1. business and industrial
2. business operations
3. business plans

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Table of contents
Word from the CBS2 Association.................................................................. 3
Program Overview .......................................................................................... 5
Detailed Program ............................................................................................ 6
Invited Speakers ............................................................................................ 15
Short Talks : Session 1 .................................................................................. 19
Short Talks : Session 2 .................................................................................. 27
Posters : Session 1 ......................................................................................... 37
Posters : Session 2 ......................................................................................... 77
Organizing commitee .................................................................................. 119
Jury and chairs ............................................................................................ 120
Thanks.......................................................................................................... 121
CBS2 Association
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Word from the CBS2 Association
It is with great pleasure that we receive you for the 13th annual meeting of the
PhD students from the CBS2 doctoral school: the CBS2 Day 2015. Following
the last years’ evolution, the Organizing Committee decided to strengthen this
occasion for all the students to meet, talk and learn from each other and other
members of the scientific community. This year will be marked by two
parallel short talks sessions to let you to choose the most interesting for you.
Obviously, like last year, there will be two poster sessions and two renowned
scientists’communications just for you!
Once again, we would like to thank all the people who made it possible:
 Sandrine Urvoy and Michel Desarmenien, head of ourdoctoral school,
supported us in our will of change,
 BioCampus with Audrey Verdier and Laurent Journot, who made
possible the reward for the best presentations,
 Genopolys with Magali Kitzmann, Géraldine Pawlak, Silke
Conquet and Marchel Méchali, who opened their doors to us,
 All the scientists who constitute the jury and chairs,
 The computing facility of the IGH, for their help with the
visioconference,
 Our partners, the University of Montpellier, MRI, IMGT, Cisbio, Pôle
BioSanté Rabelais, CNRS,
 All of you who register in such large numbers to this event!
This meeting is originally organised by PhD students, for PhD students and is
dedicated to the work we carry on every day in our labs. Enjoy the fact that
this year more than 50 other members of the scientific community registered
to the event to hear about the work done by PhD students.
Make this day your own!
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Program Overview
Genopolys/IGF
8h30-9h
Welcome of the meeting attendees
9h-9h15
Meeting opening by the doctoral school director
and the CBS2 association president
9h15-10h15
3 Short Talks of PhD students for each session
(in parallel)
Session 1A
10h15-10h45
Session 1B
11h45-12h
Coffee break
Conference of Michel Tibayrenc
«The predominant clonal evolution (PCE) concept
of microbial pathogens»
Beginning of the lunch break
12h-13h30
Poster Session 1 (with meal)
13h30-13h45
End of the lunch break
13h45-14h
CBS2 Association Presentation
(general infos, projects, report of the survey…)
14h-15h
Conference of Serge Bauin
«Decide to publish in open access: what risks for my career?»
10h45-11h45
15h-16h
3 Short Talks of PhD students for each session
(in parallel)
Session 2A
Session 2B
16h-17h30
Poster Session 2 (with coffee break)
17h30-17h45
End of coffee break
17h45-18h
Report of the PhD-Company training
18h-18h15
Award ceremony
18h15-19h
Cocktail
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Detailed Program
8h30-9h15 : Welcoming
Words from M. Désarménien (Director of the CBS2 Doctoral School) and
Vuthy Ea (president of the CBS² Association).
9h15-10h15: PhD student’s oral presentations
Session 1A : Genopolys Amphi
9h15-9h35: Efficient CRISPR-Cas9-mediated genome editing of Leishmania
parasites. Lauriane SOLLELIS
9h35-9h55: Implication of CD8+ T cells in the pathophysiology of amyotrophic lateral
sclerosis. Emmanuelle COQUE
9h55-10h15: Autophagy Restricts HIV-1 Infection by Selectively Degrading Tat in
CD4 T Lymphocytes. Coralie DAUSSY
Session 1B:IGF South Seminar Room (2nd Floor)
9h15-9h35: Characterization of P.falciparum sub-populations associated to artemisinin
drug resistance in Cambodia. Ankit DWIVEDI
9h35-9h55: Wfs1-/- mice: phenotyping and gene therapy against Wolfram Syndrome.
Jolanta JAGODZINSKA
9h55-10h15: New self-deployable, bioresorbable and anti-adhesive medical device for
the prevention of intrauterine adhesions. Salomé LEPRINCE
10h15-10h45: Coffee-break
10h45-11h45: First invited speaker
«The predominant clonal evolution (PCE) concept of microbial pathogens».
Michel Tibayrenc
Maladies Infectieuses et Vecteurs Ecologie, Génétique, Evolution et Contrôle (MIVEGEC),
Institut de Recherche pour le Développement (IRD), Montpellier, France.
12h30-14h30: Buffet and poster session 1
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Poster 1: Quantifying the pressure thresholds for tissue injury in paraplegic patients.
Marion LE GALL
Poster 2: Role of SUMOylation and Reactive Oxygen Species (ROS) in Acute
Myeloid Leukemia (AML) treatment. Hayeon BAIK
Poster 3: Global regulation of the SUMO pathway during Acute Myeloid Leukemia
treatment with chemotherapeutic drugs. Marko RISTIC
Poster 4: Identification of factors involved in proteasome-mediateddegradation of antiapoptotic Bfl-1. Loic LIONNARD
Poster 5: H4K20 methylation pathway, chromatin structure and tumorigenesis.
Fanny IZARD
Poster 6: Toxoplasma gondii AuTophaGy protein 8 has an unusual role in apicoplast
division which is essential for parasite growth. Maude LEVEQUE
Poster 7: An open access part toolbox to tune genetic expression in Bacillus subtilis.
Sarah GUIZIOU
Poster 8: Oxidative stress: key mechanism of age-related cochlear sensory hair cell
loss. Nesrine BENKAFADAR
Poster 9: Membrane dynamics and partitioning of CD9 and CD81 are differentially
regulated by the actin network. Laurent FERNANDEZ
Poster 10: High viral load in patients failing first-line ART is associated with
multidrug resistance in resource limited countries. Emilande GUICHET
Poster 11: Cardioprotection against ischemia-reperfusion injury by heart rate control.
Viviana DELGADO BETANCOURT
Poster 12: Development of a mobile application to compute food carbohydrates.
Omar DIOURI
Poster 13: Adrenergic receptor gene variants are associated with late-onset generalized
anxiety disorder. Xiaobin ZHANG
Poster 14: Role of the ribosomal protein S6 phosphorylation in the mouse brain.
Anne BIEVER
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Poster 15: Characterization of a Cancer Stem cells enriched subpopulations and role of
EMT Regulators in basal Breast Cancer Cell Plasticity. Mona HOUHOU
Poster 16: Metabotropic glutamate receptor type 7 (mGlu7) modulation of
thalamocortical activity. Benoit GIRARD
Poster 17: Modeling the role of the global regulator ShvR of Burkholderia
cenocepacia in virulence using zebrafish embryos. Margarida GOMES
Poster 18: Ubiquitin under stress: “Going hybrid with NEDD8”.Chantal MAGHAMES
Poster 19: Modulation of lateral septal neurons by oxytocin and vasopressin,
neuropeptides involved in the regulation of social behavior.Amélie BORIE
Poster 20: Potential role of P2X4R expressed by sensory neurons in BDNF-evoked
inflammatory pain. Sarah LALISSE
Poster 21: Role of RIP140 in familial colorectal cancer. Pascale PALASSIN
Poster 22: Deciphering the function of the Serine/Threonine Protein Kinase CStk
from Coxiella burnetii and its role during host infection. Solene BRELLE
Poster 23: Characterization and role of indirectly-activated human dendritic cells by
immune-complex adenovirus in vitro in immune memory response.
Thi Thu Phuong TRAN
Poster 24: BDNF knockdown induces defects in zebrafish posterior lateral line
development. Alaa YEHYA
Poster 25: Diffuse low grade gliomas: characterization and development of in vitro
model for designing innovative therapeutic approaches.Safa AZAR
Poster 26: Role of the chromatin associated proteins HP1 (Heterochromatin Protein 1)
in liver hemostasis. Shefqet HAJDARI
Poster 27: Role of PRDM9 methyltransferase activity in mouse meiotic
recombination. Boubou DIAGOURAGA
Poster 28: Dynamics and function of monocyte subsets in alcohol-dependent subjects.
Hélène DONNADIEU-RIGOLE
Poster 29: Dissecting the role of new cortical scaffolds during epithelial biology.
Elodie FOREST
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Poster 30: Role of histone modifications and non-coding RNAs in the regulation of the
imprinted Dlk1-Dio3 domain. Ildem SANLI
Poster 31: Molecular mechanisms of Notch1-induced pericyte-like transdifferentiation
of glioblastoma stem cells. Sophie GUELFI
Poster 32: A synthetic tridimensional matrix to study the migration of Glioblastoma
stem cells. Ali SALEH
Poster 33: Initiation of meiotic recombination in mouse; search for interacting partners
of PRDM9. Yukiko IMAI
Poster 34: Epigenetic modulation of intestinal cancer susceptibility. Marco BRUSCHI
Poster 35: Rad18 silences the UV-dependent DNA damage checkpoint in
early Xenopus embryos. Chames KERMI
Poster 36: Differential effect of microparticles and exosomes isolated from
mesenchymal stem cells on T cell proliferation and experimental arthritis.
Stella COSENZA
Poster 37: Epigenetics and phenotypes modulation in HMEC cells.
Amanda ABI KHALIL
Poster 38: DNA methylation and pulmonary disease in CF patients.
Milena MAGALHÃES
13h45-14h: CBS2 Association Presentation
General informations about the association, its projects. Report of the survey about
your thesis in the CBS2 doctoral school.
14h-15h: Second invited speaker
«Decide to publish in open access: what risks for my career? »
Serge Bauin
Coordinateur des politiques d’IST - COMUE Université Sorbonne Paris Cité et Chargé de
mission pour le libre accès - DIST (Direction de l'Information Scientifique et Technique) du
CNRS.
15h-16h: PhD student’s oral presentations
Session 2A: Genopolys Amphi
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15h-15h20: Generation of Alzheimer's disease (AD) genetic patients’ reprogrammed
stem cells (iPS) as tools for the diagnostic and therapeutic researches for AD.
Laura AUBOYER
15h20-15h40: Shifts in Migration route: Insights into GABAergic Interneurons
Diversity. Christelle CADILHAC
15h40-16h: Origin of the HIV-1 group O epidemic in Western Lowland Gorillas.
Mirela D'ARC
Session 2B: IGF South Seminar Room (2nd Floor)
15h-15h20: Molecular Mechanisms of Zebrafish Cardiac Regeneration.
Girisaran GANGATHARAN
15h20-15h40: Chromatin-bound MDM2 regulates serine metabolism and redox
homeostasis independently of p53. Romain RISCAL
15h40-16h: Polycomb-Mediated
Inheritance. Filippo CIABRELLI
Repression
in
Transgenerational
Epigenetic
16h-17h45: Coffee and poster session 2
Poster 39: Pro-inflammatory macrophages mediated TNFa signaling is required for
caudal fin regeneration in zebrafish larvae. Béryl LAPLACE-BUILHE
Poster 40: Role of miRNAs in the Cystic Fibrosis pathology. Jennifer BONINI
Poster 41: Evolution of meiotic recombination: variation and function of the Prdm9
gene in mice. Denis DUNOYER DE SEGONZAC
Poster 42: Biophysical interaction of nanoparticles with biological fluids.
Estelle RASCOL
Poster 43: Allosteric modulation of metabotropic glutamate receptors by chloride ions.
Amélie TORA
Poster 44: Generation of a vaccine to prevent poultry from Newcastle disease and
control viral shedding. Haijin LIU
Poster 45: Adaptation of Staphylococcus aureus to prolonged environmental stress
conditions. Christelle NGBA ESSEBE
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Poster 46: Understanding the cooperation between Notch and the polarity determinant
Scribble during neoplasia. Rémi LOGEAY
Poster 47: Identification of potential therapeutic targets regulated by Fra-1 and/or Fra2 in triple negative breast cancers. Claire TOLZA
Poster 48: Targeting Transferin Receptor 1 (TFR1) in Pancreatic Cancer.
Rana MELHEM
Poster 49: Structural characterization of Plasmodium falciparum CCT and fragmentbased drug design approach for targeting phospholipid biosynthesis pathway.
Ewelina GUCA
Poster 50: PPM1K (protein phosphatase, Mg2+/Mn2+ dependent, 1K) Gene
Association in Polycystic Ovary Syndrome for Better Understanding of the Role of
Branched-Chain Aminoacids Variation in Metabolic Disorders. Sara HAYDAR
Poster 51: Identification of new regulators of the Notch pathway in KRASG12Vdriven NSCLC. Alejandra DAMIAN
Poster 52: Identification of kinase inhibitors as alternatives for the treatment of
metastatic castration-resistant prostate cancers. Joelle AZZI
Poster 53: Assembly of proteasome and epidermal differentiation: interest in psoriasis.
Barbara ZIEBA
Poster 54: Expression of the transcriptional coregulator RIP140 in colorectal cancer.
Mouna TRIKI
Poster 55: Evolutionary Plasticity of Endodermal Gene Regulatory Networks in Ciona
intestinalis and Phallusia mammillata. Alicia MADGWICK
Poster 56: The role of primary cilia in colon homeostasis and tumor development.
Ruizhi TANG
Poster 57: The effect of dichloroacetate (DCA) on tumor cell metabolism.
Sana BELKAHLA
Poster 58: Heterochromatin factors stimulates telomere transcription. Sophie KAN
Poster 59: Common variants on XQ28 conferring risk for rheumatoid arthritis in
tunisian and french populations. Olfa KHALIFA
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Poster 60: Brucella replication in human trophoblasts: role of the eukaryotic protein
CD98hc. Beren GARCIA MENDEZ
Poster 61: Identification and characterization of Mabs4780, a new determinant
required for intracellular survival and pathogenicity of Mycobacterium abscessus.
Iman HALLOUM
Poster 62: Role of R2TP/HSP90 system in mouse development and colorectal
carcinogenesis. Chloé MAURIZY
Poster 63: Phosphoproteomics of 5-HT2A/mGlu2 heteromers: toward new insights into
the mechanism of action of hallucinogens and antipsychotics. Samy MURAT
Poster 64: Immunodepression and accumulation of cancerous cells: a role of everyday
perturbations? Camille JACQUELINE
Poster 65: E4F1 is a major regulator of pyruvate metabolism in normal skin
homeostasis and skin carcinogenesis. Berfin SEYRAN
Poster 66: Risks of in utero NSAIDs and paracetamol exposure on the early
development and maturation of the reproductive organs. Moïra ROSSITTO
Poster 67: Physiological role of APPL in Drosophila mushroom bodies’ development.
Claire MARQUILLY
Poster 68: Role of the transcription factor RIP140 in colon cancer. Nour SFEIR
Poster 69: Characterization of host cell proteins recruited at the Moving Junction
during invasion of Toxoplasma gondii. Amandine GUERIN
Poster 70: MLN4924, a NEDD8 inhibitor in a p53 based cyclotherapy approach.
Lara BOU MALHAB
Poster 71: Ribosome: Master Regulator of Cancer Cell Fate?
Laura YAZDANI
Poster 72: Pattern of drug metabolism enzyme expression in the epileptic brain: a new
mechanism of drug resistance. Badreddine BOUSSADIA
Poster 73: Determinants for UBA1 recruitment at sites of DNA damage.
Ramhari KUMBHAR
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Poster 74: The effect of microbial LPS translocation in cross-presenting dendritic cells
and in the breakdown of CD8+ T cell peripheral tolerance in irradiated mice.
Gabriel ESPINOSA
Poster 75: A new integrated platform for drug action mode determination in C.
elegans. Myriam RICHAUD
Poster 76: Transient infection induces chronic inflammation. Quang Tien PHAN
17h45-18h: Report of the PhD-Company training
18h-18h15: Award ceremony
To reward the best of you and win a large variety of gifts…
17h45-18h: Coktail
And don’t forget the party tonight… !
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Invited Speakers
Michel Tibayrenc
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« The predominant clonal evolution (PCE) concept of microbial
pathogens »
Maladies Infectieuses et Vecteurs Ecologie, Génétique, Evolution et Contrôle (MIVEGEC),
Institut de Recherche pour le Développement (IRD), Montpellier, France.
We propose that predominant clonal evolution (PCE) in microbial pathogens be
defined as restrained recombination on an evolutionary scale, with genetic exchange
scarce enough to not break the prevalent pattern of clonal population structure. The
main features of PCE are: (i) strong linkage disequilibrium (LD); (ii) the widespread
occurrence of stable genetic clusters blurred by occasional bouts of genetic exchange
(“near-clades”). We hypothesize that the PCE features are not mainly due to natural
selection, but chiefly originate from in-built genetic properties of pathogens. We show
that the PCE model obtains even in microbes that have been considered as “highly
recombining”, such as Neisseriameningitidis, and that some clonality features are
observed even in Plasmodium, which has been long described as panmictic. Lastly, we
evidence that PCE features are also observed in viruses, taking into account their
extremely fast genetic turnover.
The PCE model provides a convenient population genetic framework for any kind of
micropathogen. It makes it possible to describe convenient units of analysis (clones
and near-clades) for all applied studies. Lastly, the PCE model opens up the possibility
of revisiting the problem of species definition in these organisms, which are
responsible for devastating human, animal and crop diseases.
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Serge Bauin
« Decide to publish in open access: what risks for my career? »
Coordinateur des politiques d’IST - COMUE Université Sorbonne Paris Cité et Chargé de
mission pour le libre accès - DIST (Direction de l'Information Scientifique et Technique) du
CNRS.
After having founded and led the unit in charge of science policy indicators CNRS,
Serge Bauin has been director of the IST at the CNRS and director of the INIST from
2010 to 2013. Today, he is coordinator of IST policies within the Sorbonne Paris Cité
COMUE University and “ambassador” for open access at the CNRS. He also serves
on the scientific advisory board of the Observatory of Science and Technology.
Source : oam.biu-montpellier.fr
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Short Talks : Session 1A
Genopolys Amphi
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N°1 - Lauriane SOLLELIS
Efficient CRISPR-Cas9-mediated genome editing of Leishmania
parasites
Protozoan pathogens that cause leishmaniasis in humans are difficult to manipulate
genetically. In this work we implemented the CRISPR-Cas9 system to Leishmania
parasites and demonstrate its efficient use for genome editing. The Cas9 endonuclease
was expressed under the control of the DHFR promoter and the single guide RNA was
produced under the control of the U6snRNA promoter and terminator. As proof of
concept we chose to knockout a tandemly repeated gene family, the paraflagellar rod2 (PFR) loci. We were able to obtain null mutants in a single round of transfection. In
addition, we confirmed the absence of off-target editions by whole genome
sequencing of two independent clones. Our work demonstrates that CRISPR-Cas9mediated gene knockout represents a major improvement in comparison with the
classical method. Beyond gene knockout, this genome editing tool opens avenues for a
multitude of functional studies to speed up research on leishmaniasis.
Lauriane Sollelis1,2, Mehdi Ghorbal1,2, Cameron Ross MacPherson3, Rafael Miyazawa
Martins3, Nada Kuk1, Lucien Crobu2, Patrick Bastien1,2,4, Artur Scherf3, Jose-Juan
Lopez-Rubio1,2 and Yvon Sterkers1,2,4
1
University of Montpellier, Faculty of Medicine, Laboratory of ParasitologyMycology, 2 CNRS 5290 - IRD 224 – University of Montpellier (UMR
"MiVEGEC"), 3 Institut Pasteur – INSERM U1201 - CNRS ERL9195, Biology of
Host-Parasite Interactions" Unit, Paris, France, 4 CHRU (Centre Hospitalier
Universitaire de Montpellier), Department of Parasitology-Mycology, Montpellier,
France
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N°2 - Emmanuelle COQUE
Implication of CD8+ T cells in the pathophysiology of amyotrophic
lateral sclerosis
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by
the loss of motoneurons. ALS leads to the atrophy and paralysis of the striated
muscles, which will cause death within 3 to 5 years. Approximately 10% of ALS
cases are genetically linked, and among these, 20% are caused by mutations in SOD1
gene. Mice overexpressing human SOD1 mutations develop a motor syndrome with
features of the human disease.
A chronic inflammatory response, associated with the accumulation of blood-derived
immune cells in the CNS, is a pathological feature of ALS. Firstly, CD4+
lymphocytes invade the CNS and seem to negatively regulate the inflammatory
response. However, the early symptomatic phase is characterized by an increase of
CD8+ T cells in the CNS. Those CD8+ T cells are effectors of adaptive and innate
immunity and can promote cytotoxic effects, however, the contribution of this cell
population in the neurodegenerative process has been poorly investigated. Here, we
propose to explore the impact of the infiltration of CD8+ T cells on the development
of the disease.
Our results show that CD8+ T cells isolated from SOD1G93A, but not from wildtype
mice, trigger motoneuron death, selectively.
Our co-culture experiments showed that CD8+ T cells trigger motoneuron death in a
contact dependent-manner. Moreover, we showed that this contact requires the
recognition of the MHC-I complex exposed by motoneurons.
Our results suggest that inhibition of IFNγ, and perforin/granzyme pathway, two
classical mechanisms of CD8+ T cells cytotoxicity, saves motoneuron from the CD8+
T cell-mediated toxicity.
Beside this in vitro analysis, one of the main objective of this project is to assess the
therapeutic effect of an immunodepletion of CD8+ T cells in the SOD1G93A mice.
To this aim, we developed a protocol of long term immunodepletion of CD8 cells.
Coque, E. (Montpellier)1, Carrasco, G.E. (Montpellier)2, Salsac, C. (Montpellier)1,
Vincent, T. (Montpellier)1,3, Hernandez, J. (Montpellier)2, Raoul, C. (Montpellier)1
1 : INSERM U1051 - Déficits sensoriels et moteurs, Montpellier, France. 2 : INSERM
U844, Montpellier, France. 3 : Hopital Saint Eloi, Département d'immunologie,
Montpellier, France
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N°3 - Coralie DAUSSY
Autophagy Restricts HIV-1 Infection by Selectively Degrading Tat
in CD4 T Lymphocytes
Autophagy is a ubiquitous mechanism involved in the lysosomal-mediated
degradation of cellular components when they are engulfed in vacuoles called
autophagosomes. Autophagy is also recognized as an important regulator of the innate
and adaptive immune responses against numerous pathogens, which have, therefore,
developed strategies to block or use the autophagy machinery to their own benefit.
Upon human immunodeficiency virus type 1 (HIV-1) infection, viral envelope (Env)
glycoproteins induce autophagy-dependent apoptosis of uninfected bystander CD4 T
lymphocytes, a mechanism likely contributing to the loss of CD4 T cells. In contrast,
in productively infected CD4 T cells, HIV-1 is able to block Env-induced autophagy
in order to avoid its antiviral effect. To date, nothing is known about how autophagy
restricts HIV-1 infection in CD4 T lymphocytes.
Here, we report that autophagy selectively degrades the HIV-1 transactivator Tat, a
protein essential for viral transcription and virion production. We demonstrated that
this selective autophagy-mediated degradation of Tat relies on its ubiquitinindependent interaction with the p62/SQSTM1 adaptor. Taken together, our results
provide evidence that the anti-HIV effect of autophagy is specifically due to the
degradation of the viral transactivator Tat but that this process is rapidly counteracted
by the virus to favor its replication and spread.
Coralie F. Daussy*, Sophie Sagnier*, Sophie Borel*, Véronique Robert-Hebmann*,
Mathias Faure¤, Fabien P. Blanchet*,Bruno Beaumelle*, Martine BiardPiechaczyk*, Lucile Espert*
* : CPBS, Université de Montpellier, UMR 5236 CNRS, Montpellier, France; ¤ :
CIRI, International Center for Infectiology Research, Université de Lyon, Lyon,
France; INSERM U1111, Lyon, France;Ecole Normale Supérieure de Lyon, Lyon,
France;Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon,
France;CNRS, UMR 5308, Lyon, France
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Short Talks : Session 1B
IGF South Seminar Room (2nd Floor)
N°4 - Ankit DWIVEDI
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Characterization of P.falciparum sub-populations associated to
artemisinin drug resistance in Cambodia
Malaria is one of the most widespread parasitic infections in the world. The
undergoing WHO Malaria elimination programs are threatened by emergence of the
Plasmodium falciparum artemisinin resistant parasite in South-East Asia. These
parasites have emerged in the western part of Cambodia, where chloroquine and
pyrimethamine drug resistance emerged in the past. Based on recent reports of 1) the
presence of P.falciparum sub-populations in Cambodia (Miotto O. et al., 2013) and 2)
evidence that mutations in the K13 gene are supporting artemisinin resistance in
Cambodian parasite population (Ariey F. et al., 2014), we characterize the metabolic
properties of parasite sub-populations.
