Pleiocarpa mutica - Université Paris-Sud

Transcription

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JDR 2016 UFR PHARMACIE
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LISTE DES POSTERS
LE NUMERO CI-DESSOUS CORRESPOND AU NUMERO DE PAGE DU LIVRET ET D'AFFICHAGE DU POSTER
8. SHEDDING LIGHTS ON CANCER CELLS AND
THEIR MICROENVIRONMENT: IN VITRO 3D
TUMOR MODEL OF PANCREATIC CANCER
FOR PRECLINICAL PREDICTION OF IN VIVO
BEHAVIOR OF NANOMEDICINES
G. Lazzari, S. Mura, P. Couvreur
1. IPSIT–INSTITUT
PARIS
SACLAY
D’INNOVATION
THERAPEUTIQUE
PLATEFORMES TECHNOLOGIQUES
2. STABILIZATION
OF
ADENOSINE
TRIPHOSPHATE-LOADED, CHITOSANBASED
NANOPARTICLES BY INCORPORATION OF
IRON
M. Quaillet, H. Hillaireau, G. Giacalone, C.
Cadiou, M. Callewaert, F. Chuburu, E. Fattal
9. PROTEOMIC ANALYSIS OF MONONUCLEAR
LEUCOCYTES IN A MOUSE MODEL OF
ANXIETY/DEPRESSION IN RESPONSE TO
CHRONIC FLUOXETINE TREATMENT
I. Mendez-David, C. Boursier, V. Domergue, J.P. Guilloux, R. Colle, B. Falissard, E. Corruble,
A. M. Gardier, D. J. David
3. HUMAN BLOOD MONOCYTES ARE ABLE TO
FORM EXTRACELLULAR TRAPS
V. Granger, V. Marani, B. Noel, Y. Gallais, N.
Szely, M. Pallardy, S. Chollet-Martin, L. de
Chaisemartin
10. DEGRADABLE
PEGYLATED
POLYMER
PRODRUG NANOOBJECTS
E. Guégain, J. Tran, Q. Deguettes, J. Nicolas
4. A FLUORINE SCAN OF A TUBULIN
POLYMERIZATION
INHIBITOR
ISOCOMBRETASTATIN
A-4:
DESIGN,
SYNTHESIS, MOLECULAR MODELLING, AND
BIOLOGICAL EVALUATION
T. Naret, J. Bignon, G. Bernadat, B. Treguier,
M. Benchekroun, H. Levaique, C. Lenoir, J.
Dubois, A. Pruvost, J.D. Brion, F. Leroux, M.
Alami, A. Hamze
11. DESIGN AND INCORPORATION MECHANISM
OF FERROFLUIDS INTO LIPID-BASED JANUS
NANOPARTICLES
E. Millart, C. Ménager, S. Lesieur, V. Faivre
12. CXCR4 DESENSITIZATION REGULATES TLRMEDIATED PLASMA CELL DIFFERENTIATION
AND PERSISTENCE
N. Alouche, J. Natt, V. Biajoux, C. Freitas, N.
Fazilleau, K. Balabanian, M. Espeli
5. REGULATION OF CARDIAC PACEMAKER
ACTIVITY BY PDE4 ISOFORMS
D. Mika, A. M. Gomez, R. Fischmeister,
G.Vandecasteele
13. REGULATION DE LA DYNAMIQUE DES
MICROTUBULES PAR LA KINASE DE STRESS
JNK DANS LES CELLULES EPITHELIALES
H. Henrie, C. Poüs, A. Pilon, B. Benoit
6. CARACTERISATION DE LA SURFACE DE
NANOMEDECINES
PAR
DES
SONDES
MOLECULAIRES : DEVELOPPEMENT D’UNE
METHODE
PAR
ELECTROPHORESE
CAPILLAIRE
J.-B. Coty, F. Varenne, I. Le Potiera, C. Smadja,
C. Vauthier
14. INTEREST OF FLAGELLIN FLIC IN MUCOSAL
VACCINATION
AGAINST
CLOSTRIDIUM
DIFFICILE
J-F. Bruxelle, A. Mizrahi, S. Hoys, A. Collignon,
C. Janoir, S. Péchiné
7. RAPID ANXIOLYTIC EFFECTS OF A 5-HT4
RECEPTOR AGONIST INVOLVES PREFRONTAL
CORTEX/BRAINSTEM
NEURAL
CIRCUIT
RECRUITMENT.
C. Faye, I. Mendez-David, L. Tritschler, TH.
Pham, C. Defaix, M. A. Kheirbek, C.A. Denny,
R. Hen, A. M. Gardier, D. J. David.
15. MECHANISM
OF
SINOATRIAL
NODE
DYSFUNCTION IN A RYR2R420Q MOUSE
MODEL
OF
CATECHOLAMINERGIC
POLYMORPHIC VENTRICULAR TACHYCARDIA
Y. Y. Wang, P. Mesirca, E. Marqués-Sulé, A.
Zahradnikova Jr, O. Villejoubert, P. d’Ocon, C.
Ruiz, D. Domingo, E. Zorio, M. E. Mangoni, J.P. Benitah, A. M. Gómez
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16. NANOCAPSULES OF PERFLUOROCARBONS AS
THERANOSTIC AGENTS.
T. Boissenot, S. Houvenagel, G. Picheth, C.
Dejean, A. Paci, V. Poinsignon, B. Larrat, A.
Bordat, H. Chacun, C. Gueutin, L. Moine, O.
Diou, L. Mousnier, E. Fattal, N. Tsapis
23. ENCAPSULATION OF A LIPOPHILIC PRODRUG
OF DEXAMETHASONE INTO PLGA-PEG
NANOPARTICLES:
FORMULATION,
PHARMACOKINETICS
AND
ANTIINFLAMMATORY EFFICACY.
M. Lorscheider, R. Simón-Vázquez, N. Tsapis,
P. Calleja, L. Mousnier, E. Fattal
17. UPDATES
IN
NICKEL
ALLERGY:
IDENTIFICATION
OF
NAÏVE
T-CELLS
RECOGNIZING NICKEL AND IN VITRO
REGULATION OF THE INTERLEUKIN-12
CYTOKINE FAMILY IN HUMAN DENDRITIC
CELLS
R. Bechara, D. Antonios, M.-E. Azoury, H.
Azouri, M. Pallardy
24. SELF-ASSEMBLED
NANOPARTICLES
OF
BIOTRANSESTERIFIED CYCLODEXTRINS AND
NONLAMELLAR LIPIDS AS CARRIERS OF
WATER-INSOLUBLE SUBSTANCES
L. Zerkoune, S. Lesieur, J.-L. Putaux, L.
Choisnard, A. Gèze, D. Wouessidjewe, B.
Angelov, C. Vebert-Nardin, J. Doutch, A.
Angelova
18. AMPA RECEPTOR ACTIVATION IN DORSAL
RAPHE NUCLEUS AND GABA-A RECEPTOR
INHIBITION IN PREFRONTAL CORTEX ARE
NECESSARY
FOR
KETAMINE
ANTIDEPRESSANT-LIKE ACTIVITY
T. H. Pham, C. Defaix, A. Solgadi, P.
Chaminade, L. Tritschler, I. Mendez-David, D.
J. David, A. M. Gardier
25. PHOSPHODIESTERASE TYPE 2 LOCALIZED IN
CARDIAC
MITOCHONDRIA
REGULATES
MITOCHONDRIAL MEMBRANE POTENTIAL,
SWELLING AND CALCIUM ACCUMULATION
D. Liu, Z. Wang, D. Courilleau, G.
Vandecasteele, R. Fischmeister, C. Brenner
26. PTSA CATALYZED ANNULATION OF (E)-1,4DIARYLENYNES
INTO
(E)-3STYRYLISOCOUMARINS[1]
G. Zhao, L.-Z. Yuan, M. Roudier, J.-F. Peyrat, A.
Hamze, J.-D. Brion, O. Provot, M. Alami
19. INTEGRATION
OF
IMAC
PRECONCENTRATION, SEPARATION AND
DETECTION
OF
PHOSPHORYLATED
BIOMARKERS IN A MICROTAS AUTEURS
M. Araya-Farias, S. Dziomba, B. Carbonnier,
M. Guerrouache, N. Aboud, M. Taverna, N.
Thuy Tran
27. GENE POLYMORPHISM AND CIRCULATING
LEVELS OF MATRIX METALLOPROTEINASES
AND THEIR TISSUE INHIBITORS IN
CORONARY ARTERY DISEASE
A. Ben Braiek, B. Baudin, C. Delomenie, H.
Chahed, H. Gamra, F. Maatouk, S. Ferchichi
20. PD-CATALYZED
ANOMERIC
C(SP3)-H
ACTIVATION OF GLYCOPYRANOSIDES VIA A
CMD
(CONCERTED
METALATION
DEPROTONATION) MECHANISM
N. Probst, V. Gandon, M. Alami, S. Messaoudi
28. EXPLORATION OF OXALIPLATIN-INDUCED
NEUROPATHIC PAIN BY THE SUDOSCAN®
MACHINE
(CANALOXA
STUDY):
PRELIMINARY RESULTS AFTER 6 MONTHS
FOLLOW-UP
K.
Abdallah,
J.-B.
Delmotte,
M.
Andriamamonjy, H. Beaussier, F. Coudoré
21. NANOPARTICULES
SQUALENISEES
ET
LIPOPROTEINES
PLASMATIQUES
:
CARACTERISATION
DES
INTERACTIONS
MOLECULAIRES ET EVALUATION DE LEUR
IMPLICATION
DANS
LA
REPONSE
THERAPEUTIQUE
D. Sobot, S. Mura, D. Desmaele, P. Couvreur
29. STEREOSELECTIVE
COPPER
CATALYZED
DIRECTED THIOGLYCOSYLATION OF C(SP2)-H
BONDS OF BENZAMIDES
A. Chabrier, A. Bruneau, S. Benmahdjoub, B.
Benmerad, S. Benmerad, M. Alami S.
Messaoudi
22. CXCR4 DESENSITIZATION REGULATES TLRMEDIATED PLASMA CELL DIFFERENTIATION
AND PERSISTENCE
N. Alouche, J. Natt, V. Biajoux, C. Freitas, N.
Fazilleau, K. Balabanian, M. Espeli
4
30. CARDIO PROTECTIVE EFFECT OF ORAI1
INHIBITION AGAINST PRESSURE OVERLOADINDUCED PATHOLOGICAL REMODELING
F. Bartoli, B. Rode, D. Beech, A. M. Gomez, J.P. Benitah, J. Sabourin
37. EFFETS
METABOLIQUES
ET
NON
METABOLIQUES
DE
LA
DELETION
CARDIAQUE ET INDUCTIBLE DE L’AMPK 2
CHEZ LA SOURIS MALE.
L. Grimbert, M. N. Sanz, J. Piquereau, C.
Rucker-Martin, M. Gressette, R. VenturaClapier, V. Veksler, A. Garnier
31. SUBSTRATE
SPECIFICITY
OF
THE
CLOSTRIDIUM DIFFICILE CWP84 PROTEASE
DEPENDS ON ITS LOCALIZATION AND
MATURATION STATE.
C. Périllaud-Dubois, T. Candela, D. ChapetónMontes, C. Janoir
38. DEVELOPMENT OF A CE-MS METHOD FOR
THE ANALYSIS OF N-GLYCANS
C. Ruel, M. Taverna , C. Junot , F. Fenaille, T.
Tran
32. JANUS
NANOPARTICLES:
CHARACTERIZATION AND IMPLEMENTATION
OF SPECTROSCOPIC DESCRIPTORS AND
FOLLOW-UP SKIN APPLICATION
K. Kemel, C. Laugel, V. Faivre, A. BailletGuffroy
39. IDENTIFICATION OF SPECIES IMPLIED IN
HIAPP AMYLOID CASCADE : INTEREST IN
TYPE 2 DIABETES
C. Berardet, J. Kaffy, S. Ongeri , M. Taverna
40. WHEN A VIRAL PROTEIN SWITCHES ON
SELECTIVE
AUTOPHAGY
TO
ESCAPE
APOPTOSIS AND PROMOTE CELL SURVIVAL
G. Vilmen, G. Siracusano, L. Mouna, F.
Quignon, V. Maréchal, A. Esclatine
33. BK(CA) CHANNELS ARE KEY EFFECTORS OF
CORONARY TONE REGULATION BY PDE3
AND PDE4 IN NORMAL, BUT NOT IN FAILING
HEART.
S. Idres, G. Perrin, V. Domergue, R.
Fischmeister, V. Leblais, B. Manoury
41. PRECONCENTRATION
STRATEGIES
IN
CAPILLARY ELECTROPHORESIS. HOW TO
IMPROVE SENSITIVITY OF MOLECULAR
DIAGNOSTICS?
C. Crosnier de Lassichère, T. D. Mai, M.
Taverna
34. SKIN ALLERGY CAUSED BY AIR-OXIDIZED
TERPENES: MECHANISM OF ACTION AND
ROLE OF TRANSCRIPTION FACTOR NRF2
C. Raffalli, S. Kuresepi, E. Clouet, M.-H.
Damiens, A. Natsch, J.-P. Lepoitevin, M.
Pallardy, P.-J. Ferrt, E. Gimenez-Arnau, S.
Kerdinz-Römer
42. REGIOSELECTIVE REDUTIONS OF 1,2DIKETONES
USING
CHLOROTRIMETHYLSILANE AND SODIUM
IODIDE
L.-Z. Yuan, D. Renko, O. Provot, A. Hamze, M.
Alami
35. CAPTURE ET DETECTION DE BACTERIE A
L’AIDE DE NANOPARTICULES MAGNETIQUES
POUR
LE
DEVELOPPEMENT
DE
BIOCAPTEURS.
C. Galland, O. Lefebvre, F. Mbock Nkot, C.
Janoir, T. Candela, E. Martincic, M. Taverna,
M. Ammar, C. Smadja
43. IDENTIFICATION ET CARACTERISATION DU
ROLE DE LA MITOMYCINE C DANS LA
MALADIE VEINO OCCLUSIVE PULMONAIRE
M.-C. Chaumais, F. Perros, A.Seferian, C.
O’Connell, S. Günter, O. Sitbon, G.
Simonneau, M. Humbert, D. Montani.
36. ETUDE LIPIDOMIQUE DE L’IMPACT DE
L’ACIDE EICOSAPENTAENOÏQUE (EPA) SUR
L’EFFLUX
DU
CHOLESTEROL
DES
MACROPHAGES HUMAINS
G. Sayet, J.-P. Paul, E. Caudron, S. Tfaili, N.
Fournier, P. Chaminade
44. MOLECULAR
NETWORKING-BASED
DETECTION AND ISOLATION OF A NEW BISINDOLE ALKALOID FROM THE STEM BARK OF
PLEIOCARPA
MUTICA
BENTH.
(APOCYNACEAE)
E. Otogo N’nang, L. Evanno, K. Leblanc, P.
Grellier, B. Kumulungui, E. Poupon, M. A.
Beniddir, P. Champy
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45. MARINE NATURAL PRODUCTS AS SCAFFOLD
FOR GENERATION OF CHEMICALLY DIVERSE
LIBRARIES FOR DRUG DISCOVERY.
D. Lachkar, L. Evanno, C.Debitus, E. Poupon
49. LA TECHNIQUE D'IMAGERIE CONFOCALE
REVELE UN LIEN ETROIT ENTRE LA PROTEINE
MICROTUBULAIRE +TIP CLIP-170 ET LES
MITOCHONDRIES AU COURS DU STRESS
CELLULAIRE
D. Perdiz, V. Nicolas,C. Poüs
46. DEVELOPPEMENT D’UN GEL VAGINAL
PROBIOTIQUE POUR LA PREVENTION DE LA
GONOCOCCIE
K.C. N’Guessan Gnaman, S. Geiger, S.
Bouttier, A.A.S. Aka-Any-Grah, A. Yeo,
V.Nicolas, S. Villebrun, N. Huang, H. FayeKette, G. Ponchel, A.A. Koffi, F. Agnely
50. «ESCALADER
DES
MONTAGNES»
EN
SYNTHESE
ORGANIQUE
:ASSEMBLAGE
BIOMIMETIQUE DE LA BIPLEIOPHYLLINE ET
DE LA VOACALGINE A.
D. Lachkar, N. Denizot, G. Bernadat, K.
Ahamada, M. A. Beniddir, V. Dumontet, J.-F.
Gallard, R. Guillot, K. Leblanc, E. Otogo
N’nang, V. Turpin, C. Kouklovsky, E. Poupon,
L. Evanno, G. Vincent
47. EXPLORATION DE L’ESPACE CHIMIQUE DE
PICRALIMA NITIDA GRACE AUX RESEAUX
MOLECULAIRES,
UNE
APOCYNACEAE
OUBLIEE A L’ERE DES « BIG DATA »
C. Alcover, J.-F. Gallard, F. A. Kabran, P.
Grellier, L. Evanno, E. Poupon, M. A. Beniddir
51. INTERPLAY
BETWEEN
HUMAN
PAPILLOMAVIRUS
AND
THE
CXCR7
RECEPTOR
C. Gallego, A. Jaracz-Ros, F. Gaudin, A.
Fumagalli, P. Marin, G. Schlecht-Louf F.
Bachelerie
52. Tubulin-bound septins, microtubules and cell
motility: Study in the context of
chemoresistance
M. Saati, I. Cantaloube, C. Poüs, A. Baillet
48. LIGHT-TRIGGERED LIPOSOMAL RELEASE:
IMPACT OF LIPID COMPOSITION AND
PHOTOSENSITIZER HYDROPHOBICITY
J. Massiot, A. Makky, D. Chapron, V. Rosilio
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LISTE DES POSTERS PAR POLE THEMATIQUE DE RATTACHEMENT A L'ED569
PHARMACOTECHNIE & PHYSICO-CHIMIE PHARMACEUTIQUE
2, 6, 8, 10, 11, 16, 21, 23, 24, 41, 46, 48
PHARMACOLOGIE-TOXICOLOGIE
3, 7, 9, 17, 18, 28, 34
PHYSIOPATHOLOGIE MOLECULAIRE ET CELLULAIRE
5, 13, 15, 25, 27, 30,33, 37, 43, 49, 52
CHIMIE PHARMACEUTIQUE
4, 20,26, 29, 38, 39, 42, 44, 45, 47, 50
MICROBIOLOGIE ET THERAPEUTIQUES ANTI-INFECTIEUSES
14, 31, 40
IMMUNOLOGIE ET BIOTHERAPIES
12, 22, 51
LISTE DES POSTERS PAR POLE THEMATIQUE DE RATTACHEMENT A L'ED571
CHIMIE PHYSIQUE, BIOPHYSIQUE ET ANALYTIQUE (CPBA)
19, 32, 35, 36
7
IPSIT – Institut Paris Saclay d’Innovation Thérapeutique
9 plateformes technologiques
UPSud IPSIT, Inserm US31, Cnrs UMS3679, Université Paris-Saclay
Pôle thématique de rattachement à l'ED569 : Innovation Thérapeutique
[email protected]
L’Institut Paris Saclay d’Innovation Thérapeutique - IPSIT est une unité mixte de service qui met
ses 9 plateformes technologiques à la disposition des chercheurs de l’Université Paris Sud et des
laboratoires académiques et privés.
La plateforme de Verrerie Scientifique et Technique (VERRE Scien-Tech) a rejoint l’IPSIT en
2016 : elle propose l'étude, la conception et la réalisation d'appareils en verre et matériaux
assimilés à usage scientifique, à destination de la recherche et de l'enseignement. Elle offre
également une expertise dans le domaine des produits verriers.
