonnet (until June 2012; co‐
supervision with Prof. I. Donnay, ISV, UCL), Pierre Galka (co‐supervision with Prof. P. Soumillion, ISV, UCL), Amandine Draime (from September 2012), Lucrezia Rinaldi (from September 2012). Graduate students: Olivier Mitri, Lucrezia Rinaldi Technicians: Philippe Bombaerts (EMCA), Raphael Chiarelli (EMCA), Nathan Nguyen (EMCA‐BGMB), Secretary: Marie‐Anne Mauclet (EMCA), Maria Ruiz (EMCA‐BNUT). Research activities 2012: Hox genes define a subset of the homeobox gene family coding for homeodomain transcription factors critically involved in vertebrate embryogenesis. Aside of their roles in normal development, misregulation of Hox genes has been associated to oncogenesis. While the functions of Hox proteins have been intensively investigated since their discovery about 30 years ago, their mode of action has only very recently begun to be investigated in detail. For a couple of years, the modes of action of two Hox proteins, Hoxa1 and Hoxa2, are under detailed investigation in our group. Hoxa1 shares a conserved DNA‐binding homeodomain with all the Hox family members. However two N‐terminal amino acid residues of the Hoxa1 homeodomain are atypical. In vitro and in vivo studies have shown that these residues importantly contribute to the activity of Hoxa1. In particular, substituting these atypical residues of Hoxa1 by more canonical ones induce homeotic transformations in the axial skeleton that are reminiscent of those observed after knocking out orther genes of the Hox family. This phenotype could be related to the “phenotypic suppression” phenomenon which uncovers that some Hox proteins can functionally dominate other ones thereby establishing a functional hierarchy between Hox proteins [Remacle et al, submitted]. A genome‐wide high‐throughput screening for proteins able to interact with Hoxa1 was successfully performed in collaboration with M. Vidal (Harvard, USA). Fifty‐nine Hoxa1 interactors were revealed by a yeast two‐hybrid assay, among which 45 were confirmed by co‐precipitation from live mammalian cells. The intracellular distribution of 41 interactions was further determined by Bimolecular Fluorescence Complementation assay. Most interestingly, in addition to interactors known to be active in transcriptional regulation, several Hoxa1 interacting proteins are involved in cell signalling pathways [Lambert et al., 2012]. The functional consequences and biological relevance of the newly identified Hoxa1‐mediated interactions are currently being addressed. In particular preliminary data support a functional interaction between Hoxa1 and the TNFα/NFκB pathway which seems independent of the transcriptional activity of Hoxa1. The mode of action of Hoxa2 was approached by searching and characterizing target genes and target enhancers. New direct Hoxa2 target genes have been identified. These genes code for 32 transcription factors or signalling molecules involved in patterning the hindbrain [Nichane et al, submitted] or in the control of chondrogenic differentiation [Deprez et al., 2012; and submitted]. A handful of responsive cis‐regulatory elements were further identified in the vicinity of four targets and the regulatory interactions between Hoxa2 and partner transcription factors are under investigation in the context of these enhancers. A genome‐wide screen for Hoxa2 interactors has also been performed, in collaboration with J.C. Twizere (agrobiopole of Gembloux, ULg). Like for Hoxa1, this screen revealed interactors involved in transcription and cell signalling. A first functional group of interactors has been confirmed by co‐
precipitation [Bergiers et al, submitted]. These interactors could be linked to cell‐cycle progression, cell shape, cell migration and DNA damage response. The involvement of Hoxa2 in modulating these cellular processes is currently under investigation. Research papers: 



Deprez, P.M.L., Nichane, M.G., Rousseaux, P., Devogelaer, J.‐¨., Chappard, D., Lengelé, B.G., Rezsohazy, R., and Behets, C. (2012). Postnatal growth defect in mice upon persistent Hoxa2 expression in the chondrogenic cell lineage. Differentiation, 83, 158‐167. Hudry, B., Remacle, S., Delfini, M.‐C. Rezsohazy, R., Yacine Graba, Y. and Merabet, S. (2012) .Hox proteins display a common and ancestral ability to diversify their interaction mode with the TALE class cofactors. PLoS Biol., 10, e1001351 Lambert, B., Vandeputte, J., Remacle, S., Bergiers, I., Simonis, N., Twizere, J.‐C., Vidal, M. and Rezsohazy, R. (2012) Functional interactions of the transcription factor Hoxa1. BMC Dev Biol. 12:29. doi: 10.1186/1471‐213X‐12‐29. Sonnet, W., Rezsöhazy, R., and Donnay, I. (2012) Characterization of TALE genes expression during the first lineage segregation in mammalian embryos. Dev Dyn., 241, 1827‐1839. PhD theses : 




Wendy Sonnet: Etude de l’expression des gènes Hox et TALE lors des premières étapes de différenciation embryonnaire chez les mammifères, juin 2012. Isabelle Bergiers : Caractérisation et étude de l’interactome du facteur de transcription Hoxa2, juin 2012 Pierre Deprez : Hoxa2 and the control of chondrogenesis : insights from a gain‐of‐function model, July 2012 Delphine Paul: HOX genes and early mammalian embryogenesis: from expression prifles to loss‐of‐function strategies, October 2012 Miloud Nichane: An architect gene at work: identificiation of potential primary target genes of Hoxa2, November 2012 Master theses : 

Olivier Mitri : Hoxa1 et ses interactants‐Caractérisation des interactions d’Hoxa1 avec Tshz1, Tshz3 et MDFI, juin 2012 Lucrezia Rinaldi : Disruption de l’interaction entre Hoxa1 et Pbx1a grâce à un peptide cyclique aléatoire, septembre 2012 33 Group of Molecular Physiology (FYMO) Structure‐function of plant TSPO proteins HenriBatoko