We use a large dataset based on NGS genome sequences from ENA database to
analyze the distribution of parasite population over the country. We describe a reliable
SNP variant calling pipeline from around 200 genome sequences based on signal
parameters. The parasite sub-populations were defined based on SNP set obtained
using this pipeline. We provide genetic evidence that acquisition and transmission of
artemisinin resistance is related to parasite population structure in Cambodia. We
identify features providing functional annotation for proteins, pathways, isolates and
sub-populations. Parasite populations from western Cambodia have different
metabolic capacities than eastern populations.
We develop a barcode approach based on LUMINEX technology to rapidly screen for
the organization of the parasite population over the country. We successfully
genotypes about 300 samples. We identify a new sub-population in the south that is
associated to the emergence of the most common C580Y K13 mutation.
Also, observed admixture parasite population is a major risk because of the metabolic
capacities and ability of the parasites to cross within sub-populations and with the
members of other sub-populations. These results question the origin and the
persistence of the P.falciparum sub-populations in Cambodia.
Ankit Dwivedi[1][2], Christelle Reynes[3], Nimol Khim[4], Didier Ménard[4],
Emmanuel Cornillot[2][5]
[1] Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé (CPBS),
Montpellier, France. [2] Institut de Biologie Computationelle (IBC), Montpellier,
France. [3] Institut de Génétique Humaine (IGH), Montpellier, France. [4] Institut
Pasteur du Cambodge (IPC), Cambodia. [5] Institut de Recherche en Cancérologie de
Montpellier (IRCM), Montpellier, France
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N°5 - Jolanta JAGODZINSKA
Wfs1-/- mice: phenotyping and gene therapy against Wolfram
Syndrome
The Wolfram Syndrome (WS) is an early onset genetic disease (1/200 000) featuring
diabetes mellitus and progressive optic neuropathy ensuing mutations in the WFS1
gene. We investigated mice with deleted exon 8 of the gene to imitate the visual
aspects of the disease.
The model has been known to exhibit pancreatic β-cell atrophy but the visual function
has not yet been investigated. Therefore, we focused on assessing it via in vivo and
post mortem studies of Wfs1+/+ and Wfs1-/- mice at 3 and 7 months of age.
We examined visual acuity via changes in the optokinetic relfex, retinal ganglion cell
(RGC) function via post-scotopic treshold responce (pSTR), and eye physiology via
eye fundus observation and optical coherence tomography (OCT). We also determined
proportion of retinal ganglion cells (RGC) and axonal loss at the age of 7 months, with
anti-Brn3a immuno-labeling of retinal sections and electron microscopy of optic nerve
(ON) sections, respectively. We observed progressive loss of visual acuity
accompanied by loss of axons in the ON and pallor of the optic disc.
Next, we performed an intravitreal gene therapy (GT) with AAV-2/2-CMV-WFS1 at
1 month old Wfs1+/+ and Wfs1-/- mice. The assessment of the visual acuity and the
RGC function at 3 and 6 months of age showed no worsening of the vision originating
from GT nor the injections themselves. Importantly, the visual acuity of WFS1-/- mice
after GT was improved and the deterioration of RGC function was slower with age.
Furthermore, the controls injected with AAV-2/2-CMV-GFP showed even distribution
of its expression in the retina. The study is ongoing for histological analysis.
The presented data qualify the murine model for investigating visual aspects of WS.
Additionally, the promising preliminary results of the gene therapy encourage further
studies under a treatment for the Wolfram Syndrome patients.
JAGODZINSKA Jolanta1, BONNET-WERSINGER Delphine1, KOKS Sulev 2,
SEVENO Marie1, LENAERS Guy1, HAMEL Christian1, DELETTRE Cecile1.
1
CHU Montpellier, Institut des Neurosciences de Montpellier INSERM U1051,
Montpellier, France. 2University of Tartu, University of Tartu, Tartu, Estonia
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N°6 - Salomé LEPRINCE
New self-deployable, bioresorbable and anti-adhesive medical
device for the prevention of intrauterine adhesions
Synechiae or intrauterine adhesions result in the fibrous adherence of opposing uterine
walls, which produce partial or complete obliteration in the uterine cavity and/or the
cervical canal. Trauma to a gravid uterine cavity is known to be the main cause of
adhesions formation. Uterine curettage in the postpartum period, cesarean section,
spontaneous miscarriage, abortion or termination of pregnancy are predisposing
factors of adhesions formations. This pathology can cause menstrual abnormalities,
pelvic pain, infertility and recurrent pregnancy loss [1].
In order to prevent postsurgical adhesion formation across the cavity, a new antiadhesive and bioresorbable medical device was developed to maintain separated
uterine walls after surgical trauma. Anti-adhesive barrier from PLA50-PEO-PLA50
(polylactic acid – polyethylene oxyde – polylactic acid) triblock copolymer has been
synthesized. This copolymer presents a strong hydrophilic character to promote
swelling in water and hydrolytic degradation. Moreover high molecular weights of
PEO allow to obtain excellent filmogenic properties and provide anti-adhesive
properties. Biocompatibility and anti-adhesive effects of triblock were evaluated using
human endometrial cells. Anti-adhesive efficiency and degradation rate of triblock
copolymer were studied by implantation between cecum and peritoneal wall defects of
rats. Copolymer films provide an excellent candidate as a anti-adhesion barrier owing
to its anti-adhesion potential, as well as its flexibility and biodegradability. In order to
provide the ideal morphology of the medical device, the next step will be the
production of medical device by using 3-D printing technology (fused deposition
modeling).
This barrier aims at providing a preventive tool to limit the formation of adhesions
after an endometrial trauma and restoring normal reproductive functions. This medical
device meets the clinical requirements of the gynecologic surgeons and could become
a new tool for the management of female infertility.
[1] D. Yu. Fertility and Sterility 2008, 89, 759.
S. Leprince1*, S. Huberlant1-2, V. Letouzey1-2, I. Le Teuff1-2, C. Paniagua1, J.
Coudane1, X. Garric1.
1
Institut des Biomolécules Max Mousseron, CNRS UMR 5247, Faculté de Pharmacie,
Université Montpellier, France. 2 Service de Gynécologie et Obstétrique, CHU
Carémeau, Nîmes, France
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Short Talks : Session 2A
Genopolys Amphi
N°7 - Laura AUBOYER
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Generation of Alzheimer's disease (AD) genetic patients’
reprogrammed stem cells (iPS) as tools for the diagnostic and
therapeutic researches for AD
Amyloid precursor protein (APP) and Tau protein are two main molecular actors of
neurodegenerative affections, which are of prime importance in Human Health
(Alzheimer’s disease (AD)). Intensive research is ongoing to understand these
proteins’ metabolism, action and implication in the pathological mechanism of these
affections. They are the target of most therapeutic approaches and are used for
biological diagnosis. In the present program, our objective is to investigate neuronal
APP and Tau protein processing and metabolism using biochemical tools (single and
multiplex immunodetection system (ELISA, Luminex®, MSD®)), innovative
detection methods (mass spectrometry (MS)) and metabolic approach (incorporation
of stable isotope labelled Leucine (6C13L)) in two complementary situations: in vivo
in patients, and in vitro in cell culture. The goal is to get a comprehensive proteomic
view (synthesis, cleavage, interaction..) based on the parallel analysis of patient
isotope labelled samples (already available following perfusion of 6C13Leu and
kinetics sampling of CSF) and samples generated in neuronal differentiated human
embryonic stem cell and Induced pluripotent stem cells derived from AD-patients
where pharmacological tools (secretase/kinase inhibitors) can be tested. The final goal
is therefore to parallel the data in patients with those generated in differentiated iPS
cells and control hESC cells. This project will offer the unique opportunity to combine
state-to-the-art approaches to understand how the APP fragments and peptides are
generated as well as the modifications of the Tau protein in normal and pathological
situation.
Auboyer, L. (Montpellier)1, Radreau, F. (Montpellier)1, Monzo, C. (Montpellier)1,
Gabelle, A. (Montpellier)1, Lehmann, S. (Montpellier)1, Crozet, C. (Montpellier)1
1Institut de Médecine Régénératrice et de Biothérapie (IRMB), Montpellier, France
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N°8 - Christelle CADILHAC
Shifts in Migration route: Insights into GABAergic Interneurons
Diversity
GABAergic cell diversity is critical for proper function of neural circuitry. In the
developing brain, GABAergic interneurons use stereotyped migration route and
schedule to reach their specific laminar position. Whether and how their migration
journey impacts their stereotyped differentiation is not known. This is quite
exemplified by cerebellar Molecular Layer GABAergic Interneurons (MLGI), which
are the Basket and the Stellate cells. MLGI display major morphological differences
but present no discriminating genetic marker. It has been proposed that these
differences are due to distinct laminar position underlying distinct environmental cues,
however there is no clear evidence to support this view.
In this study, we provided insights on how local control of migratory pathways may
contribute to specific cell-type differentiation program by acting as a time-shifting
device. By using in vivo grafts experiments of GABAergic progenitors, we identified
two distinct integration paths of MLGI. We conducted a series of time-lapse
experiments on acute slices and uncovered that all MLGI entered the molecular layer
using radial migration while only a proportion of interneurons performed an additional
step of tangential migration in the external granule cell layer. In addition, we showed
that this unexpected tangential migration took place in a define window of cerebellar
development along a subpopulation of pre-migratory granular cells expressing the
cell-adhesion molecule TAG1. TAG1 blocking antibody disrupts the tangential
migration in acute slices and important granular cell axons reorganization revealed by
TAG1 gain of function in organotypic slices favors ectopic migration of MLGI. These
results show the implication of granular cell axons as a physical support for this
particular migratory process. Finally, we demonstrated, using hybrid organo-grafts
experiments, that interneurons capable of tangential migration belong to the Stellate
cell population.
Together, these findings reveal how shifts in migration itinerary of neural progenitors
impacted their cell fate differentiation program.
Christelle Cadilhac, Fabrice Ango (IGF, UM, CNRS, INSERM)
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N°9 - Mirela D'ARC
Origin of the HIV-1 group O epidemic in Western Lowland
Gorillas
HIV-1 and HIV-2 are the result of multiple viral cross-species transmissions from
Simian Immunodeficiency Viruses (SIVs) infecting Non Human Primates (NHP) to
humans. HIV-1 is the principal responsible for the global AIDS epidemic. HIV-1 is
divided into four phylogenetic lineages,called groups M, N, O, and P, which resulted
each from an independent cross-species transmission event of SIVs infecting African
apes. Although groups M and N have been previously traced to distinct chimpanzee
communities in southern Cameroon, the reservoirs of groups O and P remained
unknown. Here, we present the results recently published in PNAS journal, where we
screened fecal samples from western lowland (n = 2,611), eastern lowland (n = 103),
and mountain (n = 218) gorillas for gorilla SIV (SIVgor) antibodies and nucleic acids.
SIVgor was identified at only four sites in southern Cameroon. Amplification of
partial and full-length SIVgor sequences revealed extensive genetic diversity, but all
SIVgor strains were derived from a single lineage within the chimpanzee SIV
(SIVcpz) radiation. Two fully sequenced gorilla viruses from southwestern Cameroon
were very closely related to, and represent the source population of, HIV-1 group P.
Most of the genome of a third SIVgor strain, from central Cameroon, was very closely
related to HIV-1 group O, again pointing to gorillas as the immediate source.
Functional analyses identified the cytidine deaminase APOBEC3G as a barrier for
chimpanzee-to-gorilla, but not gorilla-to-human, virus transmission. These data
indicate that HIV-1 group O, which spreads epidemically in west central Africa and is
estimated to have infected around 100,000 people, originated by cross-species
transmission from western lowland gorillas. Thus, both chimpanzees and gorillas
harbor viruses that can that are capable of crossing the species barrier to humans and
causing major disease outbreaks.
Mirela D’arca,b, Ahidjo Ayoubaa, Amandine Estebana, Gerald H. Learnc, Vanina Bouéa,d,
Florian Liegeoisa,d, Lucie Etiennea, Nikki Tagge, Fabian H. Leendertzf, Christophe Boeschg,
Nadège F. Madindaf,g,h, Martha M. Robbinsg, Maryke Grayi, Amandine Cournila, Marcel
Oomsj,k, Michael Letkoj,k, Viviana A. Simonj,k,l, Paul M. Sharpm, Beatrice H. Hahnc, Eric
Delaportea, Eitel Mpoudi Ngolen, and Martine Peetersa,o
aUnité Mixte Internationale 233, Institut de Recherche pour le Développement, INSERM
U1175, and University of Montpellier, 34394 Montpellier, France; bLaboratory of Human
Virology, Universidade Federal do Rio de Janeiro, 21949-570 Rio de Janeiro,
Brazil; cDepartments of Medicine and Microbiology, Perelman School of Medicine, University
of Pennsylvania, Philadelphia, PA 19104; dCentre International de Recherches Médicales,
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Franceville, Gabon; eProjet Grands Singes, Center for Research and Conservation, Royal
Zoological Society of Antwerp, 2018 Antwerp, Belgium; fEpidemiology of Highly Pathogenic
Microorganisms, Robert Koch Institute, 13353 Berlin, Germany; gDepartment of Primatology,
Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany; hInstitut de
Recherche en Ecologie Tropicale, Libreville, Gabon; iInternational Gorilla Conservation
Programme, Kigali, Rwanda; jDepartment of Microbiology, kGlobal Health and Emerging
Pathogens Institute, andlDivision of Infectious Diseases, Department of Medicine, Icahn School
of Medicine at Mount Sinai, New York, NY 10029; mInstitute of Evolutionary Biology, and
Center for Immunity, Infection, and Evolution, University of Edinburgh, Edinburgh EH9 3JT,
United Kingdom; nInstitut de Recherches Médicales et d’Études des Plantes Médicinales,
Prévention du Sida au Cameroun, Yaoundé, Cameroon; and oComputational Biology Institute,
34095 Montpellier, France
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Short Talks : Session 2B
IGF South Seminar Room (2nd Floor)
N°10 - Girisaran GANGATHARAN
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Molecular Mechanisms of Zebrafish Cardiac Regeneration
The human heart’s inability to replace ischemia-damaged myocardium with
regenerated muscle results significantly in the worldwide mortality associated with
coronary artery disease. Remarkably, certain vertebrate species, such as the zebrafish,
achieve complete regeneration of the amputated or injured myocardium through the
proliferation of cardiomyocytes. Here, we report a bHLH transcription factor as a
critical regulator of the cardiomyocyte mediated heart regeneration. This gene is
known to be a key regulator of erythroid cell differentiation and endocardium
formation, but its role in cardiomyocytes has not been reported previously.
Immunohistochemical studies reveal its presence in cardiomyocytes. Using transgenic
fish, we discovered that suppression of this gene specifically in the cardiomyocyte
profoundly impairs cardiac regeneration. Next, we also hypothesized that genes
involved in cardiac development could be reutilized during cardiac regeneration.
Using a drug inhibition approach, we have identified upto 7 genes involved in this
process. Specifically, we are characterizing the role of a family of genes in scar
regression during cardiac regeneration.
Girisaran Gangatharan and Chris Jopling
Institut de Génomique Fonctionnelle, Montpellier, France.
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N°11 - Romain RISCAL
Chromatin-bound MDM2 regulates serine metabolism and redox
homeostasis independently of p53
Although the Mouse Double Minute 2 (MDM2) oncoprotein is a major negative
regulator of the p53 tumor suppressor growing evidence supports p53-independent
functions of MDM2. Here we describe a new p53-independent function for MDM2 in
cancer cell metabolism. We show that MDM2 is recruited to chromatin independently
of p53 to regulate a transcriptional program implicated in amino acid metabolism and
redox homeostasis. Genome-wide MDM2 ChIP-seq experiments combined with gene
expression profiling identified MDM2-responsive genes and highlighted an important
role for chromatin-associated MDM2 in serine/glycine and glutathione metabolism.
MDM2 recruitment to its target genes required members of the ATF family of
transcription factors and this event is modulated by the M2 isoform of pyruvate kinase
(PKM2), a rate-limiting metabolic enzyme that controls the glycolytic flux and its
branched biosynthetic pathways. Interestingly, MDM2 recruitment to chromatin
increases in specific stress conditions known to impinge on PKM2 activity, including
oxidative stress and serine deprivation. Moreover, chromatin-bound MDM2 controls
the pool of reduced and oxidized glutathione that impacted on reactive oxygen species
(ROS) levels. Interfering with MDM2 expression and exogenous serine availability
decreases cell survival in vitro and tumor growth in vivo, the latter being rescued, at
least partly, by the ROS scavenger N-acetyl cysteine. Our data indicate that MDM2 is
a key regulator of the balance between de novo serine synthesis and serine auxotrophy
and of the redox status of cancer cells independently of p53.
Romain Riscal1,2,3,4, Emilie Schrepfer1,2,3,4, Giuseppe Arena1,2,3,4, Frédérique Sabourdy5,6,
Charles Vincent1,2,3,4, Imade Ait-Arsa1,2,3,4, Thierry Levade5,6, Jean-Christophe Marine7,8, JeanEmmanuel Sarry6, Laurent Le Cam1,2,3,4,9* and Laetitia K. Linares1,2,3,4,9*.
1IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, F-34298, France.
2INSERM, U1194, Montpellier, F-34298, France. 3Université Montpellier, Montpellier, F34298, France. 4Institut régional du Cancer Montpellier, Montpellier, F-34298. 5Laboratoire
de Biochimie Métabolique, IFB, CHU Purpan, Toulouse, France. 6 INSERM UMR 1037, CRCT,
Université Paul Sabatier Toulouse-III, Toulouse, France.
7Laboratory for Molecular Cancer Biology, Center for the biology of disease, VIB, 3000
Leuven, Belgium. 8Laboratory for Molecular Cancer Biology, Center for human genetics, KU
Leuven, 3000 Leuven, Belgium. 9Corresponding authors: [email protected] (L.L.C.) and
[email protected] (L.LK)
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N°12 - Filippo CIABRELLI
Polycomb-Mediated Repression in Transgenerational Epigenetic
Inheritance
Transgenerational epigenetic inheritance is a hotly debated phenomenon whereby a
non-genetically determined phenotype can be transmitted to the next generation. So
far, this mode of inheritance has been described in few cases and it was suggested that
chromatin components might be involved, including Polycomb group proteins, which
act as repressors of key developmental genes and coordinate cell differentiation and
proliferation. The molecular mechanisms linking Polycomb-mediated silencing to
transgenerational epigenetic inheritance are far from being understood. Therefore, we
developed an experimental system in Drosophila melanogaster to induce stable
transgenerational epigenetic inheritance, in which alternative gene expression states
can be inherited in the presence of the same genetic sequence. Starting from these
highly stable epilines, we could dissect some of the genetic properties of the induced
epialleles, such as their quantitative inheritance and their ability to trans-communicate.
Moreover, the epialleles displayed synergy in their expression and transmission. One
of the molecular signatures of the epialleles is the differential presence of the
Polycomb repressive complexes and their related epigenetic marks. This diverse
distribution is independent of the transcriptional activity of the downstream genes, at
least in an early developmental stage, and could influence the three-dimensional
organization of the locus involved. Intriguingly Ago2, an RNAi pathway component,
has been found to genetically interact with the epialleles and to be directly bound on
their chromatin, indicating a possible role for the ncRNAs in the expression of the
epialleles and possibly in their transmission. Our results make a case for strong and
stable transgenerational epigenetic inheritance in metazoan and provide a model that is
amenable for the molecular dissection of this phenomenon.
Filippo Ciabrelli 1, Frederic Bantignies 1, Boyan Bonev 1, Simon Fellous 2, Anne
Xuereb 2, Giacomo Cavalli 1.
IGH, CNRS UPR 1142, 141 rue de la Cardonille, 34090
Montpellier.CBGP, Avenuedu Campus Agropolis, 34980 Montferrier-sur-Lez.
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Posters : Session 1
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N°1 - Marion LE GALL
Quantifying the pressure thresholds for tissue injury in paraplegic
patients
A pressure ulcer (PU) is a localized injury to the skin and/or the underlying tissue
mainly affecting people who lost their mobility or with sensory impairment. It is
associated with morbidities, a decrease of the patient life quality and increase of the
hospital length of stay. Nowadays prevention is still the best way to prevent PU.
However, no damage threshold is unanimously accepted by professionals and the
patient specificities would make any average limit level irrelevant for clinical use. The
main goal of this research project is to gain a thorough quantitative understanding of
the relationship between interface pressures (IP), microvasularisation and tissue
inflammation involved in PU genesis. The objective is to detect, for each patient, the
early stage of irreversible cell damage. Interface pressures between the patient body
and the air-mattress and local microvascularisation measurement are continuously
recorded on paraplegics for 3 hours (corresponding to the routine frequency
of pressure points mobilization). Furthermore, specific inflammation markers can be
quantified from muscle biopsies and correlated with those two parameters. Depending
on how long the patient is lying on the air-mattress and how high the IP intensity is, a
tissue damage threshold could be identified for each patient. The results of this PhD
study will gather new clinical data to improve the PU detection sensibility and the
prevention efficiency.
M. Le Gall M.Sc.1, A. Lacampagne Ph.D.1, L. Teot M.D. Ph.D.2, S. Matecki M.D.
Ph.D.1, C. Trial M.D.2
1
French Institute of Health and Medical Research U1046, CNRS 9214, Physiology
and Experimental Medicine: Heart and Muscles, F-34295 Montpellier, France.
2
Department of Wound Healing Unit, Montpellier Regional University Hospital,
Montpellier, France
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N°2 - Hayeon BAIK
Role of SUMOylation and Reactive Oxygen Species (ROS) in Acute
Myeloid Leukemia (AML) treatment
Acute myeloid leukaemias (AML) are severe haematological malignancies
characterized by the accumulation of malignant immature myeloid precursors in the
bone marrow. Their treatment mainly relies on chemotherapy. However, relapses are
frequent, which explain the poor prognosis. We have recently shown that SUMO, a
post-translational modifier of the ubiquitin family, and its regulation by Reactive
Oxygen Species (ROS) plays a critical role in AML response to chemotherapeutic
drugs.
A first part of my thesis project aims at charactering the role of SUMO and ROS in
AML chemoresistance. To this aim, I generated AML cell lines (HL-60 and U937)
resistant to clinically used drugs (cytarabine and daunorubicin). We could show that
the chemoresitant AML cells present large increase in ROS production, which are due
to the activation of the Nox2 NADPH oxidase at the transcriptional level. Knock
down of Nox2 could sensitize AML cells to chemotherapeutic drugs. In addition,
these cells present higher levels of SUMO. My project now aims at continuing the
characterization of the role of Nox2 and SUMO in chemoresistance, in particular
through the use of patient samples and animal models.
Differentiation therapies constitute a promising alternative to chemotherapy for AML
treatment. However, their clinical use is currently limited to acute promyelocytic
leukaemia (APL), a subtype of AML, that is successfully treated by all-trans-retinoic
acid (ATRA) induced-differentiation. So far, ATRA or other differentiating agents
such as Vitamin D3 have not been successful for the treatment of non-APL leukemias.
The second part of my project is to study whether the inhibition of sumoylation could
promote the ATRA or Vitamine D3 induced differentiation of non APL AML. To this
aim, the role sumoylation in AML differentiation, in particular in the regulation of
gene expression, will be investigated both by pharmacological and genetic
approaches.
Hayeon Baik, Tamara Salem, Yosr Hichri, Guillaume Cartron, Jean-Emmanuel Sarry,
Christian Récher, Marc Piechaczyk Guillaume Bossis
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N°3 - Marko RISTIC
Global regulation of the SUMO pathway during Acute Myeloid
Leukemia treatment with chemotherapeutic drugs
Acute Myeloid Leukemia (AML) is a severe hematological malignancy with poor
prognosis, whose treatments have not significantly changed during the past 30 years.
The standard induction chemotherapy relies on a combination of the nucleoside
analogue cytarabine (Ara-C) with an anthracyclin, such as daunorubicin (DNR). We
have recently reported that one of their early effects is the deconjugation of the
ubiquitin-like protein SUMO from its targets. This desumoylation is due to Reactive
Oxygen Species(ROS)-dependent inhibition of the SUMO-conjugating enzymes
through the formation of a disulfide-bond between E1 and E2 catalytic cysteines. This
desumoylation is involved in transcriptional activation of specific genes and
participates in the induction of apoptosis. In chemoresistant AML cells,
chemotherapeutic drugs do not induce this ROS/SUMO axis. However, its
reactivation by pro-oxidants or inhibition of the SUMO pathway restores apoptosis in
chemoresistant cell lines and patient samples. Finally, inhibition of the SUMO
pathway decreases tumor growth in mice xenografted with chemoresistant AML cells.