Et toujours :
Animalerie et Exploration Fonctionnelle – ANIMEX : Située sur deux sites Châtenay-Malabry &
Clamart, AnimEx propose des structures d’hébergement d’animaux et des équipements pour
l’exploration fonctionnelle du petit animal. Membre de CAPSud (Consortium des Animaleries
Paris-Sud), AnimEx est rattachée au Comité d’éthique CEEA n°26.
Service d'Analyse des Médicaments et Métabolites – SAMM- : plateforme instrumentale de
spectrométrie de masse couplée aux méthodes séparatives spécialisée dans l'analyse structurale
et la quantification des petites molécules et le profilage lipidomique.
Criblage haut débit – CIBLOT : a pour objectif d’identifier, par criblage à haut débit, de nouvelles
entités chimiques douées d’activités biologiques afin de développer des – outils moléculaires
pour la recherche – des molécules à visée thérapeutique.
Interactions Moléculaires – INTERMOL : offre un outil intégré dédié à la conception et l’étude
fine de nouveaux principes actifs. Les interactions moléculaires jouent un rôle central dans le
monde vivant, qu’il s’agisse d’interactions entre des protéines, des glycoprotéines, des
lipoprotéines ou encore entre des molécules d’ADN ou d’ARN.
Transcriptomique & Protéomique – TRANSPROT : propose ses compétences et son équipement
(PCR quantitative, biopuces à ADN, électrophorèse bidimensionnelle et spectrométrie de masse)
pour mener la quantification comparative de transcriptomes et/ou de protéomes.
Histologie & Immunologie du petit animal – PHIC : fournit son expertise dans le domaine de
l’analyse immune-pathologique de souris génétiquement modifiés et dans celui des modèles
animaux de pathologies humaines inflammatoires et/ou hématologiques.
Imagerie cellulaire – MIPSIT : propose une expertise et un accès à des outils technologiques de
pointe dans le domaine de la microscopie photonique et de l’analyse d’images.
Immunomonitorage –PLAIMMO: offre son expertise en cytométrie en flux et de masse pour la
réalisation de projets de recherche fondamentale et préclinique sur des modèles expérimentaux
animaux ainsi que pour des protocoles de recherche clinique chez l’Homme.
1
STABILIZATION OF ADENOSINE TRIPHOSPHATE-LOADED, CHITOSANBASED NANOPARTICLES BY INCORPORATION OF IRON
Marion Quaillet1, Hervé Hillaireau1, Giovanna Giacalone1, Cyril Cadiou2, Maïté Callewaert2,
Françoise Chuburu2, Elias Fattal1
1
Institut Galien Paris-Sud, Univ. Paris-Sud, CNRS, Université Paris-Saclay,
92296 Châtenay-Malabry, France
2
Institut de Chimie Moléculaire de Reims, UFR des Scicences Exactes et Naturelles,
51687 Reims Cedex 2, France
Pôle Pharmacotechnie et Biopharmacie
E-mail: [email protected]
Nanoparticle-based drug delivery has emerged as a promising tool to improve the stability and the
cellular uptake of hydrophilic drugs, which is of particular importance for nucleic acids but also
nucleotides such as adenosine triphosphate (ATP). Indeed the cellular delivery of ATP holds some
promise for the treatment of various pathologies including atherosclerotic lesions [1] and ischemia [2].
Our group has developed ATP-loaded, chitosan-based nanoparticles prepared by ionic gelation with a
high drug loading (45% w/w) [3]. Despite the high interest of such nanoparticle, their dispersion in
physiological media was found to promote their dissociation, which was correlated to the ionic
strength. In this context, it is necessary to develop new strategies to improve stability of this class of
nanoparticles. To do so, we propose the complexation of chitosan with iron (III) ions prior to
nanoparticle formation. The rationale of this approach lies in the ability of iron to strongly bind both
chitosan and phosphate groups [4].
The aim of this study was to synthesize and characterize a series of CS-Fe complexes and evaluate
their ability to increase the resistance of ATP-loaded, chitosan-based nanoparticles towards ionic
strength.
CS-Fe complexes were synthesized by dissolving chitosan (83% deacetylated) in a concentrated iron
nitrate (Fe(NO3)3) aqueous solution at acidic pH. The association of iron to chitosan was monitored by
phenanthroline-based assay, Inductively Coupled Plasma – Optical Emission Spectrometry (ICP-OES)
and IR. We obtained CS-Fe complexes with iron/glucosamine molar ratios ([M]/[L]) between 0.1 to
0.5, corresponding to an iron content of 2 to 20% w/w. All complexes successfully formed ATPloaded nanoparticles, as proved by DLS measurements and confirmed by microscopic observations.
The stability of these nanoparticles in isotonic solution (NaCl 0.9% w/v) was evaluated by turbidity
measurement, which showed that nanoparticle dissociation was dramatically reduced. This
nanoparticle stabilization effect increased with the iron content of CS-Fe complexes used.
In conclusion, CS-Fe complexes have demonstrated their potential to improve the stability of ATPloaded, chitosan-based nanoparticles.
References
[1] O. Leppanen, T. Bjornheden, M. Evaldsson, J. Boren, O. Wiklund and M. Levin, Atherosclerosis
2006, 188, 323-330.
[2] I. H. Chaudry, Ann N Y Acad Sci 1990, 603, 130-140; discussion 140-131.
[3] G. Giacalone, A. Bochot, E. Fattal and H. Hillaireau, Biomacromolecules 2013, 14, 737-742.
[4] S. C. Bhatia and N. Ravi, Biomacromolecules 2000, 1, 413-417.
2
Human blood monocytes are able to form extracellular traps
V. Granger*†, V. Marani*, B. Noel*, Y. Gallais*, N. Szely*, M. Pallardy*, S. Chollet-Martin*†and L. de
Chaisemartin*†
*
UMR996 - Inflammation, Chemokines and Immunopathology -,INSERM, Univ Paris-Sud, Université
Paris-Saclay, 92296, Châtenay-Malabry, France ;†APHP, Bichat Hospital, Immunology Department,
Paris, France
Pôle thématique de rattachement à l'ED569 : Toxicologie-Immunologie
[email protected]
Background
Neutrophil extracellular traps (NETs) are extracellular DNA filaments formed during
neutrophil activation, a process called netosis and originally associated with neutrophil
antibacterial properties. Several lines of evidence now suggest a major role for netosis in
thrombosis, auto-immune diseases and cancer. A similar mechanism, called etosis, has
been reported in other immune cells such as eosinophils. We investigated if such a
mechanism could exist in human monocytes.
Materiel and Methods
Magnetically sorted CD14+ monocytes and monocyte-derived dendritic cells were
stimulated with phorbol-12-myristate-13-acetate, calcium ionophore (A23187), plateletactivating factor and zymosan A. Extracellular traps release was quantified by Sytox
Green fluorescence. NETs were visualized by immunofluorescence with antibodies
against myeloperoxidase, lactoferrin, citrullinated histones, elastase, and tissue factor.
DNA-myeloperoxidase complexes were measured by ELISA and histone citrullination
was assessed by western blot.
Results
We demonstrate that human blood monocytes are capable of extracellular trap (ET)
release in response to several chemical and biological stimuli. In contrast, monocytederived dendritic cells are not capable of etosis but rather undergo necrotic cell death
upon stimulation. By microscopy, we show that monocyte ETs display a morphology
analogous to NETs, and are associated with myeloperoxidase, lactoferrin, citrullinated
histones and elastase. Monocyte etosis depends on oxidative burst via NADPH oxidase
activation but not on myeloperoxidase activity, in contrast to neutrophils. Finally, we
provide evidence of tissue factor on monocyte ETs, a feature that could be relevant to
monocyte thrombogenic properties.
Conclusion
We demonstrate that upon ex vivo stimulation, human blood monocytes can release
extracellular traps bearing several active proteins. This new cellular mechanism is likely
to improve our understanding of monocytes’ role in multiple pathological contexts such
as inflammatory disorders, infection or thrombosis.
3
A FLUORINE SCAN OF A TUBULIN POLYMERIZATION INHIBITOR
ISOCOMBRETASTATIN A-4: DESIGN, SYNTHESIS, MOLECULAR MODELLING,
AND BIOLOGICAL EVALUATION
Timothée Naret,a Jérôme Bignon,b Guillaume Bernadat,a Bret Treguier,a Mohamed
Benchekroun,a Helene Levaique,b Christine Lenoir,b Joelle Dubois,b Alain Pruvost,c JeanDaniel Brion,a Frederic Leroux,d Mouad Alami,*,a Abdallah Hamze*,a
a
BioCIS, Univ. Paris-Sud, CNRS, équipe labellisée Ligue Contre le Cancer, Université ParisSaclay, 92290, Châtenay-Malabry, France
b
Institut de Chimie des Substances Naturelles, UPR 2301, CNRS avenue de la terrasse, F91198 Gif sur Yvette, France
c
CEA, DRF/IBITECS, Service de Pharmacologie et d’Immunoanalyse, SMArt-MS,
Université Paris Saclay, F-91191 Gif sur Yvette, France
d
CNRS-Université de Strasbourg (ECPM), UMR 7509, COHA, 25 Rue Becquerel, 67087
Strasbourg, France
Chimie Pharmaceutique
[email protected] and [email protected]
This presentation reported the synthesis of a novel series of tubulin polymerization inhibitors,
based on fluorinated derivatives of isocombretastatin A-4 with the goal of evaluating the effect
of these compounds on the proliferative activity. The introduction of fluorine atom was
performed on the phenyl ring or at the linker between the two aromatic rings. The modification
of isoCA-4 by introduction of difluoromethoxy group at the para-position (3i) and substitution of
the two protons of the linker by two fluorine atoms (3l), produced the most active compounds in
the series, with IC50 values of 0.15–2.2 nM (3i) and 0.1-2 nM (3l) respectively, against a panel of
six cancer cell lines. Compounds 3i and 3l had greater antiproliferative activity in comparison
with references CA-4 or isoCA-4, the presence of fluorine group leads to a significant
enhancement of the antiproliferative activity. Analyses of cell cycle distribution, and
morphological microtubules organization showed that compound 3l induced G2/M phase arrest
and, dramatically disrupted the microtubule network. Molecular docking studies indicated that
compounds 3i and 3l occupy the colchicine binding site of tubulin.1)
1)
Submitted paper.
4
REGULATION OF CARDIAC PACEMAKER ACTIVITY BY PDE4 ISOFORMS
Delphine Mika, Ana M. Gomez, Rodolphe Fischmeister, Grégoire Vandecasteele
INSERM UMR-S 1180, Univ. Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France
Pôle thématique de rattachement à l'ED569 : Physiopathologie
[email protected]
Background
Numerous epidemiological and clinical studies have revealed a positive correlation
between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous
system is the major extracardiac determinant of HR. During sympathetic stimulation, the
activation of β-adrenergic receptors (βAR) induces an increase in cAMP levels, leading
to a positive chronotropic effect. Among the 5 cAMP-PDE families expressed in the
heart, PDE4 is critical for controlling excitation-contraction coupling (ECC) during βAR
stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. 3
genes encode for cardiac PDE4s: pde4a, pde4b and pde4d. Their respective
contribution to the regulation of pacemaker activity remains ill-defined.
Methods
The total enzymatic PDE activity was determined in mouse sinoatrial node (SAN) tissue
as the cAMP hydrolytic activity measured in the absence of PDE inhibitor and the
fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor
Ro-20-1724 (10 µM). The in vitro pacemaker activity was assessed by measuring the
spontaneous Ca2+ transients in Fluo4-loaded-SAN intact tissue. Images were obtained
using confocal microscopy.
Results
Ro-20-1724 increased the beating rate of intact mouse SAN and increased PKAphosphorylation levels of key ECC actors (ryanodine receptor, phospholamban). PDE4
enzymatic activity was found to account for 60% of the total cAMP-PDE activity in SAN.
The 3 isoforms PDE4A, 4B and 4D were found to be expressed in mouse SAN. In
PDE4D-, but not in PDE4B-deficient mice, Ca2+ homeostasis was altered in control
conditions and after βAR stimulation. Indeed, ablation of PDE4D induced a decreased
beating rate and an increased Ca2+ spark frequency in control and βAR-stimulated
conditions.
Conclusion
Our preliminary results reveal that PDE4 controls pacemaker function in mice and that
PDE4D ablation strongly perturbs normal SAN activity.
5
Caractérisation de la surface de nanomédecines par des sondes moléculaires :
développement d’une méthode par électrophorèse capillaire
J-B. Cotya,b, F. Varennea,b, I. Le Potiera,c, C. Smadjaa,c, C. Vauthiera,b
a
Institut Galien Paris-Sud, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 92290 ChâtenayMalabry, France
b
Equipe 6: Amélioration du passage des barrières par les molécules biologiquement actives
Equipe 4: Protéines et Nanotechnologies en Sciences Analytiques
Pôle thématique de rattachement à l'ED569 : Pharmacotechnie & Physico-chimie Pharmaceutique
c
[email protected]
Les nanomatériaux destinés à un usage thérapeutique souffrent aujourd’hui d’un manque
de connaissance de leurs propriétés à l’échelle moléculaire. Or, il devient de plus en plus
clair que l’étude de ces propriétés permettrait d’améliorer notre compréhension de leur
comportement dans les milieux biologiques [1, 2]. Jusqu’à présent, une grande attention
est prêtée à la caractérisation de l’identité synthétique des nanomatériaux (taille,
morphologie, charge de surface), alors qu’il apparaît que c’est l’identité biologique
acquise après administration qui gouverne leur devenir (biodistribution, temps de
circulation, toxicité) [3]. Il est aujourd’hui important de comprendre comment les
propriétés synthétiques des nanomédecines influent sur leur comportement dans les
milieux biologiques. Des modèles prédictifs du devenir in vivo à partir de l’identité
synthétique tendent à se développer [4]. Pour cela, des méthodes d’étude des interactions
des nanoparticules avec les éléments biologiques sont nécessaires. Une étape délicate lors
de l’étude de tels systèmes est la séparation des nanomatériaux des protéines restées
libres. L’électrophorèse capillaire constitue un outil de choix permettant de réaliser cette
étape de séparation in situ sans perturbation de l’échantillon, tout en dosant la quantité de
protéine adsorbée sur les nanomédecines.
La présente étude vise à développer une méthode de caractérisation de la surface de
nanomédecines via des sondes protéiques. Le but est ainsi d’établir une possible
corrélation entre la masse molaire d’une protéine et sa capacité à s’adsorber sur la surface
d’une nanoparticule stabilisée par un polymère hydrophile. Des protéines de tailles
différentes ont été sélectionnées, et leur capacité à interagir avec différentes
nanoparticules sera testée. Nous pourrons ainsi établir le profil d’adsorption relatif à
chacune de ces nanomédecines et possiblement le corréler à leur identité synthétique
initiale. Les conditions de séparation des protéines libres des nanoparticules ont été
optimisées. Les premiers résultats montrent des tendances dans la sélectivité de
l’adsorption des protéines qui sont nanoparticules dépendantes.
Cut-off 60kDa ?
Cut-off 600kDa ?
Références :
[1] S. Behzadi et al., Colloids and Surfaces B: Biointerfaces 2014, 123, 143–149
[2] C. D. Walkey et al., J.Am.Chem.Soc. 2012, 134, 2139−2147
[3] C. D. Walkey et al. ACS Nano, 2014, 8 (3), 2439–2455
[4] A. Bigdeli et al. ACS Nano, 2016, 10 (3), 3723–3737
6
Rapid anxiolytic effects of a 5-HT4 receptor
cortex/brainstem neural circuit recruitment.
agonist
involves
prefrontal
Faye C1, Mendez-David I1, Tritschler L1, Pham TH1, Defaix C1, Kheirbek MA2, Denny CA3,
Hen R4, Gardier AM1, David DJ1
Affiliations : 1Université Paris-Saclay, University Paris-Sud, Faculté de Pharmacie, CESP, INSERM
UMRS1178, Chatenay-Malabry 92296, France.. 2Department of Psychiatry, University of California, San
Francisco, San Francisco, CA 94158, USA; Center for Integrative Neuroscience, University of California, San
Francisco, San Francisco, CA 94158, USA.3Department of Psychiatry, Columbia University, New York, NY
10032, USA; Division of Integrative Neuroscience, New York State Psychiatric Institute, New York, NY 10032,
USA. 4Department of Psychiatry, Columbia University, New York, NY 10032, USA; Division of Integrative
Neuroscience, New York State Psychiatric Institute, New York, NY 10032, USA; Department of Neuroscience,
Columbia University, New York, NY 10032, USA; Kavli Institute for Brain Science, Columbia University, New
York, NY 10032, USA.
Pôle thématique de rattachement à l'ED569 : Pharmacologie-toxicologie
[email protected]
Generalized Anxiety Disorder (GAD) is one of the most prevalent mental health
conditions. Although benzodiazepines are very effective in reducing acute anxiety, their
adverse effects limit their use chronically. Selective serotonin reuptake inhibitors
(SSRIs), usually prescribed in depression, are considered to be the first line of therapy
for GAD, even though they display a delayed onset of action of several weeks. The
development of new anxiolytics, therefore, is of considerable importance, and
understanding the mechanisms underlying this delayed onset should offer insights into
new approaches. Recent studies indicate that 5-HT4 receptor agonists are faster acting
than SSRIs to treat anxiety/depression-like behavior.
Here, we explored the neural circuit recruitment in acute 5-HT4 receptor stimulationinduced fast anxiolytic-like effects in mice. Unlike fluoxetine (18 mg/kg, i.p), but similar to
diazepam (1.5 mg/kg, i.p), acute 5-HT4 receptor agonist RS 67333 (1.5 mg/kg, i.p)
induced fast anxiolytic-like effect in male BALB c/J mice in behavioral tests such as the
Elevated Plus Maze (EPM) and in the Novelty Suppressed Feeding (NSF). Since the 5HT4 receptor is expressed in the medial prefrontal cortex (mPFC) and these cells project
to the brainstem dorsal raphe nucleus (DRN), a serotonergic nucleus implicated in
emotional behavior, we evaluated the recruitment of this neural circuit in 5-HT4 receptor
stimulation-induced fast anxiolytic-like activity of RS 67333. To this end, the behavioral
consequences of local acute intra-mPFC injection of RS 67333 in the EPM and NSF in
serotonin–depleted para-chlorophenylalanine (p-CPA, a tryptophan hydroxylase inhibitor)
mice were tested. Interestingly, acute intra-cortical injection of RS 67333 (0.5 g/side)induced anxiolytic-like effect in the EPM and NSF was abolished in serotonin–depleted
p-CPA mice pointing out the critical role of cortical 5-HT release in these effects. By
inhibiting optogenetic mPFC projection-targeting to the brainstem DRN with a AAV5CaMKII-ArchT-green fluorescent protein (GFP) virus, we are currently evaluating this
neural circuit recruitment in cortical 5-HT4 receptor stimulation in the anxiolytic-like
effects of RS 67333. In summary, 5-HT4 receptor stimulation via prefrontal
cortex/brainstem neural circuit recruitment could represent an innovative and rapid onset
therapeutic approach to treat anxiety disorders.