Postdoctoral fellows: Celine Vanhee, Neha Nigam PhD students: Samuel Gengler, Yordan Muhovski, Stéphane Julié‐Galau Graduate students: Benjamin Briquemont, Pawel Jurkiewicz (Erasmus) Staff shared within the FYMO research group: Hervé Degand, Belkacem El Amraoui, Abdelmounain Errachid, Anne‐Marie Faber, Monique Leloup, Danièle Masquelier, Régeane Mathieu, Michèle Rochat. Research activities 2012: Plants are sessile organisms and their growth and development can be detrimentally affected by different environmental cues including salinity, drought or freezing, generically referred to as abiotic stresses. These conditions can be perceived by the plant at least in part, as a transient or a permanent physiological water deficit. This water deficit increases the level of active form of the stress phytohormone abscisic acid (ABA), which regulates the expression of ABA‐responsive genes. Some of the ABA‐induced proteins are only transiently expressed, suggesting that their biological role is only required temporally by the plant cell under stress. Therefore, the plant cells need an efficient regulatory mechanism for transcriptionally and/or post‐translationally regulate their expression/degradation. We’ve demonstrated that one such protein is AtTSPO (Arabidopsis thaliana Translocator protein‐related), encoded by a single intronless gene. AtTSPO belongs to the Tryptophan‐rich Sensory Protein/Mitochondrial Benzodiazepine Receptor (TspO/MBR) protein family found, with few exceptions, in organisms ranging from Archaea to metazoans. We showed previously that AtTSPO degradation in vivo is enhanced by boosting the tetrapyrrole biosynthetic pathway. AtTSPO binds heme (and its biosynthetic intermediate protoporphyn IX, albeit with relatively lower affinity) in vitro and in vivo. This binding requires the histidine residue at position 91 (H91) as heme axial coordinator. H91A substitution seems to stabilize the mutant form in vivo, in contrast to the wild type protein which is strictly regulated and was shown to be actively down‐regulated when constitutively expressed. However, overexpression of AtTSPO can alleviate pharmacologically‐induced porphyria in plant tissues. These finding suggest that AtTSPO may act as a porphyrin scavenger during stress. We used fluorescence protein tagging and constitutive expression of the chimera in planta to demonstrate that indeed the H91A substitution is stabilized in vivo. The wild type chimera under the control of an enhanced constitutively promoter failed to readily accumulate in most cells of the various transgenic lines generated. In contrast, the H91A form of the chimera was detected in every cell imaged, and Western blotting confirmed the distinguishable characteristic of this particular point mutation. The Arabidopsis multi‐stress regulator AtTSPO is transiently induced by abiotic stress and abscisic acid treatment. This polytopic membrane protein localizes in organelles of the early secretory pathway and is strictly regulated and degraded through the autophagic pathway. TSPO‐related 34 proteins can regulate the physiology of the cell by generating functional proteins complexes. A split‐
ubiquitin screen for AtTSPO interacting partners uncovered a plasma membrane aquaporin, AtPIP2;7, as one potential candidate. Pull‐down assays and fluorescence imaging approaches were used to demonstrate the physical interaction of AtTSPO and AtPIP2;7 in planta. We show that developmental accumulations of AtTSPO and AtPIP2;7 are mutually exclusive. Overexpression of fluorescent protein‐
tagged AtPIP2;7 in AtTSPO overexpressing transgenic line resulted in patchy distribution of the fluorescence, reminiscent of the expression of AtTSPO chimera in the wild type background. Mutational stabilization of AtTSPO or pharmacological inhibition of the autophagic pathway affected the stability of AtPIP2;7, suggesting that the complex containing both proteins may be degraded by the same pathway. The co‐expression of AtTSPO and PIP2;7 resulted in decreased PIP2;7 in the plasma membrane accompanied by a 2‐fold reduction of water transport activity mediated by PIP2;7. Taken together, these data support a physiological role of AtTSPO in regulating the amount and activity of AtPIP2;7 in the plasma membrane during abiotic stress through protein‐protein interaction and degradation of the complex. Whether AtTSPO may regulate the plant cell water activity by protein‐protein interaction involving a defined set of plant aquaporin remains to be clarified, as the residues involved in this interaction. Research papers: ‐Babé A., Lavigne T., Séverin J.‐P., Nagel K.A., Walter A., Cahumont F., Batoko H., Beeckman T., Draye X. (2012) Repression of early lateral root initiation event by transient water deficit in barley and maize. Phil Trans R Soc B, 367, 1534‐1541 ‐ Muhovski Y., Batoko H., Jacquemin J.‐M. (2012) Identification, characterization and mapping of differentially expressed genes in a winter wheat cultivar (Centenaire) resistant to Fusarium graminearum infection. Molecular Biology Reports, 39, 9583‐9600 ‐ Klionsky D.J. Batoko H. and 1268 others. (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy, 8, 445‐544 PhD theses : ‐Yordan Muhovski: Molecular and genetic characterization of Fusarium head blight resistance in winter wheat Centenaire Master theses : ‐Benjamin Briquemont : Etude de la régulation transcriptionnelle d’AtTSPO (Arabidopsis thaliana TSPO) 35 Group of Molecular Physiology (FYMO)
Plant Primary transporters MarcBoutry