Thus, targeting the ROS/SUMO axis might constitute a novel therapeutic strategy for
AML patients resistant to conventional chemotherapies. To understand, at a global
scale, which proteins are changing their sumoylation upon AML chemotherapeutic
drug treatment, we recently performed, in the model AML cell line HL60, a SILAC
Mass spectrometry approach. We could identify around 1000 sumoylated
proteins.Among them, around 100 were desumoylated upon short DNR or Ara-C
treatment (2 hours). These desumoylated proteins were mostly involved in the
regulation of gene expression, confirming the role of genotoxics-induced
desumoylation in the control of transcription. Interestingly, a small group of proteins
showed up-sumoylation upon DNR and Ara-C treatment. These proteins are mostly
involved in DNA Damage Response suggesting that their up-sumoylation could be
involved in DNA repair. Our ongoing work is aimed at characterizing the role of their
sumoylation in AML response to chemotherapeutic drugs.
Jon Otti Sigurdsson and Jesper V. Olsen - Center for protein research, Novo Nordisk
Foundation, Copenhagen
Marko Ristic and Guillaume Bossis - IGMM, CNRS, Montpellier
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N°4 - Loic LIONNARD
Identification of factors involved in proteasomemediateddegradation of anti-apoptotic Bfl-1
Apoptosis or programmed cell death plays a crucial role in tissue homeostasis and is
regulated by the Bcl-2 proteins, which control mitochondria membrane permeability
and cytochrome c release, two events that precede cell demise. Anti-apoptotic Bcl-2
family members can contribute to tumorigenesis and cause resistance to anti-cancer
regimens, therefore representing important targets for novel therapeutics. Bfl-1, an
anti-apototic Bcl-2 protein is often overexpressed in poor prognosis lymphoma and
chemoresistant melanoma. Since no inhibitor blocks efficiently Bfl-1-mediated
prosurvival activity in tumor cells, alternative strategies have to be found to inhibit it.
Previous studies have shown that Bfl-1 is a short-lived protein, whose expression is
tightly controlled by ubiquitination. Indeed, Bfl-1 can be ubiquitinated on its Cterminal part and degraded by the proteasome. Moreover, the phosphorylation status
of proximal residues 152S and/or 156T inhibits proteasome-mediated Bfl-1
degradation. Our preliminary results of in vitro phosphorylation assay identified
GSK3 as a protein kinase involved in the phosphorylation of Bfl-1 C-terminal part. In
addition, this work identified TRIM28 and TRIM17, two members of the TRIM
protein family (one of the subfamilies of RING type E3 ubiquitin ligases), as
interactors that may influence polyubiquitination and proteasomal turnover of Bfl-1.
Overall, the thesis project aims at characterizing the mechanisms through which these
factors control Bfl-1 stability. This work may open new avenue to promote Bfl-1
degradation and thus reactivate apoptosis in tumors that are reliant on Bfl-1
overexpression.
Loïc Lionnard1, Aurélie Cornut-Thibaut2, Abdel Aouacheria2, Claude Cochet3,
Solange Desagher1 and Jérôme Kucharczak1,2.
1 Institut de Génétique Moléculaire de Montpellier, IGMM UMR 5535 CNRS, 1919
route de Mende, 34293 Montpellier cedex 5, France.2 Laboratoire de Biologie
Moléculaire de la Cellule, Faculté de Médecine Lyon-Sud, LBMC UMR 5239 CNRS –
UCBL – ENS Lyon - HCL, 46 Allée d’Italie, F-69364 Lyon Cedex 07, France.3 CEA
Grenoble, iRTSV/BCI INSERM U1036, 17 rue des Martyrs, 38054-Grenoble, France
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N°5 - Fanny IZARD
H4K20 methylation pathway, chromatin structure and
tumorigenesis
Assembled with DNA to form nucleosomes, histone proteins are the basic building
blocks of chromatin. A striking feature of histones is that they are subject to a large
number of post-translational modifications, including phosphorylation, acetylation,
and methylation. These histone modifications are thought to contribute to the
regulation of all DNA-templated processes by mediating both alterations in chromatin
structure and recruitment of non-histone proteins to specific regions of the genome.
Lysine methylation is one of the most intriguing histone modifications in view of its
remarkable complexity. It is detected on many histone lysines, each of which can be
mono-, di-, or tri-methylated. These modifications occur at highly conserved positions
and often cluster within specific regions of the genome to organize chromosomes into
distinct structural and functional domains.
PR-Set7 is the sole enzyme responsible for the monomethylation of histone H4 at
lysine 20 (H4-K20me1), which is required for subsequent catalysis of H4-K20 di- and
tri-methylation (H4-K20me2/me3) via the activity of SUV4-20h enzymes. Loss and
gain-of function experiments have shown that the concerted activity of H4-K20
enzymes is essential for the regulation of chromatin structure, DNA replication and
repair mechanisms, thereby establishing this chromatin-signaling pathway as central
in the maintenance of genome integrity. In line with this, breast, colorectal and
prostate tumors are often characterized by alterations in levels of different H4-K20me
states, which are suspected to play a role in both genesis and progression of cancer
cells. The main objective of my thesis is to unravel the functions of H4-K20 enzymes
and their marks in the regulation of chromatin structure and determine how alterations
in the activity of these enzymes contribute to tumorigenesis. Here, I will show my
most promising results and the characterization of new chemical inhibitors of PR-Set7
activity.
Fanny IZARD Eric JULIEN (équipe Eric JULIEN, IRCM)
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N°6 - Maude LEVEQUE
Toxoplasma gondii AuTophaGy protein 8 has an unusual role in
apicoplast division which is essential for parasite growth
Autophagy is a self-degradative process which is conserved in most Eukaryotes. It
involves a double membrane compartment, called the autophagosome, to sequester
and degrade intracellular components. The protein ATG8 (LC3 in mammals) can
associate with autophagosomal membranes and it occupies a central position in the
autophagy process. Toxoplasma gondii is an obligate intracellular protozoan parasite
that can cause congenital toxoplasmosis and a severe pathology in immunocompromised individuals. This parasite possesses an apparently reduced autophagic
machinery but is nevertheless able to generate TgATG8-decorated autophagosomes
upon stress conditions. Surprisingly in normal growth conditions, this protein mainly
localizes to the apicoplast, a non-photosynthetic plastid which hosts essential
metabolic pathways. This organelle has been acquired through secondary
endosymbiosis and is thus surrounded by four membranes. Biochemical analyses,
together with super resolution microscopy imaging, suggest an outer membrane
localization of TgATG8 at the apicoplast. With the aim of elucidating the function of
this protein, we have generated a TgATG8 conditional mutant. This cell line is
severely affected in cell-growth and in the maintenance of the apicoplast. The loss of
the organelle occurs rapidly upon parasite division and remaining plastids accumulate
in the residual body within the parasitophorous vacuole. This phenotype seems to be a
consequence of a replication and/or targeting defect that prevents proper segregation
of the organelle into daughter cells during division. Taken together, these results
suggest an unusual and essential role for TgATG8 in apicoplast division which is
likely independent from canonical autophagy. To decipher this function, we have
developed a strategy to label in situ, purify, and identify by mass spectrometry,
specific TgATG8 apicoplast partners.
Maude LEVEQUE1, Laurence BERRY1, Michael CIPRIANO3, Martial SEVENO2,
Boris STRIEPEN3, Sébastien BESTEIRO1
1 DIMNP- UMR5235 CNRS, Université de Montpellier. 2 Plate-forme de
Protéomique Fonctionnelle, IGF - UMR5203, CNRS, Université de Montpellier. 3
Center for Tropical and Emerging Global Diseases and Department of Cellular
Biology, University of Georgia, Athens, USA
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N°7 - Sarah GUIZIOU
An open access part toolbox to tune genetic expression in Bacillus
subtilis.
Bacillus subtilis is the Gram-positive model and is highly used in industry for enzyme
and antibiotic production, yet tools to precisely tune gene expression levels are not
widely available. Here we engineered a toolbox of regulatory components with
variable strengths (e.g. promoters, RBS) for precisely tuning gene expression in B.
subtilis.
We first implemented a modular and standardized cassette for genetic circuit
construction in B. subtilis to make part constructions and modifications easier and to
standardize genetic context. We then selected several promoters found to be
constitutive over 104 conditions in the Basysbio project, along with several RBS of
various ranks. We then used 3 divergent sequences as templates for randomization and
obtained libraries of parts with high variability of strength (range of 500 for promoters
and 20 for RBS). We also characterized part activities at the single molecule level
with 2-photon microscopy using the scanning number and brightness method, pushing
the limits of precision measurements of standard parts activities.
This open source toolbox of regulatory components will support the engineering of
complex genetic circuits in B. subtilis. All constructs and data will be released in the
public domain.
Sarah Guiziou1, Vincent Sauveplane2, Hung-Ju Chang1, Nathalie Declerck1,
Matthieu Jules2, Jerome Bonnet1.
1 Centre de Biochimie Structurale, INSERM U1054, CNRS UMR5048, University of
Montpellier, France. 2 INRA, AgroParisTech, UMR1319 Micalis, Thiverval-Grignon,
France.
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N°8 - Nesrine BENKAFADAR
Oxidative stress: key mechanism of age-related cochlear sensory
hair cell loss
Background and Introduction: In our aging society, age-related hearing loss or
presbycusis is increasingly important. Based on observations of temporal bones from
patients with presbycusis, Schuknecht (Schuknecht and Gacek, 1993) proposed the
classification into three major forms, namely sensory, neural, and strial presbycusis
according to the location of damage (sensory epithelium, spiral ganglion neuron, or
stria vascularis). To date, the mechanisms underlying the age-related hearing loss
remain unclear. Based on our previous study (Menardo et al., 2013) showing that the
premature age-related hearing loss observed in senescence-accelerated mouse prone 8
(SAMP8) mice was correlated with altered levels of anti-oxidant enzymes and
decreased activity of mitochondrial functions, we hypothesis that the oxidative stress
may play a key role in presbycusis.
Methods: To investigate the contribution of the oxidative stress in presbycusis, we
exposed the p3 mouse cochlear explants to hydrogen peroxide (H2O2) in vitro. The
cochlear cell senescence or degeneration was evaluated using the specific biomarkers.
In addition, the role of endogenously-produced ROS in age-related hearing loss was
assessed in adult p66KO mice which have a decreased ROS production.
Results: Our results provide the evidence that the oxidative stress plays a key role in
age-related hearing loss and cochlear sensory hair cell apoptosis. We demonstrate that
H2O2 exposure induced a premature occurrence of cochlear sensory hair cell
senescence and apoptosis, illustrated by the massive increase of the cell senescence
and apoptosis biomarkers such as SA-betagal, gH2AX, AnnexinV and TUNEL,
mainly in the cochlear sensory hair cells, but, not in the spiral ganglion neurons.
Interestingly, our in vivo results from p66 KO and wt mice provided the functional
and morphological evidence that the targeting of oxidative stress by pharmacological
or genetic interventions protect the cochleae against age-related sensory hair cell death
and hearing loss.
Conclusion: Our results suggest that oxidative stress plays crucial role in age-related
cochlear sensory hair cell degeneration and hearing loss. The use of anti-oxidants may
be an attractive therapy to slowdown or stop the sensory presbycusis.
Nesrine Benkafadar1,2*, Florence François1,2*, Jean-Luc Puel1,2 and Jing
Wang1,2
1: INSERM - UMR 1051, Institut des Neurosciences de Montpellier, 34295
Montpellier, France. 2: Université Montpellier, 34295 Montpellier, France.* : these
authors contributed equally
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N°9 - Laurent FERNANDEZ
Membrane dynamics and partitioning of CD9 and CD81 are
differentially regulated by the actin network
Tetraspanins CD9 and CD81 are transmembrane proteins forming a network of
interactions at the plasma membrane of eukaryotic cells and often described as
membrane organizers. This network, composed of tetraspanins and of non-tetraspanin
partners, is very dynamic, e.g. most of the tetraspanin CD9 molecules can display free
lateral diffusion although some are confined in tetraspanin-enriched areas.
Using single molecule tracking experiments, this study shows that CD81 molecules
can freely diffuse within the membrane but are more confined and less dynamic than
tetraspanins CD9. Although both proteins are often co-localized and share similar
partners like EWI-2 and CD9P-1, we have demonstrated that their differential
behavior is mainly due to a preferential interaction of CD81 with the cytoskeleton that
restricts its diffusion at the plasma membrane. This link is mediated by the C-terminal
moiety of CD81 through its interaction with ezrin/moesin/radexin (ERM) proteins and
also implicates EWI-2 and CD9P-1. Interestingly, our work also reveals the functional
relevance of this association with the cytoskeleton in the recruitment of CD81 and
CD9 during the assembly of viral Gag proteins.
Laurent Fernandez1,2, Patrice Rassam1,2 , Selma Dahmane1,2, Patrice Dosset1,2,
Markus Thali3,4, Eric Rubinstein5,6, and Pierre-Emmanuel Milhiet1,2
1
Inserm, Unité 1054, Montpellier, France. 2 Université de Montpellier, CNRS, UMR
5048, Centre de Biochimie Structurale, Montpellier, France.3Graduate Program in
Cell and Molecular Biology, and 4Department of Microbiology and Molecular
Genetics, University of Vermont, Burlington, VT 05405 USA. 5 Inserm, U602, Villejuif,
France.6 Université Paris 11, Institut André Lwoff, Villejuif, France
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N°10 - Emilande GUICHET
High viral load in patients failing first-line ART is associated with
multidrug resistance in resource limited countries
Background: Access to ART expands, emergence of HIV drug resistance is inevitable,
especially in resource limited countries where biological monitoring is limited. The
objective of this study was to describe drug resistance mutations in patients that where
eligible for a second line treatment in the 2LADY-ANRS12269/EDCTP trial
conducted in Africa.
Methods: Genotypic HIVDR testing was performed on 454 patients with virological
failure (i.e. two plasma HIV-RNA viral load (VL) >1000 copies/ml after adherence
intervention) in Cameroon (n=304), Burkina-Faso (n=91) and Senegal (n=59). They
were taking first-line ART composed by two nucleoside (3TC+AZT/d4T) and one
non-nucleoside reverse transcriptase inhibitors (EFV/NVP). HIVDR was determined
using the ANRS (v24.2014) algorithm.
Results: Genotyping was successful for 446/454 patients and 440(97.7%) were
resistant to at least one drug. The M184IV mutation causing resistance to 3TC was
present in 433(97.1%) patients and a similar number was resistant to EFV/NVP. In
addition, 242(54.3%) mutations associated with resistance to AZT/d4T were found. A
subset of patients also accumulated mutations leading to cross-resistance to ABC
(85/446 (19.1%)), DDI (32/446 (7.2%)), TDF (6/446, 1.3%)) or ETR (56/446 (12.6%).
Importantly, multi-resistant strains were observed in patients with high viral loads:
95/122 (77.9%), 125/219 (57.6%) and 24/106 (22.6%) of patients with VL>5 log10, 45 log10, and 3-4 log10, respectively were resistant to the 3 drugs of their first line
regimen. Median VL in the 242 multi-resistant patients was 4.7 log10 (IQR 4.3-5.2)
versus 4.2 log10 (IQR 3.7-4.7) in the other 198 resistant strains.
Conclusions: This study highlights the importance of early detection of treatment
failure. The extensive accumulation of HIVDR may compromise second-line
regimens. High viral loads in multi-resistant patients increase the probability of
transmission of these strains. These data stress the need of biological monitoring and
advocate for more robust first-line regimens and continuous surveillance of HIVDR in
resource limited countries.
E. Guichet1,2, A. Aghokeng1,2, L. Serrano1, G. Bado3, C. Toure Kane4, A. Sawadogo4, C. Tidiane
Ndour5, S. Koulla-Shiro6, S. Eymard-Duvernay1, E. Delaporte1, L. Ciaffi1, M. Peeters1 and the
2LADY-study group.
1. UMI233-TransVIHMI/ INSERM U1175/ UM, IRD,, Montpellier, France.
2. IMPM/CREMER, Yaoundé, Cameroun. 3. Hôpital de jour de Bobo-Dioulasso, BoboDioulasso, Burkina-Faso. 4. Laboratoire de Bactériologie-Virologie, CHU Le Dantec, Dakar,
Sénégal. 5. UCAD, Dakar, Sénégal. 6. Université Yaoundé 1, Yaoundé, Cameroun
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N°11 - Viviana DELGADO BETANCOURT
Cardioprotection against ischemia-reperfusion injury by heart rate
control
Background: Acute myocardial infarction (AMI) is the major cause of mortality
worldwide. Early reperfusion is the only treatment recommended to reduce infarct
size. However, reperfusion induces also deleterious secondary effects called ischemiareperfusion (IR) injury due to irreversible apoptotic death of cardiomyocytes. Most
ischemic episodes are triggered by an increase in heart rate that induces an imbalance
between myocardial oxygen delivery and consumption. The BEAUTIFUL clinical
trial has demonstrated that moderate heart rate reduction diminishes the frequency of
AMI episodes in patients with stable coronary artery disease having increased heart
rate at rest. The HCN-mediated If current and the Cav1.3-mediated L-type Ca2+
currents play important roles in the generation of automaticity and heart rate, therefore
they are interesting targets for selective control of heart rate and cardioprotection
during AMI. The aim of this study was to investigate if Cav1.3 channels could be a
putative target to reduce infarct size.
Methods: Anesthetized C57BL/6J, Cav1.3-/- and Girk4-/- mice were subjected to a
surgical protocol of myocardial IR (40 min ischemia-60 min reperfusion). Heart rate
was measured with a one-lead surface ECG recording, and infarct size with triphenyl
tetrazolium chloride staining.
Results: Selective heart rate decrease (-26%) in an in vivo mouse model of AMI is
associated with reduced IR injury. Ivabradine administration before ischemia
significantly reduced infarct size (-33%). Cav1.3-/- mice presented reduced infarct
size (-30%) compared to WT mice. In addition, Girk4-/- mice, a genetic model of
moderate tachycardia (10%) displayed increased infarct size (+30%) compared to
control
mice.
Conclusions: These results show a direct relationship between heart rate and IR injury.
Heart rate reduction by inhibition of Cav1.3 channels constitutes a promising strategy
to reduce infarct size.
Delgado Betancourt V1; Covinhes A1; Mesirca P1; Bidaud I1; Nargeot J1 ; Piot C1;
Striessnig J2;Mangoni M.E1; Barrère-Lemaire S1.
(1) Institut de Génomique Fonctionnelle, CNRS UMR 5203, Inserm U 661, Université
de Montpellier, Montpellier-France, (2) Center for Molecular Biosciences, University
of Innsbruck, Innsbruck-Austria
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N°12 - Omar DIOURI
Development of a mobile application to compute food
carbohydrates
Background: Estimating correctly the right amount of carbohydrates (CHO, carb)
present in the meal is important for type 1 diabetic patients. We developed an Android
application (app) to help them in this task, and this paper presents a preliminary study
on it.
Methods: 10 patients used our application for one week, they typed each meal on the
application in blind mode, and they wrote on a logbook their own decisions. Data
collected concerned mainly meal compositions and CHO content, time needed for
entering meals in the applications, and when possible postprandial blood glucose.
Results: 187 meals were analysed, with an average CHO quantity of 74.12 ± 32.51
grams, with a mean delta of 2.773 ± 8.91 grams, and an overall underestimation of
patients compared to the APP. The average CHO quantity of lunches and dinners is
significantly higher than in breakfast, with a Delta-SD twice times higher. CHO
estimation errors are correlated to post-prandial blood glucose (p=0.0015).In fourth of
hypoglycaemias, CHO content was underestimated by more than -9 grams, and for
hyperglycaemias it was overestimated by more than 13.5 grams. The mean time to
enter a meal diminished between the beginning of the week and the end (p=0.0003).
Conclusion: Lunches and Dinners are meals with a high CHO content, and presents a
higher discrepancy estimation. Those false estimations results in out-of-range BG
excursions. A long-term clinical study will be led to measure more precisely the
impact of the application on patient’s health.
O. Diouri , J. Place , M. Traverso , E. Renard
Institut de Génomique Fonctionnelle, UMR CNRS 5203/INSERM U1191, Université
de Montpellier. Département d’Endocrinologie, Diabète, Nutrition, CHRU de
Montpellier, Montpellier
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N°13 - Xiaobin ZHANG
Adrenergic receptor gene variants are associated with late-onset
generalized anxiety disorder
Background: Generalized anxiety disorder is a common chronic condition that is
understudied compared to other psychiatric disorders. Evidence from family studies
suggests a relatively high heritability for GAD but few candidate genes have so far
been identified. An altered adrenergic function has been reported in GAD however,
direct evidence for genetic susceptibility is missing.
Objectives: The current study aimed to evaluate the associations of gene variants in
adrenergic receptors (ADRs) with late-onset GAD.
Methods: Data were obtained from 844 French community-dwelling elderly aged 65
or over. Anxiety disorders were assessed using the Mini-International
Neuropsychiatry Interview, according to DSM-IV criteria. Eight single-nucleotide
polymorphisms (SNPs) involved with adrenergic function were genotyped; 2 alpha(1)adrenergic receptor subtypes, alpha(1A) (ADRA1A), alpha(2A) (ADRA2A) and beta2
(ADRB2) receptor and the transcription factor TCF7L2. Multivariate regression
analyses adjusted for age, sex, physical and mental health comorbidity and adverse
life events.
Results: Significant associations were found for five SNPs and for ADRA1A
rs17426222 and rs573514, the associations remained significant after correction for
multiple testing. No significant associations were found with the 2 ADRA2A variants.
Gene-environment interaction was found between certain variants and stressful life
events in influencing risk of GAD. All associations appeared specific to GAD; in posthoc analysis no significant associations were found between ADR variants and
phobia.
Conclusions: This is the first study showing that ADR variants are susceptibility
factors for late-onset GAD, but not the other major anxiety disorder, especially
phobia. These data support the critical role of the adrenergic nervous system in GAD
and in particular in response to stressful adverse life events.
Inserm U1061
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N°14 - Anne BIEVER
Role of the ribosomal protein S6 phosphorylation in the mouse
brain
The 40S subunit ribosomal protein S6 (rpS6) has attracted much attention over the last
three decades since it is the first ribosomal protein shown to undergo inducible
phosphorylation upon a wide variety of stimuli. However, despite the profound
elucidation of the respective kinases and extracellular stimuli triggering phosphorpS6, little is known about the physiological role of the phosphorylation. In this study,
we aimed to charactherize the functional relevance of the in vivo rpS6
phosphorylation in the brain. For this purpose, we performed a behavioral
characterization of rpS6P-/- knock-in mice, in which all phosphorylatable serine
residues are substituted by alanines. Interestingly, these mice display a reduced
locomotor response to d-amphetamine, which markedly increases phospho-rpS6 in the
striatum of wild-type mice. Moreover, they show impaired synaptic plasticity of
GABAergic medium-sized spiny neurons in the nucleus accumbens. Finally, since
rpS6 phosphorylation has been widely correlated with changes in protein synthesis,
we performed polysome profile and puromycin incorporation analyses in rpS6P-/knock-in as well as in wild-type mice treated with d-amphetamine. Intriguingly, we
found no differences in global translational rates in the striatum, hippocampus and
frontal cortex. Altogether, our results indicate that neither basal nor drug-induced
transient rpS6 phosphorylation correlate with changes in overall mRNA translation.
Nevertheless, despite the lack of causal relationship between both events, rpS6
phosphorylation plays an important role in striatal plasticity and behavioral responses.
Anne Biever1,2,3*, Emma Puighermanal1,2,3*, Oded Meyuhas9, Emmanuel Valjent1,2,3
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094,
France. 2INSERM, U661, Montpellier, F-34094, France. 3Universités de Montpellier 1
& 2, UMR-5203, Montpellier, F-34094, France .9Department of Biochemistry and
Molecular Biology, The Institute for Medical Research – Israel-Canada, The Hebrew
University-Hadassah Medical School, Jerusalem 91120, Israel.
1
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N°15 - Mona HOUHOU
Characterization of a Cancer Stem cells enriched subpopulations
and role of EMT Regulators in basal Breast Cancer Cell Plasticity
Breast cancer is not a single disease, but rather is composed of different subtypes
associated with different clinical outcomes and molecular characteristics. A better
understanding of the heterogeneity is the key to the development of targeted
therapeutic interventions. This heterogeneity may be explained by the concept of
cancer stem cells (CSC). In breast cancer, a number of markers have been proposed to
isolate and characterize breast CSC. A panel of markers such as CD44/CD24/EpCAM,
ALDH, and capacity of mammospheres formation is used, but the question on CSC in
ER (-) breast cancer remains to be clarified and the mechanism underlying the
aggressive behavior of triple negative breast cancer (TNBC) is not well understood
yet.
To this purpose, SUM 159, MDA-MB-436 and MDA-MB-468 cell lines are used as
ER (-) models to search for the cell fractions enriched for CSC. It has been found that
the CD44+/CD24- cell population is enriched in TNBC tissues and cell lines, with a
higher capacity of proliferation, migration, invasion and tumorigenicity as well as
adhesion ability. In addition, a high level of CD44+/CD24- and ALDH+ cells in
primary breast tumors was correlated with the presence of distant metastasis.