7
SHEDDING LIGHTS ON CANCER CELLS AND THEIR MICROENVIRONMENT: IN
VITRO 3D TUMOR MODEL OF PANCREATIC CANCER FOR PRECLINICAL
PREDICTION OF IN VIVO BEHAVIOR OF NANOMEDICINES
Gianpiero Lazzari, Simona Mura, Patrick Couvreur
Institut Galien Paris-Sud, UMR 8612, CNRS, Univ. Paris-Sud, Université Paris‐Saclay,
Faculté de Pharmacie, 5 rue JB Clément, 92296 Châtenay-Malabry, France
Pôle thématique de rattachement à l'ED569 : Pharmacotechnie et Physico-chimie
Pharmaceutique
[email protected]
Nanomedicines offer the possibility to improve the efficacy of anticancer drugs, however
progresses in pancreatic cancer therapy have remained exceedingly slow mainly as consequence
of an inefficient drug delivery to cancer cells. 1 The extensive desmoplastic reaction is the
hallmark of this tumor and acts as a physical barrier sequestrating nanomedicines, blocking their
diffusion and limiting the effectiveness of the treatment.2,3 Thus, in vitro models, which reliably
mimic the clinical conditions, are highly required for an appropriate preclinical screening of
nanomedicines. Herein attention focuses on 3D in vitro tumor spheroids, which have been
already successfully constructed using the Panc-1 cell line. Viable spheroids have been
maintained in culture up to 17 days while only aggregates of poorly viable cells that quickly
disassembled were obtained using the BxPc3 cells. Co-culture of Panc1 cells and fibroblasts has
been also achieved. Multiple culture with endothelial and stellate cells is currently investigated
in order to reproduce the structural and functional integration of the various cell types
constituting the pancreatic tumor. 4 Once validated, the model will be used for screening
nanomedicines. Information gathered by this research will enable to identify the strategies that
would prompt their penetration in the tumor mass making shorter the step for their clinical
translation.
References
Torosean et al, Nanoparticle uptake in tumors is mediated by the interplay of vascular and
collagen density with interstitial pressure, Nanomedicine, 2013.
2
Kadaba et al, Imbalance of desmoplastic stromal cell numbers drives aggressive cancer
processes, J Pathol, 2013.
3
Sutherland et al, Cell and environment interactions in tumor microregions: the multicell
spheroid model, Science, 1988.
1
Figure 1. Panc-1 spheroids at d4, 7, 13 and 17 post seeding. Scale bar: 200µm
8
Proteomic analysis of mononuclear leucocytes in a mouse model of anxiety/depression in response
to chronic fluoxetine treatment
Indira MENDEZ-DAVID1, Céline BOURSIER2, Valérie DOMERGUE3, Jean-Philippe GUILLOUX1 Romain
COLLE, Bruno FALISSARD1, Emmanuelle CORRUBLE1, Alain M. GARDIER1, Denis J. DAVID1
1
CESP/UMRS1178, Univ. Paris-Sud, Fac Pharmacie, INSERM, Université Paris-Saclay, 92296 ChatenayMalabry, France.
2
Proteomic facility, UMS IPSIT (Institut Paris Saclay d'Innovation Thérapeutique), Univ. Paris-Sud,
Université Paris-Saclay, 92296 Châtenay-Malabry, France.
3
Animal Facility, UMS IPSIT (Institut Paris Saclay d'Innovation Thérapeutique), Univ. Paris-Sud,
Université Paris-Saclay, 92296 Châtenay-Malabry, France.
Pôle thématique de rattachement à l'ED569 : Pharmacologie
Adresse électronique de l'auteur principal : [email protected]
Major depressive disorder (MDD) is a common mental disorder, which has a major impact in
terms of disability, well-being and overall cost to society. Selective inhibitors of serotonin
reuptake (SSRIs) and mixed reuptake inhibitor of serotonin and norepinephrine (SNRIs) are the
most prescribed antidepressant drugs for treating MDD. Unfortunately, 60% of MDD patients do
not have an optimal response to antidepressant drugs, and from those who are responding, 30%
present relapse. Moreover, 20-30% of patients are resistant to antidepressant therapies,
defining treatment-resistant depression (TRD). To improve remission and limit TRD, studies of
biological predictors of response and non-response to antidepressants are needed. As of today,
little is known about the mechanism, biomarkers and biologic predictors, moderators and
mediators of non-response and resistance to antidepressants. Thus, the development of a panel
of biomarkers devices constituting a biological signature of depressive disorders, but also the
response to treatment is of great interest.
The aim of our study consisted in a proteomic approach in a mouse model of anxiety/depression
(the so-called “CORT” model). Our goal was to identify which proteins were differentially
regulated between responders and non-responders in peripheral blood mononuclear cells
(PBMC) following a chronic (SSRI) fluoxetine administration in these mice. Followed by 4 weeks
of chronic corticosterone (CORT, 35 g/ml) treatment in C57BL/6NTac male mice (7-8 weeks
old), anxiety/depressed-like behavior was assessed by z-normalization. The emotionality related
data of the Z-score was measured in three different behavioral tests (Elevated Plus Maze,
Novelty Suppressed Feeding and Splash Test). Among 58 C57BL/6NTac male mice with an
anxiety/depressed phenotype, 46 were treated with chronic fluoxetine (18 mg/kg/day) and 12
pursued CORT treatment for 4 weeks. A significant reduction of the emotionality score in
fluoxetine-treated animals in comparison to CORT/vehicle group was found. However, only 65%
(40 animals) of fluoxetine-treated animals presented a 50% decrease in their initial emotionality
score, defining fluoxetine responders. PBMC were isolated from whole blood after each
behavioral session. Using a proteomic approach in 7 fluoxetine responders and 6 fluoxetine nonresponders animals, we identified 51 proteins differentially expressed (48 proteins were
overexpressed in fluoxetine responder’s animals). We analyzed the differentially expressed
proteins onto the global molecular network of the Ingenuity Knowledge Database. The top
associated diseases and disorders included neurological diseases such as Inflammation and
Neurological disorders. We are currently investigating whether this peripheral biological
signature in PBMC’s predicts changes in the hippocampus (a key structure involved in
depression) as well as gene expressions (by qPCR).
9
DEGRADABLE PEGYLATED POLYMER PRODRUG NANOOBJECTS
Elise Guégain, Johanna Tran, Quentin Deguettes, Julien Nicolas
Institut Galien Paris-Sud, UMR CNRS 8612, Univ. Paris-Sud, Faculté de Pharmacie,
5 rue Jean-Baptiste Clément, 92290 Châtenay-Malabry, France
Pharmacotechnie et Physico-chimie Pharmaceutique
[email protected]
Drug-loaded nanocarriers (e.g., nanoparticles, micelles) hold great hope for the
treatment of severe diseases, such as cancer. They are generally obtained from the
encapsulation of a drug during the formulation of the nanocarrier. However, important
limitations still remain (e.g., burst release, poor drug loading, etc.) which may explain the low
number of marketed nanomedicines. Another interesting class of drug nanocarriers that
overcome the aforementioned issues are polymer prodrugs, for which the drug is covalently
linked to a polymer scaffold.
In this context, we recently reported a new method for the synthesis of polymer prodrug
nanoparticles, termed “drug-initiated”. Such approach relies on the controlled growth of
hydrophobic vinyl polymer chains from a drug-functionalized initiator using controlled/living
radical polymerization. For instance, by using gemcitabine (Gem) as the anticancer drug and
isoprene (I) as the monomer, well-defined Gem-PI conjugates were obtained. Their
nanoprecipitation in aqueous solution led to narrowly dispersed nanoparticles of ~140 nm in
diameter which displayed significant anticancer activity both in vitro and in vivo.
However, considering that the carbon-carbon backbones of vinyl materials resist
degradation, which may cause prohibitive toxicity in vivo, our aim was to develop a strategy to
confer degradability to polymer prodrugs obtained from the drug-initiated method. The use of
cyclic monomers (cyclic keten acetals, CKA) which are ester moiety precursors, enables insertion
of labile groups in the main chain.
The general strategy is based on the use of a Gem-functionalized alkoxyamine to initiate
the nitroxide-mediated radical ring-opening polymerization (NMrROP) of hydrophilic PEG-based
macromonomer (PEGMA) with hydrophobic 2-methylene-4-phenyl-1,3-dioxolane (MPDL), a
cyclic ketene acetal monomer. Herein we performed a comprehensive study focused on the
synthesis of PEGylated degradable polymer prodrugs, their self-assembling properties, and their
hydrolytic degradation. Their in vitro anticancer activity on human lung cancer model was also
evaluated.
PEGylated prodrug nanoparticles
Hydrophilic
Hydrophilic
rROP
MPDL
Nanop°
Gem
PEGMA
MPDL
Hydrophobic
Hydrophobic
10
PEGMA
Hydrophilic
Nanop°
Design and incorporation mechanism of ferrofluids into lipid-based Janus nanoparticles
Elodie Millart a, Christine Ménager b, Sylviane Lesieur a, Vincent Faivre a
a
Institut Galien Paris-Sud, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 5 rue Jean-Baptiste
Clément, 92296 Châtenay-Malabry, France
b
Laboratoire PHENIX, Sorbonne Universités, UPMC, Université Paris 06, UMR CNRS 8234, 4 place
Jussieu 75005 Paris, France
Pôle thématique de rattachement à l'ED569 : Pharmacotechnie et Physico-chimie Pharmaceutique
[email protected]
In recent years, the team Physico-Chimie des Systèmes Polyphasés (Institut Galien ParisSud) has developed original compartmented lipid nanometer-sized particles produced
by high pressure homogenization, a scalable process, with marketed and
pharmaceutically approved excipients. The particles actually belong to the family of
Janus nano-objects as they are organized in two juxtaposed substructures : one half is a
droplet of liquid-state lipids while the other half is vesicle-like and encloses an aqueous
core delimited by a phospholipid-containing bilayer shell. Added to the intrinsic
biocompatibility of the constituting lipids, such a system provides a potentially very
valuable tool in pharmaceutical and biomedical fields, able to separately incorporate
and co-convey hydrophilic and lipophilic substances with distinct activities, for example,
a medical imaging agent and a drug for coupling diagnosis and therapy. Here, we are
interested in loading Janus nanoparticles with a magnetic fluid composed of
superparamagnetic iron oxide nanocrystals (ferrofluid, FF), indeed as efficient contrast
agent for magnetic resonance imaging (MRI), being magnetically targetable and
providing ability for hyperthermia treatment. Alternately, hydrophilic or lipophilic FF
compatible with the production process have been developed by investigating different
stabilization pathways of the nanocrystals depending on the encapsulation
compartment. Furthermore, some optical microscopy images and X-ray diffraction
experiments have helped to better understand the formation mechanism of Janus
nanoparticles and the influence of FF on it.
11
12
Régulation de la dynamique des microtubules par la kinase de stress JNK dans les
cellules épithéliales
Hélène HENRIE1, Christian POÜS1, 2, Antoine PILON1, 3, Béatrice BENOIT1
1. INSERM UMR-S-1193. Equipe Mécanisme Cellulaires et Moléculaires de l’Adaptation au
Stress et Cancérogenèse, Tour D4 1er étage, Faculté de Pharmacie, 5, rue J.B. Clément, 92296
Châtenay-Malabry Cedex
2. Biochimie-Hormonologie, APHP, Hôpitaux Universitaires Paris-Sud, Antoine Béclère,
Clamart
3. Biochimie, APHP, Hôpitaux Universitaires de l’Est parisien, site St Antoine, Paris
Pôle thématique de rattachement à l'ED569 : Physiopathologie Moléculaire et Cellulaire
[email protected]
Les microtubules sont des constituants dynamiques du cytosquelette qui contrôlent à la
fois la polarité, la migration et la division cellulaire. Notre laboratoire a précédemment
démontré que la kinase de stress JNK (c-Jun N-terminal Kinase) régule la dynamique des
microtubules (MTs) dans les cellules épithéliales, en augmentant les vitesses de
polymérisation et de dépolymérisation ainsi que les fréquences de repolymérisation
(événements de sauvetage). Nous avons ici identifié la protéine CLIP-170, un facteur
majeur de sauvetage, comme un nouveau substrat de JNK. La phosphorylation de CLIP170, sur deux sites indépendants, augmente son activité cellulaire. Nous testons
actuellement si JNK phosphoryle directement ces sites in vitro et si ces phosphorylations
augmentent l'affinité de CLIP-170 dans les cellules pour les MTs et/ou son partenaire de
sauvetage CLASP. Ce travail permettra de caractériser le mécanisme de sauvetage des
MTs, essentiel dans le contrôle de la dynamique microtubulaire. La dynamique des
microtubules est une cible majeure des thérapies anti-cancéreuses. De plus, ces
chimiothérapies sont connues pour induire du stress cellulaire. Ainsi, la régulation de
CLIP-170 par JNK pourrait s’avérer importante dans l’efficacité de ces traitements.
13
Interest of flagellin FliC in mucosal vaccination against Clostridium difficile
J-F. Bruxelle, A. Mizrahi, S. Hoys, A. Collignon, C. Janoir, S. Péchiné.
EA 4043 “Unité Bactéries Pathogènes et Santé” (UBaPS), Univ. Paris-Sud, Université ParisSaclay, 92290 Châtenay-Malabry, France
Clostridium difficile (CD), a flagellated anaerobic enteropathogen, is responsible for intestinal
symptoms ranging from mild diarrhea to severe pseudomembranous colitis, particularly after
antibiotic treatment. Flagellin immunogenicity has been reported and thanks to its close
interaction with the immune system through the innate and adaptive immunity, flagellin can
be used as a vaccine candidate or an adjuvant as well. Salmonella typhymurium’s flagellin (FlAST) has already been tested as adjuvant. Here we assessed the interest of flagellin (FliC) of CD
as a mucosal adjuvant first with ovalbumin as antigen, second with a C. difficile surface protein.
First, we compared the mucosal adjuvant capacity of a CD recombinant FliC to FlA-ST and
cholera toxin (CT) with ovalbumin (OVA) as antigen. After rectal immunizations in a mouse
model, we analyzed the humoral response through detection of immunoglobulin A and G
against OVA in caecal contents and sera. Mice immunized with OVA and FliC produced a
significantly higher level of secretory IgA than mice immunized with OVA and CT (p= 0.030) and
OVA only (p=0.015). So, the use of FliC as adjuvant in immunization via the rectal route is
susceptible to stimulate a mucosal immune response.
Then, in a vaccination assay to prevent CD intestinal colonization, we assessed the effect of FliC
as adjuvant co-administrated with the C. difficile precursor of the surface layer proteins (SlpA)
as antigen. Rectal immunizations with recombinant SlpA with FliC or CT as adjuvant were
performed in a mouse model. After challenge, a significant decrease of CD intestinal
colonization, as measured by fecal shedding, was observed in immunized groups compared to
the control group, with a more pronounced tendency for the one immunized with SlpA with
FliC as adjuvant (p<0.05).
Our results showed that FliC could be used as an adjuvant in mucosal vaccination strategy
against CD infections.
14
Mechanism of Sinoatrial node dysfunction in a RyR2R420Q mouse
model of Catecholaminergic Polymorphic Ventricular
Tachycardia
Yue Yi Wang1, Pietro Mesirca2, Elena Marqués-Sulé1,3, Alexandra Zahradnikova Jr1*, Olivier
Villejoubert1, Pilar d’Ocon4, Cristina Ruiz5, Diana Domingo5, Esther Zorio5, Matteo E. Mangoni2,
Jean-Pierre Benitah1, Ana María Gómez1
1
UMR-S 1180, Inserm, Univ. Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France.
2
UMR-5203, CNRS, UMR-S 1191, INSERM, Université de Montpellier, Montpellier, France
4
Pharmacy School, University of Valencia, Valencia, Spain
5
3
Hospital Universitario y Politécnico La Fe, Valencia, Spain
Physiotherapy Department, University of Valencia, Valencia, Spain
ED569 : Pôle Physiopathologie Moléculaire et Cellulaire
[email protected]
[email protected]
Sinoatrial node (SAN) is the primary cardiac pacemaker. Ca2+ release via the ryanodine
receptor (RyR2) plays a critical role in maintaining automaticity. RyR2 mutations lead to
catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a lethal
inherited disease characterized by exercise/stress-induced syncope and/or sudden death.
CPVT patients frequently bear SAN dysfunction. In order to find out its underling
mechanisms, we created a KI mice model carrying a mutation in the N-terminal portion
of the RyR2 found in a CPVT family, RyR2 (R420Q). All KI mice showed sustained
bidirectional ventricular tachycardia and other arrhythmias after pharmacologic
(epinephrine+caffeine) or emotional (hair dryer) stress, validating the model, and a slower
resting heart rhythm was denoted in KI female mice during day time, underlying
alteration in SAN function. For further investigation, SAN tissues were dissected from
WT and KI mice and Ca2+ movements followed by confocal microscopy. The cycle
length of spontaneous [Ca2+]i transients was significantly longer in KI, and [Ca2+]i
transient amplitude was significantly shortened. Funny current (If) in isolated SAN cells
are not altered but L-type Ca2+ current (ICa,L) was reduced in absence of intracellular Ca2+
buffer. In KI mice compared to WT littermates, Ca2+ sparks frequency between
spontaneous beats and the Ca2+ spark mass was augmented pointed out an increase in
diastolic Ca2+ leak. In summary, our data show that KI mutation: 1) induces CPVT
phenotype in mice, 2) decreases the SAN rhythm without affecting If and, 3) has more
[Ca2+]i leak during diastole as Ca2+ sparks. The latter may account for ICa inactivation. In
conclusion, RyR2R420Q N-terminal mutation causes [Ca2+]i leak during diastole and leads
to the SAN dysfunction.
15
NANOCAPSULES
CAPSULES OF PERFLUOROCARBONS AS THERANOSTIC AGENTS.
T. Boissenot1, S. Houvenagel1, G. Picheth1, C. Dejean2, A. Paci3, V. Poinsignon3, B. Larrat4,
A. Bordat1, H. Chacun1, C. Gueutin1, L. Moine1, O. Diou1, L. Mousnier1, E. Fattal1, N.
Tsapis1.
1
Institut Galien Paris-Sud,
Sud, CNRS, Univ. Paris-Sud,
Paris
Université Paris-Saclay,
Saclay, 92296 Châtenay
Châtenay2
Malabry, France.
BioCIS, CNRS, Univ. Paris-Sud,
Paris Sud, Université Paris
Paris-Saclay, 92296
Châtenay-Malabry, France. 3 Gustave Roussy Cancer Campus - Service interdépartemental de
Pharmacologie
logie et d’Analyse du Médicament (SIPAM), 94800 Villejuif, France. 4
Commissariat à l’Energie Atomique (CEA), Institut d’Imagerie Biomédicale (I²BM),
Neurospin, Saclay, France.
Pôle thématique de rattachement à l'ED569 : Pharmacotechnie
[email protected]
The need to detect cancer at its early stages, as well as, to deliver chemotherapy to targeted site
motivates many researchers to build theranostic platforms which combine diagnostic and therapy.
Among imaging modalities, ultrasonography and MRI are widely available, non invasive and
complement each other. Both techniques
techniques often require the use of contrast agents.
We have developed nanocapsules of perfluorocarbons as dual contrast agent for both imaging
modalities. The soft, amorphous polymer shell provides echogenicity, while the high
high-density
19
perfluorinated liquid
d core allows detection by
F MRI. We have used mainly a shell of
poly(lactide-co-glycolide)
glycolide) (PLGA) since this polymer is biodegradable, biocompatible and can be
loaded with drugs. These capsules were shown to be efficient in vitro as contrast agents for both 19
F MRI and ultrasonography1. In addition, for in vivo applications a poly(ethyleneglycol) (PEG)
coating promotes stability and prolonged circulation. Being stealth, nanocapsule can accumulate
passively into implanted tumors by the EPR effect. We will
ll present nanocapsule formulation and
characterization, and will show promising in vivo results obtained for both ultrasonography and 19
F MRI2. The accumulation by EPR effect favors the efficacy of a chemotherapeutic agent co
coencapsulated, proving the feasibility
asibility of the theranostic approach. The design of fluorinated
polymers to promote better encapsulation of perfluorcarbons will be presented.
Figure 1: Schematic representation of the project
References
1.