Postdoctoral fellows: Catherine Navarre, Antoine Champagne, Désirée Bienert Graduate students: Nicolas Jacquet, Raphaëlle Laterre, Bertrand Magy, Marta Niczyj, Adrienne Sallets, Jérémie Tollet, Frédéric Toussaint. Undergraduate students: Jérémie Tollet (2001‐2002), Baptiste Pierman (2012‐2013), François Lefèvre (2012‐2013) Technician: Joseph Nader Research activities 2012: The research projects of this team are focused on two types of plant membrane primary transporters: H+‐ATPases and Pleiotropic Drug Resistance transporters. The objective is to understand their physiological and biochemical properties. In this report, two examples of recent achievement will be summarized. As a side project, this team is involved in developing plant culture cells as an expression system for heterologous proteins. 1. Regulation of the plasma membrane H+‐ATPase The plant plasma membrane H+‐ATPase builds a transmembrane pH and potential difference. This in turn activates a whole range of secondary transporters that move nutrients in and out of the cell, sometimes against huge concentration gradients. H+‐ATPases are highly regulated at the protein level. In particular, this enzyme possesses a C‐terminal auto‐inhibitory domain which can be deactivated by phosphorylation of the penultimate residue, a Thr, and the subsequent binding of regulatory 14‐3‐3 proteins. To better identify the physiological of this activation, we have obtained transgenic Nicotiana tabacum cell lines that express an H+‐ATPase variant lacking the auto‐inhibitory domain and thus constitutively activated. Constitutive expression was found to be lethal. We then obtained transgenic lines that express the truncated H+‐ATPase under the control of a heat shock promoter. Upon incubation at 37°C for 2 h, the transgenic lines were found to express at high level the mutant H+‐ATPase. These lines acidified the external medium faster than the wild‐types lines, indicating that the activated H+‐ATPase is functional in vivo. These cell lines can now be used to better understand how transport of metabolites and ions is activated by the H+‐ATPase. 2. Role of NtPDR5 in the plant response to insects Pleiotropic Drug Resistance (PDR) transporters form a group of membrane proteins belonging to the ABCG subfamily of ATP binding cassette (ABC) transporters. There is clear evidence for the involvement of plant ABC transporters in resistance to fungal and bacterial pathogens, but not in the biotic stress response to insect or herbivore attack. We have identified a novel PDR transporter, ABCG5/PDR5, from Nicotiana tabacum. GFP fusion and subcellular fractionation studies revealed that ABCG5/PDR5 is localized to the plasma membrane. Transgenic plants expressing the GUS reporter gene under the control of the ABCG5/PDR5 transcription promoter and immunoblotting showed that, under standard growth conditions, ABCG5/PDR5 is highly expressed in roots, stems, and flowers, but is only expressed at marginal levels in leaves. Interestingly, ABCG5/PDR5 expression is induced in leaves by methyl jasmonate, wounding, pathogen infiltration, or herbivory by Manduca sexta. To address the physiological role of ABCG5/PDR5, N. tabacum plants silenced for the expression of ABCG5/PDR5 were obtained. No phenotypic modification was observed under 36 standard conditions. However, a small increase of susceptibility to the fungus Fusarium oxysporum was observed. A stronger effect was observed in relation to herbivory: the silenced plants allowed better growth and faster development of M. sexta larvae than wild type plants, indicating an involvement of this PDR transporter in resistance to M. sexta herbivory. 3. Improving expression of heterologous proteins in Nicotiana tabacum culture cells The N. tabacum BY2 cell line is used by this team to express plasma membrane transporters and pharmacological proteins. A limitation of this system is that recombinant proteins secreted from plant suspension cells into the medium are susceptible to degradation by host proteases secreted during growth. Some degradation phenomena are inhibited in the presence of various protease inhibitors, such as EDTA or AEBSF/PMSF, suggesting the presence of different classes of proteases in the medium. We performed a proteomic analysis of the extracellular medium of a Nicotiana tabacum bright yellow 2 cells. Several serine proteases belonging to a Solanaceae‐specific subtilase subfamily were identified and the genes for four were cloned. Their expression at the RNA level during culture growth varied depending on the gene. An in‐gel protease assay (zymography) demonstrated serine protease activity in the extracellular medium from cultures. This was confirmed by testing the degradation of an antibody added to the culture medium. This particular subtilase subfamily, therefore, represents an interesting target for gene silencing to improve recombinant protein production. Research papers: 
Bienert MD, Delannoy M, Navarre C, Boutry M (2012) NtSCP1 from tobacco is an extracellular serine carboxypeptidase III that has an impact on cell elongation. Plant Physiol. 158, 1220‐1229 Bienert MD, Gerlitz Siegmund SE, Drozak A, Trombik T, Bultreys A, Baldwin IT, Boutry M (2012) A Pleiotropic Drug Resistance transporter in Nicotiana tabacum is involved in defense against the herbivore Manduca sexta. Plant J. 72, 745‐757 Navarre C, De Muynck B, Alves G, Vertommen D, Magy B, Boutry M (2012) Identification, gene cloning and expression of serine proteases in the extracellular medium of Nicotiana tabacum cells. Plant Cell Rep 31, 1959‐1968 Champagne A, Rischer H, Oksman‐Caldentey KM, Boutry M (2012) In‐depth proteome mining of cultured Catharanthus roseus cells identifies candidate proteins involved in the synthesis and transport of secondary metabolites. Proteomics, 12, 3636‐3547 


Theses : 
Amandine Baijot: Expression and functional characterization of plant pleiotropic drug resistance transporters in heterologous and homologous hosts 