Furthermore, CD44+ positivity has been clearly associated to the acquisition of a
mesenchymal phenotype, suggesting a link between EMT and stemness.
All different cell populations will then be tested for mammosphere formation and
differentiation in Matrigel in comparison with the “negative” fractions. In addition,
RNA profiling analysis will allow us to study various factors involved in EMT in
different cell fractions. The idea is to identify a signature of genes defined as "stem"
that we can analyze by qPCR and in particular by recent techniques of single-cell
qPCR (Fluidigm). It would indeed be interesting to test variations of expression of
these genes in the different cellular fractions enriched in CSC.
Keywords : Triple negative breast cancer (TNBC), Cancer stem cells (CSC),
CD44+/CD24-, ALDH+, Epithelial-mesenchymal transition (EMT), dedifferentiation,
regulators of EMT
Mona Houhou, directeur de thèse: Charles Theillet, Equipe: Plasticité phénotypique
et génétique du cancer, IRCM U1194.
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N°16 - Benoit GIRARD
Metabotropic glutamate receptor type 7 (mGlu7) modulation of
thalamocortical activity.
Thalamocortical activity depends on the balance between glutamatergic and
GABAergic activity in order to guarantee correct somatosensory computation and
sleep/wake cycle. The metabotropic glutamate receptor type 7 (mGlu7) is expressed at
the presynapse where it inhibits neurotransmitter release. We have previously shown
that mGlu7AAA KI mice, in which the receptor intracellular signalling is lost, develop
spontaneous absence-like seizures similar to the human pathology, absence epilepsy.
The present study aimed at understanding the function of mGlu7 at specific thalamic
synapses using in vitro patch-clamp recording on thalamocortical slices and in vivo
EEG recording in both wild-type and mGlu7AAA knock-in mice. We found that
mGlu7 inhibits the synaptic inputs onto thalamic reticular nucleus (TRN) neurons,
coming from either the ventroposteromedial nucleus (VPM) or within the TRN itself.
Specific activation of long-range thalamocortical projections was achieved by using
viral-mediated stereotaxic expression of ChannelRhodopsin 2, a light-sensitive
cationic channel. This approach showed mGlu7-mediated inhibition of cortical layer 4
fast-spiking interneurons, as opposed to regular spiking interneurons or pyramidal
cells. Remarkably, on top of the conventional, agonist-dependent activity, the receptor
showed but also a constitutive, agonist-independent activity both in vitro and in vivo.
This constitutive activity was suppressed by the negative allosteric modulator (NAM),
ADX71743. In vivo administration of the NAM altered thalamocortical EEG
recording, with spike-and-wave discharges typical of absence seizures and a general
induction of drowsiness in the animal. The results suggest a major role of the mGlu7
receptor in the tonic control of thalamocortical function.
Benoit Girard1,2,3*, Valériane Tassin1,2,3* , Apolline Chotte1,2,3, Pierre Fontanaud1,2,3,
Delphine Rigaud4, Julie Perroy1,2,3, Francine Acher4, Laurent Fagni1,2,3, Federica
Bertaso1,2,3
1
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094,
France, 2 INSERM, U1191, Montpellier, F-34094, France, 3 Universités de
Montpellier, UMR-5203, Montpellier, F-34094, France, 4 Université Paris Descartes,
CNRS UMR-8601, Paris, F-75006, France.
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N°17 - Margarida GOMES
Modeling the role of the global regulator ShvR of Burkholderia
cenocepacia in virulence using zebrafish embryos
Burkholderia cenocepacia has been demonstrated to be capable of surviving and
multiplying inside cells by evading host cell defense mechanisms. In our laboratory,
we have developed a zebrafish model to study virulence caused by bacteria belonging
to the Burkholderia cepacia complex, and have shown that macrophages provide an
intracellular niche for Bcc in vivo. The model allowed us to distinguish strains that
cause an acute, rapidly fatal infection, including the B. cenocepacia ET12 lineage
strains, and strains that were persistent.
In this study zebrafish embryos were used to further understand a role in virulence for
the LysR-type transcriptional regulator ShvR. Initially identified based on its role in
colony morphology, previous infection experiments in alfalfa seedlings and rats have
shown that this regulator is important for virulence, and inflammation in rat lungs.
Interestingly, bacterial numbers were, paradoxically, sometimes higher in animals
infected with the mutant compared to wildtype, suggesting the shvR mutant was
highly persistent.
Here, we show, in agreement with these earlier results, that the shvR mutant is
attenuated in virulence in zebrafish embryos, with a marked reduction in proinflammatory responses and tissue inflammation. Using real time non-invasive
imaging, we observed that the shvR mutant persists in macrophages and, in contrast to
the wildtype, is unable to disseminate from infected host cells. This persistent
phenotype is reminiscent to those we described earlier for strains including B. stabilis
LMG14294 and B. vietnamiensis LMG14942. We will present our results on the host
phagocyte and immune response to infection with the B. cenocepacia shvR mutant,
and further describe our recent efforts to show a role for this global regulator in
persistent versus virulent infections.
Margarida Gomes1,2, Sujatha Subramoni3, Pamela Sokol3 and Annette Vergunst1,2
1
INSERM, U1047, UFR Médecine Site de Nîmes, 30908 Nîmes, France. 2 Université
de Montpellier, U1047, UFR Médecine, 30908 Nîmes, France. 3 Department of
Microbiology, Immunology, and Infectious Diseases, University of Calgary, Calgary
T2N 4N1, Canada
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N°18 - Chantal MAGHAMES
Ubiquitin under stress : “ Going hybrid with NEDD8”.
Cells constantly respond to stress signals that damage proteins and/or DNA. Defects in
repair or elimination of damage can lead to many diseases such as cancer or
neurodegeneration. Ubiquitin and Ubiquitin-like molecules are implicated in the
control of myriad biological processes including detection and repair of damaged
proteins/DNA. This family of proteins covalently modifies proteins through the action
of E1, E2 and E3 enzymes. Historically, it was believed that each pathway employ its
own and unique set of enzymes to post-translationally modify their substrates.
We discovered that upon proteotoxic stress including proteasome inhibition, heat
shock and oxidative stress, the Ubiquitin-like molecule NEDD8, is activated by the
Ubiquitin E1 enzyme Ube1. This results in a global increase in NEDDylation and the
formation of ploy- NEDD8 and mixed chains between NEDD8 and Ubiquitin. The
process is reversible and cell recovery is accomplished once stress is alleviated.
Our goal is to identify the cellular components that participate in this stress response,
the mechanisms mediating the recovery process and to understand the biological
significance for this increase in protein NEDDylation.
Our results show that this accumulation of NEDDylated/Ubiquitinated substrates is
dependent on the heat shock proteins 70/90, on protein synthesis and is characterized
by a progressive translocation of the modified substrates from the cytosol to the
nucleus leading to a nuclear aggregation after a severe stress. As for the recovery
process, we found that nuclear proteasomes directly interact with and eliminate these
conjugates.
Future steps will include the identification of substrates that aggregate upon heat
shock through the formation of mixed chains and the potential biological significance
of this increased NEDDylation. In addition, we aim to characterize the recovery
process by proteasomal degradation.
Chantal MAGHAMES and Dimitris XIRODIMAS
Centre de Recherche de Biochimie Macromoléculaire - UMR 5237, CNRS, Route de
Mende
34 293, Montpellier,
Cedex 5, France
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N°19 - Amélie BORIE
Modulation of lateral septal neurons by oxytocin and vasopressin,
neuropeptides involved in the regulation of social behavior.
Central oxytocin (OT) and vasopressin (VP) have almost opposite roles in mammal
behavior: OT facilitates social interactions while VP promotes aggressivity and
anxiety, but their mechanism of action is unknown.
We study the mouse lateral septum, an exception where both OT and VP can increase
social interactions and social memory. This structure expresses significant amount of
OT and VP receptors but electrophysiological consequences of their activation have
not been studied in detail so far.
We used patch clamp recordings from acute brain slices to characterize
electrophysiological responses of mouse lateral septum neurons to TGOT (specific OT
receptor agonist) and vasopressin. Strikingly, the firing frequency of almost all
recorded neurons was significantly modulated by at least one of these peptides.
Accordingly, we classify lateral septal neurons into three populations: i) inhibited by
both peptides; ii) activated by oxytocin; iii) activated by vasopressin.
Because the septo-hippocampal network is involved in theta rhythm regulation, we
analyzed the effect of OT and VP on spike patterning. We observed that modulation of
electrical activity results from a modification of inter-burst intervals (1.5-5 s) rather
than intra-burst frequency (close to the Theta rhythm, 3-5 Hz).
With the aim of deciphering a local microcircuitery involving the three abovementioned neuronal categories, we studied spontaneous synaptic events and
demonstrated that they are mostly GABAergic. Their frequency was increased by
TGOT and VP, opening the possibility that these peptides act locally by regulating
interactions between interneurons.
In the lateral septum, we observe similar effects of OT and VP, consistent with their
behavioral action. We will then compare electrophysiological data obtained in the
lateral septum of WT mice and mice in which the OT or VP system are perturbed and
which constitutes animal models of social diseases.
Work supported by CNRS, INSERM and ANR. A B supported by University of
Montpellier
A M Borie1,2, G Guillon1,2, F Muscatelli3 and M G Desarménien1,2
1
Institut de Génomique Fonctionnelle, CNRS UMR5203, Inserm U1191, Montpellier,
France. 2 Université de Montpellier, Montpellier, France. 3 INMED, Inserm U910,
Marseille, France
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N°20 - Sarah LALISSE
Potential role of P2X4R expressed by sensory neurons in BDNFevoked inflammatory pain
P2X receptors (P2XR) are ATP-activated ion channels. Among the different P2X
subunits, P2X2 and P2X3 expressed by sensory neurons are known to be involved in
both physiological and pathological pain processing. In addition, it has been recently
demonstrated that P2X4R expressed by macrophages and microglial cells play a
crucial role in both neuropathic and inflammatory pain through the release of
proinflammatory molecules or microglial BDNF release.
While P2X4R mRNA is expressed in dorsal root ganglia neurons, the functional
expression of these receptors and their potential role in sensory information processing
is still unexplored. Here we have investigated the functional expression of P2X4
receptors in sensory ganglia and their potential involvement in pain processing.
Our results show that in physiological conditions, P2X4R are functionally expressed
by a subpopulation of sensory neurons, including small and intermediate nociceptive
neurons, suggesting a potential role of P2X4R in pain transmission. In inflammatory
condition, 24h after the injection of Complete Freund Adjuvant in the hind paw, a
subset of nociceptive neurons co-express P2X4R and BDNF. We also demonstrate
that P2X4R are present in central afferents suggesting a potential role of the receptor
in pre-synaptic BDNF release. Indeed, our results show that following peripheral
inflammation several BDNF-dependent signaling pathways are activated in the spinal
cord: activation of ERK1/2, phosphorylation of NR1 subunit and downregulation of
KCC2 cotransporter, while these modifications are not observed in P2X4-deficient
mice.
In inflammatory conditions P2X4R expressed in sensory neurons appear to shape
dorsal horn spinal network excitability by triggering BDNF release by sensory
afferents, thus contributing to inflammatory pain processing.
Lalisse, S. (Montpellier)1, Rassendren, F. (Montpellier)1, Ulmann, L. (Montpellier)1
1
Institut de Génomique Fonctionnelle - CNRS UMR 5203 - INSERM U1191 Université de Montpellier, Montpellier, France
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N°21 - Pascale PALASSIN
Role of RIP140 in familial colorectal cancer
Colorectal cancer (CRC) is a common disorder with familial forms such as Lynch
syndrome which exhibit microsatellite instability (MSI) due to a loss of function of
DNA mismatch repair system (MMR). However, about a quarter of clinically
diagnosed families do not exhibit MMR gene alterations (Lynch Like Syndrome or
LLS) thus implicating new candidate genes. The team has already shown that the
transcription factor RIP140 is involved in sporadic colorectal carcinogenesis. In
addition, preliminary results indicate that RIP140 regulates the expression of some
MMR genes. Furthermore, a mutation in the RIP140 coding sequence (RIPMSI)
which generates a truncated protein has been detected in cells and tissues from MSI
CRC.
The aim of this thesis project is to decipher the role of RIP140 in familial colon
cancer. I will study the regulation of MMR gene expression by RIP140 using different
cell lines and tissues displaying a deregulated expression (overexpression or knockdown) of the RIP140 gene. I will analyze the impact of this regulation on mismatch
repair activity and on the susceptibility to various cytotoxic drugs. In parallel, I will
study the effects of RIPMSI on parameters previously described and I will search for
this mutation at the DNA and protein levels in a cohort of MSI CRC patients.
By decreasing MMR gene expression, the inactivation of RIP140 could affect DNA
repair and promote colorectal tumorigenesis. The RIPMSI mutation might explain the
microsatellite instability in patients where no MMR gene mutation is found and
become a useful biomarker for LLS diagnosis.
Keywords : Colorectal cancer, Lynch syndrome, RIP140, Mismatch repair, DNA
repair.
Pascale PALASSIN, Marion LAPIERRE, Carole CORSINI, Sandrine BONNET,
Stéphan JALAGUIER, Audrey CASTET-NICOLAS, Vincent CAVAILLES
IRCM, INSERM U1194, Montpellier
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N°22 - Solene BRELLE
Deciphering the function of the Serine/Threonine Protein Kinase
CStk from Coxiella burnetii and its role during host infection.
Coxiella burnetii is an intracellular Gram negative bacterial pathogen. It is the
causative agent of Q fever, an emerging zoonosis with severe health and economic
impact. The symptoms of Q fever range from pneumonia, fatigue and hepatitis in the
acute form of the disease, to severe endocarditis and encephalitis in its chronic form.
After internalisation by the host cell, C. burnetii uses a Dot/Icm type IV secretion
system (T4SS) to translocate effector proteins and divert the cell machinery to
generate its own parasitophorous vacuole (PV). Screening of a C. burnetii transposon
mutant bank indicated that expression of a unique Ser/Thr Protein Kinase (STPK),
CStk (CBU_0175) is important for the intracellular development of the bacteria.
Furthermore, a previous study has shown that CStk could be translocated into the host
cytoplasm using Legionella pneumophila as a surrogate host.
In our study, we will characterise the Ser/Thr protein kinase activity of CStk, confirm
its secretion by Coxiella and identify host targets.
S. Brelle(1), E. Martinez(2), F. Cantet(2), F. Letourneur(1), M. Bonazzi(2) and V.
Molle(1)
1:CNRS-UMR5235-Dynamique des Interactions Membranaires Normales et
Pathologiques, Université de Montpellier, France.2:CNRS/UM-UMR5236-Centre
d'études d'agents Pathogènes et Biotechnologies pour la Santé, Montpellier, France.
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N°23 - Thi Thu Phuong TRAN
Characterization and role of indirectly-activated human dendritic
cells by immune-complex adenovirus in vitro in immune memory
response
Dendritic cells (DCs) are specialized antigen-presenting cells (APCs), and critical
establisment of immunological memory. They also re-stimulate T and B cells and play
a role in the maintenance of their function. Human adenovirus type 5 (HAd5), nonenveloped DNA viruses that primarily cause self-limiting disease, are used as a vector
for vaccination, long-term gene transfer and cancer treatment. Immune complexHAd5 (IC-HAd5) induce functional maturation of human DCs which includes
cytokine and chemokine secretion. Moreover, IC-HAd5 induce a pro-inflammatory
cell death of DCs called pyroptosis. DCs maturation includes up-regulation of major
histocompatibility complex (MHC) class II, costimulatory CD80/CD86 molecules,
and production of pro-inflamatory cytokine and chemokine. Immature DCs express
receptors for inflammatory molecules (eg. type I IFN) released by DCs or other cells
directly activated by pathogen associated ligand. Indirectly activated DCs acquire a
mature phenotype, increasing expression of MHC II and co-stimulatory molecules.
Our question is what happens to immature DCs in environment created by IC-HAd5
stimilated DC? Our goal is to characterize indirectly-activated DCs and their roles in
memory immune response. In addition, we want to identify influential factors in
indirect DCs maturation. We use biochemistry and fluorescences test to characterize
indirectly- stimulated DCs in transferwell. Our results indicated that indirectly
activated DCs increased surface marker expression after 12 hours. Moreover, we
found that the percentage of cell death by IC-HAd5 of the indirectly- activated DCs is
less than directly activated DCs.
Thi Thu Phuong Tran, Karsten Eichholz, Eric J . Kremer(1)*
(1)Adenovirus Laboratory, IGMM, CNRS, Montpellier, France.
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N°24 - Alaa YEHYA
BDNF knockdown induces defects in zebrafish posterior lateral line
development
Derived Neurotrophic Factor (BDNF) the most abundant member of the neurotrophin
family in the human brain, BDNF signaling is mediated by two principal
transmembrane receptors, receptor tyrosine kinase B (TrkB) and p75NTR
neurotrophin receptor (p75NTR). BDNF has been studied extensively for its major
roles, in neuronal differentiation, survival, plasticity, as well as in cognition. In this
study we aimed to investigate further BDNF signaling actions, we used zebrafish
posterior lateral line (pLL) system as a model, which is a mechanosensory system in
fish and amphibians that evolved to detect local water flow and pressure. PLL
development includes different physiological processes including, morphogenesis,
collective migration, and neuronal development. Our results show that Knockdown of
BDNF using antisense morpholino oligonucleotides resulted in abnormal primordium
migration and a significant reduction in the number of deposited neuromasts. In
addition, morphant embryos had a defect in PLL nerve and neuromasts hair cells
regeneration compared to complete regeneration in controls. Collectively these
findings indicate that BDNF signaling has an essential role in the regulation of PLL
development. In addition, the phenotypes observed in response to BDNF knockdown
show the utility PLL system in studying defects in BDNF signaling that might
resemble some conditions in human disorders.
Alaa Yehya, Michelle Silhol, Nicolas Cubedo, Jean-Michel Verdier, Mireille Rossell
1Université Montpellier, Montpellier F-34095, France; Inserm U1198, Montpellier F34095, France; EPHE, Paris F-75014, France
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N°25 - Safa AZAR
Diffuse low grade gliomas: characterization and development of in
vitro model for designing innovative therapeutic approaches
Diffuse low-grade glioma (DLGG) are slow growing grade II tumors that develop in
different functional places in the brain and affect young patients between the second
and the fourth decades of life. Although, they are clinically stable over a long period
of time, these tumors can ineluctably progress to a higher grade of malignancy leading
to anaplasia, which then rapidly compromises patient survival. Most grade II gliomas
(around 80%) have a false sense mutation (R132H) in the IDH1 gene (isocitrate
dehydrogenase) combined with a 1p19q codeletion, mutations in p53 or ATRX. IDH1
mutations induce major dysregulations of DNA methylation leading to defects in cell
differentiation. These tumors, classified as astrocytoma or oligodendroglioma, are
probably derived from neural progenitors which remain in the adult brain. Today, one
important obstacle preventing the development of new therapies for DLGG is the lack
of understanding of the cellular composition of these tumors and the absence of an in
vitro model due to their low proliferation rate. The first aim of our study is to
characterize by immunohistochemistry the different pools of cells causing the
heterogeneous profile of the tumor and to understand the different signaling pathways
that are activated and/or inhibited, as well as the interactions between the cancerous
cells and their environment. The second part is to use different approaches in order to
enhance the cell proliferation including cell immortalization via expressing or
inhibiting the different signaling pathways that are involved in the cell cycle.
Safa Azar(1),Aurélie Gennetier(1), Frédérique Lorcy(2), Valérie Rigau(2), Hugues
Duffau(1) (2), Bernard Rothhut (1), Jean-Philippe Hugnot(1).
(1): Institut for neurosciences of Montpellier, INSERM U1051, Team "plasticity,
neural stem cells and glioma. (2): Hospital Guy De Chauliac, Montpellier
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N°26 - Shefqet HAJDARI
Role of the chromatin associated proteins HP1 (Heterochromatin
Protein 1) in liver hemostasis
DNA is wrapped in a complex structure called chromatin that is known to play
essential roles in establishment and maintenance of cellular identity and to be involved
in cancer development. HP1 are chromatin associated proteins that are believed to
play crucial roles in chromatin dynamics. Using animal models with specific
inactivation of each of the HP1, we have obtained results indicating that HP1 are
important for liver homeostasis and we are now trying to uncover the mechanisms
underlying these functions of HP1.
Analysis of gene expression in 5 weeks old mice with liver specific inactivation of
either HP1g or HP1b in an HP1aKO background (HP1agKO, abKO) show
deregulation of expression of some genes involved in different pathways. Expression
of genes involved in the p53/cell cycle control pathway (p53 and p27) and B-catenin
pathway (Ctnnb1 and Birc5) were increased in the HP1ag mutant as compared to WT
mice. Interestingly, the expression of several cancer related genes such as Tert and AR
is up-regulated in HP1ag or HP1abKO mice respectively. Additionally, genes
deregulated in HP1agKO mice display a strong enrichment in genes of the KRABZFP family, suggesting a loop of auto-regulation between HP1, TRIM28 and KRABZFP. By using TMA (Tissue MicroArray), we observed that the number of AFP (liver
cancer marker) positive cells increased on HP1KO histological sections as compared
to WT. Analysis of Ki67 positive cells as marker of proliferation shows that HP1aKO
liver cells tend to proliferate more. Additionally, livers from both HP1agKO and
HP1abKO mice display hyper-proliferation as compared to WT animals.
Our results demonstrate that HP1 are involved early during the development of mouse
liver and suggest that these early molecular and functional deregulations in liver of
mice lacking HP1 may serve as a basis for cancer development through cell cycle and
aberrant expression of cancer related genes.
Shefqet HAJDARI, Nelly PIROT, Florence CAMMAS
Team of Epigenetics, Cell Differentiation and Cancer, Institute of Cancer Research of
Montpellier (IRCM – U1194)
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N°27 - Boubou DIAGOURAGA
Role of PRDM9 methyltransferase activity in mouse meiotic
recombination
During meiosis, genetic recombination between homologous chromosomes promotes
accurate chromosome segregation and generates new combinations of alleles. DNA
double-strand breaks introduced by the SPO11 protein are repaired by homologous
recombination. This event occurs in 1 to 2 kb-long regions, recombination hotspots.
Recently, PRDM9 has been shown to specify hotspots by targeting specific DNA
sequences through its zinc finger array. The PR/SET-domain of PRDM9 possesses an
H3K4me3 activity, which correlates with a PRDM9-dependent enrichment of
H3K4me3 at active hotspots.
Using in vitro and in vivo strategies, we characterized the catalytic activity of PRDM9
and determined its role in mouse meiosis. We have characterized the structure and the
activity of the PR/SET domain (Wu et al., Cell reports 2013). The crystal structure of
this PR/SET domain in complex with a histone H3 peptide and the cofactor revealed a
topology similar to that of canonical SET domains. To get insights into the substrate
specificity of PRDM9, we performed an in vitro methylation assay on a histone Nterminus tail peptide array with various post-translational modifications.
Methyltransferase activity was detected on H3K4, H3K9, and, with a lower efficiency,
on H3K36. To determine the role of PRDM9 methyltransferase activity in vivo, we
used the G278A mutation, inactivating the catalytic activity of PRDM9 (Hayashi et
al., Nature 2005). Taking advantage of the specificity of two Prdm9 alleles (Prdm9b
and Prdm9wm7), which target each a specific set of hotspots, we generated transgenic
mouse strains expressing the mutant allele Prdm9wm7-G278A and the endogenous
wild-type Prdm9b. We showed that H3K4me3 mark is not enriched and there is no
recombination at Prdm9wm7-specific hotspots in this strain. In mice expressing solely
the mutant allele, meiosis does not progress beyond early stages. Our results show that
PRDM9 methyltransferase activity is required for recombination and completing
meiosis.
Boubou Diagouraga, Frédéric Baudat, Bernard de Massy
Institute of Human Genetics, UPR 1142, CNRS, 141 rue de la Cardonille, 34396
Montpellier, France
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N°28 - Hélène DONNADIEU-RIGOLE
Dynamics and function of monocyte subsets in alcohol-dependent
subjects
Chronic alcohol consumption has a modulating effect on immune functions that may
contribute to decreased immunity and host defense. Monocytes constitute the first line
of host defense against pathogens and act as key regulators of both inflammation and
tissue homeostasis. Human monocytes are heterogeneous and comprise several
subtypes committed to different functions. Whether and how the chronic use of
alcohol impairs blood monocyte subsets in alcohol-dependent patients remains
unknown. Thus, we investigated the phenotypic and functional characteristics of blood
monocyte subsets in alcohol-dependent patients (AD), before and after alcohol
withdrawal. Compared to healthy controls (HC), changes in the distribution of
monocyte subsets was found in AD subjects before withdrawal, as well as altered
functional characteristics. The classical CD14+CD16- subset was decreased whereas
the non-classical CD14dimCD16+ subset was expanded (p<0,001). The frequencies of
TLR2- and TLR4-expressing monocytes were reduced in AD patients compared to
HC. Althought the steady state production of TNF by monocytes was comparable to
HC, the percentage of TNF-producing cells was reduced following LPS challenge.