E. Pisani, N. Tsapis, B. Galaz, M. Santin, R. Berti, N. Taulier, E. Kurtisovski, O.
Lucidarme, M. Ourevitch, B.T. Doan, J.C. Beloeil, B. Gillet, W. Urbach, S.L. Bridal, E.
Fattal, Adv. Funct. Mat. 2008, 18(19), 2963-2971.
2.
O. Diou, N. Tsapis, C. Giraudeau, J. Valette, C. Gueutin, F. Bourasset, S. Zanna, C.
Vauthier, E. Fattal, Biomaterials 2012, 33(22), 5593-5602.
3.
O. Diou, A. Brûlet, G. Pehau-Arnaudet,
Pehau Arnaudet, E. Morvan, R. Berti, K. Astafyeva, N. Taulier, E.
Fattal, N. Tsapis. Coll. Surf. B. 2016, 146, 762-769.
16
UPDATES IN NICKEL ALLERGY: IDENTIFICATION OF NAÏVE T-CELLS
RECOGNIZING NICKEL AND IN VITRO REGULATION OF THE INTERLEUKIN-12
CYTOKINE FAMILY IN HUMAN DENDRITIC CELLS
Rami Bechara 1, 2, Diane Antonios 2, Marie-Eliane Azoury 1, Hayat Azouri 2 and Marc
Pallardy 1
1
INSERM UMR996, Université Paris-Sud, Université Paris-Saclay, 92290, ChâtenayMalabry, France;
2
Saint Joseph University, Laboratory of Toxicology, Beirut, Lebanon
Pharmacologie-Toxicologie
[email protected]
Allergic contact dermatitis (ACD) is a major cause of occupational skin disease and nickel is
among the most prevalent contact allergen. Dendritic cells play an important role in the type of
the ensuing immune response through differential cytokines production. In particular, the IL-12
cytokine family plays a major role in the generation of allergen-specific T cell responses.
Moreover, the manifestations of nickel-induced ACD has been ascribed to T-cells activation,
demonstrating the presence of nickel-specific T-cells.
In this work, we studied the effect of nickel on IL-12 cytokine family and evaluated the frequency
of human naïve T-cells specific to nickel in peripheral blood.
Immature human monocyte-derived dendritic cells (Mo-DC) were differentiated from monocyte
CD14+ and at day 5, they were stimulated with NiSO4. Moreover, naïve CD4+ T-cells were
stimulated once a week with human autologous dendritic cells loaded with NiSO4. Activation of
nickel-specific T-cells was detected using an interferon-γ EliSpot assay.
First, we showed that nickel induced the production of IL-12p40, IL-23 and IL-27, in Mo-DC, and
its effect on these cytokines correlated with the expression of their subunits. Nickel-treated
MoDCs induced an increase in the percentage of IL-17A+ allogeneic CD4+ T cells, an effect
reduced by IL-23. In the second part, naïve CD4+ T-cell specific to nickel were detected in 8/9 of
the tested donors. Frequency of nickel-specific naïve CD4+ T-cells varied from 0 to 2.03 nickelrecognizing naïve CD4+ T-cells per million of naïve CD4+ T-cells with a mean value of 0.69.
Finally, our results describe a novel effect of nickel on IL-12 cytokine family and show, for the
first time, a quantification of pre-existing naive CD4+ T-cells recognizing nickel.
17
AMPA RECEPTOR ACTIVATION IN DORSAL RAPHE NUCLEUS AND GABA-A RECEPTOR
INHIBITION IN PREFRONTAL CORTEX ARE NECESSARY FOR KETAMINE
ANTIDEPRESSANT-LIKE ACTIVITY
1
1
Thu Ha Pham , Céline Defaix , Audrey Solgadi2, Pierre Chaminade2,3, Laurent Tritschler1, Indira MendezDavid1, Denis J David1, Alain M Gardier1
1
CESP/UMR-S 1178, Univ. Paris-Sud, Fac. Pharmacie, INSERM, Université Paris-Saclay, Châtenay-Malabry,
France
2
UMS IPSIT SAMM, Univ. Paris-Sud, Université Paris-Saclay, F-92290 Châtenay-Malabry, France
3
Chimie Analytique Pharmaceutique (FKA EA4041 Groupe de Chimie Analytique de Paris-Sud), Univ. ParisSud, Université Paris-Saclay, F-92290 Châtenay-Malabry, France
Pôle thématique de rattachement à l'ED569 : Pharmacologie-Toxicologie
[email protected]
Summary
Ketamine, a non-competitive, glutamatergic NMDA receptor antagonist has been found to
relieve symptoms within hours when administered at sub-anesthetic doses in treatment-resistant
depressed patient. Since this discovery, many studies have confirmed ketamine’s efficacy in humans and
rodents. However, the mechanism of action underpinning this rapid antidepressant-like response is still
unclear. We found that ketamine-induced increase in the extracellular serotonin levels [5-HT]ext in the
medial prefrontal cortex (mPFCx) following its local injection was correlated with the swimming
duration in the forced swim test (FST), suggesting that activation of the cortical 5-HT system is required
for its antidepressant activity. Knowing that mPFCx projections to the dorsal raphe nucleus (DRN) have
been shown to affect antidepressant drug response, we determined whether this neuronal circuit underlies
ketamine antidepressant-like activity in BALB/cJ mice, a strain developing a highly anxious phenotype.
We compared the effects of a low sub-anesthetic ketamine dose (10 mg/kg, i.p.) with fluoxetine
(a classical selective serotonin reuptake inhibitor, 18 mg/kg, i.p.), 24h post-administration, in the FST, a
test used to predict antidepressant-like potential of a drug in rodents. Concomitant measures of 5-HText,
Glutamineext, Glutamateext and GABAext using in vivo microdialysis coupled with Liquid
Chromatography and Mass Spectrometry (LC-MS/MS) were assessed in the mPFCx and DRN and
correlated to the swimming duration measured in the same mice. Unlike fluoxetine, ketamine displayed
an antidepressant-like activity in the FST, associated with an increase in Glutamateext (>300%) and a
decrease in GABAext (-80%) in both the mPFCx and DRN. The local mPFCx ketamine injection (0.25 µg
each side) confirmed its systemic effect in the FST with a trend to increase mPFCx Glutamateext.
Given the AMPA receptor-dependent antidepressant-like effects of ketamine, we also assessed
herein the role of cerebral glutamate and its precursors/products: Glutamine and GABA in ketamine
activities. To this aim, we microinfused either NBQX (an AMPA receptor antagonist), bicuculline (a
GABA-A receptor direct antagonist) in the DRN or muscimol (a GABA-A receptor direct agonist)
bilaterally in the mPFCx. Interestingly, intra-DRN NBQX (0.1 µg) and intra-mPFCx muscimol (0.1 µg),
but not intra-DRN bicuculline, blocked ketamine antidepressant-like activity 24hr post-injection,
suggesting a role of DRN AMPA receptor and mPFCx GABA receptor in ketamine-induced persistent
antidepressant-like effects.
Then, we are currently using optogenetic inactivation of the mPFCx-DRN circuit at the time of
FST and microdialysis, using intra-mPFCx injection of the AAV5-CaMKIIa-ArchT-GFP vector, to
decipher the role of this neuronal circuit in ketamine-induced antidepressant-like effects.
In conclusion, our results bring the first connection between ketamine antidepressant-like
activity in the FST and the brain glutamatergic system. Overall, these data suggest that ketamine-induced
long-lasting antidepressant-like effects require indirect activation of AMPA receptor in the DRN and
inhibition of GABA-A receptor in the mPFCx.
18
INTEGRATION OF IMAC PRECONCENTRATION, SEPARATION AND DETECTION OF PHOSPHORYLATED
BIOMARKERS IN A MICROTAS AUTEURS
Monica Araya-Farias, Szymon Dziomba, Benjamin Carbonnier, Mohamed Guerrouache, Nacera
Aboud, Myriam Taverna, N. Thuy Tran
Institut Galien Paris Sud, UMR 8612, Protein and Nanotechnology in Analytical Science (PNAS)
Pas rattaché à l'ED569 mais à l’ED 2MIB
[email protected]
Despite the success of μTAS, one issue is still the too low concentration sensitivity. Sample
preconcentration is a crucial step to achieve biomarker analysis in biological fluids. Porous
polymer monoliths present interesting properties (high specific surface area, in-situ
polymerization) making them good candidates for preconcentration. In a previous work, we
reported the successful synthesis of an ethylene-glycol methacrylate phosphate-co-bis-acrylamide
(EGMP-BAA) monolith in a one step procedure allowing both efficient photosynthesis and
anchoring inside native PDMS microchannels [1]. Here we extended the use of this monolith in
glass microchips to develop an Immobilized-Metal Affinity Chromatography (IMAC) module to
preconcentrate phosphopeptides and phosphoproteins. Indeed, abnormal phosphorylation of
proteins may lead to various diseases (cancer, neurodegenerative diseases) and those
phosphoproteins can be envisioned as potential disease biomarkers. Many efforts have been made
for phosphopeptide enrichment on microfluidic devices but mostly by packing functionalized
particles into microchannels. Here we propose a new approach based on a simple preparation of
monolith from a precursor bearing phosphate groups, without the need of frits. Only two groups
proposed an IMAC-based monolith into simple straight microchannel for phosphopeptide
enrichment but off-line coupled to MALDI-MS [2, 3].
Here, we present for the first time a real μTAS integrating the EGMP-BAA monolith-based
IMAC enrichment, electrokinetic separation and fluorescence detection of phosphorylated
biomarkers. A new method for synthesizing the EGMP-BAA monolith under UV irradiation,
which differs from that of Dong [4] from the support format (chip vs capillaries) and irradiation
conditions (UV vs heating), is presented. We also demonstrated that this synthesis could be
achieved using a very simple UV irradiation method using an inverted epifluorescence
microscope. To date only two groups reported microscope-based syntheses but using either LED
[5] or laser beams [6]. We could produce hundreds micrometer long monolith with a very good
homogeneity. The addition of hydroquinone to the prepolymerization mixture was beneficial to
yield sharp edges of well-localized monolith segments. The monolith length was easily tuned by
simply changing the magnification objective and was used as an IMAC support after
immobilization of Zr4+ ions. Very high efficiency (92%) and reproducibility (RSD=2.9%) for
phosphopeptide extraction using the EGMP-BAA-Zr4+ monolith were demonstrated. A MCE
method was developed to analyze a mixture of nonphosphorylated and three phosphorylated
peptides differing only by their phosphorylation degree and site. The obtained separation was
highly resolutive. Then the potential of this µTAS to on-line preconcentrate, separate and detect
phosphopeptides was demonstrated. The signal enhancement factor obtained was estimated higher
than 910, 340 and 840 for the phosphopeptides pT, pY and pTpY, respectively. It is the first time
that a so high enrichment factor for phosphopeptides is obtained in microchips.
This is the first fully integrated device performing an automated sequence of IMAC-based
preconcentration, elution, injection, electrophoretic separation and detection of phosphopeptides.
This device lays the foundation for analysis of phosphorylated biomarkers in Alzheimer’s disease
or Parkinson’s disease.
19
Pd-catalyzed anomeric C(sp3)-H activation of glycopyranosides via a CMD (concertedmetalation deprotonation) mechanism
Nicolas Probst, Vincent Gandon, Mouad Alami, Samir Messaoudi
BioCIS CNRS UMR 8076 Faculté de Pharmacie-Université Paris Sud
Pôle thématique de rattachement à l'ED569 : Laboratoire de Chimie Thérapeutique
[email protected]
Résumé : The Pd-catalyzed cyclisation of glycosides by activation of the anomeric C(sp3)-H position is
described.
Unprecedented fused glycosylquinolinones formation from N-methylanilideglycosides has been
investigated using a combination of a catalytic amount of palladium and ligand. The fused
glycosylquinolinones can be obtained in good to excellent yields from N-methylanilideglycosides. The
effect of substitution on the aryl ring containing the halide was explored by various examples affording up
to 90% yield and is extended to other glycosides such as D-galactoside, and D-mannoside.
Interestingly, in the case of 2-deoxyglucoside R = H), spiroglycosides can be obtained under our optimized
conditions (publication under preparation).
O
AcO
O
AcO
N
Pd/L cat.
AcO
R=H
H2
O
AcO
OAc
AcO
H1
O
O
R
Pd/L cat.
N
H2
Br
R
O
AcO
N
AcO
R = OAc
R
OAc
C(sp3)-H
anomeric
quaternization
Double C(sp 3)-H1 - C(sp3)-H2
activations
12 examples
41-90% yield
3
The mechanism we suggested starts from the anomeric C(sp )-H activation via a concerted metalation
3
deprotonation (CMD) pathway, followed by a second CMD on the C(sp )-H2 through a palladium migration
at the C2 position and a β-OAc elimination (if R = OAc). This mechanism is currently under investigation by
DFT calculations.
H
O
AcO
AcO
Br
O
R
N
Me
Br
L 2 Pd
H O
Oxydative Addition
O
AcO
O
N
O
AcO
OAc
1
AcO
Me
2
R
N
Me
CMD - C(sp3)-H1 activation
OAc
Pd(0)L 2
AcO
OAc
O
Reductive Elimination
O
+Cs -O
O
Me
H O
N
O
AcO
O
AcO
AcO
L2
Pd
Pd
OAc L 2
AcO
R
N
Me
OAc
CMD - C(sp 3)-H2 activation
HOAc
O
AcO
O
AcO
OAc
CsHCO3
Me
N
L2
Pd
Me
N
O
AcO
H O
O
O
Pd
L2
AcO
R
OAc
Me
N
L2
Pd
O
AcO
O
H
AcO
OAc
O
O
if R = OAc
if R = H
Reductive Elimination
O
AcO
AcO
H
OAc
20
Me
N
O
Nanoparticules squalenisées et lipoprotéines plasmatiques : caractérisation des
interactions moléculaires et évaluation de leur implication dans la réponse
thérapeutique
Dunja Sobot*, Simona Mura*, Didier Desmaele*, Patrick Couvreur*
* Institut Galien Paris-Sud, UMR CNRS 8612, Université Paris-Sud, Faculté de Pharmacie
Pôle thématique de rattachement à l'ED569 : Pharmacotechnie et Biopharmacie
[email protected]
Après administration intraveineuse, un nanovecteur va interagir avec de nombreuses molécules
endogènes, notamment celles présentes dans la circulation sanguine. En fonction de la
composition chimique du nanovecteur, ces molécules vont conférer à celui-ci une signature
spécifique qui va orienter sa biodistribution et sa reconnaissance par certaines cellules de
l’organisme. Plusieurs études sur l'identification des protéines adsorbées à la surface des
nanovecteurs ont été menées alors que moins d'attention a été consacrée à l'interaction avec les
lipoprotéines (LPs). Or, un nombre élevé de récepteurs aux LPs a été observé dans les cellules à
croissance rapide et des études ont démontré que certaines cellules cancéreuses surexpriment ces
récepteurs. De ce fait, l’utilisation des LPs comme vecteurs de médicaments anticancéreux a été
proposée afin de favoriser le ciblage des cellules tumorales. Ce projet de thèse repose sur
l’utilisation d’un bioconjugué (SQGem) issu du couplage chimique de la gemcitabine (Gem), une
molécule anticancéreuse, au squalène (SQ) (un lipide naturel et précurseur de la biosynthèse du
cholestérol), dont l’auto-organisation sous forme de nanoparticules à préalablement été décrite au
laboratoire. Nous avons pu mettre en évidence la capture et le transport spontané de la SQGem
par les LPs plasmatiques. Les résultats in vitro et in vivo que nous avons obtenus démontrent
parfaitement que l’association préférentielle de la SQGem aux LPs est directement corrélée avec
la quantité de cholestérol présente dans ces derniers. Par la suite, cette interaction spontanée a été
mise à contribution pour effectuer le ciblage indirect des cellules cancéreuses ayant une
expression élevée de récepteurs aux LPs, ce qui a été confirmé in vitro sur cellules ainsi qu’in vivo
sur un modèle de tumeur expérimentale chez la souris. L’ensemble de ces résultats suggère
l’originalité de notre approche, basée sur le ciblage des tumeurs de manière indirecte via les LPs
qui constituent ainsi des « vecteurs » endogènes de SQGem. La « squalénisation » évite ainsi la
préparation fastidieuse de vecteurs à base de LPs reconstitués et représente, par l’utilisation des
LPs endogènes, une stratégie innovante et potentiellement révolutionnaire dans le traitement
expérimental du cancer.
21
CXCR4 DESENSITIZATION REGULATES TLR-MEDIATED PLASMA
CELL DIFFERENTIATION AND PERSISTENCE
1
1
1
1
2
Nagham ALOUCHE , Jessica NATT , Vincent BIAJOUX , Christelle FREITAS , Nicolas FAZILLEAU , Karl
1
1
BALABANIAN , Marion ESPELI
1
UMR996 - Inflammation, Chemokines and Immunopathology, Inserm, Univ Paris-Sud, Clamart F-92140, France
2
Centre de Physiopathologie de Toulouse, Université Toulouse III Paul-Sabatier, Toulouse F-31300, France
[email protected], http://mac-gratuit.fr/site/U996/Presentation.html
During a humoral immune response in the secondary lymphoid organs, B cells differentiate into plasmablasts
that migrate, partially via CXCR4, to the bone marrow (BM) where they can become long-lived plasma cells
(PCs). Heterozygous gain-of-function mutations of CXCR4 affecting its desensitization in response to the
chemokine CXCL12 were reported in a severe immunodeficiency disorder called the Warts,
Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) syndrome (WS). Following vaccination, WS
patients mount a normal immune response but fail to maintain antibody (Ab) titers with time. We used
+/1013
[1]
Cxcr4
knock-in mice
that phenocopy WS-related pan-lymphopenia to assess how a gain-of-Cxcr4function impacts PC biology and Ab production. We showed in vitro that the gain-of-Cxcr4-function leads to
enhance PC differentiation after both BCR and TLR activation with no alteration of cell survival. Interestingly,
this effect was particularly strong when the TLRs and Cxcr4 were stimulated together. Accordingly we showed
+/1013
in vivo that Cxcr4
mice produced more antigen-specific PCs in the spleen and lymph nodes after a Tdependent immunization suggesting that Cxcr4 desensitization is an important regulatory mechanism
[2]
controlling PC differentiation . Despite this enhanced production of PCs in the secondary lymphoid organs,
+/1013
antigen-specific long-lived PCs were not detected in the BM of Cxcr4
mice and Ab titers were not
maintained with time. Moreover an aberrant accumulation of immature plasmablasts was observed in this
tissue potentially hampering the homing or maintenance of the antigen-specific long-lived PCs. Interestingly,
+/1013
this population was also induced in Cxcr4
mice after a type I T-independent immunization, suggesting that
the gain-of-Cxcr4-function may control the homing of immature plasmablasts to the BM after TLR activation.
We are now investigating the migration and localization of those plasmablasts in the BM of Cxcr4 mutant mice
to identify the possible niches accounting for their function and survival.
References:
[1] Balabanian K, Brotin E, Biajoux V, et al, Blood.2012, 119:5722-5730
[2] Biajoux V, Natt J, Freitas C, Alouche N et al. Cell Rep. 2016, 27;17(1):193-205
22
ENCAPSULATION OF A LIPOPHILIC PRODRUG OF DEXAMETHASONE INTO
PLGA-PEG NANOPARTICLES: FORMULATION, PHARMACOKINETICS AND ANTIINFLAMMATORY EFFICACY.