Julien Roland: Identification of substrates of two plant pleiotropic drug resistance transporters, NtPDR1 from Nicotiana tabacum and MpPDR1 from Mentha x piperita Master theses : 
Jérémie Tollet: Expression d’un anticorps de type IgG dans des cultures cellulaires d’Arabidopsis thaliana 37 Group of Molecular Physiology (FYMO)
Aquaporins FrançoisChaumont
Research associate: Marie‐Christine Flamand Postdoctoral fellows: Achref Aloui, Arnaud Besserer, Patrick Bienert, Charles Hachez, Linda Jeanguenin, Alicia Pou Mir PhD students: Marie Berny, Adrien Chevalier, Thomas Milhiet, Hagen Reinhardt Graduate students: Agnieska Czekaj (Erasmus), Elsa Ghurburun Visitors: Gloria Barzana (Estacion experimental del Zaidin, CSIC, Granada, Spain), Henrique Noronha (Universidade do Minho, Braga, Portugal), Cecilio Frois and Adel Meziane (INRA, Montpellier, France) Technician: Marie‐Christine Eloy Staff shared within the FYMO research group: Hervé Degand, Belkacem El Amraoui, Abdelmounain Errachid, Anne‐Marie Faber, Monique Leloup, Danièle Masquelier, Régeane Mathieu, Michèle Rochat. Research activities 2012: Plant growth and development are dependent upon the tight regulation of water uptake and transport across cell membranes and tissues. Water can pass through cell membranes via aquaporins, which are small (23‐34 kDa) channels containing six membrane‐spanning alpha helices connected by five loops (A to E) and that assemble as tetramers in the membrane. The research projects developed in my laboratory aim at understanding the function and regulation plant aquaporins. During the year 2012, we pursued our characterization of PIP (plasma membrane intrinsic proteins) trafficking to the plasma membrane. We identified a new trafficking motif located in the transmembrane 3 of ZmPIPs that is required for their endoplasmic reticulum exit (Chevalier et al., in preparation). SNARE proteins regulate membrane fusion and contribute to protein targeting and delivery in all eukaryotic cells. We showed that the maize syntaxin SYP121, also involved in the regulation of K+ channels, regulates the trafficking and possibly the gating of ZmPIP, suggesting that SYP121 could be a main regulator of the maintenance of overall cell osmotic homeostasis (Besserer et al., 2012). This molecular process seems to be conserved between monocots and dicots since expression of Arabidopsis AtSYP121‐Sp2 negatively alters AtPIP2;7 plasma membrane delivery. We also showed using BiFC and immunoprecipitation that a Trans Golgi Network syntaxin, namely SYP61, interacts with PIP2;7 in Arabidopsis (Hachez et al., in preparation). All these data show that the trafficking of PIPs involves specific signals and interaction mechanisms. In silico 3D modeling of maize ZmPIP1;2 and ZmPIP2;5 heterotetramers was carried out and highlighted putative interacting residues in all the transmembrane domains (TM). Single mutation of some of them had an important impact on the water channel activity of the homo‐ and/or hetero‐
tetramers as well as the protein subcellular localization and/or expression level, as demonstrated after expression in Xenopus oocytes (Berny, Gilis, Rooman, and Chaumont, in preparation). We also identified a conserved cysteine residue located in an extra‐cytosolic loop of PIPs which is involved in a disulfide bridge formation resulting in dimerization of two monomers. This disulfide bond was not required for PIP assembly, trafficking and activity (Bienert et al., 2012). To investigate in more details PIP physiological role in planta, we prepared genetic constructs to deregulate the expression of selected PIP genes in maize specific tissues and set up a biological assay based on protoplast swelling to perform a chemical genetic screen and identify compounds 38 specifically inhibiting or modulating PIP trafficking or activity. The genetic screen will be performed in 2013. The XIP aquaporin subfamily has been recently discovered in the genomes of moss and many eudicots but not in Arabidopsis and monocots. The physiological roles of tobacco NtXIP1;1 are currently investigated in tobacco deregulated in XIP1;1 expression (over‐expression or silencing) (Bienert and Chaumont, unpublished data). Interestingly, a strong phenotype resembling a boron deficiency syndrome was observed in the XIP1;1 overexpressing plants. It includes arrested growth of the apical shoot meristem, formation of side shoots and arrested growth of the secondary apical shoot meristems, resulting in a bushy phenotype. Furthermore, the young leaves were malformed and cupped downwards. ICP‐MS analyses are currently performed to analyse the boron content of these plants. Finally, in the frame of the European DROPS project, we analyzed the expression of PIP genes in leaf of maize lines diverging in drought tolerance. In the frame of the European EURoot project, we started the measurement of the root hydraulic conductivity in different maize lines in response to nitrogen stress. Using microsatellites approaches, Marie‐Christine Flamand studied the genetic phylogeny of trout (Salmo trutta) populations in the Walloon rivers, the genetic diversity of red deer and wild boar populations as well as the reproductive success of male red deer (funded by the Walloon Region). Research papers: ‐ Hachez, C., Veselov, D., Ye, Q., Reinhardt, H., Knipfer, T., Fricke, W. and Chaumont, F. (2012) Short term control of maize cell and root water permeability through plasma membrane aquaporin isoforms. Plant, Cell & Environment, 35, 185‐198 ‐ Bárzana, G., Aroca, R., Paz J.A., Chaumont, F, Martinez‐Ballesta,, M.C., Carvajal, M. and Ruiz‐Lozano, J.M. (2012) The arbuscular mycorrhizal symbiosis increases apoplastic water flow in roots of the host plant under both well‐watered and drought stress conditions. Annals Botany, 109, 1009‐1017 ‐ Babé, A., Lavigne, T., Séverin, J.