Importantly, 14 days of sobriety restored the distribution of CD14dimCD16+
monocytes and the frequency of TNF-producing cells in response to LPS stimulation
to levels comparable to those in HC. Our findings indicate that chronic alcohol
consumption alters the distribution as well as the phenotypic and functional
characteristics of blood monocyte subsets, which are restored following 2 weeks of
withdrawal.
Key words: monocytes, TLR, alcohol, wthdrawal, cytokines
Hélène DONNADIEU-RIGOLE1,2,3, Isabelle DUROUX-RICHARD1,2, Pierre
PORTALES4, Martine BOUTHIER4, Thibault MURA5, Jean-François ELIAOU4,
Christian JORGENSEN6, Pascal PERNEY3, Florence APPARAILLY1,2,6
1 INSERM, U1183, IRMB, University Hospital Saint Eloi, 80 rue Augustin Fliche, 34295
Montpellier, France. 2 University of Medicine, Boulevard Henri IV, 34090 Montpellier,
France. 3 Department of addiction, University Hospital Saint Eloi, 80 rue Augustin Fliche,
34295 Montpellier, France. 4 Department of immunology, University Hospital Saint Eloi,
80 rue Augustin Fliche, 34295 Montpellier, France. 5 Department of information,
University hospital of Montpellier. 6 Clinical department for osteoarticular diseases,
University hospital Lapeyronie, 371 Avenue Gaston Giraud, 34295 Montpellier, France.
Corresponding author : Hélène Donnadieu-Rigole @mail : [email protected] : + 0033 4 67 33 70 20
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N°29 - Elodie FOREST
Dissecting the role of new cortical scaffolds during epithelial
biology
Epithelial cells form the most abundant cell type in the human body. They are highly
polarised along an apical-basal (A/B) axis, where the apical side faces the lumen. This
A/B polarity is achieved through the asymmetric segregation of highly conserved
protein scaffolds, many of which containing PDZ domains. A/B polarity controls then
key features of epithelial cells such as adhesion, trafficking, proliferation or signaling.
The observation that polarity is always lost in the late stages of cancers of epithelial
origin highlights the important tumour-suppressive role of proper A/B polarity, and
hence of the protein scaffolds controlling it.
Using both the fruit fly Drosophila melanogaster and human cancer cell lines, my
project is to characterise two neglected multi-PDZ scaffolds: bbg / PDZD2 and the
MAGI scaffolds.
First, using genetics and in-vivo and in-cellulo phenotypic analysis focusing on cell
architecture, proliferation and adhesion, I will determine the function of these two
families of scaffolds.
Second, using massive proteomic approaches, I will identify the partners of these
scaffolds to identify new protein complexes controlling and/or implementing the A/B
polarity program in epithelial cells.
As preliminary results, we identified that, the MAGI scaffolds are implicated in the
dynamics of cell junction formation and that bbg / PDZD2 are involved in cell number
and cell size control.
Elodie FOREST - IRCM
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N°30 - Ildem SANLI
Role of histone modifications and non-coding RNAs in the
regulation of the imprinted Dlk1-Dio3 domain
Genomic imprinting is an epigenetic phenomenon that mediates parent-of-origin
specific mono-allelic gene expression. Most known imprinted genes are organized in
clusters and their expression is regulated by differentially methylated regulatory
sequence elements called ‘imprinting control regions’ (ICRs). One of these imprinted
clusters, Dlk1-Dio3 domain is important for development. This domain, located on
mouse chromosome 12, contains three protein-coding genes, Dlk1, Rtl1 and Dio3
expressed from the paternal chromosome and several non-coding RNAs including
Gtl2, C/D box snoRNAs and many microRNAs expressed from the maternal
chromosome. The imprinted gene expression at this domain is controlled by a
paternally methylated ICR, called the IG-DMR. In the mouse, deletion of the IG-DMR
on the maternal chromosome results in loss of expression of all the maternallyexpressed ncRNAs, and de-repression of the protein-coding genes, on the maternal
chromosome. Paternal deletion of the IG-DMR has no effect. This indicates that the
yet-unknown mechanisms that control imprinted expression at this domain act on the
maternal chromosome.
Our aim is to elucidate which mechanism(s) bring about the silencing of the
paternally-expressed genes of the Dlk1-Dio3 domain on the maternal chromosome. To
distinguish between the parental chromosomes, I use intra-subspecific hybrid ES cells
between M. m. domesticus strain C57BL/6J and M. m. molossinus strain JF1 that
were previously generated in our lab. In hybrid ES cells and neural cells differentiated
in vitro, I have been investigating the repressive histone modifications and the
recruitment of repressive complexes at Dlk1 and Dio3 on the maternal chromosome. I
am particularly interested in the role of Gtl2 ncRNA in the repression of Dlk1 and
Dio3. For this purpose, I will perform studies on the recruitment and allele-specificity
of the repressive complexes and the repressive modifications in Gtl2 deficient cells.
Ildem Sanli, Sébastien Lalevée, Robert Feil
Institute of Molecular Genetics (IGMM), CNRS UMR5535 and University of
Montpellier, 1919 Route de Mende, Montpellier 34293, France
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N°31 - Sophie GUELFI
Molecular mechanisms of Notch1-induced pericyte-like
transdifferentiation of glioblastoma stem cells
Glioblastoma Multiforme (GBM, WHO grade IV) is the most aggressive and
infiltrative of primary brain tumors. Extensive vascularization is a major hallmark of
GBM, and current therapeutic strategies focus on targeting tumor angiogenesis. GBM
origin is still unknown but it was shown that subpopulations of multipotent stem-like
cells exist within the tumor, and could be responsible for relapse.
The Notch signaling pathway is central in the maintenance and proliferation of these
cells within the perivascular niche, where GBM stem cells closely interact with
endothelial cells. Here, we questioned whether Notch1 pathway activation in such
cells could influence the extent of tumoral vascularization and dissemination, using an
activated form of the Notch1 receptor (NICD). Although Notch1 receptor is activated,
target transcription factors (HES5, HEY1, HEY2) are not or barely expressed in our
GBM stem cell cultures.
Notch1 activation in these cells alters cell morphology, reduces their growth rate and
migration. This is accompanied by a transcriptional switch where OLIG2 and SOX2
expression is reduced while HEY1/2, KLF9 and SNAI2 transcription factors are
upregulated. Further analyses showed that NICD induces the expression of vascular
adhesion proteins (ICAM1, VCAM1), angiogenic cytokines (IL8, PLGF), and
angiogenic metalloproteinase MMP9. Remarkably, NICD expression also induces the
expression of pericyte markers in vitro (NG2, PDGFRβ and αSMA). When
xenotransplanted, Notch1-stimulated cells resulted in poorly disseminating but highly
vascularized tumors containing round and normalized vessel structures. Cells also
express pericyte markers in vivo and closely associated with host endothelial cells,
which confirmed a pericyte-like differentiation of GBM stem cells.
Our current project focuses on deciphering the transcriptional mechanisms underlying
this phenotypic switch. We are especially interested in how SNAI2 and KLF9 are
involved in Notch1-induced dormancy and pericyte-like features, by using in vitro
mechanistic approaches as well as studying their function in the context of GBM
perivascular niches.
Guelfi, S1, Guichet, P.-O.1, Teigell, M.2, Hoppe, L.1, Bakalara, N.1, Bauchet, L.1,
Duffau, H.1, Lamszus, K.3, Rothhut, B.1 and Hugnot, J.-P.1
1
INSERM U1051 - INM, Montpellier, France, 2RHEM-INM, BioCampus, CNRS
Montpellier, Montpellier, France, 3Laboratory for Brain Tumor Biology, Department
of Neurosurgery, University Medical Center Hamburg- Eppendorf, Martinistrasse 52,
Hamburg, Germany
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N°32 - Ali SALEH
A synthetic tridimensional matrix to study the migration of
Glioblastoma stem cells
Glioblastoma Multiforme (GBM) is the most common and aggressive primary brain
tumor. After decades of fundamental and clinical research, the prognostic of this
disease remains dismal and this is due to the high infiltrative capacity of a
subpopulation of tumor cells called Glioblastoma Stem Cells (GSC).
In our laboratory, using patient-derived primary cultures of GSC, we have developed
different models to study the migration of this tumor cells at the cellular and
molecular levels. In vivo, this has been possible with an orthotopic xenograft of
human cancerous stem cells into the brain of immunocompromised mice. In vitro, the
study was developed using a new synthetic tridimensional matrix of nanofibers.
At a time when different scientific publications have demonstrated the prominence of
the extracellular matrix in the control of tumor progression, the bidimensional models
of cell migration are becoming obsolete. Our tridimensional support aims at
mimicking the cerebral microenvironment and thus constitutes a valuable model to
unravel the migratory characteristics of GSC.
Using this matrix, different aspects of the migration of GSC have been determined.
First of all, we have identified different modes of cell migration, collective or
individual imitating then the variety of tumor cells displacement in the brain. Cell-cell
cohesion, anteroposterior asymmetry and hierarchical repartition of cells with the
presence of leader and follower cells have been determined. We have also identified
the importance of the physical organization of the matrix in the control of the
directional migration of tumor cells, a phenomenon known as physical topoinduction.
In fact, by producing aligned nanofibers, we have induced a cell migration in the
direction of those fibers. This topology mimics the routes used by cancerous cells to
invade the brain, especially the myelinated axons and the basal lamina of blood
vessels.
Ali Saleh, Zahra Hassani, Marisa Teigell, Soumaya Turpault, Aziz Cherifi, David
Cornu et Norbert Bakalara.
Institut des Neurosciences de Montpellier - Hôpital Saint Eloi - 80, rue Augustin
Fliche - 34091 Montpellier.
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N°33 - Yukiko IMAI
Initiation of meiotic recombination in mouse; search for interacting
partners of PRDM9
Homologous recombination during meiosis is an essential event for faithful
segregation of homologous chromosomes and production of genetic diversity. Meiotic
recombination is initiated by the induction of programmed DNA double strand breaks
(DSBs). Meiotic recombination takes place in specific regions of a genome, called
hotspots, where initiation occurs preferentially. Recently, PRDM9 was reported as a
protein involved in the specification of recombination hotspots in mouse and human.
PRDM9 contains a PR/SET domain with histone methyltransferase activity that
catalyzes the formation of H3K4me3 and a DNA binding domain. Our recent working
model includes PRDM9 as a key component for the initiation of meiotic
recombination: PRDM9 binds DNA via its zinc fingers and modifies chromatin
structure locally. Through an unknown process, SPO11 is recruited and catalyzes DSB
formation near PRDM9 binding sites. Our goal is to address the question: how does
PRDM9 recruit DSB machinery to hotspots? To gain insight in this mechanism, we
focused on characterization of PRDM9 interacting proteins. Here, we report potential
interactors of PRDM9 identified by yeast two hybrid screens with testis cDNA
libraries, as well as complex purifications followed by mass-spectrometry analysis.
Yukiko IMAI and Bernard DE MASSY, Institut de Genetique Humaine CNRS UPR
1142
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N°34 - Marco BRUSCHI
Epigenetic modulation of intestinal cancer susceptibility
The interplay between genetic and epigenetic lesions leading to the initiation and
progression of cancer has been extensively studied, and the epigenetic alterations are
nowadays considered as hallmarks of malignancies. We now aim at shedding light on
the correlation between the interindividual epigenetic polymorphisms and the relative
risk to develop malignancies.
To investigate the molecular aspects related with tumor initiation in the intestinal
epithelium we are using mice carrying a germline heterozygous mutation on the
gatekeeper gene Apc (Apcd14/+). Upon the second mutational hit, these mice
invariably develop intestinal adenomas during their adult life. Adult mice in our
colony display an outstanding variability in terms of number of intestinal adenomas. A
remarkable degree of such variability cannot be ascribed to major genetic changes,
and the determinants of such genetic-independent heterogeneity are to be researched
elsewhere. By mean of epigenome-wide analyses (i.e. RNA-sequencing, methylome
profiling) we are therefore attempting to identify the molecular signature associated
with such heterogeneity by analyzing the tumor-free tissue of bona fide syngeneic
Apcd14/+ mice. To ascertain the predictive value of our results we defined an
innovative strategy based on intestinal microsurgery performed on young mice to
collect intestinal biopsies before cancer initiates.
We are also investigating the remodeling occurring in the epigenome of intestinal cells
early after the deletion of one or both the Apc alleles, whose loss represents the most
common initiating event in colorectal cancer. To this aim we are taking advantage of
mouse models in which the loss the Apc alleles is selectively induced in the intestinal
epithelium, or specifically in the stem cell compartment.
Our ultimate aims consist in defining the very early events occurring in the epigenome
during malignant transformation and in identifying powerful epigenetic biomarkers,
providing evidences of their value in the early prediction of cancer-associated risk
within the population.
Marco BRUSCHI(1), Stanislas QUESADA(1), Pierre CESSES(1), Maxime MAHE(2),
Michael HELMRATH(2), Michael WEBER(3), Philippe JAY(1)
1: Institut de Génomique Fonctionnelle, Montpellier, UMR5203, INSERM U1191,
UM. 2: Cincinnati Children Hospital Medical Center, Cincinnati, USA. 3: École
supérieure de biotechnologie de Strasbourg, 67411 Illkirch
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N°35 - Chames KERMI
Rad18 silences the UV-dependent DNA damage checkpoint in
early Xenopus embryos
In early embryos the DNA damage checkpoint is silent until the midblastula transition
(MBT) due to maternal-limiting factors of unknown identity. Here, we identify the
Rad18 ubiquitin ligase as one such factor in Xenopus. We show in vitro and in vivo,
that inactivation of Rad18 function leads to DNA damage-dependent checkpoint
activation, monitored by Chk1 phosphorylation. Moreover, we show that both Rad18
and PCNAmUb abundance are developmentally-regulated. Increased DNA abundance
limits availability of Rad18 close to MBT thereby reducing PCNAmUb and inducing
checkpoint derepression. Further, we show that this embryonic-like regulation can be
reactivated in somatic mammalian cells by ectopic Rad18 expression thus conferring
resistance to DNA damage. Finally, we find high Rad18 expression in cancer stem
cells highly resistant to DNA damage. Altogether these data propose Rad18 as a
critical embryonic checkpoint-inhibiting factor and suggest that Rad18 deregulation
may have an unexpected DNA damage-dependent oncogenic potential.
Chames Kermia, Susana Prietob, Siem van der Laanc, Nikolay Tsanovb, Bénédicte
Recolina, Emmanuelle Uro-Costed, Bernadette Delisled and Domenico Maioranoa
a
Genome Surveillance and Stability laboratory, CNRS-UPR1142, Institute of Human
Genetics. Montpellier. France ; b Present address: Institute of Molecular Genetics of
Montpellier ; c Present address: CNRS UMR3145. Parc Euromedicine Cap Delta.
Montpellier ; d Laboratoire Universitaire d'Anatomie Pathologique. Neuropathologie
humaine et expérimentale. CHU Toulouse Rangueil. INSERM CRCT - Equipe 51,
Toulouse.France
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N°36 - Stella COSENZA
Differential effect of microparticles and exosomes isolated from
mesenchymal stem cells on T cell proliferation and experimental
arthritis
Mesenchymal stem cells (MSC) are multipotent cells that possess immunomodulatory
functions that are of high interest for cell therapy in various pathologies. MSC
functions are primarily mediated by soluble mediators released in the extracellular
milieu or within extracellular vesicles (EV). Even if the use of MSC is highly
controlled, the safety of MSC injection in long term is still controversial. Thus, the use
of EV derived from MSC could be a good alternative for therapeutic approaches.
EVs consist of three main populations: exosomes, microparticles and apoptotic bodies
that differ by their size, their composition and their secretion way. EVs seem to mirror
the effect of parental cells but little is known about the respective role of the various
subsets of EVs (exosomes and MP) secreted by a specific cell type. The aim of this
project is to compare in vitro and in vivo the immunomodulatory effects of exosomes
and microparticles secreted by MSCs.
After purification of the two EV subsets by differential centrifugations, we
characterized exosomes and microparticles on their size (less than 100nm and 400nm
respectively), their structure (bilayer phospholipids) and their specific markers
(endosomal markers, membrane cell markers). Then, we studied the functional effects
of EVs in vitro in proliferative assays. Our analysis indicated that addition of
microparticles significantly inhibited the proliferation of total splenocytes and CD4+
T cells in a dose-dependent manner whereas exosomes were not able to exert a
suppressive effect. This immunomodulatory function of microparticles was also
observed in vivo in the CIA model of inflammatory arthritis with a reduced incidence
and reduction of clinical symptoms: paw swelling and inflammation.
Our in vitro and in vivo data indicated that the immunosuppressive effect of MSCs is
at least in part mediated by EVs and microparticles seemed to play a major role in T
cell proliferation inhibition.
Cosenza Stella1,2, Toupet Karine1,2, Luz-Crawford P1,2, Jorgensen Christian1,2,3, Noël
Danièle1,2
1
Inserm U1183, Montpellier; 2Université Montpellier 1, Montpellier; CHU
Montpellier, Unité Clinique d'Immuno-Rhumatologie, Montpellier, France
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N°37 - Amanda ABI KHALIL
Epigenetics and phenotypes modulation in HMEC cells
In tumor cells epithelial-mesenchymal transition (EMT) has been associated to
increased invasiveness and stem like properties. EMT can be induced by various
cytokines, as well as by ectopic expression of transcriptional regulators (EMT genes).
A link between inactivation of p53 and EMT has been described and recent work has
shown a significant link between EMT and epigenetics modifications.
We developed a cellular model of stepwise transformation based on the sequential
transduction of primary HMECs cells with defined genetic elements. The general idea
was to create models representing different steps of transformation. In step 1 cells are
immortalized, upon inactivation of p53 (shp53), in step 2 cells are transformed by
ectopic overexpression of known oncogenes (WNT or RAS), which belong to distinct
signaling pathways.
Independent biological replicates were established for each genetic variant.
Genetically modified cells were characterized at the phenotypic, genetic (CNC,
mRNA and miRNA expression) and epigenetic (whole genome DNA methylation) to
monitor changes occurring at each step of progression in our cellular model.
Our data show for the first time to our knowledge that: (1) inactivation of p53 result in
a drift in DNA methylation; (2) different epigenetics patterns determined by the
oncogenic pathway that was activated.
We isolated different shp53 cell clones that bore different phenotypes. Some kept
epithelial characteristics while the others had acquired a fully mesenchymal
phenotype.
The aims of my project are to determine:
1) the link between DNA methylation and phenotypic changes in shp53 cells 2) if
changing the methylation state of cells will revert their phenotypes 3) the impact of
the oncogenes in the relation between methylation and phenotype 4) determine if
EMT is essential to cell transformation.
Amanda Abi khalil, thesis director: Charles theillet at "Institut de recherche en
cancerologie de Montpellier U1194"
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N°38 - Milena MAGALHÃES
DNA methylation and pulmonary disease in CF patients
Cystic fibrosis (CF) is a multivisceral disorder caused by mutations in CFTR (Cystic
Fibrosis Transmembrane Conductance Regulator), where p.Phe508del is the most
frequent mutation. Morbidity and mortality in CF are mainly due to lung disease.
Decrease in lung function is very variable among patients and depends on: the residual
activity of the mutated CFTR protein, the genetic background (CF modifier genes) and
the environmental exposure. Because environmental factors influence the phenotypes
of living organisms by shaping the epigenome, we hypothesize that DNA methylation
of modifier genes modulates the severity of pulmonary disease in CF patients. To
provide comprehensive profiles of CF patients, we analyze DNA methylation (a) in 13
candidate modifier genes and CFTR by Bisulfite and NGS (454 Roche) and (b)
genome-wide using the 450K BeadChip (Illumina). The study is carried out with
METHYLCF, a multi-center prospective cohort specifically built for this epigenetic
study of 50 p.Phe508del homozygous CF patients and 25 healthy volunteers. We
collected patient and control nasal epithelial and whole blood cells to establish a
biobank of DNA and RNA. DNA methylation analysis of candidate genes is almost
achieved. By combining global methylation data in a Partial Least Square Regression,
we correctly classified 74% of CF patients providing a CF-specific molecular
signature. The majority of differentially methylated CpG were hypermethylated in
NEC and hypomethylated in blood samples of CF patients. Also, we identified two
DMR in genes involved in sodium transport and oxidative stress. DNA methylation of
three genes correlated with the severity of pulmonary disease. The genome-wide DNA
methylation analysis is in progress. Besides explaining the extreme phenotypes of CF
patients characterized by the same mutation, this study may reveal modifier genes that
were underscored by previous genetic and transcriptomic studies and highlight
predictive markers of lung disease useful for CF follow-up.
M. Magalhães1, R. Chiron2 , I. Rivals3, J. Varilh1,2, A. Bergougnoux1,2, E.
Beyne1,2, L. Mely4, S. Leroy5, T. Ha3, D. Caimmi2, I. Vachier2, M. Claustres1,2, A.
De Sario1.
1EA Université de Montpellier; 2Montpellier Hospital; 3ESPCI Paris; 4Hyères
Hospital; 5Nice Hospital
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N°39 - Béryl LAPLACE-BUILHE
Pro-inflammatory macrophages mediated TNFa signaling is
required for caudal fin regeneration in zebrafish larvae.
Macrophages play an important role in the immune response to injury. However, a
broader role in repair and regeneration has been reported. While macrophage ablation
impairs epithelialisation and reduces scar formation in skin repair model in mammals,
it impairs the regeneration of the limb in axolotl and of the fin in adult zebrafish. This
discrepancy is likely due to the high plasticity of macrophages, which can adopt
various phenotypes and functions. Here we used the zebrafish larva to study the role
of pro-inflammatory, M1-like macrophages during caudal fin regeneration. Using 4D
confocal microscopy and double transgenic larvae to visualise macrophages and TNFα expressing cells, we showed that initially most recruited macrophages expressed
TNF-α (referred as M1-like). However TNF-α expression was transient and decreased
from 24 hours post amputation, due to the recruitment of a second wave of noninflammatory macrophages (referred as M2-like) and to a local phenotypic conversion
of macrophages. We demonstrated that TNF-α expressing macrophages were crucial
for fin regeneration as early depletion of macrophages blocked blastema formation
and regeneration process, while the depletion of late M2-like macrophages did not.
We then turned our attention to the trophic control exerted by M1-like macrophages
on the regeneration process and investigated the role of TNF-α signaling. Using a
combination of morpholino knock down strategy and parabiosis experiments, we
showed that TNFR1 played a necessary and direct role in the induction of fin
regeneration by expanding blastemal cell. In summary the present data strongly
suggest that TNF-a producing macrophages stimulate blastemal cell proliferation
through the TNFR1 activation, revealing a new insight into the mechanism of caudal
fin regeneration.
1,2*
Laplace-Builhe B., 1,2*Nguyen-Chi M.,3Travnickova J., 1,2Luz-Crawford
P., 1,2Tejedor G., 3Kissa K, 3Lutfalla G., 1,2Jorgensen C.,1,2Apparailly F 1,2Djouad F.
1
Inserm U 844, Montpellier, France; 2Université Montpellier 1, Montpellier,
France; 3Université Montpellier 2, Montpellier, France ; *equally contributing
authors
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N°40 - Jennifer BONINI
Role of miRNAs in the Cystic Fibrosis pathology.
Material & Methods: To identify targets in Cystic Fibrosis (CF), we assessed the
miRNAs expression profile in ALI cultures from CF patients (p.Phe508del
homozygous) or healthy individuals. We compared three epithelium models from
nasal, polyps or bronchial biopsies (n=4 per condition).From total RNAs, a library was
prepared according to the TruSeq Small RNA protocol and samples were sequenced
by using a MiSeq sequencer (Illumina). Results were analysed by sRNAbench
software. Then, deregulated miRNAs that act on the CFTR 3’UTR region were
studied by luciferase assays. Results:We found that 9 miRNAs are deregulated in CF
compared to non CF in the three ALI models. We also identified miRNAs which have
been previously described in other studies to be deregulated in CF, such as miR-145,
or involved in the ciliogenesis such as miR-449 family. Then, we studied miRNAs
that putatively act on the CFTR 3’UTR region. Functional analysis showed that mir100 deregulated in CF have a repressive effect on CFTR stability in bronchial
cells. Conclusion: Finding new regulatory players involved in CF physiopathology
and/or controlling the CFTR mRNA level help us to envision new tools for Cystic
Fibrosis therapy.