Mathilde Lorscheider1, Rosana Simón-Vázquez1,2, Nicolas Tsapis1, Patricia Calleja1, Ludivine
Mousnier1, Elias Fattal1
1
Institut Galien Paris-Sud, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 92296 ChâtenayMalabry, France
² Immunology, Biomedical Research Center (CINBIO) and Institute of Biomedical Research
of Orense, Pontevedra and Vigo (IBI), University of Vigo, Campus Lagoas Marcosende,
Pontevedra, 36310, Spain
Pôle thématique de rattachement à l'ED569 : Pharmacotechnie et Biopharmacie
The use of glucocorticoids (GC) in the treatment of degenerative inflammatory diseases, such as
rheumatoid arthritis (RA), is hampered because of the significant side effects induce by their
unfavorable pharmacokinetics (PK) and the high dose needed to reach a therapeutic effect. The
encapsulation of dexamethasone (DXM), a GC suitable for the treatment of RA, into liposomes
or polymer nanoparticles (NPs) has already shown an improvement in the accumulation in the
inflamed tissues and controlled release of the DXM. However, in most of the cases the drug
loading was very low. To overcome the limited encapsulation efficiency and improve the PK
profile, the prodrug dexamethasone palmitate (DXP) has been encapsulated into PLGA-PEG NPs.
The DXP-loaded PLGA-PEG NPs, with a diameter of about 150 nm and a negative Z-potential,
have shown high encapsulation efficiency and drug loading and a safe profile for the systemic
administration in vitro. Nanoparticles were able to inhibit the LPS-induced release of
inflammatory cytokines in macrophages demonstrating that release and hydrolysis of the
prodrug occurred intracellularly.
The PK profile was also significantly improved. The concentration of DXM in plasma of healthy
mice was almost constant up to 18 hours, much longer than the commercial soluble drug
dexamethasone phosphate (DSP). Regarding the biodistribution, there were not relevant
differences with the free drug and in some organs the encapsulated DXP was even accumulated
to a higher extent. However, the encapsulated GC remained mostly as the prodrug, which is not
active. Efficacy results in a murine model of rheumatoid arthritis are very promising with a
reduction of both inflamed paw volume and of the arthritis score at an equivalent DXM dose of
1mg:kg whereas the same dose of dexamethasone sodium phosphate was inefficient.
23
SELF-ASSEMBLED NANOPARTICLES OF BIOTRANSESTERIFIED
CYCLODEXTRINS AND NONLAMELLAR LIPIDS AS CARRIERS OF WATERINSOLUBLE SUBSTANCES
Leïla Zerkoune1, Sylviane Lesieur1, Jean-Luc Putaux2, Luc Choisnard3, Annabelle Gèze3,
Denis Wouessidjewe3, Borislav Angelov4, Corinne Vebert-Nardin5, James Doutch6, and
Angelina Angelova1
1
Institut Galien Paris-Sud, CNRS UMR 8612, Univ. Paris-Sud, Université Paris-Saclay,
LabEx LERMIT, 5 rue J.-B. Clément, 92296 Châtenay-Malabry cedex, France,
2
Université Grenoble Alpes, CNRS, Centre de Recherches sur les Macromolécules Végétales
(CERMAV), F-38000 Grenoble, France,
3
Université Grenoble Alpes, CNRS UMR 5063, Département de Pharmacologie Moléculaire
(DPM), F-38000 Grenoble, France,
4
Institute of Physics, ELI Beamlines, Academy of Sciences of the Czech Republic,
CZ-18221 Prague, Czech Republic,
5
IPREM/EPCP, Technopole Helioparc, 2 Av. Pdt Angot, 64053 PAU cedex 09, France,
6
Diamond Light Source Ltd., Didcot, Oxfordshire, OX11 0DE, UK.
Pôle thématique de l'ED 569: Pharmacotechnie et Physico-chimie Pharmaceutique
[email protected]
Hierarchically structured particles were created by self-assembly of an amphiphilic deep
cavitand cyclodextrin CD-nC10 (degree of substitution n = 7.3), with a nanocavity grafted by
multiple alkyl (C10) chains on the secondary face of the CD macrocycle through enzymatic
biotransesterification, and the nonlamellar lipid monoolein. The effect of the non-ionic
dispersing agent polysorbate 80 (P80) on the liquid crystalline organization of the nanocarriers
and their stability was studied in the context of the vesicle-to-cubosome transition. The
coexistence of small vesicular and nanocubosome membrane objects with bigger nanoparticles
with inner multicompartment cubic lattice structures was established as a typical feature of the
employed dispersion process. The cryogenic transmission electron microscopy (cryo-TEM)
images and small-angle X-ray scattering (SAXS) structural analyses revealed the dependence of
the internal organization of the self-assembled nanoparticles on the presence of embedded
CD-nC10 deep cavitands in the lipid bilayers. The obtained results indicated that the
incorporated amphiphilic CD-nC10 building blocks stabilize the cubic lattice packing in the lipid
membrane particles, which displayed structural features beyond the traditional CD
nanosponges. UV-Vis spectroscopy was employed to characterize the nanoencapsulation of a
model hydrophobic dimethylphenylazo-naphthol guest compound (Oil red) in the created
nanocarriers. In perspective, these dual porosity carriers should be suitable for co-encapsulation
and sustained delivery of peptide, protein or siRNA biopharmaceuticals together with small
molecular weight drug compounds or imaging agents.
References:
Zerkoune, L.; Lesieur, S.; Putaux, J.-L.; Choisnard, L.; Gèze, A.; Wouessidjewe, D.; Angelov, B.; VebertNardin, C.; Doutch, J.; Angelova, A. Soft Matter, 2016, 12, 7539-7550.
Angelov, B.; Angelova, A.; Filippov, S.; Drechsler, M.; Štěpánek, P.; Lesieur, S., ACS Nano, 2014, 8, 5216.
Zerkoune, L.; Angelova, A.; Lesieur, S. Nanomaterials , 2014, 4, 741-765.
24
Phosphodiesterase type 2 localized in cardiac mitochondria regulates mitochondrial
membrane potential, swelling and calcium accumulation
LIU D1,WANG Z1, COURILLEAU D2, VANDECASTEELE G1, FISCHMEISTER R1,2, and
BRENNER C1,2
1
INSERM UMR-S 1180, Faculty of Pharmacy, Univ Paris-Sud, University Paris-Saclay,
Chatenay-Malabry, France
2
IPSIT-US31-UMS3679, Faculty of Pharmacy, Univ Paris-Sud, University Paris-Saclay,
Chatenay-Malabry, France
Pôle thématique de rattachement à l'ED569: Physiopathologie Moléculaire et Cellulaire
[email protected]
Mitochondria plays a role in energy production as well as a role in many other essential
cellular processes including ATP and metabolite synthesis, redox balance, calcium
homeostasis and cell death. Recently, a cardiac mitochondrial cAMP production was
shown to stimulate respiration, and inhibit calcium accumulation, permeability transition
and cell death. Here, we identified PDE2A protein in rat cardiac mitochondria by
western-blot. Functional assay showed that upon HCO3- stimulation, inhibition of PDEs
delays the mitochondrial membrane potential loss and matrix swelling induced by
calcium. In addition, we found that PDE2A expression is altered in heart failing
mitochondria. Interestingly, in mitochondria isolated from transgenic PDE2A mice, the
OXPHOS activity was significantly lower than wild-type mice. Thus, our study identify a
new regulation of cAMP levels and signaling cascade in mitochondria by PDE2A, which
may have implications for the metabolic control of cardiac function.
25
PTSA CATALYZED ANNULATION OF
(E)-1,4-DIARYLENYNES INTO (E)-3-STYRYLISOCOUMARINS[1]
Guangkuan Zhao, Ling-Zhi Yuan, Mylène Roudier, Jean-François Peyrat, Abdallah Hamze, Jean-Daniel Brion,
Olivier Provot, Mouad Alami
BioCIS, Univ. Paris-Sud, CNRS, équipe labellisée Ligue Contre le Cancer, Université Paris-Saclay, 92290, Châtenay-Malabry,
France
[email protected]
[2]
In the continuation of our work dedicated to the synthesis of isocoumarins, we now presented a rapid metalfree procedure for the cyclization of ortho-substituted-(E)-1,4-diarylenynes. The reaction promoted by a
catalytic amount of PTSA (way A) takes place in EtOH at 160 °C under microwave irradiation and afforded
stereoselectively a variety of (E)-3-styrylisocoumarins in good to excellent yields. Moreover, (E)-chloenynes
having an ortho-ester function underwent cyclization (way B) into isocoumarin derivatives using a catalytic
combination of PTSA and PdCl2.
We believe that PTSA catalyzed annulation-reaction process proceeds (way A), as shown in the following
scheme, by a sequence involving: (i) activation of the triple bond by PTSA, (ii) nucleophilic attack by the oxygen
[3]
atom of the ester group to the more electrophilic acetylenic carbon atom according to a 6-endo-dig process,
[4]
and (iii) loss of the methyl group of the oxonium species promoted by the solvent to afford the desired 3styrylisocoumarins together with EtOMe.
We demonstrated that ortho-substituted-chloroenynes constitute common starting materials for the synthesis
of various 3-styrylisocoumarins having several elements of structural diversity on the styryl unit. The strategy
presented in the communication is general and involves two different and complementary pathways: (i) a
Suzuki-coupling reaction between boronic acids and (E)-chloroenynes followed by cyclization in the presence of
catalytic amounts of PTSA, or (ii) a PTSA/PdCl2–catalyzed cyclization of (E)-chloroenynes into
(chlorovinyl)isocoumarins followed by Suzuki-couplings. Some of these novel 3-styrylisocoumarins derivatives
[5]
having a great resemblance to biologically active heteroaromatic analogues of combretastatins, will be
evaluated in our lab for possible antitumor-activities.
References:
[1]
[2]
[3]
[4]
[5]
Zhao, G, K.; Yuan, L.-Z.; Roudier, M.; Peyrat, J.-F.; Hamze, A.; Brion, J.-D.; Provot, O.; Alami, M. Synthesis 2016,
DOI: 10.1055/s-0036-1588608.
Le Bras, G.; Hamze, A.; Messaoudi, S.; Provot, O.; Le Calvez, P.B.; Brion, J.-D.; Alami, M. Synthesis 2008, 1607.
Baldwin, J. E. J. Chem. Soc., Chem. Commun. 1976, 734.
Jacubert, M.; Provot, O.; Peyrat, J.-F.; Hamze, A.; Brion, J.-D.; Alami Tetrahedron 2010, 66, 3775.
Penthala, N.R.; Thakkar, S.; Crooks, P.A. Bioorg. Med. Chem. Lett. 2015, 25, 2763.
26
Gene polymorphism and circulating levels of matrix metalloproteinases
and their tissue inhibitors in coronary artery disease
1,2,4
Ben Braiek Assia , Baudin Bruno1,3, Delomenie Claudine4, Chahed Hinda2, Gamra Habib,
Maatouk Faouzi, Ferchichi Salima2.
1- Biochemical service, Saint-Antoine Hospital, HUEP, Paris 12ème; 2-Biochemical service
CHU Farhat Hached, 3- UMR INSERM 1193, Faculty of Pharmacy Paris Sud, 4“Transcriptome” Plate-forme, Faculty of Pharmacy Paris Sud
Pôle thématique de rattachement à l'ED569 : Hôpital Saint Antoine, Catheterisation service
CHU Fattouma Bourguiba
[email protected]
Abstract:
±
Background:
matrix metalloproteinases (MMPs) play an important role in inflammation and matrix
degradation involved in atherosclerosis and plaque rupture. They may have importance as risk
markers for coronary artery disease (CAD). The present study aims to assess the influence of MMPs
and their inhibitors gene polymorphisms and their circulating levels on CAD.
Methods: based on coronary angiography and electrocardiography results, 472 patients and 285
healthy controls were enrolled in the study. Total plasma levels of MMP-9,-3 and their inhibitor TIMP1,-2 were measured using enzyme linked immunosorbent assay (ELISA). Polymerase chain reactionbased restriction fragment length polymorphism (RFLP-PCR) and amplification-refractory mutation
system (ARMS-PCR) were used to determine the rs11568818 in the MMP-7 gene and the rs17576 in
the MMP-9 gene, respectively.
Results: plasma levels of MMP-9 (502.1 ± 37.2ng/ml) and MMP-3 (435 ± 50.19ng/ml) were
significantly higher in CAD group than in control group (236.5 ± 28.07ng/ml and 179.84 ± 32.04ng/ml,
respectively, p < 0.05), with a wide variation between different CAD subgroups. But the plasma levels
of TIMP-1 (269.45 ± 39.08ng/ml) and TIMP-2 (187.32±46.5ng/ml) were statistically lower in CAD
group than in control group (407.3±26.5ng/ml and 357.2±19.2ng/ml, respectively, p < 0.05) showing
conditions favorable for plaque disruption.
The risk for A/A, G/G genotypes versus AA genotype of MMP-7 rs11568818:181A/G for CAD was
higher mainly in patients with ST-segment elevation myocardial infraction (STEMI) or non-ST-segment
elevation myocardial infraction (NSTEMI) than in patients with stable angina. The single nucleotide
polymorphism (SNP) of MMP-9 rs-17576:836A/G was amplified with 270pb using ARMS-PCR technics;
we observed that this mutation was present in most of CAD patients.
Conclusion: ours results suggest that higher MMPs plasma levels and low levels of TIMPs are
associated with CAD and we suggest that the AA and AG genotypes and A allele of MMP-7 Rs11568818 polymorphism is correlated with an increased risk of coronary artery disease and to
improve if single nucleotide polymorphism of rs-17576 of MMP-9 is associated with CAD.
Key words: CAD: coronary artery disease, MMP: matrix metalloproteinase, STEMI: ST-segment
elevation myocardial infarction, NSTEMI: non ST-segment elevation myocardial infarction.
27
Exploration of oxaliplatin-induced neuropathic pain by the SUDOSCAN® machine
(Canaloxa study): preliminary results after 6 months follow-up.
1
1
1
1
2,3
Kahina Abdallah , Jean-Baptiste Delmotte , Mihary Andriamamonjy , Hélène Beaussier , François Coudoré
2
Centre de Recherche Clinique, Service de Biologie, Groupe Hospitalier Paris Saint Joseph, 185 rue Raymond
3
Losserand Paris, Laboratoire de Neuropharmacologie (UMR-S 1178), Faculté de Pharmacie, Université Paris
Sud
e-mail: [email protected]
1
Introduction:
Sudoscan® is a medical device validated for the exploration of peripheral neuropathy in diabetic patients.
It measures the skin conductance of chloride ions related to sweat function and intraepidermal small fiber
damage. Because oxaliplatin, an anti-cancer drug widely used in the treatment of colorectal cancer,
produces a dose-limiting peripheral neurotoxicity with peripheral fiber damages, we studied the possible
use of the Sudoscan® apparatus in this peripheral neuropathy, classically characterized by an acute
hyperesthesia/distal cold hyperesthesia/ allodynia and a chronic hypoesthesia (CANALOXA study, NCT
02827916).
Method:
We included patients treated with oxaliplatin-based chemotherapy (over 5 courses) and suffering from
neuropathy. Small ﬁber neuropathy was assessed using Sudoscan® (IMPETO, Paris, FRANCE) to quantify
objectively the neuropathy through evaluation of Electrochemical Skin Conductance of chloride (ESC).
Thermal sensitivity ((Quantitative Sensory Testing, QST) was measured with the Thermotest® (SOMEDIC,
Hörby, Sweden) and the questionnaire NPSI (Neuropathic Pain Symptom Inventory) was administered.
Both tests were performed in all patients after inclusion and results were analyzed using chi-squared test
to correlate the quantitative changes in the thermal sensitivity to hot and cold with any changes in the
ESC.
Results:
From April 13, 2016 to October 24, 2016, 24 patients (9 men, 15 women; mean age: 63.8 years) treated
with oxaliplatin (85 mg/m² every 2 weeks for 6 months) (18 FOLFOX; 5 FOLFIRINOX; 1 GEMOX) were
included (23 patients in clinical grade 1 and 1 patient in clinical grade 2).
- Regarding the NPSI scale, 16 patients (66.6%) experienced pain triggered by contact with a cold object.
- Thermotest® results: 15/24 patients (62.5%), 5/24 patients (20.8%) and 2/24 patients (8.3%) experienced
pathological values for cold detection thresholds (CDT), warm detection thresholds (WDT) and cold pain
thresholds (CPT), respectively. No patient had pathological heat pain thresholds (HPT) (normal values from
Rolke et al., 2006).
- Sudoscan® results: the chloride conductance was modified in 8 patients (33.3 %) with ESC < 40 µS and 2
with 40 < ESC < 60 µS. There is no statistically significant difference between ESC of hands and feet: 65.7 ±
20.6 µS vs 62.5± 17.9 µS (p= 0.2008), respectively.
-There was no relationship between ESC and each individual thermal threshold. P values were 0.67, 0.13,
0.14 and non-calculable for CDT, WDT, CPT and HPT, respectively (chi-squared test).
Discussion/Conclusion:
Unfortunately, statistical results of the chi-squared test for 24 patients do not show a significant
relationship between conductance to chloride ions and thermal sensory parameters on hand area. This
assessment of small fiber neuropathy through a quantitative measurement of sweat function, despite the
occurrence of neuropathic pain symptoms, hypoesthesia and cold allodynia in our population as in
literature (Kus T et al., 2016) (Attal et al., 2009) needs to explore more peripheral areas than hands
because: i) the clinical grade of the neuropathic patients is low, ii) more peripheral nerve endings are
often affected in oxaliplatin-induced thermal neuropathy.
28
STEREOSELECTIVE COPPER CATALYZED DIRECTED THIOGLYCOSYLATION OF
C(SP2)-H BONDS OF BENZAMIDES
Amélie Chabrier,1 Alexandre Bruneau,1 Sara Benmahdjoub,2 Berkacem Benmerad,2 Sabrina
Benmerad,2 Mouad Alami,1 and Samir Messaoudi,1
1
BioCIS, Univ. Paris-Sud, CNRS, University Paris-Saclay, Châtenay-Malabry, France
2
Laboratoire de Physico-Chimie des Matériaux et Catalyse, Faculté des Sciences Exactes,
Université de Bejaia, 0600 Bejaia, Algeria
Pôle thématique de rattachement à l'ED569: Chimie Pharmaceutique
[email protected]
Thioglycosides constitute an important class of compounds that find widespread use as
pharmaceuticals [a] as well as versatile intermediates in organic synthesis [b]. Our work presents
an efficient and practical thiolation of C(sp2)-H bonds with thiosugars [c]. Using only Cu(OAc)2.H2O
as a catalyst and Ag2CO3 as an additive in DMSO, the protocol proved to be general, and a variety
of thioglycosides have been prepared in good to excellent yields with exclusive β-selectivity. The
value of this transformation has been highlighted via the synthesis of complex trithioglycosylated compounds (easily prepared via our previously reported Pd-G3-catalyzed
coupling reaction [d]), as well as the late stage thioglycosylation of biologically active compounds.
Bibliographic references:
H. Driguez, Thiooligosaccharides, in Glycobiology, Glycoscience Synthesis of Substrate
Analogues and Mimetics, Springer, Heidelberg 1997, 187, 85-116.
(b)
J. D. C. Codee, R. E. J. N. Litjens, L. J. van den Bos, H. S. Overkleeft, G. A. van der Marel, Chem.
Soc. Rev. 2005, 34, 769-782.
(c)
A. Chabrier, A. Bruneau, S. Benmahdjoub, B. Benmerad, S. Benmerad, M. Alami, S. Messaoudi,
Chem. Eur. J. 2016 (VIP), DOI: 10.1002/chem.201602909
(d)
A. Bruneau, M. Roche, A. Hamze, J.-D. Brion, M. Alami, S. Messaoudi, Chem. Eur. J. 2015, 21,
8375-8379.
(a)
29
Cardio protective effect of Orai1 inhibition against pressure overload-induced
pathological remodeling
Fiona Bartoli1, Baptiste Rode2, David Beech2, Ana Maria Gomez1, Jean-Pierre Benitah1 and Jessica
Sabourin1
1
INSERM UMR-S1180, Université Paris-Sud, Chatenay-Malabry, France
2
School of medicine, University of Leeds, Leeds, United Kingdom
[email protected]
Cardiac hypertrophy and dysfunction in response to sustained mechanical stress are central
features of most forms of heart disease. Whereas increasing evidences support a role for the
Orai1-dependent Store-Operated Ca2+ entry (SOCE) in this pathophysiology, the cardiac protective
or deleterious effect of Orai1 after pathological insults is still under debate. Using a genetic mouse model
with cardiac specific expression of the dominant negative Orai1R91W mutant (dn-Orai1R91W), we
assessed whether and how Orai1-mediated SOCE contributes to cardiac Ca2+ signaling and contractility in
a model of pressure overload-induced pathological remodelling in mice.
The littermates controls (WT) and dn-Orai1R91W Tg mice at 2 months of age were subjected to 3
weeks of pressure overload by transverse aortic contriction (TAC). Echocardiograms and
morphometrics parameters show similar level of cardiac hypertrophy in WT and dn-Orai1R91W Tg
mice with increased cardiac mass (heart weight to body weight ratio), left ventricular mass and
septal thickness. Consistent with these observations, we found similar induction of
prohypertrophic markers and collagen expression at mRNA level in both model. Nevertheless,
although WT mice display a decline in fractional shortening as an indicator of decreased
myocardial contractility, dn-Orai1R91W Tg mice maintain preserved systolic function after TAC.
Protein quantification by western-blot in ventricle tissue reveals increased Orai1, Orai3, TRPC6
and STIM2 expression following TAC in WT mice. This is correlated with enhanced SOCE
activated by Ca2+ stores depletion measured by Mn2+-quenching method in isolated adult WT
ventricular myocytes. Importantly, the increased SOCE is blunted in dn-Orai1R91W
cardiomyocytes. While prolonged action potential duration after TAC is observed independently
of the genotype, impairement of Ca2+ homeostasis and contraction with a decrease in [Ca2+]i
transients amplitude with slower kinetics and decreased cell shortening and SR Ca2+ content
observed in WT mice after TAC are prevented in dn-Orai1R91W Tg mice.
In conclusion, our findings highlighted an upregulation of Orai1 expression and function after
hypertrophic insults. Furthermore, dn-Orai1R91W Tg mice are partially protected from loss of cardiac
functional performance following long-term pressure overload stimulation. Thus, inhibition of
Orai1-dependent Ca2+ entry specifically in heart could represent a good strategy to prevent systolic
dysfunction and will be a promising strategy for the treatment of heart disease.
30
Substrate specificity of the Clostridium difficile Cwp84 protease depends on its
localization and maturation state.
Claire Périllaud-Dubois1, Thomas Candela1, Diana Chapetón-Montes1, Claire Janoir1
EA 4043, Unité Bactéries pathogènes et Santé, Faculté de Pharmacie, Univ. Paris-Sud,
Université Paris-Saclay
Pôle thématique de rattachement à l'ED569 :Microbiologie
[email protected]
Substrate specificity of the Clostridium difficile Cwp84 protease depends on its localization and
maturation state.
Clostridium difficile is a Gram positive anaerobic bacteria causing several intestinal pathologies.
The main virulence factors are two toxins but several other factors are involved in the infectious
process, especially surface components that mediate first bacterial interactions with the host.
Among those, the main surface component is the S-layer, which derived from the cleavage of
their precursor SlpA by the Cwp84 cysteine protease. The Cwp84 is found mainly as a 77 kDa
form at the bacterial surface, anchored to a cell wall polysaccharide via its C-terminal domain.
Cwp84 is also found as a 47 kDa form in the extracellular fraction. We hypothesized that
substrate specificity of Cwp84 would depend on its localization and/or its maturation state: the 77
kDa form would be responsible for the cleavage of the precursor SlpA and the extracellular 47
kDa form would degrade proteins of the host's extracellular matrix. In addition, Cwp84 has been
described as a negative modulator of biofilm production in vitro. The increased biofilm formation
in the mutant knockout for cwp84 could be explained either by the immaturity of the S-layer or by
the absence of the catalytic activity of the protease.
In the mutant CD Cwp841-506 (expressing Cwp84 deleted from its C-terminal anchoring domain),
the truncated protease was only detected by immunoblotting in the extracellular fraction and was
no more anchored to the surface. In addition, in this mutant, only the precursor SlpA was detected
in the supernatant, not the two subunits of the S-layer. These results showed that the truncated
form of the protease, when not associated to the bacterial surface, is not able to cleave the
precursor SlpA. In addition, we observed that biofilm formation was increased in mutant CD
Cwp841-506, despite the presence of the truncated protease. This result argues for that the increase
in biofilm would therefore be due to the immaturity of the S-layer.
On the other hand, we showed that the recombinant form rCwp8430-518 was able to degrade the
laminin in the ratio 1/1 and 1/10 (protease/substrate).
To conclude, these results suggested that substrate specificity of Cwp84 depends on its
localization and maturation state.
31
Janus nanoparticles: characterization and implementation of spectroscopic descriptors
and follow-up skin application
Kamilia Kemel, Cécile Laugel, Vincent Faivre, Arlette Baillet-Guffroy
Groupe de Chimie Analytique de Paris-Sud - Lip(Sys)2, Institut Galien Paris-Sud:PhysicoChimie des Systèmes Polyphasés
Faculté de Pharmacie Paris Sud, 5, rue J.B. Clément 92296 Châtenay-Malabry
[email protected]
L'ED n°571: Sciences Chimiques : Molécules, Matériaux, Instrumentation et Biosystèmes
(2MIB)
Pôle : Chimie Physique, BioPhysique et Analytique (CPBA)
Résumé
This is a study of Janus-type bi-compartmental lipid nanoparticles. They are a dispersion
of objects characterized by the presence of a lipid compartment associated with an
aqueous compartment delimited by a phospholipid bilayer between 150 and 300 nm in
size and protected by patent FR 3 008 900-A1. The technique of attenuated total
reflection by Fourier transform infrared (ATR-FTIR) was used to define spectroscopic
descriptors of these objects, in order to follow their behavior after topical cutaneous
administration. Descriptors were then used to obtain a spectral signature of nanoparticles
before and after cutaneous application, in order to better understand the mechanisms of
interaction that may exist between nanoparticles and the skin. The fate of Janus
nanoparticles after topical administration in fact remains to be elucidated.
32
BKCa channels are key effectors of coronary tone regulation by
PDE3 and PDE4 in normal, but not in failing heart
1
2
1
Sarah IDRES , Valérie DOMERGUE-DUPONT , Rodolphe FISCHMEISTER , Véronique LEBLAIS
1
and Boris MANOURY .
1
2
1
INSERM UMR-S 1180, Faculté de Pharmacie, Université. Paris-Sud, Université Paris-Saclay, ChâtenayMalabry, France.
Plateforme ANIMEX, UMS IPSIT, Faculté de Pharmacie, Université. Paris-Sud, Université Paris-Saclay, France.
Pôle : physiopathologie moléculaire et cellulaire
[email protected],
Background: Regulation of vascular tone by 3'-5'-cyclic adenosine monophosphate
(cAMP) involves many cellular effectors, including large conductance, Ca2+-activated K+
(BKCa) channels. In arteries, cAMP concentrations are mainly controlled by type 3 and 4
phosphodiesterases (PDE3 & PDE4). Here, we sought to clarify whether BKCa channels
are key players in the regulation of tone by PDE activities in rat coronary arteries (CA),
and if this contribution is altered in heart failure (HF).
Material & methods: CA were isolated from rats with HF sacrificed 22 weeks after
surgical stenosis of the ascending aorta. Age-matched "sham" animals were used as
controls. Tension measurements were conducted on CA contracted using the thromboxan
analogue U46619.
Results: PDE4 inhibitor (Ro-20 1724, Ro, 10 µM) or a PDE3 inhibitor (cilostamide, Cil,
1 µM) partially relaxed sham CA. These effects were blunted by the BKCa channels
blocker iberiotoxin (IBTX, 100nM). Simultaneous addition of Cil and Ro synergistically
relaxed sham CA, an effect also reduced by IBTX. CA isolated from rats displaying
robust signs of HF showed poor maximal contractile capacity (-82%) and reduced
relaxation to ACh (-41%) compared to CA from sham rats. Cil clearly relaxed HF CA,
while effect of Ro was modest. Cil and Ro together synergistically relaxed CA to a
degree equivalent to that in sham. In contrast, IBTX did not modify responses to PDE
inhibitors in HF CA. Amount of BKCa channel alpha-subunit displayed a 2-fold decrease
in HF CA extracts compared to sham. A 70kDa-PDE4B was increased in HF, while other
PDE3 or PDE4 isoforms expressed in rat CA displayed no difference.
Conclusion: Our data show that BKCa channels are key effectors in the relaxation of rat
CA induced by PDE3 and PDE4 inhibition in rat CA. In HF, the contribution of BKCa
channels to these responses is lost, suggesting that PDE-BKCa channel coupling is altered
in diseased arteries.
33
SKIN ALLERGY CAUSED BY AIR-OXIDIZED TERPENES: MECHANISM OF
ACTION AND ROLE OF TRANSCRIPTION FACTOR NRF2
Chloé RAFFALLI1, Salen KURESEPI2, Elodie CLOUET3, Marie-Hélène DAMIENS1,
Andreas NATSCH4, Jean-Pierre LEPOITTEVIN2, Marc PALLARDY1, Pierre-Jacques
FERRT3, Elena GIMENEZ-ARNAU2, Saadia KERDINE-RÖMER1
1
INSERM UMR-S 996, Université Paris-Saclay, Châtenay-Malabry, France
2
CNRS UMR 7177, Laboratoire de Dermatochimie, Université de Strasbourg, Strasbourg,
France
3
Pierre Fabre Dermo-Cosmétique Research & Development, Toxicology Division, Safety
Department, Toulouse, France
4
Givaudan Schweiz AG, Duebendorf, Switzerland
Pharmacologie - toxicologie
[email protected]
Allergic Contact Dermatitis (ACD) to fragrance ingredients affects about 1 to 3% of the
population in Europe. It occurs when an individual has been exposed on the skin to a
sufficient dose of a fragrance sensitizer, for example through its presence in a cosmetic
product. Many fragrance terpenes are considered as low skin sensitizers in ACD on Local
Lymph Node Assay (LLNA) tests results. On air exposure, those prehaptens autoxidize to
form different oxidation products such as epoxides, diols and allylic hydroperoxides.
Allylic hydroperoxides are classified as strong skin sensitizers in LLNA tests results. To
become immunogenic, allylic hydroperoxides need to bind to skin proteins through
radical reactions. This complex will be captured by Dendritic Cells (DCs), a crucial cell
type in the development of ACD.
In this work, we studied how two terpenes, linalool and limonene, and their respective
allylic hydroperoxides (LINA-OOH and LIMO-OOH) could induce the activation of DCs.
The THP-1 model cell line, used in the h-CLAT in vitro test, was used to test those
molecules in this study. Linalool, limonene, LINA-OOH and LIMO-OOH were tested at
subtoxic concentrations (1, 1.5, 1 and 1.5 mM respectively). With flow cytometry, our
results show that after 30 minutes and 1 hour of treatment, linalool and limonene
hydroperoxides oxidized cell surface thiols. Those thiols are then recycled 2 hours post
exposure. This leads to a decrease of GSH/GSSG ratio after 1 hour of treatment. GSH is
involved in membrane proteins recycling. This oxidative stress leads to the activation of
Nrf2, measured by western blot, after 6 hours of exposure. Nrf2 activity, measured by
an array assay, is directly correlated with its target genes induction (ho-1, nqo-1 and il-8)
in response to linalool and limonene hydroperoxides. MAPKs (P38 MAPK, JNK, ERK) and
NF-KB pathways are also activated. In fine, in response to allylic hydroperoxides, cellsurface markers CD54 and CD86 are expressed after 24 hours of exposure.
To conclude, our results show that linalool and limonene need to be oxidized to activate
THP-1 cells. Those oxidative products decrease THP-1 surface thiols, resulting in a
cascade of events which lead to cell maturation.
34
Magnetic Extraction and detection of bacteria in complex media : Innovative methods for the
integration of immunosensors based on magnetic nanoparticles in lab-on-chip
ENDEMIQUE project
Titre : Capture et détection de bactérie à l’aide de nanoparticules magnétiques pour le développement de
biocapteurs.
2
1
2
3
3
Claire GALLAND , Olivier Lefebvre , Fabrice Mbock Nkot , Claire Jannoir ,Thomas Candela Emile
1
1,
2
Martincic , Myriam Taverna, Mehdi Ammar Claire Smadja
1
Institut d’Electronique Fondamentale, CNRS UMR 8622, Université Paris Saclay, Orsay F-91405, France
Institut Galien Paris-Sud, CNRS UMR 8612, Université Paris Saclay, Chatenay-Malabry F-92296, France
3
Département de microbiologie, Université Paris-Saclay, Chatenay-Malabry, France
2
Pôle thématique de rattachement à l'ED 571 (Sciences Chimiques : Molécules, Matériaux,
Instrumentation et Biosystème) : Chimie Physique, BioPhysique et Analytique
L’identification et la quantification des bactéries est un enjeu majeur dans différents
contextes ; du contrôle qualité à la détection de contaminations environnementales ien
incluant la bio-défense. Les méthodes actuelles de détection reposent sur la culture en
boites de petri, des tests immuno-enzymatiques ou de la PCR et sont longues et couteuses.
Un test permettant une détection rapide et sensible serait d’un grand intérêt. Aussi, nous
nous proposons de développer un biocapteur original, à magnéto-impédance géante,
hautement spécifique basé sur l’utilisation de micro-bobines intégrées dans un canal
microfluidique. Celui-ci repose sur l’utilisation de nanoparticules magnétiques, à base
d’oxyde de fer, biofonctionnalisées par des anticorps combinant une extraction sélective de
la bactérie d’un milieu complexe, ainsi que sa détection par le biocapteur en une seule
étape. Celui-ci sera appliqué dans un premier temps à la détection de l’Escherichia Coli (E.
Coli) non pathogène afin d’établir la preuve de concept. Dans un premier temps nous avons
vérifié, qu’il était possible d’effectuer une capture sélective des E.Coli par les nanoparticules
magnétiques fonctionnalisées par des anticorps. Cette expérience a été réalisée en
« batch » à l’aide de bactéries fluorescentes et caractérisée par microscopie à fluorescence.
Nous avons ensuite testé l’interaction nanoparticules magnétiques bio-fonctionnalisées/
bactéries en microsystème. Les expériences réalisées ont montré qu’il est possible de
capturer les bactéries en conditions micro-fluidique. L’étape suivante consistera à détecter
les bactéries capturées à l’aide de micro-bobines enchâssées dans le biocapteur.
35
ETUDE LIPIDOMIQUE DE L’IMPACT DE L’ACIDE EICOSAPENTAENOÏQUE
(EPA) SUR L’EFFLUX DU CHOLESTEROL DES MACROPHAGES HUMAINS
Guillaume Sayet, Jean-Louis Paul, Eric Caudron, Sana Tfaili, Natalie Fournier, Pierre
Chaminade
Affiliations : Lip(Sys)² -Chimie Analytique Pharmaceutique (FKA EA4041 Groupe de
Chimie Analytique de Paris-Sud)
Pôle thématique de rattachement à l'ED571 : 2MIB pole CPBA
[email protected]
Une alimentation riche en acides gras polyinsaturés de la famille 3 (AGPI n-3) est considérée
comme protectrice vis-à-vis de l’athérosclérose. Les AGPI en s’incorporant dans les
phospholipides (PL) membranaires en modifient la composition et influencent les fonctions des
protéines insérées dans la membrane. Nous avons récemment étudié l’impact de l’incorporation
d’AGPI dans la membrane de macrophages humains sur l’efflux du cholestérol dépendant de
l’ABCA1, première étape du transport inverse du cholestérol qui est une voie métabolique
essentielle anti-athérogène. Nous avons montré que l’incorporation d’acide eicosapentaénoïque
(EPA), (C20 :5, 70M) provoquait une diminution moyenne d’efflux de 28%. L’EPA ne modifie ni
l’expression de l’ABCA1 ni sa localisation membranaire mais réduit la fonctionnalité du
transporteur par une voie dépendante de la PKA. Afin de mieux cerner le rôle de l’EPA, nous
avons procédé à une analyse plus complète des modifications éventuelles des PL membranaires.
Les monocytes ont été séparés du sang total de 7 sujets sains. Après différenciation, les
macrophages sont incubés 34h dans du milieu enrichi en EPA (70M). Au terme de cette
incubation, l’efflux du cholestérol est mesuré et les lipides totaux sont extraits et analysés par 3
méthodes complémentaires : i) une analyse par chromatographie gazeuse couplée à la
spectrométrie de masse pour déterminer la composition globale en AG, ii) la quantification des
classes de PL par Corona, iii) l’analyse des espèces moléculaires des classes de PL par
chromatographie en phase normale couplée au LTQ-Orbitrap. Les données ont été analysées par
chimiométrie au moyen d’analyses discriminantes. Les résultats obtenus confirment que l’EPA
s’incorpore dans les membranes sous forme d’EPA mais aussi sous forme d’acide
docosapentaénoïque (DPA, C22 :5) qui est son produit d’élongation au détriment de l’acide
arachidonique (AA, C20 :4 n-6) et à un degré moindre des AG monoinsaturés. L’analyse
quantitative des différentes classes de PL ne montre pas de différence significative. L’analyse
chimiométrique a été effectuée sur une matrice de 1832 variables. L’analyse chimiométrique et
l’étude des schémas de fragmentation a mis en évidence 22 espèces moléculaires de PL
surexprimées dans les cellules incubées avec de l’EPA. Ces espèces moléculaires se caractérisent
par la présence d’EPA ou de DPA en position sn-2 et concernent essentiellement la
phosphatidylcholine (PC), le phospatidylinositol (PI) et les alkényls de phosphatidyléthanolamine
(PE). Quinze espèces moléculaires sont sous-exprimées dans ces cellules touchant
principalement le PI estérifié en sn-2 par de l’AA ou par de l’acide eicosatriénoïque (C20 :3) et
des alkyls et alkényls de PE et de PC. Afin de rechercher l’existence d’une relation entre la
diminution d’efflux et les variations des espèces moléculaires des PL, une analyse de régression
PLS a été effectuée et a permis de révéler que la surexpression du PI 18 :0/22 :5 est l’espèce
moléculaire la plus fortement reliée avec la diminution d’efflux. Ce résultat original met ainsi en
évidence un rôle potentiel de la modification du PI sur l’efflux dépendant de l’ABCA1.
36
Effets métaboliques et non métaboliques de la délétion cardiaque et inductible de
l’AMPK2 chez la souris mâle.
Grimbert L1, Sanz MN1, Piquereau J1, Rucker-Martin C2, Gressette M1, Ventura-Clapier R1, Veksler
V1, Garnier A1.
1
Inserm, UMR-S 1180, Univ. Paris-Sud, Université Paris-Saclay, F-92296, Châtenay-Malabry, France
2
Inserm UMR-S 999, Labex LERMIT, Hypertension Artérielle Pulmonaire: Physiopathologie et
Innovation Thérapeutique, Centre Chirurgical Marie Lannelongue, Le Plessis-Robinson, France
[email protected]
La dysfonction mitochondriale joue un rôle majeur dans la physiopathologie de l’insuffisance
cardiaque (IC). La kinase dépendante de l’AMP (AMPK), activée lors d’une augmentation du ratio
AMP-ADP/ATP, régule de nombreuses voies métaboliques. Plusieurs études ont mis en évidence
un rôle protecteur de l’AMP dans le myocarde défaillant, mais l’absence de modèles animaux
appropriés ne permet pas de conclure quant à la spécificité cardiaque et la composante
métabolique de ces effets. Nous avons donc développé un modèle murin de délétion spécifique
du cœur et inductible à l’âge adulte de l’AMPK2, l’isoforme majoritaire cardiaque. Nous avons
obtenu ces souris en croisant une lignée portant des sites lox-P sur l’exon 6 de l’AMPK codant
pour la sous-unité catalytique avec des souris présentant une cre recombinase inductible par la
tamoxifène et sous le contrôle du promoteur de l’-MHC. La délétion de l’exon 6 a été effectuée
par injection intrapéritonéale de 40 mg/kg/j de tamoxifène sur 2 jours. Quatre semaines après
injection, nous avons observé une diminution de l’expression cardiaque de l’AMPK de 70% chez
les souris KO. A partir de 7 semaines après injection, les souris KO présentent une diminution de
la fraction d’éjection du ventricule gauche (VG, -10%) ainsi qu’une dilatation du VG. A 4 mois, les
cœurs des souris KO présentent une augmentation significative de la fibrose totale (+64%). De
plus, la consommation d’oxygène par la mitochondrie via le complexe I de la chaîne respiratoire
mesurée sur des fibres perméabilisées de VG est réduite de 28% chez les souris KO. Aucun effet
délétère n’est observé pour le complexe II.
Ainsi, la délétion de l’AMPK à l’état basal dans le cœur adulte, mène à une fonction contractile
altérée qui peut être corrélée à des atteintes morphologiques et/ou métaboliques. Des
expériences complémentaires sont nécessaires afin d’étudier la relation de cause à effet de ces
résultats.
37
Development of a CE-MS method for the analysis of N-glycans
C. Ruel 1,2, M. Taverna 1, C. Junot 2, F. Fenaille 2 et T. Tran 1
Affiliations : 1 Institut Galien Paris Sud, UMR8612, Protein and Nanotechnology in
Analytical Science (PNAS), CNRS, Univ. Paris-Sud, Université Paris-Saclay, 5 rue Jean
Baptiste Clément, 92290 Châtenay-Malabry, France ; 2 CEA, iBiTec-S, Service de
Pharmacologie et d’Immunoanalyse, Laboratoire d’Etude du Métabolisme des Médicaments,
Gif-sur-Yvette 91191, France
Pôle thématique de rattachement à l'ED569 : chimie thérapeutique
[email protected]
1
Glycosylation is one of the most complex types of post-translational modifications of proteins .
Disease-associated modifications in protein glycosylation constitute a major source of biomarkers
2
and are often exploited for diagnosis and monitoring in various pathologies .
The goal of my PhD thesis is to implement an analytical platform for the analysis of free
oligosaccharides in urine and/or plasma protein-bound oligosaccharides. Starting from biological
fluids, the first step will be the on-line enrichment of glycoproteins on a functionalized monolithic
column. Then an enzymatic deglycosylation of preconcentrated glycoproteins followed by the
derivatization of released glycans with a fluorophore will be performed. The obtained glycans will
be separated on-line using orthogonal techniques such as high-performance liquid
chromatography and capillary electrophoresis for the efficient resolution of isomeric
oligosaccharide structures. Such separation system will be coupled to high resolution mass
spectrometry (Orbitrap or Q-TOF).
I will present here the first step of my work which is the development and the optimization of the
separation method of derivatized N-glycans using capillary electrophoresis coupled to mass
spectrometry (Q-TOF). A new fluorophore, the Rapifluor-MS, developed in 2015 by Waters for Nglycan analysis, is supposed to increase MS sensitivity and is currently only used after HPLC
3
separations . The objective of this study is to investigate the usefulness and relevance of the
Rapifluor-MS reagent for the CE-MS analysis of N-glycans.
For this purpose, different conditions of CE-MS (background electrolyte, sheath liquid, mass
parameters…) were tested in order to evaluate this derivatization.
References
S. C. Bunz et al, Capillary Electrophoresis/Mass Spectrometry of APTS-Labeled Glycans for the
Identification of Unknown Glycan Species in Capillary Electrophoresis/Laser-Induced
Fluorescence Systems, Analytical chemistry, 2013
2
S. Bekesova et al, N-glycans in liver-secreted and immunoglobulin-derived protein fractions,
Journal of Proteomics, 2012
3
M. Lauber et al. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling
Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection. Analytical chemistry.
2015
1
38
Identification of species implied in hIAPP amyloid cascade : interest in type 2 diabetes
Corentin Berardet a,b, Julia Kaffy b, Sandrine Ongeri b, Myriam Taverna a
Affiliations : a Proteins and Nanotechnology in Analytical Science, Institut Galien Paris Sud, Faculté de
Pharmacie, Université Paris Saclay, Châtenay-Malabry
b
Molécules Fluorées et Chimie Médicinale, BioCIS UMR CNRS 8076, Labex LERMIT,
Faculté de Pharmacie, Université Paris Saclay, Châtenay-Malabry
Pôle thématique de rattachement à l'ED569 : Chimie thérapeutique
[email protected]
Type II diabetes (T2D) and associated cardiovascular risks are now considered as major
concerns of public health, affecting more than 300 million people in the world. Human Islet
Amyloid Polypeptide (hIAPP) is a 37 amino acids peptide co-secreted with insulin by pancreatic 
cells and involved in glucose homeostasis. Its aggregation under certain conditions leads to 
cells degeneration and promotes T2D by inhibiting insulin secretion. Both mechanisms and
involved species of hIAPP toxicity are still unknown. 1
Here we propose to develop a capillary electrophoresis method to monitor in vitro
oligomerization and aggregation of hIAPP in order to get more insight on this pathological
process and also to develop a screening method to test for new therapeutic agents. Due to its
high pI (8.59), hIAPP is cationic under physiological conditions which leads to its adsorption onto
the silica capillary walls and reproducibility issues. A polybrene coating of the capillary was
developed in order to inhibit this phenomenon. Identification of most of the different species
involved in this process is under progress by Ion Mobility Spectroscopy-Mass Spectrometry2
through a collaboration with the LCP (Pr G. Van der rest) and by Capillary Electrophoresis- QTOF
Mass Spectrometry.
This method will allow us in the future to test peptidomimetics inhibitors of hIAPP aggregation in
order to target specifically toxics species for  cells. Preliminary tests to select candidates are
conducted with Thioflavin-T fluorescence assays.
1
Mukherjee et al, Type 2 diabetes as a protein misfolding disease, Trends in Molecular
Medicine, 2015.
2
Young et al, Ion Mobility Spectrometry–Mass Spectrometry Defines the Oligomeric
Intermediates in Amylin Amyloid Formation and the Mode of Action of Inhibitors, Journal of the
American Chemical Society, 2014.
39
WHEN A VIRAL PROTEIN SWITCHES ON SELECTIVE AUTOPHAGY TO
ESCAPE APOPTOSIS AND PROMOTE CELL SURVIVAL
G. Vilmen1, 2, G. Siracusano2, L. Mouna1, F. Quignon2, V. Maréchal2, A. Esclatine1
(1) Institute for Integrative Biology of the Cell (I2BC) Univ Paris-Sud Equipe Virulence et
latence des Herpesvirus, Faculté de Pharmacie 92290 Chatenay-Malabry (2) CIMI. Hôpital
La Pitié Salpêtrière. Equipe Infections virales persistantes, 75013 Paris
Pôle thématique de rattachement à l'ED569 : Microbiologie
[email protected]
Epstein-Barr virus (EBV), a member of the Herpesviridae family, is associated with
infectious mononucleosis and with several types of cancers including Burkitt’s
lymphoma, post-transplant B-cell lymphoma disease and nasopharyngeal carcinoma.
This virus is able to establish persistent infection and to undergo lytic cycle after
reactivation. BHRF1, an EBV transmembrane protein homolog of cellular protein Bcl-2, is
able to inhibit apoptosis avoiding cell death and therefore promoting cell survival.
Autophagy is a cellular process involved in recycling of cellular components and
promoting cell survival. The aim of this study was to identify whether BHRF1 can
modulate autophagy and the consequence of this modulation.
We demonstrated that BHRF1 expression leads to an accumulation of autophagosomes,
which are double-membrane vesicles positive for the autophagic marker LC3. Using
tandem-fluorescent-tagged LC3 (mRFP-GFP-LC3), which is based on different pH stability
of GFP and mRFP fluorescent proteins, for monitoring autophagic flux, we clearly
demonstrated that BHRF1 stimulates autophagy, whereas Bcl-2 blocks the autophagic
process. By co-immunoprecipitation, we demonstrated that BHRF1 can interact with
Beclin1, a key protein of the autophagic machinery.
We characterized the subcellular localization of BHRF1, and observed that BHRF1 is
localized in mitochondria and ER membranes. Moreover, expression of BHRF1 leads to a
complete reorganization of the mitochondria network to form juxtanuclear
mitochondrial aggregates close to the MTOC (Microtubules Organizing Center). Based on
the importance of microtubules on both autophagy and mitochondria transport, we
explored microtubule dynamics and tubulin post-translational modifications after BHRF1
expression. We observed a specific requirement of the microtubule network to form the
mito-aggresomes and a clustering of acetyl-tubulin around them.
In accordance to mitochondrial phenotype of BHRF1-expressing cells, we observed that
BHRF1 can induce mitophagy, a process that promotes selectively the clearance of
impaired mitochondria by autophagy. We hypothesized that mitophagy induction is a
mean to interfere with apoptosis, allowing viral persistence within cells. We are
currently working on characterizing the importance of mitophagy for apoptosis
inhibition by BHRF1.
The understanding of interactions between this protein and autophagy, and in a wide
way EBV and autophagy, can give new tools for therapeutics targeting the virus.
40
PRECONCENTRATION STRATEGIES IN CAPILLARY ELECTROPHORESIS
HOW TO IMPROVE SENSITIVITY OF MOLECULAR DIAGNOSTICS?
Cédric Crosnier de Lassichère*, Thanh Duc Mai, Myriam Taverna
Institut Galien Paris Sud,PNAS, UMR8612
Pôle thématique de rattachement à l'ED569 :
[email protected]
46 million people in the world are living with dementia [1]. Nowadays, no treatment for
dementia is available and the different associated pathologies (Alzheimer’s disease, Parkinson’s
disease, Amyotrophic lateral sclerosis…) can be detected only at a late stage when the brain
damages are irreversible. Diagnosing those diseases earlier and enrolling well diagnosed patients
for testing new treatments during clinical trials are therefore major societal needs. In this
context, scientists are developing new methods of molecular diagnostics to early distinguish a
pathology from another one. However most of the developed methods need a preconcentration
step because of the low abundance of biomarkers in biological fluids, mostly relying on
immunoprecipitation which implies the availability of specific antibodies. Capillary
electrophoresis (CE) is an alternative and fast method for separation but also for sample
preconcentration. All preconcentration methods in CE exploit velocity differences between the
sample zone and background electrolyte [2,3,4]. The challenge is how this can be done with
biological fluids.
Thus, the objective of the project is to develop analytical setups based on either lab-on-a-chip or
mass spectrometry to diagnose different neurodegenerative diseases. One important aspect of
the project is the realization of new strategies for sample enrichment and matrix removal as an
alternative to the conventional preconcentration methods such solid phase extraction.
We present here 3 different approaches (FASS, LVSEP and ITP) to enhance the detection
sensitivity of validated biomarkers of Alzheimer’s disease. Thanks to those methods we could
detect and quantify some Amyloid β peptides in real cerebrospinal fluid. Downscaling one of the
method into a microchip is under progress.
[1] Prince et al., https://www.alz.co.uk, 2015
[2] Verpillot et al., Analytical chemistry, 2011
[3] Oukacine et al., Analytical chemistry, 2014
[4] Mai et al., J of Chromatography A, 2016
41
REGIOSELECTIVE REDUTIONS OF 1,2-DIKETONES USING
CHLOROTRIMETHYLSILANE AND SODIUM IODIDE
Ling-Zhi Yuan, Dolor Renko, Olivier Provot, Abdallah Hamze, Mouad Alami
BioCIS, Univ. Paris-Sud, CNRS, équipe labellisée Ligue Contre le Cancer, Université Paris-Saclay, 92290, Châtenay-Malabry,
France
[email protected]
We found that the TMSCl/NaI combination was used as a selective reducing reagent in diaryl diketones (benzil
[1]
derivatives) . This combination which has good tolerance for various functional groups was successfully
evaluated with several unsymmetrically benzil derivatives 1, and the reduced products (deoxybenzoins) with a
total -regioselectivity were obtained at room temperature in CH2Cl2 with good to excellent yields:
After the success obtained with the -regioselective reduction of benzils (diaryl system), we tested this
reducing combination with a variety of aryl-alkyl-1,2-diketones which were reduced in moderate to good yields
again with a total -regioselectivity at room temperature in CH2Cl2. On the contrary to benzils which needed
the help of EDGs on ortho- or meta-position, the deoxygenation of arylalkyldiketones works well with all aryl
groups containing either donating- or withdrawing-groups in ortho, meta, or para-positions.
In conclusion, we have demonstrated that using TMS-Cl together with NaI, it is now possible to prepare deoxydiketones 2 and 4 with a total -regioselectivity and moderate to good yield. Moreover, a lot of
functional groups were tolerated during this novel reaction, which makes this reduction method very practical,
“green” and easy to handle (no use of classical metal-reducing species ).
[1]
L-Z. Yuan, D. Renko, I. Khelifi, O. Provot, J.-D. Brion, A. Hamze, M. Alami, Org Lett., 2016, 18, 3238.
42
IDENTIFICATION ET CARACTERISATION DU ROLE DE LA MITOMYCINE C DANS LA MALADIE VEINO
OCCLUSIVE PULMONAIRE
Marie-Camille Chaumais, Frédéric Perros, Andrei, Seferian, Caroline .O’Connell, Sven Günter, Olivier
Sitbon, Gérald Simonneau, Marc Humbert, David Montani.
UMR_S 999
[email protected]
Dans la communauté scientifique et médicale, il est reconnu que l’exposition à certaines drogues ou
toxines peut favoriser le développement d’hypertension pulmonaire (HP) : hypertension artérielle
pulmonaire ou maladie veino-occlusive pulmonaire (MVOP). Ces drogues peuvent avoir un effetclasse (« amphetamine-like ») ou non (dasatinib) et n’induisent une HP que chez une faible
proportion (<1%) des patients exposés. Il est essentiel d’identifier précocement d’autres
médicaments susceptibles de favoriser le développement d’une HP. Par ailleurs, la compréhension
des mécanismes physiopathologiques mis en jeu est un élément fondamental pour améliorer la
compréhension de la maladie et identifier d’éventuelles cibles thérapeutiques. De ce rationnel a été
mis en place le projet VIGIAPATH, soutenu par l’ANSM depuis le 1er janvier 2014.
L’objectif est ici de présenter les résultats issus du projet VIGIAPATH concernant la MVOP, forme
rare d’HP, caractérisée par une atteinte veinulaire et capillaire pulmonaire prédominante et dont le
pronostic est extrêmement sombre.
Le projet VIGIAPATH à permis de mettre en lumière un lien entre l’exposition aux agents alkylants,
notamment la mitomycine C (MMC), dans le développement de la MVOP chez l’homme.
Actuellement, 11 cas de MVOP associée à la MMC, prescrite dans un contexte de cancer anal, ont
été rapportés dans le cadre du programme VIGIAPATH. Le délai médian entre le début de
l’exposition à la MMC et la survenue de la MVOP était de 5 mois (min-max 2-30). Parmi ces cas, il
existe une nette prédominance féminine (9/11) ce qui est classique dans les maladies vasculaires
pulmonaires mais ne reflète pas l’épidémiologie du cancer anal. Cela pourrait laisser suggérer un
facteur hormonal ou un lien pharmacologique. Par manque de données sur le nombre de patients
exposés en France à la MMC, il est impossible d’estimer avec précision une incidence de la MVOP
chez les patients exposés à la MMC. En revanche, le traitement du cancer du canal anal reposant sur
la MMC associée au 5FU, nous avons pu estimer que l’incidence de la MVOP chez les patients
atteints de cancer anal était de 3,9/1000 patients/an, ce qui est largement supérieur à l’incidence de
la MVOP idiopathique (0,5 cas/millions/an). De plus, nous avons pu démontrer que l’administration
de MMC chez le rat induisait après 3 semaines, un remodelage veinulaire et capillaire pulmonaire
sévère associé à une HP, reproduisant les caractéristiques de la MVOP humaine. L’administration
d’amifostine dans ce modèle animal prévenait la MVOP induite par la MMC.
L’ensemble de ces données humaines et expérimentales issues du projet VIGIAPATH, ainsi que la
publication de nouveaux cas dans la littérature, convergent sur l’imputabilité forte de la MMC dans
le développement de la MVOP. La mise au point du premier modèle animal de MVOP par
l’utilisation de la MMC représente une opportunité unique de mieux comprendre la
physiopathologie de la MVOP et d'évaluer des thérapies innovantes dans cette maladie sans
traitement à ce jour.
43
Molecular networking-based
networking based detection and isolation of a new bisindole alkaloid from the stem bark of Pleiocarpa mutica Benth.
(Apocynaceae)
Elvis Otogo N’nang1,2, Laurent Evanno1, Karine Leblanc1, Philippe Grellier3, Brice
Kumulungui2, Erwan Poupon1, Mehdi A. Beniddir1, Pierre Champy1
BioCIS, Univ. Paris-Sud,
Sud, CNRS, Université Paris-Saclay,
Paris
92290 Châtenay-Malabry,
Malabry, France,
2
Laboratoire de microbiologie, Université des Sciences et Technique de Masuku, Franceville, Gabo
Gabon,
3
Unité Molécules de Communication et Adaptation des Microorganismes (MCAM, UMR 7245),
1
Pôle thématique de rattachement à l'ED569 : Chimie
psud.fr
In the search for antiplasmodial molecules from plants used in traditional African me
medicine,
the study of the alkaloidic extract of the stem bark of Pleiocarpa mutica (Apocynaceae), col
collected in Gabon, was carried out by a structure-driven
structure
HPLC-ESI-Q/TOF
Q/TOF dereplication strate
strategy, using the “molecular network” approach.1 This strategy led to
o the rapid detection of over
twenty known compounds,2 several of which were isolated for means of biological
evaluation, such as the heterosidic monomer pleiocarpinine (1)) and the aspidofractine /
eburnamine dimer pleiomutine (2).
( Analysis of the alkaloidic
ic extract network also revealed
the presence of several new and unusual bisindole alkaloid of the aspidofractine series.
Spectral data (1D, 2D NMR and MS) of purified evidenced a homodimeric aspidofractine
skeleton, with an original methylene linker. Pleiokomenine
Plei
(3)) showed an interesting activity
against Plasmodium falciparum (FcB1 strain), with an IC50 of 3.7 μM. Structural elucidation
of several analogues is underway.
References:
king as a
[1] Yang JY, Sanchez LM, Rath CM, Liu X, Boudreau PD. Molecular Networking
Dereplication Strategy. J Nat Prod 2013, 76: 1686-1699.
1686
[2] Thomas DW, Achenbach H, Bieman K. A new dimeric indole alkaloid from Pleiocarpa
mu-tica Benth. J Am Chem Soc 1966, 88: 1537-1544.
1537
44
Marine Natural Products as scaffold for generation of chemically diverse
libraries for drug discovery.
David Lachkar,a Laurent Evanno,a Cécile Debitus,b Erwan Poupon a
a
Chimie des substances naturelles, BioCIS, Univ. Paris-Sud, Université Paris-Saclay, 92290,
Châtenay-Malabry, France. b Institut de Recherche pour le Développement, UMR
Ecosystèmes Insulaires Océaniens, Papeete, French Polynesia.
Pôle thématique de rattachement à l'ED569 : Chimie Pharmaceutique
Résumé :
The use of simple reactions to distort the core architecture of natural products provide libraries of
new complex compounds that could be used as biological probes or drug candidates(1).
In the present communications, we will apply this ring-distortion strategy to ilimaquinone(2) and its
epimer(3). Diverse ring transformation will be discussed :
Marine Natural Products
Ilimaquinone
O
O
OMe
OMe
HO
HO
5 epi-Ilimaquinone
O
O
Extraction and
Chemical modifications
Ilimaquinone
O
Dactylospongia metach
HO
romia
O
Biological tests
O
O
HO
O
Drug candidates
OMe
O
O
O
O
OH
New libraries of molecules
(1)
Hergenrother, P. J. et al. Nature Chem. 2013, 5, 195. Hergenrother, P. J. et al. Angew. Chem. Int. Ed. 2014, 53, 220.
Kumar, K. et al. Angew. Chem. Int. Ed. 2016, 55, 7586.
(2)
Luibrand, R. T. et al. Tetrahedron. 1979, 35, 609. Capon, R. J., MacLeod, J. K. J. Org. Chem. 1987, 52, 5059.
(3)
Carté, B.; Rose, C. B., Faulkner, J. D. J. Org. Chem. 1985, 50, 2785.
45
46
Exploration de l’espace chimique de Picralima nitida grâce aux réseaux moléculaires,
une Apocynaceae oubliée à l’ère des « Big Data »
Charlotte ALCOVER1, Jean-François
François GALLARD2, Faustin A. KABRAN3, Philippe
4
GRELLIER , Laurent EVANNO1, Erwan POUPON1, Mehdi A. BENIDDIR1
Affiliations :
1
BioCIS, Univ. Paris-Sud,
Sud, CNRS, Université Paris Saclay, 92290 CHATENAY MALABRY,
France ;
2
Centre de Recherche de Gif, ICSN, CNRS, LabEx LERMIT, 1, avenue de la Terrasse,
91198 GIF SUR YVETTE, France ;
3
Laboratoire de Chimie Organique Biologique, UFR Sciences des Structures de la Matière et
Technologie, Université Félix Houphouët-Boigny,
Houphouët Boigny, 22 BP 582 Abidjan 22, Côte d'Ivoire
4
UMCAM, UMR 7245, Museum d’Histoire Naturelle, CNRS, Sorbonne
Sorbonne Universités, 57 rue
Cuvier, 75005 PARIS, France.
Pôle thématique de rattachement à l'ED569 : Chimie Pharmaceutique
Picralima nitida est une plante de la famille des Apocynaceae, originaire d’Afrique tr
tropicale,
dont les extraits possèdent une forte activité antipaludique.
Pour cette raison, elle a été l’objet de nombreuses études phytochimiques, de 1910 à 1990
principalement. Cependant, parmi la vingtaine d’alcaloïdes indolo-monoterpéniques
indolo monoterpéniques qui en ont
étéé isolés, aucune molécule n’a montré d’activité antiplasmodiale claire.
C’est pourquoi, dans le cadre de l’intérêt historique du laboratoire pour les alcaloïdes indolo
indolomonoterpéniques, et dans le but d’identifier des composés à activité antiprotozoaire
antiprotozoaire, nous
avons décidé de réexplorer la diversité chimique de P. nitida,, avec une approche plus
contemporaine.
En effet, le développement des techniques modernes de déréplication – notamment celles
basées sur la spectrométrie de masse, comme le « Molecular Networking » – a provoqué une
véritable révolution dans le domaine de la chimie des substances naturelles, en permettant de
rapidement visualiser la diversité chimique d’un mélange, comme un extrait végétal par
exemple.
Grâce à cet outil, agrémenté d’une base de
de données spectrales mise au point au laboratoire,
plusieurs nouveaux composés, dont au moins un actif contre Plasmodium falciparum
falciparum, ont ainsi
pu être isolés de Picralima nitida.
nitida
N
H
N
H
H
O
O
MIADB
N+
O
N
H
O
O
47
Light-triggered liposomal release: impact of lipid composition and photosensitizer
hydrophobicity
Julien Massiot, Ali Makky, David Chapron, and Véronique Rosilio
Affiliations : Institut Galien Paris Sud, UMR 8612, Univ Paris-Sud, CNRS, Université ParisSaclay, 5 rue J.B. Clément, F-92290 Châtenay-Malabry, France
Pôle thématique de rattachement à l'ED569 : Pharmacotechnie et biopharmacie
[email protected]
Photo-triggerable liposomes are considered nowadays as promising drug delivery systems
due to their potential to release their drug into spatial and temporal manner. These lightresponsive liposomes offer the advantage of being composed of biocompatible molecules
(i.e. phospholipids) with efficient drug loading capacity. Typically, in photo-oxidative
liposomes, a photosensitive (PS) molecule that absorbs in the NIR is embedded in a
matrix constituted of phospholipids with monounsaturated or polyunsaturated fatty acyl
chains. Upon illumination, the photosensitive molecules generate reactive oxygen species
(ROS), such as singlet oxygen (1O2), that oxidize the phospholipids (PLs) unsaturated
chains. Lipids peroxidation provokes remarkable alterations of the PLs molecular
organization, composition of the vesicle membrane and membrane permeability.
In this work, we have investigated the photopermeation efficiency of three
photosensitizers, verteporfin, pheophorbide a and m-THPP when incorporated in
liposomes with well defined lipids composition SOPC, DOPC or SLPC. By changing the
nature of phospholipids and photosensitizers, we could determine quantitatively that the
illumination of the studied systems altered significantly their lipid bilayer properties and
that the extent of a system efficiency was dependent on the ability of the PS to peroxidize
the acyl chain but also photosensitizer / phospholipid combination. Our results
demonstrated quantitatively the possible use of three PSs clinically approved (or under
investigation) PSs as potential candidates of interest for photo-triggerable liposomes
conception.
48
La technique d'imagerie confocale révèle un lien étroit entre la protéine
microtubulaire +TIP CLIP-170 et les mitochondries au cours du stress cellulaire
D., Perdiz1, V., Nicolas2 et C., Poüs1,3
1
UMR1193 INSERM Equipe 4 Mécanismes cellulaires et moléculaires de l'adaptation au stress et
cancérogénèse. Faculté de Pharmacie, Université Paris-Saclay
F-92290 Châtenay-Malabry, France
2
Institut Paris Saclay d'Innovation Thérapeutique (IPSIT). UMS IPSIT Université Paris-Sud US 31 INSERM - UMS 3679 CNRS. Plate-forme d'imagerie cellulaire – MIPSIT
Les mitochondries sont des organites doués d'une dynamique propre qui, aux
conditions physiologiques, se caractérise par un équilibre entre un phénotype
tubulaire et un phénotype fragmenté. En revanche, lorsque la cellule est exposée à
un stress, cet équilibre peut être rompu en faveur d'une fragmentation (ou fission) du
réseau mitochondrial, de sorte à éliminer les portions de mitochondries
endommagées. Nous avons récemment montré (Perdiz et al. soumis) que les
microtubules (MTs) qui sont notamment impliqués dans le déplacement des
mitochondries, interviennent aussi dans la régulation de la fission mitochondriale
provoquée par un stress cellulaire. Parmi les protéines microtubulaires, CLIP-170 est
une protéine de liaison entre les MTs et des protéines ou complexes protéiques
présents dans le cytoplasme. Cette protéine se localise plus spécifiquement à
l'extrémité + des MTs en croissance. Ces deux propriétés, CLIP pour Cytoplasmic
Linker Protein et +TIP pour + end tracking protein permettent à CLIP-170 de réguler
des interactions réciproques entre les extrémités des MTs en croissances et
certaines protéines ou organites cellulaires.
En associant l'imagerie confocale et l'utilisation de protéines CLIP-170
recombinantes, nous constatons qu'un stress NaCl entraine un arrêt quasi-immédiat
de la progression de CLIP-170 de type "sauvage" à l'extrémité du MT en croissance
et ce contrairement à d'autres +TIPS comme la protéine EB1 (End Binding 1). De
plus, l'expression d'une CLIP-170 tronquée (CLIP-170 H1) qui exprime uniquement le
domaine N-ter de liaison aux MTs abolit cet arrêt, suggérant que le domaine opposé
C-ter de liaison à d'autres protéines est impliqué dans ce mécanisme.
Récemment, il a été montré que CLIP-170 en interagissant avec la formine
mDia1 favorisait l'élongation de l'actine (Henty-Ridilla et al. 2016). Or, la polymérisation
d'un filament d'actine entre la mitochondrie et le réticulum endoplasmique participe à
la fragmentation mitochondriale. De fait, nous avons émis l'hypothèse selon laquelle
CLIP-170 pouvait être impliquée dans la fission mitochondriale. Au cours d'un stress
NaCl, nous observons que CLIP-170 et les mitochondries interagissent et que CLIP170 semble être localisée préférentiellement sur des sites de fission. Nous
confirmons cette observation en réalisant un triple marquage, celui des
mitochondries, de la protéine de fission Drp1, et de CLIP-170.
Perdiz et al. Stress-induced acetylation of microtubule modulates mitochondrial fission. Soumis.
Henty-Ridilla et al. (2016) Accelerated actin filament polymerization from microtubule plus ends.
Science 352, 6288.
49
« Escalader des montagnes » en synthèse organique :
Assemblage biomimétique de la bipléiophylline et de la voacalgine A.
David Lachkar,a Natacha Denizot,b Guillaume Bernadat,a Kadiria Ahamada,a Mehdi A.
Beniddir,a Vincent Dumontet,c Jean-François Gallard,c Régis Guillot,b Karine Leblanc,a Elvis
Otogo N’nang,a Victor Turpin,a Cyrille Kouklovsky,b Erwan Poupon,a Laurent Evanno,a
Guillaume Vincentb
a
Chimie des substances naturelles, BioCIS, Univ. Paris-Sud,
Sud, Université Paris
Paris-Saclay, 92290,
b
Châtenay-Malabry, France. Institut de Chimie Moléculaire et des Matériaux d’Orsay
(ICMMO), Univ. Paris-Sud,
Sud, CNRS, Université Paris-Saclay,
Paris Saclay, UMR 8182, 91405 Orsay CEDEX
c
France. ICSN, Institut de chimie des substances naturelles, CNRS UPR CNRS 2301, Université
Paris-Saclay, 1 avenue de la Terrasse, 91198 Gif-surGif
Yvette CEDEX France
Pôle thématique de rattachement à l'ED « innovation thérapeutique » : chimie
[email protected]
Les alcaloïdes indolomonoterpéniques constituent une classe de molécules rassemblant près de
3000 composés.(1)L’un des plus complexes, la
l bipléiophylline,(2) a été isolé en 2008 des écorces
d’Alstonia angustifolia (2 mg.. kg-1) par l’équipe de Kam.
Ce bis-indole est constitué de deux unités indoliques (pléiocarpamine) séparées par une
plateforme aromatique (de type acide dihydroxybenzoïque). Représentée comme « une
montagne à escalader » par Hanessian(3), cette molécule est à ce jour considérée comme un
incroyable défi synthétique pour le chimiste organicien.
Notre équipe (en collaboration avec l’équipe de Dr. Guillaume Vincent de l’ICMMO) a entrepris
de synthétiser la bipléiophylline. Une méthode de couplage oxydatif entre indo
indoles et
plateformes aromatiques catalysées à l’oxyde d’argent mimant ainsi la voie biosynthétique
postulée a été mise au point.
Sur la base d’une étude méthodologique, confortée par de la modélisation moléculaire (DFT),
toutes les conditions ont pu être réunies
réunies pour réaliser avec succès l’assemblage biomimétique
de la bipléiophylline. Au décours de nos études, nous avons même pu réviser la structure d’une
substance naturelle tout aussi complexe : la voacalgine A.(4)
(1)
(2)
(3)
(4)
Szabỏ, L. F. Molecules 2008, 13,
13 1875-1896.
Kam, T.-S.; Tan, S.-J.; Ng, S.-W.;
W.; Komiyama, K. Org. Lett. 2008, 10, 3749-3752.
Hanessian, S.; Giroux, S.; Merner, B. L. Design and Strategy in Organic Synthesis : From the Chiron Approach to Catalysis
(Wiley-VCH, 2013).
Lachkar, D.; Denizot, N.; Leblanc, K.; Beniddir, M. A.; Dumontet, V.; Gallard, J.-F.;
J. F.; Evanno, L.; Vincent, G.; Kouklovsky, C.;
Poupon, E. et al. publication soumise.
50
INTERPLAY BETWEEN HUMAN PAPILLOMAVIRUS
AND THE CXCR7 RECEPTOR
1
1
2
3
3
Carmen GALLEGO , Agnieszka JARACZ-ROS , Françoise GAUDIN , Amos FUMAGALLI , Phillippe MARIN ,
1
1
Géraldine SCHLECHT-LOUF* and Françoise BACHELERIE*
1
UMR996-Inflammation, Chemokines and Immunopathology, INSERM, Univ Paris-Sud,
Université Paris-Saclay, Clamart, France.
2
UMR996-Inflammation, Chemokines and Immunopathology, INSERM, Univ Paris-Sud, Université Paris-Saclay, Clamart,
France; US31-UMS3679-Plateforme PHIC, Institut Paris-Saclay d'Innovation Thérapeutique (IPSIT), Inserm, CNRS, Univ ParisSud, Université Paris-Saclay, Clamart, France.
3
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, F-34000 Montpellier, France.
* these authors have equally contributed to this work.
[email protected] http://mac-gratuit.fr/site/U996/Presentation.html
Human papillomavirus (HPV) infection can cause benign lesions such as warts but viral persistence can
result in cancer development, especially in immunocompromised individuals, and according to virus subtypes
(high or low risk for cancer development). So far, there is no treatment targeting directly the virus. Therefore, a
better understanding of the host/virus interactions including immune responses and host susceptibility factors
is needed. Previous work of our group suggested an interplay between the CXCL12 chemokine signalling axis
[1]
and HPV, which was observed both at the HPV life cycle and HPV-associated carcinogenesis levels . This
interplay was further supported by studies on the WHIM syndrome, a rare immunodeficiency caused by gain-of
function mutation in the CXCR4 gene, in which patients display a selective susceptibility to HPV infection. Such
[2]
WHIM-associated dysfunctions in the CXCL12/CXCR4 axis lead to an altered cell cycle progression , increased
[3]
[4]
tumorigenic potential of HPV-infected keratinocytes
as well as increased inflammation . These results
support the notion that CXCL12 and receptors have both keratinocyte-intrinsic and immune effects on the
[5]
virus. More recently, CXCR7 (also known as ACKR3) has been described as the second CXCL12 receptor ,
which can signal upon CXCL12 engagement and also modulate CXCR4 signalling via CXCL12 scavenging function.
Our group has shown that HPV oncogene expression leads to CXCR7 and CXCL12 up-regulation, which resulted
[3]
in PI3K/AKT activation in accordance with the reported role of CXCR7 in cell survival and proliferation.
Our aim is to further investigate the mechanisms of the interplay between HPV18 (high risk subtype)
infection and CXCR7 function either as a decoy or signalling receptor. By characterizing intracellular partners of
CXCR4 and CXCR7 using mass spectrometry strategy, our collaborators have identified connexin43 (Cx43), a gap
junction protein, as a selective intracellular partner of CXCR7 (A. Fumagalli & P. Marin, unpublished data). We
have thus assessed Cx43 levels in 3D organotypic cell cultures, the only replicative cell system for this
epitheliotropic virus, and we show a reduced Cx43 expression only when cultures were infected with HPV18.
The relevance of these results with regard to HPV infection and replication are further being investigated
notably through the generation of CXCR7-knockout keratinocyte cell lines by CRISPR-Cas9 methodology.
Furthermore, the role of the CXCL12-signaling axis in host immune responses toward HPV will be assessed in in
vivo mice models upon infection at the skin or mucosal levels followed by mass cytometry (CyTOF) analyses.
References:
[1] Balabanian, K., Lagane, B., Pablos, J.L. et al. Blood, 2005, 105, 2449-2457.
[2] Meuris, F., Carthagena, L., Jaracz-Ros, A., Gaudin, F. et.al. Plos Path., in revision.
[3] Chow, K. Y., Brotin, E., Ben Khalifa, Y., Carthagena, L. et al. Cell Host Microbe., 2010, 8, 523-533.
[4] Meuris, F., Gaudin, F., Aknin, ML., Hémon, P. et.al. J. Invest. Dermatol. 2016, 136(2), 473-480.
[5] Balabanian, K., Lagane, B., Infantino, S., Chow, K.Y. et al. J. Biol. Chem., 2005, 280, 35760-35766.
51
Tubulin-bound septins, microtubules and cell motility: Study in the context of
chemoresistance
Mahasen Saati1,2, Isabelle Cantaloube1, Christian Poüs1,3 and Anita Baillet1
1
Laboratoire de Biochimie et Biologie Cellulaire, INSERM UMR-S 1193, Université Paris Saclay, Faculté de
Pharmacie, Châtenay-Malabry, France
2
Present address : Laboratoire de radiopathologie, UMR 967, CEA, Fontenay-aux-Roses, France
3
Laboratoire de Biochimie-Hormonologie, Hôpital Antoine Béclère, AP-HP, Clamart, France
Pôle thématique de rattachement à l'ED569 : Physiopathologie cellulaire
[email protected]
Acquired resistance to the microtubule (MT)-stabilizing agent Taxol® is a major
obstacle for successful chemotherapy and limits its use as an anticancer drug. We have
evidenced a new mechanism of Taxol® resistance acquired by MDA-MB 231 breast
cancer cells which involves i) MT enrichment in retyrosinated and long-chain
polyglutamylated tubulin and ii) overexpression and relocalization from the actin
cytoskeleton to the MT network of several septins, a family of filamentous GTPases
implicated in cytokinesis and membrane compartmentalization. Altogether, these
modulations enhance CLIP-170 and MCAK recruitment to MTs, which would in turn
compensate Taxol®-mediated inhibition of MT dynamics (Froidevaux-Klipfel et al.,
2015). We also showed that the overexpression of TTL, TTLL5 and 11 together with that
of a panel of septins that comprised the SEPT9_i1 isoform were necessary and sufficient
to make septin filaments localize to MTs, and allowed a variety of sensitive cells (not
only MDA-MB 231 but also Hela, CHO or RPE-1) to partially resist Taxol® (Targa et
al., in preparation).
As a first step for studying tumor progression, we show here that septin filament
coalignment to MTs that we observed in the resistant MDA-MB 231 cells perturbs the
directionally persistent migration and the subcellular localization of paxillin in focal
adhesions. Also, while the overall level of tubulin acetylation did not differ between
Taxol®-sensitive and -resistant MDA-MB 231 cells, resistant cells that migrate in
response to wounding failed to polarize tubulin acetylation on the MTs oriented toward
their leading edge.
These data shed light on the importance of the interplay between septin filaments and
the MT cytoskeleton in cell motility and provide new insights into how post-translational
tubulin modifications and septins may be involved in metastasis, which may represent
potential therapeutic targets to address in the future.
52

Pleiocarpa mutica - Université Paris-Sud

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