‐P., Nagel, K.A., Walter, A., Chaumont, F., Batoko, H., Beeckman, T., Draye, X. (2012) Repression of early lateral root initiation events by transient water deficit in barley, Phil. Trans. R. Soc. B, 367, 1534‐1541 ‐ Bienert, G.P*., Cavez, D*., Besserer, A., Berny, M.C., Gilis, D., Rooman, M. and Chaumont, F. (2012) A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers, Biochemical Journal, 445, 101‐111 ‐ Frantz, A., Bertouille, S., Eloy, M.‐C., Licoppe, A., Chaumont, F. and Flamand, M.‐C. (2012) Comparative landscape genetic analyses show a Belgian motorway to be a gene flow barrier for red deer (Cervus elaphus), but not wild boars (Sus scrofa). Molecular Ecology, 21, 3445‐3457 ‐ Besserer, A., Burnotte, E., Bienert, G.P., Chevalier, A.S., Errachid, A., Grefen, C., Blatt, M.R. and Chaumont, F. (2012) Selective regulation of maize plasma membrane aquaporin trafficking by the SNARE SYP121. The Plant Cell, 24, 3463‐3481 ‐ Hachez, C*., Besserer, A*., Chevalier, A.S., and Chaumont, F. (2012) Insights into plant plasma membrane aquaporin trafficking. Trends in Plant Science, in press Master theses: ‐ Elsa Ghurburun (BBMC21‐22) : Etude de l’implication de la protein de transport Sec24 dans l’adressage des aquaporines d’Arabidopsis thaliana vers la membrane plasmique 39 Group of Molecular Physiology (FYMO) Regulatory mechanisms controlling multiple drug resistance in yeast MichelGhislain
Staff shared within FYMO: Hervé Degand, Belkacem El Amraoui, Abdelmounain Errachid, Anne‐
Marie Faber, Monique Leloup , Danièle Masquelier, Régeane Mathieu, Michèle Rochat. Research activities 2012: Multiple drug resistance is the cellular ability to survive to lethal doses of more than one drug. This phenomenon constitutes an obstacle not only for the treatment of human infection caused by bacteria, protozoa and fungi, but also for the chemotherapy treatment of cancer. The cellular mechanism that leads to drug resistance is conserved from yeast to man, and its related to four main events: detoxification by enzymatic modification or destruction of the drug, genetic alteration of intra or extracellular targets, diminished permeability of the cellular membrane and active extrusion of the drug by altered expression of transporter proteins. Resistance to hydrophobic drugs is often associated with increased production of ATP‐dependent transporters with low drug specificity. The plasma membrane from the budding yeast Saccharomyces cerevisiae contains several ABC‐
transporters, including the well‐characterized Pdr5 protein. Gene expression of Pdr5p is regulated by the Pdr1/Pdr3 transcriptional activators. Our research focuses on the posttranscriptional regulatory mechanisms, in particular protein phosphorylation and degradation. It has been shown that, in the yeast Kluyveromyces lactis, the Sit4 Ser‐Thr phosphatase is a negative modulator of multiple drug resistance, through downregulation of the activity of Pdr5p. Contrary to the physiological effect caused by the SIT4 deletion in K. lactis, the abundance of Pdr5p in the plasma membrane is decreased by the deletion of the S. cerevisiae SIT4 gene, resulting in impaired multidrug resistance. These results indicate a positive regulatory role of the protein Ser‐Thr phosphatase on Pdr5p expression. A study of the regulation of carbohydrate metabolism in S. cerevisiae has shown that the expression of the Hxt1 glucose transporter is regulated by Sit4p (collaborative project with the laboratory of Montero‐Lomeli, Brazil). Research papers: Souza A.A., Miranda M.N., da Silva S.F., Bozaquel‐Morais B, masuda, C.A., Ghislain M. and Montero‐
Lomeli, M.M. (2012) Expression of the glucose transporter HXT1 involves the Ser‐Thr protein phosphatase Sit4 in Saccharomyces cerevisiae, FEMS Yeast Res. 12, 907‐917 Master theses : Joanna Kowalczyk : Expression of yeast WSS1 gene in Escherichia coli cells 40 Group of Molecular Physiology (FYMO) Intracellular protein and membrane transport PierreMorsomme
PhD students: Joanna Dodzian, Bérengère Guerriat, Aleksandra Szopinska, Didier Demaegd, Julien De Block, Jennifer Villers, Anne‐Sophie Colinet Graduate students: Romain Perick, Robin Tyberghein, Antoine Deschamps Staff shared within the FYMO research group: Hervé Degand, Belkacem El Amraoui, Abdelmounain Errachid, Anne‐Marie Faber, Monique Leloup, Danièle Masquelier, Régeane Mathieu, Michèle Rochat. Research activities 2012: The Congenital Disorders of Glycosylation (CDG) are a heterogeneous group of inborn errors affecting directly or indirectly the glycosylation pathway. Mutations in a new human gene have been identified as a novel cause of CDG. This gene is highly conserved throughout evolution and orthologs of the protein can be found in many bacteria and all eucaryotes, including the yeast Saccharomyces cerevisiae. We have shown that the yeast protein (called Gdt1p) is localized in the Golgi apparatus and is involved in calcium tolerance. The calcium‐sensitivity phenotype is highly enhanced when known calcium transporters are deleted together with our target gene. This genetic interaction may suggest a shared function in the transport and homeostasis of calcium in the Golgi apparatus. In 2012, we have developped an assay to measure the concentration of cytosolic calcium in vivo. This assay is based on the chemiluminescence response of genetically‐encoded sensor (aequorin) upon calcium binding. This assay has been used to show the variation of cytosolic calcium after salt stress. This variation depends on known calcium transporters like the Golgi‐localized P‐ATPase Pmr1p, the vacuolar channel Yvc1p and the vacuolar H+/Ca2+ antiporter Vcx1p. We have shown that Gdt1p could also influence de calcium response suggesting that Gdt1p plays an important role in calcium homeostasis. In a second project, we developed a quantitative gel‐free proteomic approach to monitor membrane proteins changes in biological membranes. We used an optimized plasma membrane purification procedure and a quantitative gel‐free proteomic approach based on iTRAQ (isobaric Tags for Relative and Absolute Quantitation) labeling to monitor changes in the plasma membrane proteome in cells exposed to a sudden change of nitrogen source in the medium. Quantitative analysis revealed that 18 plasma membrane proteins were significantly less abundant after growth on ammonium than on proline, and 2 were more abundant on ammonium. In order to follow the evolution over time of the plasma membrane proteome after ammonium induction, a ‘kinetic experiment’ was performed. Our study confirmed the nitrogen‐sensitive feature of a great number of plasma membrane transporters like Gap1p, Put4p, Dal5p, Mep2p, and allowed the identification of new targets of the nitrogen regulation. Interestingly, the kinetic experiment indicated that the internalization rate after ammonium induction varies among the proteins. Some proteins are internalized very rapidly while others much more slowly. This demonstrates that plasma membrane proteome is a highly dynamic system that rapidly reacts in response to different external stimuli. These results have been validated by biochemical experiments. 41 Finally, we studied the molecular function of small membrane proteins called Sna1p, Sna2p, Sna3p and Sna4p. These proteins belong to the same family but are located in different subcellular compartments. The molecular function is unknown for Sna2p, Sna3p and Sna4p. Interestingly, Sna1p has been shown to be involved in cations transport across the plasma membrane. However the molecular mechanism is still unknown. We have found that Sna1p is important for the endocytosis of plasma membrane transporters and could influence the recruitment of endocytic machinery to the plasma membrane. Interestingly, we also found some connections with sphingolipids metabolism suggesting that Sna1p could be involved in the lipid composition of the plasma membrane, which in turn could influence endocytosis and cations transport. Research papers : 


Prabhakaran A., Yin J., Nysten B., Degand H., Morsomme P., Mouhib T., Yunus S., Bertrand P. and Delcorte A. (2012) Metal condensates for low‐molecular‐weight matrix‐free laser desorption/ionization, Int. J. Mass Spec., 315, 20‐32 Foulquier F., Amyere M., Jaeken J., Zeevaert R., Schollen E., Race V., Bammens R., Morelle W., Rosnoblet C., Legrand D., Demaegd D., Buist N., Cheillan D., Guffon N., Morsomme P., Annaert W., Freeze H., Van Schaftingen E., Vikkula M. and Matthijs G. (2012) TMEM165 deficiency causes a Congenital Disorder of Glycosylation, Am. J. Hum. Gen., 91, 15‐26 Galka P., Santabarbara S., Khuong T., Degand H., Morsomme P., Jennings R.C., Boekema E.J. and Caffari S. (2012) LHCII loosely bound to PSII becomes a very efficient plant PSI light‐
harvesting antenna in State II conditions, Plant Cell, 24, 2963‐78 Theses : 
Aleksandra Szopinska: Differential quantitative analysis of the plasma membrane proteome of the yeast Saccharomyces cerevisiae Master theses : 


Romain Perick : Identification d’acides aminés essentiels de la protéine de levure Gdt1p par mutagenèse aléatoire Robin Tyberghein : Etude des fonctions de PIL1 et PMP3 dans Saccharomyces cerevisiae Antoine Deschamps : Utilisation de peptides amphiphiles comme outils dans la solubilisation de protéines membranaires 42 Scientific platforms The Proteomic/Mass Spectrometry facility platform was founded in 2009 as part of the Institut des Sciences de la Vie (ISV) and the de Duve Institute. The platform provides proteomics services principally through 2D‐gel electrophoresis and mass spectrometry. Our tools are ESI‐IT and MALDI‐
TOF‐TOF mass spectrometers equipped for online and offline LC experiments. We specialize in the identification and quantification of soluble and membrane proteins from complex samples. We can also provide data on the location of post‐translational modifications. The experimental farm of the UCL (center A. de Marbaix) situated at Corroy‐le‐Grand is used for both research and teaching purposes. ISV users of the platform are Yvan Larondelle (feeding strategies of dairy cows aiming at lowering methane production and for a sustainable production of CLA‐rich milk) and Isabelle Donnay (ram semen collection for the constitution of the Walloon cryobank ; bovine embryo transient transfers in recipient cows in order to evaluate the quality of in vitro embryos produced in different conditions). The "Marcel Huet" platform is concentrating its efforts on aquaculture research and training activites. A major part of the research conducted by ISV members deals with fish physiology and nutrition, as well as with the impact of toxic compounds on these animals. Members of the ELI institute are also using the facilities of the platform to study the genetic influence of reared Fario trouts introduced in the nature on the behaviour of the wild trouts in Belgium. The “Greenhouses and Phytotrons” technology platform (SEFI) provides a logistic support for research and teaching to the scientists and academics belonging to the ISV and ELI institutes. This platform has been approved by the University authorities in 2012. IMABIOL is an imaging platform dedicated mainly to biology and bringing together expertise and equipment from 3 institutes, namely ELI, IMCN and ISV. The platform offers state‐of‐the‐art