- This work is supported by the association Vaincre la Mucoviscidose –
J. Bonini (1,2), J. Varilh (3), R. Chiron (4), E. Brochiero (5), M. Claustres (1, 2, 3),
M. Taulan-Cadars (1,2)
1. Université Montpellier I, France; 2. INSERM U827,
ontpellier, France; 3. Service de Génétique Moléculaire, CHRU Montpellier,
France; 4. Département des maladies respiratoires,
CHRU Montpellier, France; 5. Département de physiologie moléculaire et
intégrative, Université de Montréal.
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N°41 - Denis DUNOYER DE SEGONZAC
Evolution of meiotic recombination: variation and function of the
Prdm9 gene in mice
In humans and mice, the genomic positions of DNA Double Strand Breaks (DSBs)
initiating meiotic recombination appear tightly controlled by the DNA binding domain
of PRDM9, made of a tandem array of C2H2 zinc fingers (ZnF). This domain has
been shown to be extremely polymorphic; phylogenic studies suggest that Prdm9 is a
fast evolving gene with residues involved in interaction with the DNA under positive
selection. Prdm9 is also a major determinant of male sterility in hybrids (HS) between
certain genotypes from two subspecies (musculus and domesticus) of the house
mouse.
We first tested the generality of the implication of Prdm9 in hybrid sterility and
speciation by examining fertility of F1 males from crosses between various wild
derived strains. These strains were harboring very different Prdm9 ZnF haplotypes:
the size and the dissimilarity of their Prdm9 ZnF region guided us in the selection. 32
crosses involving four different M.m. musculus and four M.m. domesticus were
analyzed, none of them lead to sterility. However, using the laboratory mice C57BL/6
crossed with a M.m. musculus, a phenotype of sterility was observed. The M.m.
musculus Prdm9 allele is already implicated in hybrid sterility when crossed with
C57BL/6 progenitor exclusively. In our case, the sterility phenotype does not depends
of the direction of the crosses. We conclude that only a very specific combination of
Prdm9 and/or genetic background leads to the hybrid sterility phenotype.
In addition, as hybrid sterility is associated with an arrest in meiotic prophase at the
stage of DSB repair, we started to monitor in detail the DSB repair activity. We are
performing this by following by ChIP-SEQ the association of Dmc1, the protein
involved in strand exchange during DSB repair, to single strand DNA. We aim to test
whether DMC1 localization and or enrichment differs between fertile and sterile
hybrids.
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N°42 - Estelle RASCOL
Biophysical interaction of nanoparticles with biological fluids
Mesoporous silica nanoparticle (MSN) is a promising material as drug delivery system
or nanovector. This biocompatible material associated with porous, magnetic and
fluorescent
properties
forms
a
multifunctional
platform.
The first challenge of my thesis was to study MSN biological stability and membrane
cell interaction depending of different surface coatings, such as lipid bilayer or
polyethylene glycol, to limit aspecific internalization.
We characterized MSN in suspension with biologic media, using dynamic light
scattering associated with transmission electron microscopy and zeta potential
measurements. This physicochemical study shows low stability of MSN in
suspension. This poor dispersibility of MSN is largely depending of ionic strenght and
medium composition, such as protein content. MSN coating with lipid bilayer is a
strategy to enhance MSN stability.
We used cell membrane model to investigate MSN - cell membranes interaction. The
model used is supported lipid bilayer on gold surface. This model membrane is simply
composed of EggPC phospholipids. Interaction between MSN and cell membrane
model is followed by surface plasmon resonance (SPR). This method, based on energy
absorption of gold at the nanometer scale, is able to detect, with high sensitivity, little
changes in refractive index at the near vicinity of gold surface. By varying MSN
medium, lipid MSN coating (both influence MSN aggregation), and transmembrane
potential, different responses were observed. For example, MSN coated with lipid
bilayer interacts lower with membrane model than bare MSN.
As a conclusion, the large choice of phospholipids, proteins and medium composition
is a powerful tool to depict specific and aspecific interactions of bare, protein or lipid
coated MSN with cell membrane.
Premier auteur :Estelle RASCOL, Dernier auteur, Joël CHOPINEAU, Entre les deux
: Jonas CROISSANT, Jeff NYALOSASO, Clarence CHARNAY, Yannick GUARI,
Christophe DORANDEU,Jean-Marie DEVOISSELLE
Affiliation de ces personnes : ICGM (Institut Charles Gerhardt, UMR 5253)
Également :Magali GARY BOBO, Marie Maynadier, Marcel GARCIA.
Affiliation de ces personnes : NanoMedSyn
Et :Cédric PISANI, Odette PRAT, Jean ARMENGAUD
Affiliation de ces personnes : CEA SBTN toxicologie (Marcoule)
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N°43 - Amélie TORA
Allosteric modulation of metabotropic glutamate receptors by
chloride ions.
Metabotropic glutamate receptors (mGluRs) play key roles in the modulation of many
synapses and are considered promising targets for the treatment of various CNS
disorders. Chloride (Cl-) is known to directly bind and regulate the function of
different actors of neuronal activity and several studies have pointed to the possible
modulation of mGluRs by Cl-. Through the use of innovative biosensors and secondmessenger assays, we demonstrate herein that Cl- behaves as positive allosteric
modulator of mGluRs. At mGlu4 receptors, for example, Cl- could increase glutamate
potency by more than 900-fold. Cl- potency was 78.6±3.5 mM, close to the estimated
resting value of extracellular Cl-. Cl- possesses a very high positive cooperativity with
glutamate (Hill slope ≈ 6 on mGlu4), meaning that small variations in [Cl-] lead to
large variations in glutamate action. Interestingly at mGlu3 receptors, not only Clpotentiates glutamate action but also increases basal activity of the receptors. Using
molecular modeling and site-directed mutagenesis, we have identified two wellconserved Cl- binding pockets in the extracellular domain of mGluRs. Moreover,
modeling of activity-dependent Cl- variations at GABAergic synapses suggests that
these variations may be compatible with a dynamic modulation of the most sensitive
mGluRs present in these synapses. Taken together, these data reveal a necessary role
of Cl- for the glutamate activation of many mGluRs. Exploiting Cl- binding pockets
may yield to the development of innovative regulators of mGluRs activity, as already
exemplified by compounds like LSP4-2022 that occupy both the glutamate binding
site and one of the Cl- sites.
Amélie S Tora (1,2), Xavier Rovira (1,2), Ibrahima Dione (3), Hugues-Olivier
Bertrand (4), Isabelle Brabet (1,2), Yves De Koninck (3), Nicolas Doyon (3), JeanPhilippe Pin (1,2), Francine Acher (5) and Cyril Goudet (1,2)
(1) Institut de Génomique Fonctionnelle, CNRS UMR5203, Université de Montpellier,
F-34094 Montpellier, France.(2) INSERM, U1191, F-34094 Montpellier, France.(3)
Centre de recherche de l’Institut universitaire en santé mentale du Québec and
UniversitéLaval, G1J 2G3 Québec, Canada.(4) BIOVIA Dassault Systèmes, 20 rue
Jean Rostand, F-91898 Orsay Cedex, France.(5) Laboratoire de Chimie et Biochimie
Pharmacologiques et Toxicologiques, CNRSUMR8601, Université Paris Descartes,
Sorbonne Paris Cité, F-75270 Paris Cedex 6, France.
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N°44 - Haijin LIU
Generation of a vaccine to prevent poultry from Newcastle disease
and control viral shedding
Newcastle disease (ND) is the major viral infection of poultry inducing high
morbidity, mortality, and significant economic impacts on the poultry industry. ND is
caused by virulent strains of avian paramyxovirus serotype 1 (aPMV-1) which have
the capacity to spread over long distances. All strains of aPMV-1 belong to a single
serotype and current vaccines have demonstrated their efficacy in terms of clinical
protection. However, recent studies have shown that the virus has undergone
significant evolution, which has led to the progressive emergence of new genotypes
with potential antigenic drifts. The recently described genotype XI in Madagascar
(Maminiaina et al, 2010) contains original amino acid substitutions on F and HN
proteins, some of those clustered in the head of the proteins, presumably exposed to
the host immune system, suggesting that these substitutions may account for antigenic
drifts. Indeed, we showed in vivo, under controlled conditions, that current vaccines
(genotypes II and III) induced equal clinical protection against genotypes II and XI,
but were unable to prevent viral shedding of genotype XI comparing genotype II. In
order to generate a vaccine preventing chicken from viral shedding and to try to
understand the forces that trigger aPMV-1 evolution and correlate with protection,
reverse genetics has been applied. NDV minigenomes expressing eGFP under two
promoters (T7 and CMV) have been constructed and compared in vitro. Subsequently,
full genomes of the genotype XI and II (NDV MG-725 and NDV LaSota) strains,
have been assembled under CMV promoter. In addition, the F and HN genes of a live
attenuated old-genotype virus (the genotype II Lasota strain) have been replaced by
the corresponding genes of the recent Madagascar genotype XI. These viruses
including the chimeric genotype XI-II will be characterized in vitro and finally
evaluated in immunization/challenge trials.
Haijin Liu(1,2), Renata S D Almeida(1,2), Patricia Gil(1,2), Minet Cécile(1,2),
Emmanuel Albina(1,3)
1. CIRAD, UMR CMAEE, F-34398 Montpellier,France; 2. INRA, UMR 1309
CMAEE, F-34398 Montpellier, France; 3. CIRAD, UMR CMAEE, F-97170 PetitBourg, Guadeloupe, France.
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N°45 - Christelle NGBA ESSEBE
Adaptation of Staphylococcus aureus to prolonged environmental
stress conditions
Objectives: Staphylococcus aureus (SA) is the most prevalent pathogen isolated in
diabetic foot ulcers (DFU). Two SA populations are distinguished in DFU: one with
low virulence present in colonizing ulcers and one with much higher virulence
isolated in infecting ulcers. The difference between these two strains is the presence in
the colonizing strain of a phage (Rosa-like), which alters the metabolism of the strain
and induces a low virulence. Moreover the deletion of the phage restored the virulence
of the strain. The purpose of this study is to determine the adaptation of the colonizing
strain under different prolonged environmental stress conditions encountered during
DFU and the potential of phage excision in these environments.
Methodology: During 16 weeks, the colonizing SA strain NSA1385 were grown in
different conditions: glucose 10%, antibiotics at 0,75x MICs (linezolid, vancomycin),
anaerobia, associations between sugars and antibiotics (glucose + linezolid, glucose +
vancomycin). Excision of the ROSA-like phage was evaluated by a multiplex PCR.
The expression of the two main virulence genes (hla, spa) and the virulence regulatory
genes (agr, rot, sae, sarA) were evaluated by qRT-PCR.
Results: Our results showed that the ROSA-like phage is stable in the colonizing strain
and cannot be excised whatever the stress condition tested after 16 weeks. Concerning
the adaptation of NSA1385 during stress conditions, we observed that vancomycin
increased the expression of hla after 16 weeks. Linezolid caused overexpression of agr
and rot. Anaerobia downregulated agr and saeP. Glucose downregulated all genes
except sarA and spa. These effects are almost entirely offset by the addition of
antibiotics.
Discussion: This study demonstrates the stability of the phage inserted in the
colonizing strain. It also showed the adaptation of the strain to prolonged stress
environmental conditions. All these results allow a best comprehension of the SA
virulence in chronic wound conditions.
Key words: Staphylococcus aureus, sugar, antibiotic, stress, expression, adaptation
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N°46 - Rémi LOGEAY
Understanding the cooperation between Notch and the polarity
determinant Scribble during neoplasia
One classic model of solid tumour development is a progressive model. A first set of
mutations triggers hyperplastic growth of the epithelial cells: cells remain polarised,
become hyperproliferative, but the tissue is not capable of invasion. A second set of
mutations later transforms this hyperplasia in neoplasia: cell polarity is lost and cells
become invasive. In the wing imaginal discs of Drosophila melanogaster, this
progression can be mimicked by constitutive activation of the Notch pathway to
trigger hyperplasia and then by removing the epithelial polarity to trigger neoplasia.
The Notch pathway is implicated in development and homeostasis of many
proliferating epithelia. Upon activation, the receptor Notch is cleaved to release its
intra-cellular domain which then enters the nucleus and binds the transcription factor
Suppressor of Hairless to activate the transcription of the Notch pathway direct
targets. The loss of polarity can be accomplished by mutating Scribble, a key
component of the cortical complexes that establish and maintain epithelial polarity.
However scribble mutation alone does not lead to hyperproliferation or invasion. It is
actually the cooperation between Notch activation and loss of polarity that leads to an
invasive neoplasia. The aim of my project is to dissect this cooperation including
finding out if there is addition or synergy between the two processes. By comparing
RNAseq and ChIP analysis for the transcription factor Suppressor of Hairless we will
be able to identify the Notch direct targets in the different conditions: wild type,
hyperplasia and neoplasia. We will then be able to determine if neoplasia and invasion
are due to an additive effect (the loss of polarity adds its effects to the proliferation
induced in the hyperplasia) or a synergic effect (the loss of polarity redirects the Notch
pathway to new targets responsible of the invasion).
Rémi Logeay, Alexandre Djiane
Institut de Recherche en Cancérologie de Montpellier (IRCM U1194)
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N°47 - Claire TOLZA
Identification of potential therapeutic targets regulated by Fra-1
and/or Fra-2 in triple negative breast cancers
Triple negative breast cancers (TNBC) are of poor prognosis and there is currently no
available targeted therapy. Fra-1 and Fra-2, two members of the Fos family involved
in the formation of the AP-1 transcriptional complex, are frequently overexpressed
in aggressive TNBC where they contribute to the tumorigenic phenotype. However,
the panel of genes under the control of Fra-1 and/or Fra-2 in TNBC is still not clearly
identified and the molecular mechanisms through which Fra-1 and Fra-2 control
their expression are mostly unknown. Our aim is to identify and characterize the
network of genes controlled by Fra-1 and/or Fra-2 in TNBC for a better understanding
of the biology of these cancers and to identify and study potential druggable
therapeutic targets.
To identify the panel of genes regulated by Fra-1 and/or Fra-2, I carried out
transcriptomic analysis in MDA-MB231 cells in presence or in absence of Fra-1
and/or Fra-2 by RNAi approach. The data were then crossed with our ChIP-seq data
obtained for Fra-1 and Fra-2 in the same cell line, to select putative direct target genes.
I am currently crossing this data with breast cancer data banks (CIT and TCGA) to
select genes whose expression is also correlated to that of Fra-1 and/or Fra-2 in human
tumors. Functional analysis of the selected genes will be carried out using RNAi
approach. The attenuation of various properties promoting tumor aggressiveness
(proliferation, cell viability, cell cycle, apoptosis, migration, invasion) will be studied.
The observations will be extented to other TNBC cell lines. If inhibitors for the
protein product of the studied genes are avalaible, they will be included in our tests.
As Fra-1 and Fra-2 are also overexpressed in many epithelial aggressive carcinomas,
our observations may have repercussion beyond breast cancer.
Claire TOLZA, Marc Piechaczyk and Isabelle Jariel-Encontre. IIGMM, UMR 5535
CNRS, Montpellier, France.
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N°48 - Rana MELHEM
Targeting Transferin Receptor 1 (TFR1) in Pancreatic Cancer
Pancreatic cancer is a highly aggressive disease associated with poor diagnosis and
high mortality. It is therefore necessary to search for new therapeutic targets or
treatments. One of the interesting options would be targeting iron metabolism. Indeed,
cell transformation is generally accompanied with increased needs for iron together
with increased expression of the transferin receptor 1, TfR1, the major receptor
involved in cellular iron supply via the internalisation of plasma transferin loaded with
iron. We have generated a fully human anti-transferin receptor antibody, namely
3GH7, capable of blocking the entrance of iron bound transferin to cells thus
exhibiting inhibitory effects on various pancreatic cancer cell lines. On the BxPC3
pancreatic cancer cell line, we show that the antibody decreases cellular viability,
inhibits proliferation, and induces apoptosis. These effects are likely due to a drastic
decrease in the labile iron pool which is a consequence of the blockage of transferin
uptake. At the protein level, the antibody upregulates surface transferin receptor 1 and
induces the expression of the metastasis suppressor NDRG1 as well as its
phosphorylated form (Ser330, Thr346). NDRG1 has a widely reported anti-tumor
function and is considered as a promising therapeutic target against pancreatic
cancer.These results are to be completed with in vivo studies to further elucidate the
possibility of using the anti-transferin receptor antibody as a therapeutic target in
pancreatic cancer.
Rana Melhem, Marie-Alix Poul, Thiery Chardes, Andre Pelegrin
IRCM, INSERM U1194, ICM, Universite de Montpellier 1
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N°49 - Ewelina GUCA
Structural characterization of Plasmodium falciparum CCT and
fragment-based drug design approach for targeting phospholipid
biosynthesis pathway.
Phospholipid synthesis metabolic pathways in Plasmodium falciparum are validated
drug targets for new type of antimalarials. In the de novo Kennedy pathway of
phosphatidylcholine biosynthesis, the second step catalyzed by CTP:phosphocholine
cytidylytransferase [EC 2.7.7.15] is rate limiting. We are focused on the structural
characterization of this enzyme, the identification of effectors by fragment-based drug
design approach (FBDD) and then their optimization to eventually design a lead. We
solved the X-ray crystal structure of the catalytic domain of the enzyme target
(PfCCT) at resolution 2.2 Å and of the complexes with substrates and product. These
data allows us to give detailed images of the binding pocket, to reveal conformational
changes between apo- and holo-protein forms and to provide information about the
mechanism of the catalytic reaction at atomic level. The FBDD method uses a library
of small molecules (fragments) with molecular weight that does not exceed 300 Da to
explore target binding sites. Screenings of a fragment library (300 molecules) has been
performed by fluorescence-based thermal shift assay and Nuclear Magnetic
Resonance Saturation Transfer Difference (NMR STD). This combination of
techniques identified so far 4 fragment hits that are currently evaluated for their
binding modes and affinities. Co-crystallization of the protein-fragments complexes is
carrying out to provide accurate information on the molecular interactions. Topology
of these interactions will be used to rationally monitor every iterative round of the
optimization process allowing subsequent rational design.
Ewelina Guca1, Francois Hoh2, Jean-Francois Guichou2, Christian Roumestand2,
Henri Vial1, Rachel Cerdan1
1 - DIMNP UMR 5235, Université de Montpellier. 2 - CBS CNRS UMR 5048
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N°50 - Sara HAYDAR
PPM1K (protein phosphatase, Mg2+/Mn2+ dependent, 1K) Gene
Association in Polycystic Ovary Syndrome for Better
Understanding of the Role of Branched-Chain Aminoacids
Variation in Metabolic Disorders
Polycystic ovary syndrome (PCOS), an endocrine disorder characterized by
abnormalities in reproduction and metabolic disorders has been recently associated
with plasma variations of branched-chain aminoacids (BCAA). Moreover, BCAA
catabolism pattern has been related to insulin resistance and susceptibility to type 2
diabetes (T2D) and BCAA plasma levels were reported as different in PCOS. Many
genes, as PPM1K (protein phosphatase, Mg2+/Mn2+ dependent, 1K) are implied in
the catabolism of BCAA and are then candidates for the association to PCOS and its
components. In this context, we carried-out a case-control study in a population from
Central Europe focusing on the association of PPM1K in PCOS, using as marker the
SNP (single nucleotide polymorphism) rs1440581 C/T, previously reported as leader
in association with insulin resistance. At first we assessed the linkage disequilibrium
(LD) pattern on the genomic region by screening, 48 SNPs surrounding the leader
SNP rs1441581 in 55 cases and 48 controls, using Affymetrix technology, and
continued the genotyping of rs1440581 by KASPar in the rest of the population
(reaching 401 cases and 143 controls). LD pattern, haplotype reconstruction and
cladograms determination were obtained by HAPLOVIEW 4.2, PHASE 2.1 and
ARLEQUIN 2.000 softwares, respectively. Gene association and genotype-phenotype
correlation were calculated by logistic regression and ANOVA using STATVIEW 5.0
and Abacus. We observed LD block involving 6 SNPs and rs1440581, and
reconstructed 9 haplotypes among which H4 (TCTACTT), H6 (TTCATTC), H9
(CTCACCT), H3 (TCTACTC) and H2 (TCCGCTC) are more frequent (38.3, 35.9,
8.7, 8.3 and 5.3%, respectively). Trend of association of rs1440581 C allele with
metabolic syndrome (MetS) in PCOS was found (P = 0.07, OR 1.97 95%CI [0.94 –
4.09]) which is concordant with the literature. This allele is the ancestral one in the
cladogram. The present pilot study is in progress and further data are expected.
Sara Haydar(1), Redha Attaoua(1), Mihai Coculescu(2), Madalina Vintila(2), Nicoletta
Baculescu(2), Christophe Normand(1) and Florin Grigorescu(1)
1) IURC, Molecular Endocrinology Laboratory, Nutrition & Genomes, UMR-204
NUTRIPASS, Montpellier, France. 2) Departement of Endocrinology, University of
Medicine and Pharmacy "Carol Davila", Bucharest, Romania
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N°51 - Alejandra DAMIAN
Identification of new regulators of the Notch pathway in
KRASG12V-driven NSCLC
Previously, it has been shown that oncogenic mutated KRAS activates the Notch
pathway in Non-small Cell Lung Cancer (NSCLC) cells and that Notch is critical for
both the generation and the maintenance of NSCLC (Maraver et al., 2012; Maraver
and Serrano, 2012).
The aim of this study is to determine the mechanisms involved in how oncogenic
KRAS activates the Notch pathway in NSCLC and to identify which are the genes
mediating Notch activation only in the oncogenic setting.
In order to achieve this objective, we will perform four consecutive screenings using
different biological systems.
First, we will use NSCLC cell lines derived from mutant KrasG12V-driven
adenocarcinoma from mouse (Ambrogio et al., 2014a) to perform high-throughput
screening using the NKI 15000 library of shRNAs. As a reporter, we will use the
pGreenfire-Notch lentiviral plasmid that co-express destabilized GFP and luciferase
under the control of Notch response elements (System Biosciences).
Then, we will perform a second in vitro screen with 200 genes candidates in the
human cell lines BEAS-2B transformed with KRASG12V and in the non-transformed
parental one, to find those new key players that inhibit Notch pathway in KRASG12V
transformed cells.
To be sure that the genes that we may find in the cultured cells play a decisive role in
a 3D in vivo setting, we will perform a genetic in vivo screen using Drosophila as
model.
Finally, those putative new regulators that are consistent among all the screens,
conserved from flies to human cells, will be tested in KrasG12V-driven NSCLC in
mice models.
These key genes acting downstream of oncogenic KRAS to activate Notch would
represent very relevant targets for the development of new anti-cancer drugs. More
importantly, these genes would be selective for cancer cells not affecting the healthy
ones and avoiding the side-effects of Notch inhibition.
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N°52 - Joelle AZZI
Identification of kinase inhibitors as alternatives for the treatment
of metastatic castration-resistant prostate cancers
Prostate cancer is the most common cancer among men with an increasing incidence
with age. Localized tumors are treated with surgery or radiotherapy while the most
advanced forms are treated by androgen deprivation. However, most of these patients
become resistant to castration and are treated with taxane-based chemotherapy
(docetaxel or cabazitaxel) or second generation hormonal treatments (abiraterone
acetate or enzalutamide) with limited effectiveness. Other anticancer agents have been
tested without significant improvement of overall survival. Moreover, these studies do
not necessarily take into account the genetic characteristics of this type of tumor. With
the emergence of new sequencing techniques, it is possible to have a better idea of
specifically deregulated or mutated genes in these cancers. The aim of my thesis is to
identify new potential treatments that can be used as an alternative to taxanes based on
the specificity of mCRPC (metastatic castration-resistant prostate cancer) tumors, in
particular gene expression profiles. Using the NCI60 databases, we looked for
correlations between the expression levels of 96 genes characterizing the evolution of
prostate cancer evolution towards castration resistance and the sensitivity to kinase
inhibitors, with the goal of identifying derivatives that may have better efficacy than
taxanes. We identified several genes, the expressions of which are significantly
correlated with cell sensitivity to two inhibitors of the MAPK pathway: vemurafenib
(targeting RAF) and selumetinib (targeting MEK). We are currently in the process of
validating these correlations at the functional level by investigating whether the
modulation of their expression could indeed alter the sensitivity of prostate cancer
cells to those two kinase inhibitors. In longer terms, the signatures of expression that
will be validated by our approach could be used to identify patients most likely to
respond to vemurafenib or selumetinib, an essential step in the development of
prospective clinical trials.
Joelle AZZI, Hanane AGHERBI, Philippe POURQUIER, Nadine HOUEDE
INSERM U1194 Institut de Recherche en Cancérologie de Montpellier & CHU de
Nîmes
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N°53 - Barbara ZIEBA
Assembly of proteasome and epidermal differentiation: interest in
psoriasis
Proteasomes play a critical role in cell homeostasis through the regulated degradation
of most intracellular proteins. Previous work in our lab has shown that it is
overexpressed in the epidermis upon psoriasis, which is the most common, relapsing
skin pathology characterized by inflammation, hyperprofileration of keratinocytes and
impaired epidermal differentiation [ED]. This increase is likely to be caused by
enhanced assembly of the proteasome since no change in the transcription levels of
various subunits can be detected. Indeed one of the proteasome assembly chaperones POMP - seems to play important role in ED. It was shown that a point mutation that
silences expression of this protein is directly linked to KLICK syndrome - a rare
pathology of ED. We observed increased expression of POMP in psoriatic lesions
compared to healthy epidermis. Therefore we hypothesize that balanced proteasome
assembly plays an important role in proper keratinocyte differentiation.
To test this hypothesis we established an in vitro model of ED using immortalized
keratinocytes, HaCaT cells. We observed a peak (≈50% increase) in POMP expression
24h after [Ca2+]-induced differentiation. In order to then investigate deeper the role of
POMP in ED we are presently creating HaCaT cell lines stably expressing POMP and
we will perform POMP silencing experiments in parallel. In a different approach, we
analyzed the proteasome content of HaCaT cells during differentiation and showed by
native PAGE that the proteasome disassembles during differentiation, while
expression of its subunits remains unchanged. We believe that this disassembly is
linked to oxidative stress that is reported to participate in ED and we are planning to
study the possible causal relationship between these two phenomena.
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N°54 - Mouna TRIKI
Expression of the transcriptional coregulator RIP140 in colorectal
cancer
Receptor Interacting Protein 140 (RIP140) is a transcriptional coregulator essential for
female fertility which is involved in inflammation, lipid metabolism and cognition.
Recently, it has been shown that RIP140 is also implicated in intestinal homeostasis
and colon cancer. Moreover, LCoR (ligand-dependent corepressor) was found to be a
partner and a transcriptional target gene of RIP140.
The aim of this study is to better characterize the expression of RIP140 and LCoR in
colorectal tumors by immunohistochemistry. We have initiated the study of the two
proteinsin 161 colorectal carcinoma (CRC) specimensf rom Tunisian patients in order
to evaluate their correlation with different clinicopathological parameters.In this
cohort, the male/female ratio was 1.01 and the patient ages ranged from 17 to 92 years
(mean age: 63 years). Our preliminary results showed that RIP140 and LCoR
expression was positive in 57.8% (93 out of 161 cases) and 67.7% (109 out of 161)
respectively. Significant association of LCoR expression and tumor site was found.
Indeed, 74.4% of tumors in rectum were positive for LCoR while only 51.3% of colon
tumors expressed this protein (p=0.009). When we consider the expression of both
proteins, we found that 56% and 38.5% of specimens were respectively positive and
negative for both LCoR and RIP140. The association between RIP140 and LCoR
staining and clinicopathological parameters (tumor grade, lymph node invasion,
patient survival…) is under investigation. The correlation with the expression of
different actors of the Wnt pathway will also be investigated.In parallel, we will
analyze the methylation status of the RIP140 promoter in tumor tissues. In preliminary
experiments, the unmethylated pattern of RIP140 promoter was found in 70% of
cases, nevertheless, it needs to be confirmed on larger sample size.
Keywords:Receptor Interacting Protein 140 (RIP140), colorectal cancer, Wnt, DNA
methylation.
Mouna TRIKI1,2, Lobna AYADI3, Abdelmajid KHABIR3, Vincent CAVAILLES1, Raja
GARGOURI2
1
IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194,
Université Montpellier, Montpellier, France.2Laboratory of Cancer Genetics and
Production of Therapeutic Proteins, Center of Biotechnology, Sfax,
Tunisia.3 Department of Pathology, Habib Bourguiba Hospital,Sfax, Tunisia.
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N°55 - Alicia MADGWICK
Evolutionary Plasticity of Endodermal Gene Regulatory Networks
in Ciona intestinalis and Phallusia mammillata
Regulatory evolution of developmental genes is an important source of morphogenic
diversity. This phenomenon has been suggested to be invovled in many organisms
such as for the vulva of C. elegans, colour patterning in Heliconius wings and even the
beak shape of Darwin’s finches. However, regulatory evolution could also be a source
of morphogenic conservation.
Ascidians Ciona intestinalis and Phallusia mammillata diverged over 300 million
years ago yet they have conserved an almost identical morphology throughout
embryogenesis. Their genomes have greatly diverged which suggests that the gene
regulatory networks (GRNs) regulating embryogenesis must have also undergone
modifications. Ascidians are sessile marine invertebrates that belong to the subphylum
Tunicata and the phylum Chordata. Their small genome and their simple, invariant
early development make ascidians an invaluable tool for studying both evolution and
developmental biology. These simple organisms are ideal to study GRNs at a cellular
level and to observe inter-species regulatory evolution.
Ciona embroygenesis is well documented; much of this data has been detailed on the
ANISEED website including expression data from wild type and knockdown
experiments. In contrast, Phallusia has not been studied as extensively; therefore, I am
initially investigating the spatial and temporal expression of the homologous Phallusia
genes known to be involved in Ciona early endoderm development by in situ
hybridisation. It will therefore be possible to determine to what extent gene expression
has diverged in these two species before investigating what interactions have evolved
within the endodermal GRNs to accommodate these divergences. The endoderm is an
interesting tissue when studying morphology as it drives gastrulation during which
time the endodermal cells do not undergo division in ascidians. Once the GRNs of
both organisms have been built and compared, we will then be able to evaluate the
mechanisms that have allowed evolution of the regulation of these ascidians.
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N°56 - Ruizhi TANG
The role of primary cilia in colon homeostasis and tumor
development
The primary cilium is a sensory organelle expressed on nearly all eukaryotic cells.
Primary cilia are assembled on the basis of a microtubule component, the axoneme.
The functions of primary cilia are modulation of signaling pathways including WNT
and Hedgehog (HH), which are essential in the regulation of intestinal homeostasis.
We have recently described that glycylation, a posttranslational modification of
microtubules, is crucial in the maintenance of primary cilia. We observed a decreased
number of primary cilia and increased cell proliferation in the colon in mice deficient
for the glycylase TTLL3, which is the only glycyclase expressed in the colon. Despite
this overproliferation, TTLL3-deficient mice display a normal tissue architecture of
the colon. However, loss of glycylase activity promotes induced colon
carcinogenesis. In my PhD project we will focus on the consequences of a complete
loss of primary cilia in intestinal epithelial cells and intestinal myofibroblasts
respectively. For this, we will use villin-cre-mediated recombination to generate mice
in which components of the ciliary transport machinery (Kif3A or Ift88) are knocked
out specifically in the intestine. Mice will be analyzed for altered colon homeostasis
and altered susceptibility for induced colon carcinogenesis. In addition, we will
investigate altered WNT and HH signaling by the analysis of respective target genes
in colon and the use of WNT and HH reporter constructs in isolated colonic epithelial
cells or intestinal organoid cell cultures. My work should provide direct proof that the
primary cilia regulate proliferation of colon epithelium.
Ruizhi Tang1,2, Laura Papon1,2, Cecilia Rocha1,2,3,4,5,6, Carsten Janke3,4,5,6,
Michael Hahne1,2,7
1 IGMM CNRS UMR5535, Montpellier, France, 2 Université Montpellier Sud de
France, Montpellier, France, 3 Institut Curie, Orsay, France, 4 PSL Research
University, Paris, France, 5 CNRS UMR3306, Orsay, France, 6 INSERM U1005,
Orsay, France, 7 Academic Medical Center, Amsterdam, The Netherlands
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N°57 - Sana BELKAHLA
The effect of dichloroacetate(DCA) on tumor cell metabolism
Several clinical studies have shown the efficacy of DCA in the treatment of several
metabolic diseases and is currently in clinical trials for the treatment of cancers.
DCA reverses cancer metabolism, causes cancer cell apoptosis in some cancer cells
and works synergistically with other cancer therapies, such as radiation, gene therapy,
and viral therapy or with chemotherapy.
AMPK, is an important sensor of intracellular energy levels, targets ACC, FASN,
mTORC1 and p53, thereby inducing oxidative metabolism, propelling fatty acid
oxidation and inhibiting ATP-consuming anabolism. Indeed, metformin, which
activates AMPK, also shows anti-cancer effect. Moreover, the mitochondrial
membrane potential modulates the mitochondrial apoptotic pathway, e.g.cytochrome c
redox state regulates apoptosis.
An important partner of AMPK is the tumor suppressor p53, which is emerging as an
important regulator of metabolic homeostasis.
Therefore, a clearer understanding of the signals and mechanisms by which DCA
sensitizes cancer cells to chemotherapy is needed to understand its mode of action. In
addition, identification of this mechanism will help to elucidate metabolic pathways
involved in cancer cell survival. During our studies, we observed a link between DCA
sensitivity and p53 status.
However, it is of great interest to identify the mechanism underlying DCA-induced
leukemic cell elimination because this should help to select blood-borne cancer
patients who could effectively benefit from this treatment.
Sana Belkahla1, Ewelina Krzywinska1, Abrar Ul Haq Khan1 , Nerea Allende-Vega1 ,
Dang-Nghiem Vo1, Martin Villalba1,2.
1/ INSERM, U1183; Université de Montpellier 1, UFR Medecine, 80, av.
Augustin Fliche. 34295 Montpellier Cedex 5, France.2/ Institut
de Regenerative Medicine et Biothérapie (IRMB), CHU Montpellier, Montpellier,
34295, France.
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N°58 - Sophie KAN
Heterochromatin factors stimulates telomere transcription
Telomeres are critical regions that protect chromosome ends from degradation or
aberrant repair. These regions are assembled into heterochromatin. Here, we
investigate the function of Histone H3 lysine 9 trimethylation at the telomeres in
mouse embryonic stem cells. Heterochromatin is typically believed to repress gene
expression but we found that at telomeres, the H3K9me3 mark instructs
transcriptional elongation and/or inhibit transcriptional termination of telomeres into
the non-coding RNA TERRA. Using the quantitative PICh method, we further show
that this mark controls the recruitment of histone chaperones that are necessary to
maintain RNA polymerase II processivity on telomeres. We propose that it occurs
genome-wide, and is broadly involved in the general transcriptional response to
stressful conditions in mammals.
Sophie Kan, Nehmé Saksouk and Jérôme Déjardin, Institut de Génétique Humaine,
CNRS, France
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N°59 - Olfa KHALIFA
Common variants on XQ28 conferring risk for rheumatoid
arthritis in tunisian and french populations
Background and Objectives: Rheumatoid arthritis (RA) is a systemic autoimmune
disease characterized by a chronic destructive inflammation in synovial joints that is
about three times more common in women than in men. Investigations into RA
genetics have identified several susceptibility loci. Among these, multiple singlenucleotide polymorphisms (SNPs) at an X chromosome locus harbouring IRAK1 and
TMEM187 have previously been shown to be associated with susceptibility to RA in
Greek, Chinese, and Korean populations. The present study aimed at investigating the
association between critical polymorphisms in Xq28, from rs1059702 (C/T) and
rs1059703 (C/T) in IRAK1 through rs13397 (A/G) in TMEM187 with risk for RA in
two novel populations: Tunisian and French. In addition, the present study aimed at
comparing the differences in genotypic distribution in both populations.
Material and methods: A case–control study of 493 RA patients and 507 healthy
controls age and sex matched was conducted in both Tunisian and French. All RA
patients were ACPA positive. DNA was isolated from total blood using standard
phenol chloroform method. All subjects were genotyped for the three polymorphisms
using a direct sequencing with the BigDye Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems). The genotype distribution, haplotype analysis, and the linkage
disequilibrium (LD) were analysed using the Bayesian method, and PLINK 1.07 and
Haploview 4.2 softwares, respectively.
Results: The three polymorphisms respected Hardy-Weinberg equilibrium, both in
Tunisian and French populations. The associations between both IRAK1 rs1059703
and TMEM187 rs13397 polymorphisms and RA susceptibility were found (p<0.001
and p=0.004, respectively) in women, but not in men. The genotype CC of rs1059703
represented a protective factor for RA (OR= 0.29, 95% CI=0.17-0.50). Linkage
disequilibrium between the 3 SNPs was weak (D=8-16). There were 5 haplotypes with
a frequency higher than 5%, constructed from the Xq28 polymorphisms. Haplotype
GTC was associated with decreased risk for RA (p=0.00075), whereas the haplotype
ACC was significantly associated with increased risk for RA (p<0.001). Correlation
analyses with clinical and biological parameters are in progress.
Conclusion: Our case-control study replicated for the first time the association of the
Xq28 chromosomal region with RA risk in Tunisian and French populations, and
suggested that RA susceptibility is associated with rs1059703 polymorphism in
IRAK1 and rs13397 polymorphism in TMEM187 only for female patients. These data
further support the involvement of X chromosome in RA susceptibility.
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N°60 - Beren GARCIA MENDEZ
Brucella replication in human trophoblasts: role of the eukaryotic
protein CD98hc
Brucellosis is one of the most important bacterial zoonosis worldwide, causing huge
economic losses and long lasting health problems in many countries, mainly around
the Mediterranean basin. This disease is caused by the pathogenic bacteria Brucella. In
female animals, the main consequence of Brucella infection is abortion, but it is still
unclear if it can also cause abortion in Humans. Recently, it has been shown that
several species of Brucella can replicate within human placental cells (trophoblasts)
(Salcedo et al., JID, 2013). This observation raised the question about a link with the
ability of these bacteria to cause abortion.
The aim of this PhD project is to identify some of the factors required for Brucella
replication in human trophoblasts, from both the pathogen and the host side.
We have examined the replication profile in trophoblasts of a panel of Brucella strains
(representing different species, host or associated symptoms), including two strains
that caused abortion in non-human primates. Our data shown that there is no clear
correlation between the ability of Brucella to replicate within trophoblasts in vitro and
whether they caused abortion or not.
From the host side, we will focus on the eukaryotic protein CD98hc, which has
important functions in placental cells and that was found to be essential for Brucella
replication in other cellular models (Keriel et al., JID, 2014).
We first checked whether Brucella infection would alter either CD98hc expression or
its cellular sub-localization in trophoblasts. We found that none were affected by
Brucella infection. We are now knocking-down CD98hc expression in trophoblasts to
see if Brucella require this protein for intracellular replication in trophoblasts.
With this project we hope to have a better comprehension of how some Brucella
species can replicate in placental cells and might allow explaining why this bacteria
can cause abortion.
Karellen Beren GARCIA MENDEZ1,2; Jean-Pierre GORVEL3,4 ; David
O’CALLAGHAN1,2 ; Anne KERIEL1,2.
1
INSERM, U1047, Nîmes, France. 2 Université Montpellier, U1047, Nîmes, France.
3
Centre d'Immunologie de Marseille-Luminy (CIML), Marseille , France. 4AixMarseille University , Marseille , France
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N°61 - Iman HALLOUM
Identification and characterization of Mabs4780, a new
determinant required for intracellular survival and pathogenicity
of Mycobacterium abscessus
Background: Mycobacterium abscessus (Mabs) is an emerging rapid-growing
mycobacteria causing severe lung infections, particularly in CF patients. Unlike the
smooth (S) variant, the rough (R) variant is characterized by the loss of surface
glycopeptidolipids. Despite the involvement of Mabs R in severe clinical forms, the
underlying physiopathological mechanisms remain obscure.
Aims. Herein, we aimed to decipher the contribution of MABS4780 in Mabs virulence
and investigate the molecular paradigm for its involvement in intracellular survival in
various cellular and animal models.
Methods: A MABS4780 deletion mutant was constructed and permeability of its cell
wall and susceptibility to several antibiotics were assessed. The intracellular fate and
virulence of the mutant were investigated in murine macrophages, Acanthamoeba
castellanii and in zebrafish embryos.
Results: The mutant exhibited a higher susceptibility to thiacetazone, compared to the
parental strain and increased sensitivity to detergents, presumably due to alterations of
cell envelope composition. Concurrently, the crystal structure of the M. smegmatis
orthologue unraveled hotdog domains, characterizing dehydratases, thus potentially
linking MABS4780 to lipid metabolism. The mutant failed to replicate in
macrophages and in A. castellanii, thought to be the Mabs environmental reservoir,
and was highly attenuated in zebrafish embryos.
Conclusion: This demonstrates the unanticipated role of MABS4780 in Mabs R
virulence and persistence in environmental amoeba. Future work will address the
mechanism of attenuation of the mutant. Our results also emphasize the potential of
this dehydratase as an attractive drug target.
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N°62 - Chloé MAURIZY
Role of R2TP/HSP90 system in mouse development and colorectal
carcinogenesis
The Heat Shock Protein 90 folds the protein substrates neo-synthesized into an active
state. HSP90 inhibition results in degradation or aggregation of the substrates. Many
clients are involved in signal transduction pathways and related to tumor progression.
We know that the inhibition of HSP90 destabilizes HSP90 clients and could lead anti
tumoral effects. Until now, it was believed that this effect was due to the inhibition of
receptors and kinases.
A new HSP90 co-chaperon has been described: R2TP complex. It formed by four
proteins: Rvb1, Rvb2, Spaghetti/RPAP3 and Pih1D1. HSP90/R2TP system is
involved in the assembly of snoRNP, telomerase RNP, the nuclear RNA polymerases
and PIKKs, which all play some key functions in cellular proliferation.
On the one hand, HSP90 is involved in cancer so that more than 12 drugs inhibiting its
activity, have been tested in clinical trials. On the other hand, three components of
R2TP, Rvb1, Rvb2 and Spaghetti are overexpressed in hepatocellular and colorectal
carcinoma (our unpublished results). We hypothesize that the co-chaperon R2TP is
necessary to sustain, at least in part, the transforming activity of HSP90.
To study the role of R2TP in animal development and carcinogenesis, we generated
animals deficient for SPAGHETTI (SPAG) protein. We have generated mouse lines
harboring two different alleles: a Floxed one and one encoding a truncated protein.
We chose this subunit because (i) it interacts directly with HSP90, (ii) it
overexpressed in colorectal and hepatocellular cancer and (iii) it is not essential in
yeast.
Our preliminary data show that SPAG is expressed in all tested adult tissues.
Moreover, preliminary results suggest that SPAG is necessary for mouse embryonic
development. This correlates with the function of Spaghetti in Drosophila
Melanogaster. One fundamental interest of this work is that very few HSP90 cochaperone have been characterized in mouse model so far.
Chloé Maurizy : Institut de Génétique Moléculaire de Montpellier, université de
Montpellier, Bérengère Pradet-Balade : Centre de Recherche de Biochimie
Macromoléculaire, Edouard Bertrand : Institut de Génétique Moléculaire de
Montpellier
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N°63 –Samy MURAT
Phosphoproteomics of 5-HT2A/mGlu2 heteromers: toward new
insights into the mechanism of action of hallucinogens and
antipsychotics
The serotonin 5-HT2A receptor is the primary target of psychedelic hallucinogens such
as LSD, mescaline and psilocybin (agonists), which reproduce some of the core
symptoms of schizophrenia and of second-generation antipsychotics such as
clozapine, olanzapine and risperidone (antagonists or inverse agonists). Recent
findings demonstrate that 5-HT2A receptors form heteromers with metabotropic
glutamate mGlu2 receptors, another target of last-generation antipsychotics (agonists
or positive allosteric modulators). The association of both receptors has profound
consequences on their pharmacology and signal transduction properties as well as on
the behavioural effects of drugs that bind to either 5-HT2A receptors or mGlu2
receptors. For instance, 5-HT2A receptor/mGlu2 heteromer formation is essential for
the expression of psychotropic-like effects of hallucinogens and imbalanced activity
and coupling properties of 5-HT2A and mGlu2 receptors within the heterocomplex
might be one of the molecular substrates for a susceptibility to schizophrenia. To get
further insight into the mechanism of action of drugs acting at 5-HT2A/mGlu2
heteromers, we explored their impact upon the phosphorylation pattern of each
receptor by high-resolution mass spectrometry. We show that hallucinogenic 5-HT2A
receptor agonists (LSD, DOI) but not non-hallucinogenic 5HT2A receptor agonists
promote 5-HT2A receptor phosphorylation at Ser280 located in the i3 loop, a region
important for receptor desensitization, both in HEK-293 cells and in mice prefrontal
cortex. Correspondingly, Ser280 phosphorylation was responsible for the lower
capacity of hallucinogens to promote receptor desensitization and internalization,
compared with non-hallucinogenic agonists. Conversely, several phosphorylated
residues were identified in the C-terminal domain of mGlu2 receptors co-expressed
with 5-HT2A receptors in HEK-293 cells. Glutamate treatment increased the
phosphorylation state of some of these residues, an effect prevented by the coapplication of the synthetic hallucinogen DOI, which alone did not affect mGlu2
phosphorylation. Collectively, these findings reveal novel molecular substrates that
might underlie the behavioural effects of drugs acting at each subunit of 5HT2A/mGlu2 heteromers.
Samy Murat(1-4), Samah Karaki(1-4), Carine Becamel(1-4), Clotilde Mannoury la Cour(5), Mark J.
Millan(5), Laurent Prézeau(1-4), Joël Bockaert(1-4), Philippe Rondard (1-4), Philippe Marin(1-4*),
Franck Vandermoere(1-4*)
(1) CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier; (2) INSERM, U661, Montpellier;
(3) Université Montpellier 1,Montpellier; (4) Université Montpellier 2, F-34094 Montpellier, France ; (5)
Institut de Recherches Servier, Croissysur-Seine, France. (*) These authors equally contributed to the study
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N°64 - Camille JACQUELINE
Immunodepression and accumulation of cancerous cells: a role of
everyday perturbations?
Immune cells are a key component against cancerous cells proliferation through their
ability to detect and to destroy them. However, immune system experiences temporary
immunodepression periods combined with long-term immunosenescence process.
Thanks to a theoretical model, we wanted to show how these immunodepression
periods, by their frequency and their duration, may impact cancerous cells
proliferation. Our results suggested that long immunodepression period can have a
significant impact on cancerous cells accumulation, with a stronger effect if such
immunodepression occurs early in life. Nevertheless, for a given total duration of
immunodepression, we highlight that numerous shorts episodes have a stronger
influence on cancerous cells accumulation than a long one. Our results suggest new
approaches where immunodepression factors should be treated to prevent threatening
accumulation of cancerous cells. We can also speculate about a potential novel
indirect role of infectious diseases on cancer incidence by diverting the immune
reaction against cancerous cells.
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N°65 - Berfin SEYRAN
E4F1 is a major regulator of pyruvate metabolism in normal skin
homeostasis and skin carcinogenesis
The multifunctional protein E4F1 is an essential regulator of normal skin homeostasis.
E4F1 inactivation in embryonic or adult skin results in stem cell autonomous defects
causing exhaustion of the epidermal stem cell (ESC) pool from their niche. At the
molecular level, we have recently shown that E4F1 controls ESC maintenance through
the transcriptional regulation of components or major regulators of the pyruvate
dehydrogenase (PDH) macro complex, which metabolizes pyruvate into acetyl-coA in
order to link glycolysis to the Krebs cycle. Our data demonstrates that defective PDH
activity in E4F1KO keratinocytes results in the redirection of glycolytic flux towards
lactate secretion both in vivo and in vitro. This metabolic reprogramming correlates
with alterations of the microenvironment and affects ESC adhesion in their niche.
Thus, our data highlights for the first time how cellular metabolism impacts on ESC
maintenance and skin homeostasis. Based on these results and on growing evidences
linking pyruvate metabolism to cancer development, I now wish to investigate the
functions of E4F1 during skin carcinogenesis, more specifically in melanoma, which
is the most aggressive type of skin cancer. Melanoma, through a multistep process,
arises from melanocytes and is driven by mutations in the Ras/ Raf signaling pathway.
In the early steps of melanogenesis, nevi which are benign tumors also harboring
Ras/Raf genetic alterations, melanocytes display senescent features, which have been
described as a barrier against malignancy. Interestingly, it has been shown that PDH is
a crucial mediator of this Braf-induced senescence. Moreover, recent results from the
lab point out the importance of E4F1 in regulating senescence also through PDH
modulation. Taken altogether, these data led me to characterize a potential E4F1 and
Ras/Raf cascade interplay, its influence on PDH regulation and how it impacts
melanomagenesis.
Berfin Seyran, Perrine Goguet, Laurie Gayte, Florence Bernex, Hélène Delpech,
Charles Vincent, Nelly Pirot, Claude Sardet, Matthieu Lacroix and Laurent Le Cam.
IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194
CBS2 Association
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N°66 - Moïra ROSSITTO
Risks of in utero NSAIDs and paracetamol exposure on the early
development and maturation of the reproductive organs
The non-steroidal anti-inflammatory drugs (NSAIDs) and acetaminophen (ACE,
paracetamol) are used worldwide to reduce mild to moderate pain, fever... Some
medications are available over the counter without a prescription and are often used in
self-medication.