equipments and applications in confocal imaging and atomic force microscopy. Detailed and practical informations about this facility can be found at http://www.uclouvain.be/en‐279076.html. 43 ISV postdoctoral fellowships Dr Chloé Bonnineau from 1st September 2012 to 31th august 2013, Promoters: Prof. Jean‐François Rees and Cathy Debier 44 PhD Students’Day Program ORAL PRESENTATIONS Didier Demaegd “Characterization of Gdt1p, a yeast protein involved in calcium homeostasis” (promoter : P. Morsomme) Mary Doumit “Selection and characterisation of cyclic peptide inhibitors of the AcrAB‐TolC multidrug efflux pump” (promoter : P. Soumillion) Stéphanie Garcia “Selection of human aquaporins inhibitors within combinatorial libraries of backbone cyclic peptides biosynthesized in Escherichia coli” (promoter : P. Soumillion) Emmanuelle Ghys “Does the sex of the embryo affect developmental and apoptotic rates at the blastocyst stage?” (promoter : I. Donnay) Caroline Louis “Efficient in vitro lipolysis: a tool to study the toxicokinetics of lipophilic compounds” (promoter : C. Debier) Bertrand Magy “Antibody production in plant culture cells depends on the isotype and the host species” (promoter : M. Boutry) Caroline Mullier “Study of molecular mechanisms involved in the intracellular trafficking of Brucella abortus” (promoter : X. De Bolle) Annick Notte “Hypoxia inhibits taxol‐induced cell death by inhibiting taxol‐induced JNK activation and JNK dependent Bcl2 and BclXL phosphorylation while autophagy promotes cell survival” (promoter : C. Michiels) Adrienne Sallets “Separation of the two types of leaf glandular trichomes (hairs) from Nicotiana tabacum to unravel their respective metabolism” (promoter : M. Boutry) Anastassia Vorobieva “Dimeric isocitrate dehydrogenase: Toward engineering new platforms for protein directed evolution” (promoter : P. Soumillion) Geoffroy Walbrecq “Cell models to study the function of peroxisomal peroxiredoxin 5” (promoter : B. Knoops) Julie Winand “Obesity prevention by phenolic compounds ?” (promoter : Y.J. Schneider) POSTERS BARBIER Thibault, J.J. LETESSON “Metabolic adaptation of the intracellular pathogen Brucella”. BERNY Marie, F. CHAUMONT « Hetero‐oligomerisation of plant plasma membrane aquaporins » BRASSEUR Anne, Y.J. SCHNEIDER & L. PUSSEMIER “Detection and quantification of silver nanoparticles in food and biological matrices” CHEVALIER Adrien, F. CHAUMONT “Role of transmembrane domain 3 in the trafficking of plant aquaporins to the plasma membrane” 45 COLOMBARO Vanessa, N. CARON “Analysis of renal phenotype of hyaluronidase‐1 or hyaluronidase‐2 knockout mice” DE BLOCK Julien, P. MORSOMME “Pmp3p, a small proteolipid involved in specific plasma membrane transporters regulation in yeast” DEMINE Stéphane, T. ARNOULD “Potential role of lipophagy in lipolysis induced by mild mitochondrial uncoupling in 3T3‐L1 adipocytes” DEMOULIN Benjamin, P. DUMONT “Influence of MAGE‐A genes on p53 function” DOGNE Sophie, B. FLAMION “Hyal‐1 deficiency may protect the endothelium in a model of streptozotocin‐induced diabetes” GENGLER Samuel, H. BATOKO and PIERRE WATTIAU “Entomopathogenic nematodes as disseminating agents for yersinia pseudotuberculosis” GENIN Marie, C. MICHIELS “Study of THP‐1 macrophage M2 polarization by IL‐4 and IL‐13 and their involvement in chemotherapy resistance” HANOT‐MAMBRES Delphine, J.J. LETESSON In vivo identification of infectious cycle steps of Brucella melitensis implicated in the development of protective immunity in mouse experimental model” HARDIJ Julie, J.M. DOGNE “Atomic force Microscopy, a promising diagnostic tool for thrombotic risk encounterred by cancer patients” JACQUET Nicolas, M. BOUTRY “Transient or stable expression of a viral membrane protein in plants and plant culture cells” MATERNE Philippe, D. HERMAND “Study of role and regulation of the RNA Polymerase II carboxy‐
terminal domain phosphorylation in the induction of the sexual differentiation in the yeast Schizosaccharomyces pombe” NAUNY Philippe, P. SOUMILLION & B. HALLET « Étude la réplication du prophage plasmide pGIL01 de Bacillus thuringensis. Vers le développement de nouveaux outils pour l'évolution dirigée des protéines ». ONCLINX Cécile, B. FLAMION “Analysis of chronic haemolysis in Hyal2‐/‐ mice” PIRSON Marc, B. KNOOPS “Implication of the peroxiredoxin‐thioredoxin system in spinal cord development” SROUR Elise, O. DE BACKER “L’implication des gènes MAGE de type I dans la tumorigenèse” STAUDT Catherine, M. JADOT “Identification of Mannose 6‐Phosphate signal‐independent pathway(s) for the targeting of acid hydrolases to lysosomes” 46 ISV Scientific Day 22 décembre 2012 Yves‐Jacques Schneider (BCNT) “Food engineered nanomaterials: a major safety problem?” Yves Dufrene (BGMB) “Atomic force microscopy: a nanoscopic window on the cell surface.” Isabelle Donnay (EMCA) “Is gender equality compatible with in vitro fertilization and embryo culture? The bovine model.” Brigitte Reusens (CELL) Departure talk. “Do we prepare our health potential already in utero? Focuses on the pancreas and diabetes.” Programmed cell death, the bright side of life Dr. Paul McCabe, UCD School of Biology and Environmental Science University College Dublin “Application of a novel root hair assay to study plant programmed cell death.” Dr Peter Vandenabeele VIB Department for Molecular Biomedical Research UGent “Molecular mechanisms of necroptosis, a novel type of cell death with far‐reaching therapeutic implications” Dr. Didac Carmona‐Gutierrez Institute for Molecular Biosciences University of Graz Austria “Programmed cell death in Saccharomyces cerevisiae: pathways and subroutines.” 47 Seminars 01/02/2012 Targeting signalling pathways by dietary polyphenols in cancer Dr Sonia RAMOS (Madrid, Spain) 03/02/2012 TALE and HOX expression in neuroblastic tumors Dr Dirk GEERTS (Rotterdam, The Netherlands) 10/02/2012 Growth factor delivery by nanoparticles for tissue engineering Dr Anne DES RIEUX (UCL‐
LDRI) 17/02/2012 Metabolism and cellular functions saturated fatty acids, need of new dietary recommendations Dr Philippe LEGRAND (INRA, Rennes, France) 24/02/2012 Control of Hox transcription factor activity : insights from genetic and structural approaches Dr Yacine GRABA (Marseille, France) 02/03/2012 Exploration of the stress response for disease control in aquaculture Dr Peter BOSSIER (Gent Universiteït) 09/03/2012 Dissecting transcriptional regulatory properties of human highly conserved non‐coding regions Dr Jos‐Luis ROYO‐PALENCIA (Sevilla, Spain) 16/03/2012 Natural and laboratory evolution of dehalogenases for bioremediation and biocatalysis Dr Dick JANSSEN (Groningen, The Netherlands) 23/03/2012 Heme trafficking from the ground‐up Dr Iqbal HAMZA (Maryland, USA) 30/03/2012 Engineering trees for biofuels Dr Wout BOERJAN (Gent Universiteït) 20/04/2012 Focussing on enzyme active sites: Improving gene therapy and addressing antibiotic resistance Dr Joelle PELLETIER (Université de Montréal) 27/04/2012 Environmental epidemiology – a Flemish cohort study: prenatal contaminant exposure and health outcomes in children Dr Isabelle SIOEN (Gent universiteit) 04/05/2012 Epigenetic regulation of human telomeric non‐coding RNAs Dr Anabelle DECOTTIGNIES (UCL‐ICP) 11/05/2012 Investigations on allergic immune responses during asthma using mouse models and mould antigens Dr Olivier DENIS (ISP ‐ Bruxelles) 18/05/2012 Structure and function of intrinsically disordered proteins Dr Peter TOMPA (VIB‐VUB) 25/05/2012 PhD Students' Day ‐ Namur 21/09/2012 Structural biology of protein complexes central to hematopoiesis and the human immune system Dr Savvas Savvides (UGent) 05/10/2012 Le chercheur de l'UCL face à la propriété intellectuelle et à la valorisation de ses résultats : que faut‐il absolument savoir ? Dr Marlène Dubuisson (UCL‐ADRE) 12/10/2012 16h DNA polymerase gamma in mitochondria Dr Françoise Foury (UCL‐ISV‐FYMO) 48 19/10/2012 The HOX redox‐ molecular analysis of HOXD13 function during limb development and diseases Dr Nathalie Brison (UCL‐ISV‐EMCA) 26/10/2012 Molecular biomarkers in aquatic ecotoxicology Dr Chloé Bonnineau (UCL‐ISV‐BNTE) 16/11/2012 Journée SFBBM Dr Donald Hilvert (ETH, Zurich), Dr Gert Vriend (CMBI, Nijmegen) 23/11/2012 Understanding the neuro‐anatomy of face recognition: insights from lesion studies and neuroimaging Dr Bruno ROSSION (UCL ‐ iONS) 30/11/2012 Exome sequencing for the fast identification of novel types of congenital disorders of glycosylation (CDP) Dr Gert Matthijs (UZ Leuven) 07/12/2012 Nanobodies as tools for the structural and functional analysis of GPCR transmembrane signalling Dr Jan Steyaert (VIB/VUB) 49 PhD dissertations 13/12/2012 Amandine BAIJOT Expression and functional characterization of plant pleiotropic drug resistance transporters in heterologous and homologous hosts 26/11/2012 Julien ROLAND Identification of substrates of two plant pleiotropic drug resistance transporters, NtPDR1 from Nicotiana tabacum and MpPDR1 from Mentha x piperita 21/11/2012 Céline THIRY Influence of selenium speciation on its bioavailability from diet and food supplements 09/11/2012 Miloud NICHANE An architect gene at work: identification of potential primary target genes of Hoxa2 05/11/2012 Valérie VAN DER EECKEN Evolution of peroxiredoxin 5 mitochondrial targeting in mammals : molecular mechanism and functional consequences 30/10/2012 Marie VANDEN BERGHE PBDEs, chlorinated POPs and their metabolites in mother and pup seals: influence of the lactation period, relationships with vitamin A status and potential impact on the immune system 24/10/2012 Sophie BOUREZ Intracellular accumulation of polychlorinated bipenyls in various in vitro models of adipocytes 22/10/2012 Delphine PAUL HOX genes and early ammalian embryogenesis : from expression profiles to loss‐of‐function strategies 10/09/2012 Yordan MUHOVSKI Molecular and genetic characterization of resistance to fusarium head blight in winter wheat "centenaire" 29/08/2012 André NIVYOBIZI Evaluation de différents modèles mathématiques appliqués à la méthode in sacco dans la caractérisation de la valeur nutritive de graminées tropicales et secondaires tempérées pour l'alimentation des ruminants 30/07/2012 Laurence DELBRASSINNE The use of liquid chromatography coupled to mass spectrometry as a necessary tool for the assessment of cereulide in food 27/06/2012 Isabelle BERGIERS Caractérisation et étude de l'interacome du facteur de transcription Hoxa2 22/06/2012 Wendy SONNET Etude de l'expression des gènes Hox et TALE lors des premières étapes de différenciation embryonnaire chez les mammifères 30/05/2012 Elvis BERNARD Etude du rôle fonctionnel de l’O‐acétylation et de l’amidation du peptidoglycane chez les lactobacilles 29/05/2012 Aleksandra SZOPINSKA‐MORAWSKA Differential quantitative analysis of the plasma membrane proteome of the yeast Saccharomyces cerevisiae 19/03/2012 Anne‐Catherine SCHNEIDER Uptake and metabolism of trans and conjugated fatty acids at the intestinal level: different pathways to produce c9t11 CLA 50 16/03/2012 Gabrielle WORONOFF Development of methods for the directed evolution of penicillin G acylase by in vitro compartmentalization 01/02/2012 Sylvie HOLLEBEECK Promegranate (Punica granatum L.) juice industry by‐products are potential preventive agents against inflammatory bowel diseases 31/01/2012 Myriam GHARDI Cellular and molecular effects of spaceflight and spacefight‐simulated environments on in vitro models 51 52