In Europe and the United States, more than 50% of pregnant women take analgesic
drugs (Rebordosa et al., 2008; Werler et al., 2005). These medications are not
recommended only after 5 months of pregnancy. Very few studies have focused on the
putative effects of these drugs on the formation of embryonic gonads, knowing that
the gonads develop in humans in the first trimester of pregnancy (between 6 and 8
weeks).
The targets of NSAIDs and ACE are the cyclooxygenases (COX), the key enzymes in
the synthesis of prostaglandins from arachidonic acid. My team found that
prostaglandin D2 (PGD2) was involved in various stages of development and
maturation of embryonic testis at the somatic and germinal levels (Malki et al., 2005;
Moniot et al., 2014; Rossitto et al., 2015).
The purpose of this project is to evaluate the impact of in utero exposure to ACE and
NSAIDs on development and maturation of the reproductive organs in the mouse
embryo.
Moïra Rossitto*, Pascal Philibert*°, Candice Marchive*, Francis Poulat & Brigitte
Boizet-Bonhoure*
* Genetic and Development department, Institute of Human Genetics, CNRS
UPR1142, Montpellier, France.
° Hormonology department, Lapeyronie Hospital,
CHU Montpellier and Montpellier University, Montpellier, France
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N°67 - Claire MARQUILLY
Physiological role of APPL in Drosophila mushroom bodies’
development
The protein APPL (Amyloid Precursor Protein-Like) is the Drosophila homologue of
human APP, known to be involved in Alzheimer’s disease (AD). Despite its
implication in AD, the functional role of APP is still poorly known. Here, we
investigate the role of APPL during brain development. The mushroom bodies (MBs)
are the Drosophila center for learning and memory. APPL is highly expressed in the
MBs. This brain structure is, therefore, particularly relevant for this study. Recently,
APPL was described as a novel neuronal-specific modulator of the PCP pathway
required for the robustness of axonal outgrowth of the MB during development
(Soldano et al. PlosBiol 2013). It was shown that this protein facilitates the PCPspecific phosphorylation of the Wnt adaptor protein DSH/DVL (dishevelled) by the
Abelson kinase (Abl). We show here that APPL also interacts with ARM (armadillo)
protein, the Drosophila homologue of β-catenin, for the development of MB neurons.
This interaction seems to involve the function in adherens junction of ARM rather
than its function in the Wnt signaling pathway. Moreover, a previous study showed
that Abl regulates the planar polarized junctional dynamics through tyrosine
phosphorylation of ARM. Consequently, ABL could be a link between APPL and
ARM for the axonal outgrowth in the MBs. Only one mutant allele for the Appl gene
does exist which results from some very complex chromosomal rearrangements that
renders its analysis very difficult. In order to circumvent this problem we are creating
a molecularly defined complete deletion of the gene via the recent CRIPSR-Cas9
technic.
Claire MARQUILLY , Lee G. FRADKIN , Jean-Maurice DURA
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N°68 - Nour SFEIR
Role of the transcription factor RIP140 in colon cancer
The RIP140 protein is a transcriptional co-regulator involved in various physiological
processes. RIP140 represses the transcriptional activity of many transcription factors
such as nuclear receptors and E2F1 factor. Our recent results based on approaches that
combined the use of mouse models with molecular and cellular biology experiments,
have shown that RIP140 controls intestinal homeostasis and suggest that this gene
plays a tumor suppressor role in colorectal cancer. Indeed, in the intestinal epithelium,
RIP140 regulates cell proliferation and differentiation by inhibiting the Wnt signaling
pathway via the transcriptional regulation of the APC gene expression. In addition,
analysis of human biopsies showed that RIP140 mRNA levels are decreased in colon
cancer compared to healthy tissue (Lapierre et al, Journal of Clinical Investigation,
2014). The proposed thesis research program aims to further characterize the role of
RIP140 in intestinal tumorigenesis according to the following objectives. My first goal
will be to decipher the molecular interrelationships between RIP140 and the Wnt
signaling pathway. In addition, knowing that a crosstalk exists between the Wnt and
the Notch signaling pathways, I will study the molecular interrelationships between
RIP140 and the Notch signaling pathway and the role of the transcription factor KLF4
in these crosstalks. Finally, I will analyze the role of RIP140 in vivo in colon cancer
(generation and use of conditional transgenic mouse models). This project will clarify
the mechanisms by which RIP140 controls colon tumorigenesis and may highlight a
new biomarker for use in the diagnosis and / or treatment of this disease.
Keywords: RIP140, Wnt and Notch signalling, colon cancer, transgenic mouse
models.
Nour SFEIR, Sandrine BONNET, Marion LAPIERRE, Vincent CAVAILLES
IRCM, INSERM U1194, Montpellier Cédex 5
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N°69 - Amandine GUERIN
Characterization of host cell proteins recruited at the Moving
Junction during invasion of Toxoplasma gondii
Toxoplasma gondii is an obligate intracellular parasite which belongs to the
Apicomplexa phylum, including Plasmodium species which are responsible for
malaria. The invasion mechanism is unique among this phylum. It involves the
formation of a tight connection between the parasite and the host cell plasma
membranes called moving junction (MJ). During invasion, a complex formed of the
parasite rhoptry neck proteins RON2/4/5/8 is injected into the host cell and localized
to the MJ. RON2 spans the host plasma membrane and functions as a receptor for the
microneme protein AMA1 exposed on the parasite surface, resulting in close and
irreversible contact between the parasite and the host cell. The proteins RON4/5/8
localize to the cytosolic face of the host cell and are believed to provide a stable
anchoring point for host penetration. Apart from being located at the MJ, the precise
role of the RON4/5/8 proteins during invasion remains elusive. These proteins are
ideally positioned to carry out anchoring roles for the parasite through a connection
with host cytoskeleton proteins. Here, we present the specific recruitment of several
host cell proteins at the MJ by the RON complex.
Amandine Guérin1, Mauld H. Lamarque1, Michelle L. Tonkin2, Martin J.
Boulanger2 and Maryse Lebrun1
1
UMR 5235 CNRS, Université de Montpellier 2, 34095 Montpellier, France.
2
Department of Biochemstry and Microbiology, University of Victoria, Victoria,
British Columbia, Canada V8W 3P6
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N°70 - Lara BOU MALHAB
MLN4924, a NEDD8 inhibitor in a p53 based cyclotherapy
approach
TP53 gene also called the guardian of the genome helps maintaining our genome
integrity. Under normal conditions, p53 is kept at low expression levels by its two
negative regulators Mdm2 and Mdmx. Under stress conditions, different pathways
will be activated to induce p53 stabilisation. According to the stress level, p53 will
induce different p53-dependent genes expression involved in cell cycle arrest, DNA
damage repair or apoptosis. In 50% of cancer cases, p53 gene is found mutant. In
others, defects of its upstream regulators or downstream effectors are detected.
MLN4924, a specific inhibitor of the NEDD8 pathway (ubiquitin-like) is now in phase
I clinical trials. In order to target cancer cells specifically using MLN4924 as chemo
agent and to protect normal cells against the potential toxic effects of MLN4924, a
p53-based cyclotherapy was tested. Our results show that low activation of wild type
p53 by Actinomycin D protects normal cells to MLN4924 treatment leaving cancerous
cells with mutant p53 or no p53 to selectively commit to apoptosis upon MLN4924
treatment. Our results provide a possible combination therapy for MLN4924 based on
the p53 status. We are actually validating our findings in vivo, more specifically in
zebrafish model system.
Lara BOU MALHAB and Dimitris XIRODIMAS. Centre de Recherche de Biochimie
Macromoléculaire - UMR 5237, CNRS, Route de Mende 34 293, Montpellier, Cedex
5, France.
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N°71 –Laura YAZDANI
Ribosome: Master Regulator of Cancer Cell Fate?
It is well established that the ribosome acts as a master regulator of gene expression by
regulating protein synthesis both quantitatively and qualitatively. An emerging
concept suggests that ribosome is heterogeneous and can be "reprogrammed". These
"specialized ribosomes" would preferentially engage certain mRNA at the expense of
others and therefore drive cell phenotype and favor cell adaptation. From a more
pathological standpoint, many studies have correlated deregulation of both translation
machinery composition and activity with cancer initiation and evolution. Our goal is
to demonstrate that protein synthesis is differentially regulated depending on cancer
cell subpopulation and determine whether ribosomal heterogeneity could impact
tumoral evolution and plasticity.
Laura Yazdani1, Françoise Macari1, Oualid Ayad1, Damien Paulet2, Julie
Pannequin1, Armelle Choquet1 and Alexandre David1
1Institut de Génomique Fonctionnelle (IGF), INSERM U651 - CNRS UMR5203,
Montpellier. 2Laboratoire d'Informatique, de Robotique et de Microélectronique
(LIRMM), Montpellier
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N°72 - Badreddine BOUSSADIA
Pattern of drug metabolism enzyme expression in the epileptic
brain: a new mechanism of drug resistance
P450 metabolic enzymes are expressed in the human and rodent brain. Recent data
support their involvement in the pathophysiology of epilepsy. However, the
determinants of metabolic enzyme expression in the epileptic brain are unclear. We
tested the hypothesis that status epilepticus (SE) or exposure to phenytoin or
phenobarbital affects brain expression of the metabolic enzyme CYP2E1.
SE was induced in C57BL/6J mice by systemic kainic acid. Brain CYP2E1 expression
was evaluated 18–24 h after severe SE by immunohistochemistry. Co-localization
with neuronal nuclei (NEUN), glial fibrillary acidic protein (GFAP) and CD31 was
determined by confocal microscopy. The effect of phenytoin, carbamazepine and
phenobarbital on CYP2E1 expression was evaluated in vivo or by using organotypic
hippocampal cultures in vitro. CYP2E1 expression was investigated in brain
resections from a cohort of drug-resistant epileptic brain resections and human
endothelial cultures (EPI-EC). Immunohistochemistry showed an increase of CYP2E1
expression limited to hippocampal CA2/3 and hilar neurons after severe SE in mice.
CYP2E1 expression was also observed at the astrocyte-vascular interface. Analysis of
human brain specimens revealed CYP2E1 expression in neurons and vascular
endothelial cells (EC). CYP2E1 was expressed in cultured human EC and overexpressed by EPI-EC. When analyzing the effect of drug exposure on CYP2E1
expression we found that, in vivo or in vitro, ethanol increased CYP2E1 levels in the
brain and liver. Treatment with phenytoin induced localized CYP2E1 expression in
the brain whereas no significant effects were exerted by carbamazepine or
phenobarbital. Our data indicate that the effect of acute SE on brain CYP2E1
expression is localized and cell specific. Exposure to selected anti-epileptic drugs
could play a role in determining CYP2E1 brain expression. Additional investigation is
required to fully reproduce the culprits of P450 enzyme expression as observed in the
human epileptic brain.
Key words : CYP2E1; status epilepticus; drug exposure; biotransformation
Boussadia B1, Ghosh C2, Plaud C1, Pascussi JM3, De Bock F1, Rousset MC1,
JanigroD2, Marchi N1.
1
Laboratory of Cerebrovascular Mechanisms of Brain Disorders, Department of
Neuroscience, Institute of Functional Genomics, CNRS, Montpellier, France.
2
Cerebrovascular Research Center, Department of Biomedical Engineering and
Molecular Medicine, Cleveland Clinic, USA. 3Laboratory of Signaling, Plasticity and
Cancer, Department of Cancer Biology, Institute of Functional Genomics, Centre
National Recherche Scientifique (CNRS), Montpellier, France.
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N°73 - Ramhari KUMBHAR
Determinants for UBA1 recruitment at sites of DNA damage
Ubiquitylation is an important posttranslational modification that is necessary for
protein degradation as well as for the regulation and the localization of many cellular
factors. A number of proteins implicated in DNA replication and DNA damage
response are ubiquitylated. Ubiquitylation during the DNA damage response is
selectively dependent on the ubiquitin-activating enzyme UBA1, which functions at
the apex of the ubiquitylation cascade. Our objective is to elucidate whether and how
UBA1 is recruited at damaged sites and to uncover the role of ubiquitylation in ATR
signaling.
Using a cell free system developed in the lab that recapitulates ATR kinase-signaling
pathway, we found that UBA1 is recruited to linear DNA substrates and mediates
ubiquitylation of DNA-bound proteins. ATR-mediated Chk1 phosphorylation in cellfree extracts was dependent on UBA1 activity. Intriguingly, upon UBA1 inhibition,
Chk1 accumulated on biotinylated DNA coupled to streptavidin beads. We found that
protein ubiquitylation and the recruitment of UBA1 to DNA in cell-free extracts was
dependent on the kinase DNA-PKcs and on the poly ADP-ribose polymerase PARP1,
two sensors of DNA lesions. UBA1 exhibited affinity for PARP1 and for poly ADPribose (PAR) chains in vitro. Consistent with this, PARP1 promoted UBA1
recruitment at an inducible DNA double-strand break in living cell, as revealed by
chromatin immunoprecipitation.
These results indicate that UBA1 is recruited to DNA damaged sites in a DNA-PKcs
and PARP1 dependent-manner and that protein ubiquitylation is necessary for the
assembly of a productive ATR signaling complex. Thus, UBA1 inhibitors could be
used to target ATR signaling in cancer cells.
Ramhari Kumbhar1, Sophie Vidal-Eychenie1, Alkmini Kalousi2, Evi Soutoglou2, Cyril
Ribeyre1 and Angelos Constantinou1
1
Institute of Human Genetics (IGH), CNRS UPR-1142, Montpellier France. 2 Institute
of Genetics and Molecular and Cellular Biology (IGBMC), Strasbourg France
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N°74 - Gabriel ESPINOSA
The effect of microbial LPS translocation in cross-presenting
dendritic cells and in the breakdown of CD8+ T cell peripheral
tolerance in irradiated mice
We have utilized transgenic mice that express a well-characterized model antigen,
influenza HA, in the beta cells of the pancreas. These mice are profoundly tolerant of
the HA antigen in both the CD8+ and the CD4+ T cell compartments.
Lymphodepletion is currently used as adjuvant for adoptive cytotoxic T cell
immunotherapy in cancer. In fact mild irradiation enhance CD8+ T cell anti-self
responses and promotes the breakdown of T cell tolerance and the onset of
autoimmune diabetes. One of the effects of irradiation is the systemic translocation of
LPS from commensal bacteria. To assess whether bacterial LPS is responsible for the
breakdown of tolerance observed we have treated mice with a cocktail of antibiotics in
the drinking water in order to prevent translocation. Our results demonstrated that
antibiotic treatment can efficiently prevent the systemic translocation of LPS induced
by irradiation. Surprisingly, Beta cell-specifc CD8+ T cells proliferated extensively in
response to self-antigen cross-presentation in the draining lymph nodes of the
pancreas, differentiated into effector cells and infiltrated the islets of Langerhans
inducing disease in both antibiotic treated mice and controls. Analyses of the CD11c+
antigen presenting cells demonstrated that irradiation induces their activation as
measured by the enhanced expression of CD80, CD86, CD40 and MHC II. Although
antibiotic treatment partially prevent the activation of most of cross-presenting
dendritic cells subsets, our results indicate that commensal bacteria LPS translocation
blockade is not sufficient to prevent the breakdown of CD8+ T cell peripheral
tolerance and the onset of autoimmune diabetes.
Gabriel Espinosa-Carrasco12, Marine Villard12, Pascale Louis-Plence12 and Javier
Hernandez12*
1 Inserm U1183, Institute for Regenerative Medicine and Biotherapy, Montpellier, F34295 France. 2 Université Montpellier, UFR de Médecine, Montpellier, F-34000
France
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N°75 - Myriam RICHAUD
A new integrated platform for drug action mode determination in
C. elegans
We used the Caenorhabditis elegans model to decipher new molecular regulations of
aging that spread out or emerge from the main pathway regulating life span: the
Insulin/IGF-1/daf-2 pathway. This pathway can control many features of aging
determination process including stress resistance, metabolic control as well as
hormetic mechanisms. Growing evidence retrieved in model organisms indicates that
molecular networks regulating aging can be now extrapolated to humans.
We have developed an original set of molecular tools (Araiz et al., 2008a) as well as
innovative technical sets that allow our team to look experimentally at aging
determination and plasticity by molecular genetic, physiology (HTS-HCS screenings
robots Cellomics array-scan), imagery, transgenesis (gene gun BioRad).
We have developed new molecular and genetic toll that allow us to look at the aging
process control in relation with various treatments in order to find unidentified targets.
As for example, Chicoric acid can act on muscle mitochondria homeostasis as well as
on metabolism linked with type 2 diabetes. We have started to analyse C. elegans
aging impact of Chicoric acid and found an unexpected positive effect even at very
low (micromolar) concentrations. We are now testing by gene knockout and gene
knockdown (RNAi) the molecular pathways involved in Chicoric acid action.
Our quantitative C.elegans technology can include either soluble or insoluble drug
screenings on identified or unidentified molecular targets (or pathways) as well as
monitoring of mitochondrial, metabolism or aging impact as well.
Myriam Richaud, Pierre Cuq and Simon Galas
Laboratoire de Toxicologie, Faculté de Pharmacie, 15 Av Charles Flahault, BP 14
491, 34093 Montpellier Cedex 5
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N°76 - Quang Tien PHAN
Transient infection induces chronic inflammation
Zebrafish notochord has been used as an emerging model for the study of bone and
cartilage inflammatory diseases. We have recently shown that infection of zebrafish
embryo notochord by non-pathogenic E.coli could induce a chronic inflammation
episode, which subsequently led to severe defects of the notochord and malformation
of the vertebrae. In the present study we further identify the molecular mechanisms
that promote and sustain the inflammation after the bacterial clearance. We also reveal
the cellular interactions between bacteria, notochordal cells and the leukocytes of
innate immune system.
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N°77 - Marianne EL BAROUK
Endogenous retrovirus and mobility
Les rétrovirus appartiennent à la grande famille des rétroéléments exogènes et endogènes
qui ont en commun un mécanisme de réplication par transcription inverse de leurs ARN
génomiques. Plus précisément, la forme provirale des rétrovirus exogènes partage avec le
groupe des rétroéléments à LTR (LTR-retroelements) une structure génomique canonique :
les gènes gag ou gag-like, pol, et parfois env encadrés par deux séquences répétées
terminales (LTR : Long Terminal Repeat) contenant les séquences cis-régulatrices
nécessaires à la transcription. Les rétrovirus endogènes représentent une composante plus
ou moins importante des génomes eucaryotes selon les espèces considérées et l’étude de
ces éléments soulève de nombreuses questions. Quelles sont les protéines impliquées dans
l’endocytose et l’exocytose de ces rétrovirus endogènes? Quel est l’impact de ces
séquences sur l’intégrité et le fonctionnement de leurs génomes hôtes? Connaît-on tous les
mécanismes de régulations des rétrovirus endogènes?
Au laboratoire, nous étudions un rétrovirus endogène de drosophile, gypsy ( Marlor et al.,
1986). Ce rétrovirus est exprimé dans les cellules dans les cellules folliculaires de l’ovaire
de drosophile et rétrotranspose dans l’ovocyte. Ceci se traduit par la présence de nouvelles
copies provirales de gypsy dans la descendance des femelles. Le projet a pour but de
déterminer les protéines impliquées dans le transfert de gypsy du soma au germen. Un
début de réponse a été apporté pour un autre virus endogène de D. melanogaster nommé
ZAM dont le cycle de réplication est très similaire à celui de gypsy (Leblanc et al., 2000).
Des données expérimentales suggèrent que les particules de ZAM sont transférées aux
ovocytes grâce à un «piratage» des voies d’exocytoses et d’endocytose utilisées par
vitellogénine (Brasset et al., 2006). Est-ce un cas particulier, y a-t-il d’autres protéines
impliquées ?
Si la mobilité des rétrovirus endogènes peut s’avérer parfois bénéfique pour l’hôte, elle lui
est fatale dans la plupart des cas. Des mécanismes maintenant la transposition à des taux
compatibles avec la survie des espèces ont été sélectionnés au cours de l’évolution. Ces
mécanismes de répression font intervenir des protéines de la famille Argonaute et des
petits ARN non codants, les piRNA (Piwi-interacting RNA) (revue Senti and Brennecke
2010). À cause de cette répression, nous n’avons actuellement que peu de données
concernant le passage de ces rétrovirus d’une cellule à l’autre. En utilisant la génétique de
la drosophile, le laboratoire a développé un outil permettant d’augmenter la fréquence de
ce passage en inactivant la voie de régulation par les piRNA. Cette approche génétique,
couplée avec des approches de séquençage à haut débit, permettra de déterminer les
protéines de l’hôte impliquées dans la mobilité et le passage de cellule à cellule, et aussi
d’étudier d’éventuels modes de régulation indépendants de la voie des piRNA.
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N°78 - A. Mahdi LAAREF
Contribution of the splicing machinery to the control of
pluripotency maintenance and early embryonic development
The therapeutic potential of understanding the molecular basis of pluripotency and
differentiation has led to many studies comparing transcriptional profiles in different
human Embryonic Stem Cell (hESC) lines and the study of expression changes during
differentiation. Alternative pathways for processing the primary transcript can
profoundly affect the diversity and function of the protein products that are generated
from a single gene to set complex programs involved in pluripotency and/or
differentiation of hECS. While our knowledge about transcriptional networks
regulating pluripotency and differentiation has been intensively studied, however the
role of alternative splicing in this process is not yet understood and clear examples of
concerted switching of multiple genes from one isoform to another have not been
demonstrated. The main objective of this project is to discover splicing factors
involved in the control of pluripotency maintenance and the early differentiation
potency in the three germ layers, and to explore their role in these processes. For this
purpose, we will use the iPSC and high-throughput technologies that we developed in
the laboratory to determine the splicing profiles that are affected by RNAi knockdown
of known constitutive component of the different spliceosomal complexes. The project
constitutes a fundamental investigation into the function and mechanism of alternative
splicing in stem cells that might have a major impact in regenerative medicine. This
investigation will capitalize on exciting discoveries made recently during analysis of
high throughput data in our lab to concentrate on the growing consensus that
alternative splicing is a crucial determinant of cellular morphology.
A Mahdi LAAREF, Yacine BARECHE, Jamal TAZI and Laure LAPASSET
Institue of Molecular Genetics of Montpellier
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Organizing commitee
The CBS2 Day 2015 is the thirteenth edition of CBS2 days. This event was
organized by the CBS2 association, with the support of the CBS2 Doctoral
School and BioCampus Montpellier.
Vuthy Ea (IGMM)
Tania Louis (IGH)
Selma Damhane (CBS)
Marco Bruschi (IGF)
Laurent Fernandez (CBS)
Ramhari Kumbhar (IGH)
Emilie Aponte (CRBM)
Laura Auboyer (IRBM)
Coralie Berthoux (IGF)
Amélie Borie (IGF)
Miriam Candelas (IGF)
Romain Davaze (IGH)
Benoît Girard (IGF)
Sarah Guiziou (CBS)
Camille Jacqueline (IRD)
Pierre Lesport (IGF)
Samy Murat (IGF)
Ruba Nasri (IBMM)
Matthias Ollivier (IGF)
Naresh Yandrapalli (CPBS)
Elise Goyet (IGF)
Ankit Dwivedi (CBPS/IBC)
Bouasse Malik (IGF)
Sandrine Urvoy
Secrétariat ED CBS²
Michel Desarménien
Directeur ED CBS²
Audrey Verdier
BioCampus Montpellier
Laurent Journot
BioCampus Montpellier
Jury and chairs
The organizing board wants to thank particularly the researchers who accepted
to be the jury and chairs of this CBS2 Day 2015 :
Dr. Bonnet Jerome
(CBS : Centre de Biochimie Strcuturale)
Dr. Talarek Nicolas
(IGMM : Institut de Génétique Moléculaire de Montpellier)
Dr. Hached Khaled
(CRBM : Centre de Recherche de Biochimie Macromoléculaire)
Dr. Claeysen Sylvie
(IGF : Institut de Génomique Fonctionnelle)
Dr. Bertaso Federica
(IGF : Institut de Génomique Fonctionnelle)
Dr. Basavarajaih Poornima
(IGH : Institut de Génétique Humaine)
Dr. Gerbe François
(IGF : Institut de Génomique Fonctionnelle)
Dr. Bouschet Tristan
(IGF : Institut de Génomique Fonctionnelle)
Dr. Lamb Ned
(IGH : Institut de Génétique Humaine)
Dr. Quesada Stanislas
(Université de Montpellier)
Louis Tania
(IGH : Institut de Génétique Humaine)
Yahia Yousra
(IGMM : Institut de Génétique Moléculaire de Montpellier)
Roussel Morgane
(IGF : Institut de Génomique Fonctionnelle)
Voisin Tiphaine
(IGF : Institut de Génomique Fonctionnelle)
Thanks
In addition to the CBS² doctoral school and BioCampus Montpellier, the CBS²
association is grateful to the following organizations for financial and
logistical support: