11: 30 plenary session animal Pathology
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09:00 – 09:30 WELCOME INTRODUCTION Doctoral School Introduction DNA-Biosanté association presentation 09:30 – 11:30 PLENARY SESSION Animal Pathology 09:30 – 10:15 CIRON Carine – UMR 703 PAnTher - Nantes (FR) Animal model and translational gene therapy for Pompe Disease (Glycogenosis type II) 10:15 – 11:15 Selected short oral presentations (10 minutes + questions) ARIZA Juan Manuel – BIOEPAR - Nantes Assessment of the footbaths contamination by dairy cattle manures under fields conditions. CAMANES Guillaume – BIOEPAR - Nantes Assessing the decrease in prevalence of paratuberculosis in dairy cattle herds by regulating cattle trade movements at a regional scale POIZAT Axelle – BIOEPAR - Nantes Special feature of the French young bulls’ value chain and associated sanitary issue to control bovine respiratory diseases 11:15 – 13:00 COFEE BREAK – VISIT OF EXHIBITION – POSTER SESSION 13:00 – 14:30 LUNCH 
THURSDAY 10 DECEMBER - AFTERNOON 14:30 – 16:00 PLENARY SESSION Physiology / Physiopathology 14:00 – 14:45 LAUZIER Benjamin – Institut du Thorax - Nantes (FR) O-GlcNAc and biology, friend or foe ? 14:45 – 15:45 Selected short oral presentations (10 minutes + questions)
Lethal arrhythmias, Ca2+ handling defects and cytoskeleton disorganization are associated with dilated cardiomyopathy in a new model of type 3 long QT syndrome: Scn5a+/ΔQKP1510-1512 mice. DILASSER Florian – Institut du Thorax – UMR 1087 -Nantes Essential role of smooth muscle Rac1 in bronchoconstriction and asthma-associated hyperresponsiveness HERIVEAUX Anais – GEIHP, UPRES EA 3142– Angers Hybrid histidine kinases: role in stress adaptation and pathogenicity in Scedosporium apiospermum 16:00 – 16:45 COFEE BREAK – VISIT OF EXHIBITION – POSTER SESSION 16:45 – 19:00 PLENARY SESSION Scientific open-minding 16:45 – 17:30 DINAChristian – Institut du Thorax - Nantes Genetic epidemiology to understand human pathologies 17:30 – 19:00 Selected short oral presentations (10 minutes + questions)
Fine-scale population structure in Western France: Loire River as genetic barrier COGNE Benjamin – Service de génétique médicale - CHU de Nantes A new progeroid syndrome caused by a mutation in vimentin? MARCHAND Jérémy – LABERCA, Oniris – Nantes Combining NMR and MS fingerprinting for fine characterisation of lipid profiles. Application to chemical food safety issues PARCOURET Simon – UMR INSERM 1089 – Angers A Rapid and Robust Identity and Homogeneity Assay for AAV Preparations RMAIDI Assia – UMR INSERM 1066 – Angers – UMR 6283 – Le Mans Synthetic vectors and mechanical-biological approach to optimize the use of stem cells in neurodegenerative medicine 19:00 – 21:30 DINNER Westotel – La Chapelle sur Erdre 21:30 – 01:00 AFTER-PARTY Animation by DNA BioSanté association
FRIDAY 11 DECEMBER - MORNING 09:15 – 11:15 PLENARY SESSION Immunopathology / Infectious diseases 09:15 – 10:00 HALARY Franck – CRTI INSERM UMR1064 – Nantes (FR) Myeloid DC/virus interplays 10:00 – 11:00 Selected short oral presentations (10 minutes + questions)
Transient antibody targeting of CD45RC to induce transplant tolerance and sustained antigen-specific regulatory T cells MARIN Eros – CRTI, INSERM UMR1064 – Nantes Human autologous tolerogenic dendritic cells impair T-cell proliferation through contact-independent mechanisms DAGNELIE Marie-Ange – CRCNA – UMR 892 - Nantes Deciphering Propionibacterium acnes phylotypes according to body localization in acne patients versus healthy humans. PRASANNA Maruthi – UFIP – Nantes Designing the semisynthetic glyco-vaccines for effective immunization against pneumonia 11:00 – 11:30 COFEE BREAK – VISIT OF EXHIBITION – POSTER SESSION 11:30 – 13:00 PLENARY SESSION Osteoarticular diseases 11:30 – 12:15 Invited speacker 12:15 – 13 :00 Selected short oral presentations(10 minutes + questions) CHICHIRICCO’ Pauline – LIOAD INSERM UMRS 791 – Nantes Photocrosslinked hydrogel for Periodontal Tissue Regeneration DARRIEUTORT- LAFFITE Christelle –INSERM U957 – Nantes Composition of tendinous calcific deposits: a histological and spectroscopic study GLUAIS Maude – LIOAD INSERM UMRS 791 – Nantes Design and evaluation of electrospun structured biomaterial for intervertebral disc regeneration 13:00 – 14:00 LUNCH
FRIDAY 11 DECEMBER - AFTERNOON 14:00 – 15:40 PLENARY SESSION Cancerology 14:00 – 14:45 BIDERE Nicolas – CRCNA – UMR 892 -Nantes Deciphering antigen receptor-mediated NF-kB activation and lymphoma survival. 14:45 – 16:15 Selected short oral presentations (10 minutes + questions) BLONDY Thibault – CRCNA – UMR 892 - Nantes Development and characterization of nanovectors with theranostic properties: Application in HDACi carrier for malignant mesothelioma treatment BOUSSEAU Simon – INSERMU1083, INSERM U1063 – Angers Phostine implication in angiogenesis, mitochondrial functions and associated pathologies CLEMEN- COLMOU Karen – CRCNA – UMR 892 - Nantes Tumor vascular remodeling is modified according with radiotherapy dose and fractionation HALA Estephan – CRCNA – UMR 892 - Nantes Improving the response of breast cancers to radiotherapy by modulating the p38MAPK / Hsp27 pathway following ceramide secretion by endothelial cells ORVAIN Corentin – CRCNA – UMR 892 - Nantes GATA2 Expression Level in Chronic Myeloid Leukemia (CML) Patients Correlates with Their Prognostic Scores and Is Associated with Disease Stage at Diagnosis 16:15 – 16:45 COFEE BREAK – BEST PRESENTATIONS AND POSTERS AWARDS 16:45 – 17:00 MEETING CLOSURE 17:00 RETURN TO NANTES OR ANGERS ORAL COMMUNICATIONS ARIZA JUAN MANUEL 2nd year PhD student – BIOEPAR Assessment of the footbaths contamination by dairy cattle manures under fields conditions. JM Ariza*, N. Bareille, K. Oberle, A. Gaugain, R. Guatteo Objectives Infectious diseases, such as Bovine digital dermatitis (BDD), became one of the most important causes of lameness in dairy cattle. Among medical actions, footbaths represent a useful alternative to treat concomitantly an important numbers of animals for a given period. Nevertheless, the active substances used in footbaths can be challenged against different amounts of organic matter (OM), temperatures and/or pH changes, which could alter their bactericidal activity. The aim of our experiment was to determine under field conditions the variations of the temperatures, pH, and OM amounts in footbaths challenged against different numbers of cows which walk through them. Materials and methods The experiment was carried in 5 dairy cattle farms from western France. The herds had to be (i) in zero grazing system to avoid natural cleaning of the feet on pasture, (ii) be housed in cubicles where the cleanliness of the feet is expected to be worse than in strawyard housing system, and finally, (iii) the herd size was limited to a minimum of 100 cows milked twice a day, to homogenize the sampling period to 24 hours. The hygienic status of the farms was determined by the overall feet hygienic score of the farm based on a specific grid focusing on the feet. A footbath filled with 250 liters of water was placed at the parlour exit for 24 hours. The temperature was continuously monitored through a capture device located in the footbath and in the barn (to monitor ambient temperature). After an homogenization of the footbath content with a shovel, 3 samples (1L/sample) were taken throughout the footbath at different places after the cow passages, to analyze the evolution of OM content and pH. Additionally, the number of cows that defecated into the footbaths was recorded. Results Preliminary results indicate that the OM content rise together with the increased number of animals that walk through, and mostly, by the number of defecations into the footbath, varying from 0.086 g/L to 40.3 g/L. The temperature measured in the footbath varies across the day from 12.5 °C to 18°C with less than 2% of variation in relation to the ambient temperature. Finally, the pH of the footbath varied within the 24 hours period from 6.70 to 7.76. After the first defecation the pH increased from 6.70 to 7.48, and then the measures varied in-between 7.32 to 7.76. The mean number of animals that had defecated into the footbaths rose to 10% after 200 cows passages. We are currently performing the data analysis in order to provide practical advices such as renewal frequencies or concentration of the product according to the hygienic status of the farm. Conclusion The results of the present finding support the need for further studies to test the effectiveness of disinfectant in presence of different OM content or pH conditions to adapt, if necessary, the concentration of the product in the footbath or the frequency of renewal. BLONDY THIBAUT 2nd year PhD student - INSERM U892-CNRS 6299 CRCNA équipe 4 Development and characterization of nanovectors with theranostic properties: Application in HDACi carrier for malignant mesothelioma treatment Thibaut Blondy†*, Camille Linot†, Julien Poly‡, Lénaïc Lartigue§, Boucard Johanna§, Steven Nedellec , Philippe Hulin , Nadège Marec†, Eléna Ishow § and Christophe Blanquart,† †IRS UN, INSERM UMR 892 – CNRS UMR 6299, CRCNA, 8 quai Moncousu - BP 70721 - 44007 Nantes cedex. ‡ISM–UMR CNRS 7361, Université de Haute-Alsace, 15 rue Jean Starcky, 68057 Mulhouse, France. §CEISAM–UMR CNRS 6230, Université de Nantes, 2 rue de la Houssinière, 44322 Nantes, France. INSERM UMS 016-UMS CNRS 3556, 8 quai Moncousu, 44007 Nantes, France. Once considered as a rare type of cancer, malignant pleural mesothelioma (MPM) must now be seen as a major public health interest among other cancer types. It is a very aggressive form of cancer, arising from its poor diagnosis, prognosis but mostly its lack of effective treatment. It raises the necessity to develop new therapeutic approaches. In the past decades, drug delivery systems (DDS) for active and passive targeting strategies have been highly investigated to improve the clinical efficacy of drugs. Indeed, the defective angiogenesis occurring in tumours allows molecules of a nanoparticle size to accumulate in the tumor tissue, by means of the Enhanced Permeability and Retention (EPR) effect. Since few years, histone deacetylase inhibitors (HDACi) appear as promising drugs regarding their anti-tumor effects on numerous cancer cell lines. However, their clinical evaluation showed their limits such as short plasma half-life, weak diffusion in tumor tissue and toxicity. To overcome these defects, the laboratory previously developed a nanovector for intra-tumour HDACi delivery with success. However, it was not possible to follow the vector distribution in vivo without adding imaging probes. Our new strategy is based on the developement of nanovectors (NVs) which own fluorescent and superparamagnetic properties for multimodality imaging. Fluorescent signal is useful for cellular studies. The magnetic properties allow imaging using magnetic resonance imaging (MRI). Nude NVs and pegylated nanovectors, to exploit EPR effect, were produced for further study on in vivo behaviour and tumour accumulation. NVs (100-150 nms) are prepared by mixing fluorescent compound (FluoMag) nanohaste in an iron oxyde nanoparticle solution. The obtained structure is composed of a fluorescent organic heart surrounded by iron oxyde nanoparticles. A polymer named PAA is engrafted around the fluorescent heart to maintain the colloidal stability of the NVs. These NVs are named PAA. To improve circulation time and then, stealth property, various polyethylene-glycols (PEG) are added on the NV’s surface. The pegylated NVs are called PAA-PEG, JP09 and JP10. NV characterization was performed on mesothelioma and lung cancer cell lines, from our biocollection, established from patient pleural effusions. NVs endocytosis was confirmed in several mesothelioma and lung cancer cell lines by using flow cytometry. The pegylated form JP10 seemed to be more internalized than the others in malignant mesothelioma cell lines. Co-localisation between the NVs and the lysosomal compartments was demonstrated by using Stream X cytometer and confocal microscopy. NVs degradation in cells was demonstrated by flow cytometry. These results show that our NVs could be suitable for drug delivery in mesothelioma and lung cancer cells. In vivo studies will be performed to evaluate toxicity and biodistribution of the NVs in order to validate or not their use as drug delivery system. Keywords: theranostics nanovectors, mésothélioma , fluorescence , biodistribution , lysosomal compartment BOUCAULT LAETITIA 2nd year PhD student – CRTI, INSERM UMR 1064 Transient antibody targeting of CD45RC to induce transplant tolerance and sustained antigen-specific regulatory T cells Laetitia Boucault1*, Séverine Bézie1*, Elodie Picarda1*, Elodie Autrusseau*, Bernard Martinet*, Véronique Daguin*, Ignacio Anegon2* and Carole Guillonneau2* *INSERM UMR1064 – Center for Research in Transplantation and Immunology-ITUN, Centre Hospitalier Universitaire Nantes, Faculté de Médecine, Université de Nantes, 30 Bd Jean Monnet, 44093, NANTES Cedex 01, France; 1 E.P., S.B. and L.B. contributed equally to this report 2 both senior and corresponding author: Dr. Carole Guillonneau and Dr. Ignacio Anegon, INSERM UMR1064 – Center for Research in Transplantation and Immunology-ITUN, 30 Bd Jean Monnet, 44093, Nantes Cedex 01, France. Currently, the essential goal in transplantation is to develop therapy with specific immunosuppression to induce allograft tolerance without lifelong immunosuppression which induces important side effects. The CD45RC molecule expressed on mononuclear lymphocytes can be used to define two different subpopulations among T cells. Effector T cells express high level of CD45RC (CD45RChigh) while regulatory T cells express low level of CD45RC (CD45RClow). We have previously described in rats that CD40Ig treatment prolongs allograft survival through the induction of regulatory CD8+CD45RClow (Guillonneau, JCI, 2007). We hypothesize that targeting the CD45RC isoform with a short term anti-CD45RC MAb treatment could eliminate CD45RChigh effector cells, enrich in CD45RClow regulatory T cells and thus induce tolerance in transplantation. Rat and human CD4+ and CD8+ Tregs expressing low levels of CD45RC have strong immunoregulatory properties. CD45RC is not expressed by CD4+ and CD8+Foxp3+ Tregs, while CD45RA/RB/RO are. We evaluated the effect of a short term (10) treatment with an anti-CD45RC mAb in a heart allograft rat model and observed allograft tolerance in 80% of anti-CD45RC treated recipents (n=6). This treatment induced a transient decrease of both CD4+ and CD8+ CD45RChigh T cells (95% at day 4) through apoptosis mediated cell death. At the day 10, we observed in turn a transient 5 fold increase of the absolute number of both CD4+ and CD8+ CD45RClow Tregs. We also demonstrated the total absence of IgG humoral anti-donor immune responses in anti-CD45RC treated rats (n=3), while, primary and memory immune responses against exogenous antigens were not affected (n=3), demonstrating that anti-CD45RC MAb treatment induces a specific immunosuppression. In addition, we demonstrated that CD45RClow CD4+ and CD8+ Tregs from long-surviving anti-CD45RC-treated recipients were more suppressive in vitro and in vivo compared to those obtained from naive rats (n=4). Finally, we tested an anti-human CD45RC MAb in a GVHD model of human PBMCs infusion in humanized mice and demonstrated through cell sorting and co-transfer of CD45RClow cells or direct administration of the MAb into NSG mice that this protocol prevented GVHD occurrence in most of the mice (n=6). Our results highlighted the potential of anti-CD45RC MAb as a new innovative therapy in transplantation to induce specific immune tolerance without any other treatment. BOUSSEAU SIMON 2nd year PhD student INSERM U1063 - Stress Oxydant et Pathologies Métaboliques INSERM U1083 - Laboratoire de Biologie Neurovasculaire et Mitochondriale Intégrée Phostine implication in angiogenesis, mitochondrial functions and associated pathologies Bousseau Simon1,2,*, Lenaers Guy1, Andriantsiohaina Ramaroson2 1, INSERM U1083 - Laboratoire de Biologie Neurovasculaire et Mitochondriale Intégrée 2, INSERM U1063 - Stress Oxydant et Pathologies Métaboliques Phostines are a family of synthetic compounds analogues of hexo-pyranoses, exhibiting anti-cancer properties. In particular, the lead compound Pst3.1a has anti-glioblastoma properties both in vitro and in vivo. The objective of the present study is to assess the effect of phostine 3.1a on angiogenesis and on mitochondrial functions. Angiogenesis is a complex process characterized by the early degradation of extracellular matrix, followed by migration and proliferation of endothelial cells and the maturation of the new blood vessel in response to local pro-angiogenic factors such as vascular endothelial growth factor (VEGF) (Lavaud et al, 2012). Angiogenesis takes part in various pathological conditions such as tumor growth, diabetic retinopathy, rheumatoid arthritis, and ischemic diseases. Mitochondria is the organelle responsible for the production of adenosine triphosphate (ATP), providing energy for all physiological processes, among which angiogenesis. Primary results showed an inhibition of different steps leading to angiogenesis including migration, proliferation, adhesion and tube formation. They highlighted the implication of the VEGF type-2 receptor and the focal adhesion kinase FAK. Pst3.1a treatment does not affect mitochondrial function, such as biogenesis and respiration. Nevertheless Pst3.1a treatment inhibits a glycolytic protein, PFKFB3, reported to be one of the key component in the regulation of glycolysis and angiogenesis. These results provide information on an innovative approach to target angiogenesis related diseases including cancer. Key words : Phostine, angiogenesis, mitochondria, cancer growth CAMANES GUILLAUME 2nd year PhD student – Laboratoire BioEpAR Assessing the decrease in prevalence of paratuberculosis in dairy cattle herds by regulating cattle trade movements at a regional scale Camanes Guillaume1,2*, Alain Joly1, Pauline Ezanno2 1 – Groupement de Défense Sanitaire de Bretagne, 56019 Vannes, France 2 – BioEpAR INRA, ONIRIS, 44307 Nantes, France Paratuberculosis is an incurable disease of ruminants reported worldwide. It is caused by Mycobacterium avium subsp. paratuberculosis (Map) which is highly resistant in the environment and causes intestinal lesions. The disease is hard to detect using routine tools, the apparent prevalence being depicted as an ‘iceberg effect”. This disease causes a significant economic impact with a decreased milk production, animal weight losses, and the early culling of animals. Animal health services (AHS) from Brittany aim at helping cattle farmers to manage the disease and to find sustainable solutions to prevent the regional Map spread. At herd scale, the best way to prevent new infections is to protect young animals from adult shedders and to quickly remove heavy shedders. At between-herd scale, animal trade movements play a key role in Map spread. Therefore, AHS aim to assess new solutions based on managing regional trade movements. The objective of my PhD is to assess the efficacy of regulating cattle trade movements as a complementary measure to current ones, to reduce the regional Map prevalence, with application to Brittany. Using a modeling approach, I will evaluate a large range of epidemiological situations and management options. First, I am determining which control measures are required to prevent prevalence degradation at herd scale for various levels of prevalence. Second, I will define realistic rules to regulate trade movements among herds having contrasted within-herd prevalence, using a new between-herd epidemiological model calibrated on cattle movement and detention data available in Brittany. Third, I will assess strategies combining current measures and trade rules based on their ability to decrease the proportion of infected herds at a regional scale. CHIZELLE FRANCK 2nd year PhD student - Institut du thorax, Inserm UMR S1087 CNRS UMR6291 Lethal arrhythmias, Ca2+ handling defects and cytoskeleton disorganization are associated with dilated cardiomyopathy in a new model of type 3 long QT syndrome: Scn5a+/ΔQKP1510-1512 mice. Franck Chizelle1*, Jérôme Montnach1, Nadjet Belbachir1, Linwei Li2, Agnès Carcouët1, Jean-Pierre Benitah2, Ana Maria Gómez2, Isabelle Baró1, Flavien Charpentier1 1 Inserm UMR S1087, CNRS UMR 6291, l’institut du thorax, Nantes, France 2 Inserm UMR S1180, Paris Sud, Université Paris Saclay, Châtenay-Malabry, France INTRODUCTION: Type 3 long QT syndrome is a hereditary cardiac electrical disease causing syncope and sudden cardiac death due to gain-of-function mutations in SCN5A gene product, the main cardiac sodium channel Nav1.5. Interestingly, among known mutations, deletion of QKP1507-1509 amino acids was shown to induce not only a long QT syndrome but also a dilated cardiomyopathy (DCM). METHODS AND RESULTS: To identify the mechanisms of DCM observed in mutation carriers, we generated a knock-in model expressing the equivalent ΔQKP1510-1512 mutation: Scn5a+/ΔQKP mice. We first demonstrated that these mice exhibit a phenotype similar to the patients’ one, i.e., long QT syndrome associated with ventricular arrhythmias, DCM and high incidence of premature death. Calcium homeostasis and expression of key proteins regulating calcium cycle were investigated in 4-week-old mice. Scn5a+/ΔQKP mice exhibited enhanced sarcoplasmic reticulum Ca2+ load combined with intracellular Ca2+ transients with higher amplitude and prolonged time to peak and decay time. SERCA2 expression was not altered. However, total phospholamban expression was increased whereas the amount of calcium-calmodulin kinase II-dependent Thr17-phosphorylated form was decreased. This was associated with a lower activity of CaMKII and decreased expression of calmodulin and calcineurin. In addition, Scn5a+/ΔQKP cardiomyocytes showed a higher frequency of spontaneous Ca2+ waves. Because structural protein α-actinin 2 interacts with Nav1.5 channel in a domain close to the QKP deletion, potential impacts on the cytoskeleton organization have also been investigated. We observed that both α-actinin 2 and t-tubule networks were disorganized in Scn5a+/ΔQKP mice. CONCLUSION: Scn5a+/ΔQKP mice represent an appropriate model to study the origin of DCM in a context of type 3 long QT syndrome. Combined with arrhythmias, disturbances in both calcium cycle and cytoskeleton organization might be at the origin of DCM. Acute and chronic treatments are in progress in order to suppress electrical and/or structural cardiac phenotype in this model. Key words: long QT syndrome - Nav1.5 channel - calcium cycle - dilated cardiomyopathy - cytoskeleton disorganization. CHICHIRICCO’ PAULINE MARIE 2nd year PhD student - LIOAD INSERM UMRS 791 Photocrosslinked hydrogel for Periodontal Tissue Regeneration Pauline M.Chichiricco’*b, Raphael Rivaa, Jean-Michel Thomassina, Catherine Le Visageb, Xavier Struilloub, Pierre Weissb, Christine Jérômea*. aCenter for Education and Research on Macromolecules (CERM) Chemistry Department B6, University of Liège, Sart-Tilman, B-4000 Liège, Belgium bLaboratoire d'Ingénierie Ostéo-Articulaire et Dentaire (LIOAD-INSERM UMR_S 791), UFR d'Odontologie, Université de Nantes , 1 Place Alexis Ricordeau 44042, Nantes, France pauline.chichiricco@univ-nantes.fr Periodontitis is a set of inflammatory diseases resulting from the presence of oral bacteria biofilm in periodontal tissue, which provokes an immune-inflammatory response and destroys the tooth-supporting attachment apparatus. Left untreated, it can lead to reduce the alveolar bone level, and finally tooth loss. When periodontitis are not too advanced, a restoration of lost supporting tissue can be attempted by Guided Tissue Regeneration in infrabony defects. This procedure was developed to ensure that regenerative cells selectively repopulate the periodontal wound area using a physical barrier to exclude the unwanted re-growth of gingival epithelium and connective tissue cells. In this study, a new material applicable as a membrane in periodontal disease is developed. The purpose is to formulate a viscous solution that can be applied directly in the periodontal wound and quickly cured in situ under light irradiation. In order to avoid a second surgical intervention on the patient, the membrane is based on degradable polymer. Modified chitosan, namely carboxymethyl chitosan (CMCS), was selected as polymer precursor. Indeed, this biocompatible and biodegradable polysaccharide is water-soluble and is naturally antibacterial. CMCS was modified with Glycidil Methacrylate in order to graft on the backbone a photo sensible function. The IR and NMR analysis confirmed the chemical grafting on the polysaccharide chain at different percentage. Different polymer formulations were irradiated using a photointiator system based on vitamin B2. Gel point and the mechanical properties were analysed by rheology. A gel was obtained in less than 1 minute. These preliminary results are quite promising for the development of novel hydrogel systems for biomedical uses. Keywords: Periodontal disease, hydrogel, photocross-linkable CLEMENT-COLMOU KAREN 2nd year PhD student - Centre de Cancerologie de Nantes-Angers / Inserm U892 Tumor vascular remodeling is modified according with radiotherapy dose and fractionation Karen Clement-Colmou*, CRCNA, Nantes. karen.colmou@etu.univ-nantes.fr Vincent Potiron, CRCNA / ICO, Nantes François Paris, CRCNA, Nantes Stéphane Supiot, CRCNA, Nantes Tumor vasculature is abnormal and irregular, with low quality blood diffusion and large hypoxic zones. In these hypoxic zones, cancer cells are more aggressive and more resistant to anticancer treatments. Radiotherapy administered in daily fraction of 2 grays (classical clinical fractionation scheme) modifies quality of tumor vasculature, with better perfusion and lower hypoxia on prostatic tumor models. The aim of this study was to characterize influence of different hypofractionated radiotherapy schemes, which are increasingly used in clinical practice, on tumor vasculature. Human prostatic cancer cells (PC3) were subcutaneously injected to NMRI nude mice and grew until forming solid measurable tumors. Mice were divided into 5 groups receiving different normo or hypofractionated radiotherapy schemes during 2 weeks. The radiotherapy schemes were : 10 fractions of 2 Gy, 5 times a week / 6 fraction of 4 Gy, 3 times a week / 3 fractions of 8 Gy, 2 times a week/ 2 fractions of 12 Gy, 1 time a week / no radiotherapy (control group). After irradiation, tumors were collected and their vasculature was immunohistologically studied and compared. Ten mice of each group were used to follow tumor regrowth after radiotherapy. Despite microvascular density remained stable in all the groups, vascular hoechst perfusion significantly increased with the dose per fraction (12Gy>8Gy>4Gy>2Gy>control, p<0.001). Comparing to control, hypoxia was reduced in all radiotherapy groups, with no hierarchy (p=0.003). Tumor regrowth was more delayed in the 12 Gy group comparing to other fractionation schemes (p=0.047), and tumor regrowth delay was significantly longer in radiotherapy groups comparing to control (p<0.001). These results implicate that there is a tumor vascular remodeling after radiotherapy, which depends on the fractionation scheme. Twelve gray fractions seem to be the most effective to reduce hypoxia and improve response to anticancer treatment. Quality of vascular network and drug delivery after radiotherapy is the next research object of this phD Thesis. keywords : tumor vasculature, radiotherapy, hypoxia, hypofractionated radiotherapy, prostate cancer COGNE BENJAMIN 2nd year PhD student – Service de génétique médicale, CHU de Nantes A new progeroid syndrome caused by a mutation in vimentin? B. Cogné*, S. Küry, X. Latypova, W. Deb, M. Nizon, M. Vincent, T. Besnard, S. Mercier, B. Isidor, S. Bézieau Service de génétique médicale, CHU de Nantes Whole-exome sequencing (WES) allows the analysis of all coding sequences of the human genome. During past years, WES has enhanced the identification of new genes causing Mendelian diseases. During the analysis by WES of a patient with a progeroid syndrome, we identified a de novo missense variant in VIM (p.L387P). The patient, born in 1978, suffered from neonatal frontonasal dysplasia, hypertelorism and oligodontia, and successively appeared a severe scoliosis, a peripheral neuropathy, a progressive bilateral deafness, a lipodystrophy, an amyotrophy and strokes, altogether evocating a progeroid syndrome. VIM encodes for vimentin, a type III intermediate filament (IF) protein expressed in mesenchymal cells. IFs, along with microtubules and actin microfilaments, compose the cytoskeleton of metazoan cells. IFs share a common structure: a central alpha-helical rod domain flanked by variable head and tail non-helical domains. Vimentin monomers have a strong tendency to dimerize, forming an alpha-helical coiled coil by their rod domains, then the dimers assemble into tetramers, unit length filaments and 10nm filaments. Several genetic disorders have been linked to mutations in IFs, notably in lamins responsible for Hutchinson-Gilford progeria and other progeroid syndromes, but for vimentin, only one patient has been published with a congenital cataract caused by a mutation in 1A alpha-helical domain (p.E151K) inducing the aggregation of vimetin. Our missense variant substitutes a highly conserved Leucine in the 2A alpha-helical domain by a Proline, known to disrupt helix by its physical properties. Of note, the same substitution in desmin induces the aggregation of desmin filaments and a myopathy. We performed vimentin staining by immunofluorescence on patient’s cells and we observed a misdistribution of vimentin network in cytoplasm. Other functional studies are ongoing to ascertain that vimentin mutations are responsible for a new progeroid syndrome. Beyond the genetic syndrome, it opens a more general question about the role of vimentin IFs in aging. DAGNELIE MARIE-ANGE 2nd year PhD student - Laboratoire d’immuno-dermatologie - U892 Deciphering Propionibacterium acnes phylotypes according to body localization in acne patients versus healthy humans MA Dagnelie*1, S. Corvec1,2, M. Saint-Jean1,3, A. Khammari1,3, and B. Dréno1,3 1 French National Institute of Health and Medical Research (INSERM U892)/National Center for Scientific Research (CNRS U6299), Team 2, Nantes, France 2 Bacteriology and Hygiene Unit, Biology Institute, Nantes University Hospital, Nantes, France 3 Oncodermatology Department, Nantes University Hospital, Nantes, France Introduction Propionibacterium acnes (PA) is a major member of the human normal skin flora. Many studies highlight the differences of cutaneous flora according to body localization. Several papers revealed the importance of PA phylotypes (IA, IB, IC, II and III) in skin diseases. However, the link between PA and acne physiopathology needs further investigations to be completely understood. Acne is a common skin disease characterized by inflammatory lesions on the face, the back, and the trunk. Interestingly, PA phylotypes distribution among the human body has never been investigated before. Objectives In this first phase of the project, we investigated which PA phylotypes were found according to body localization (back and face) in acne versus healthy group. Material and methods Patients (n=24) and healthy volunteers (n=12) were selected according to medical criteria. Cutaneous floras were sampled from each group on back and face. After culture, PA strains were isolated and identified by Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight method. In total, we collected 68 PA strains and typed them according to Single Locus Sequence-based Typing (SLST) or Multilocus Sequence Typing method (MLST). Results The main preliminary results are : (i) a higher diversity of PA phylotypes on face vs back zone, in both groups ; (ii) phylotypes IA1 and II predominantly found in healthy group (respectively 39 % and 44 %) ; (iii) a large predominance of phylotype IA1 in acne group (82 %), especially on back zone (96 %). Discussion The predominance of PA IA1 phylotype in acne group is consistent with the lieterature. Our data show that PA phylotypes are differently distributed among human body. Indeed, in both groups, less than 60 % of subjects carry the same phylotype on back and on face. This observation can be related to daily hygiene cares, cosmetics and environment. Indeed, many factors can impact on face/back skin microbiome such as sweating, moisturizing creams, and UV radiations. Furthermore, we found lower phylotype diversity in acne patients, which could play a role in abnormal activation of immune system in these patients. DARRIEUTORT-LAFFITE CHRISTELLE 2nd year PhD student - Laboratoire de physiopathologie de la résorption osseuse, INSERM U957 Composition of tendinous calcific deposits: a histological and spectroscopic study Christelle Darrieutort-Laffite*(1), Aurélie Najm (1), Guy Louarn (2), Pierre Layrolle (1), Frédéric Blanchard (1), Benoit Le Goff (1) (1)Laboratoire de Physiopathologie de la résorption osseuse, Faculté de Médecine, 1 rue Gaston Veil, 44035 Nantes cedex 1, France (2) Institut des Matériaux de Nantes, 2 chemin de la Houssinière, 44300 Nantes, France Introduction: Calcific tendinopathy (CT) of the rotator cuff is a frequent cause of shoulder pain. The pathogenesis of CT remains unclear. The objective of the study was to determine the composition of calcific deposits present within the tendon. Methods: Calcific deposits were collected from patients during a treatment by ultrasound-guided needle puncture. The lavage of the calcification has been performed with saline solution until the aspirate was free of calcific particles. Then, the saline washes were centrifuged, and the calcific fraction was stored at +4°C in 70% Ethanol. Samples were dried and examined by scanning electron microscopy (SEM), Infra-Red Spectroscopy (IRS), and Raman spectroscopy (RS). Calcifying tendons were collected on cadaveric subjects in the anatomy laboratory of the University of Nantes. Samples (N=3) were fixed in formalin 4%, decalcified or not in EDTA and embedded in paraffin. Then, they were stained with hematoxylin and eosin and Von Kossa. To characterize the cells located around the calcification, anti-Runx2 and anti-Sox9 antibodies were used. Results: Fifteen calcific deposit samples have been analyzed by SEM. They contained ellipsoidal objects of size ranging from 3 to 120 µm. IRS and RS analyses of 14 samples showed a poorly crystalline carbonated apatite with no differences of composition between patients. Compared to synthetic hydroxyapatite, we observed additional peaks due to the presence of proteins. This was confirmed by Western Blot analysis. Histological analyzes showed that calcific areas were acellular. We found two different patterns: little calcifications disseminated between tendon fibers and voluminous ones surrounded by a fibrocartilaginous structure. In the peripheral zone of larger calcifications, we observed cells with round nuclei, they expressed Runx2 and Sox9 suggesting their chondrocyte phenotype. Conclusion: Calcific deposits were mainly composed of poorly crystalline carbonated apatite associated with proteins that need to be characterized. Chondrocyte-like cells were identified around the larger deposits; their implication in the development of these deposits has to be investigated. Keywords: calcific tendinopathy, carbonated apatite, chondrocyte metaplasia DILASSER FLORIAN 2nd year PhD student – L’Institut du Thorax INSERM UMR 1087 / CNRS UMR 6291 Essential role of smooth muscle Rac1 in bronchoconstriction and asthma-associated hyperresponsiveness F. Dilasser*, G. André, J. Chesné, F. Braza, A. Magnan, G. Loirand and V. Sauzeau l’institut du thorax, INSERM, CNRS, UNIV Nantes, Nantes, France Introduction. The molecular mechanisms regulating airway smooth muscle cells (aSMC) contraction and proliferation involved in airway hyperresponsiveness (AHR) are still largely unknown. Small GTPases of the Rho family (RhoA, Rac1 and Cdc42) play a central role in smooth muscle functions. Recently, we demonstrated that Rac1 play an essential role in the control of arterial pressure by modulating vascular SMC contraction (André et al., J Am Heart Assoc., 2014). Accordingly, we hypothesized that Rac1 could also be involved in aSMC contraction. Methods and Results. Ex vivo and in vitro analysis of bronchial reactivity shows that the specific SMC deletion of Rac1 (SM-Rac1-KO) in mice prevents the bronchoconstrictor response to KCl and methacholine. Our results demonstrated that the decreased expression or activity of Rac1 in aSMC impairs bronchoconstrictor-induced rise in intracellular Ca2+ concentration through a mechanism involving Rac1-dependant control of PLC activity. Experiments performed in human bronchi and aSMC reveals a similar role of Rac1 in the control of bronchoconstriction in humans. In vivo, Rac1 deficiency has no impact on the respiratory system in basal, physiological condition. However, deletion of SMC Rac1 or nebulization of the Rac inhibitor NSC23766 prevents AHR in murine models of allergic asthma (ovalbumine and house dust mite (HDM)-sensitized mice). Conclusion. These data indicate that (1) Rac1 plays a critical role in aSMC contraction, and (2) its inhibition prevents AHR. Compared to classical bronchodilators, Rac1 inhibition presents the additional advantage to simultaneously induce bronchodilation and decrease pulmonary inflammation. Inhibition of Rac1 activity or expression may represent a novel therapeutic approach for patients with AHR associated to asthma or COPD. Key words: Rac1, airway smooth muscle, asthma, airway hyperresponsiveness, calcium, PLC ESTAPHAN HALA 2nd year PhD student - « Radiobiologie et Ciblage de l'endothélium, Centre de Recherche en Cancérologie Nantes-Angers, UMR Inserm 892 CNRS 6299 » et « Ciblage Thérapeutique Anti-Tumoral, Faculté des Sciences, Université Libanaise » Improving the response of breast cancers to radiotherapy by modulating the p38MAPK / Hsp27 pathway following ceramide secretion by endothelial cells ESTEPHAN H* 1, 2; CORRE I1; HADCHITY E 2 and PARIS F1 1Endothelium Radiobiology and Targeting, UMR Inserm 892, Cancer Research Center, Nantes, France 2Anti‑Tumor Therapeutic Targeting Laboratory, Faculty of Sciences, Lebanese University, Hadath; Purpose: Radiotherapy is considered a mainstay of treatment for breast cancer, the most frequently diagnosed cancer in women. To overpass tumor radiation resistance, better understanding of the molecular and cellular factors involved in the escape of breast tumor to radiation therapy will provide more effective treatment protocols. Recent studies in our laboratory have highlighted the importance of the tumor micro-environment in the response to radiation. The endothelial cells are directly involved in tumor radiation sensitivity. After irradiation, the ceramide generation by these cells regulates the response of tumor cells to radiotherapy, but the mechanism of this connection is not yet elucidated. Two major pathways p38MAPK/Hsp27 and p53/p21 play a role in the modulation of the tumor resistance. The signaling pathway mediated by p38 MAPK promotes the growth of breast tumor cells, resulting in tumor radioresistance. Several studies have shown that inhibition of Hsp27 leads to a tumor sensitization. Objective: The aim of our work is to improve the effectiveness of radiation therapy against breast cancer and thus by the elucidation of the molecular mechanisms mediated by the key molecular players Hsp27/p38MAPK/ceramide, involved in the tumor response to radiotherapy. Results: Our preliminary results showed that the irradiation of endothelial cells HMVEC-L with a single dose of 15 Gy induces the phosphorylation of p38MAPK and Hsp27 at 1 hour post irradiation and of Akt at 2 hours post irradiation. It also induces the expression of p53 and p21 at 6 hours following irradiation. The irradiation of the breast cancer cells MCF7 with a single dose of 15 Gy showed a phosphorylation of Hsp27 at 2 hours post irradiation, and an expression of p53 and p21 at 6 hours. These results demonstrate that irradiation of endothelial cells and tumoral cells induce the activation of p38MAPK/Hsp27 signaling pathway and the expression of p53/p21 implicated in the regulation of cell cycle. Our actual studies consist on the identification of the molecular mechanisms that connect both types of cells after irradiation and the signaling pathways activated following treatment of HMVEC-L and MCF7 with ceramide and irradiation. Keywords: Breast cancer, endothelial cells, radiotherapy, p38MAPK/Hsp27, ceramide. GIEMZA JOANNA 2nd year PhD student - l’institut du thorax, Inserm UMR 1087 / CNRS UMR 6291 Fine-scale population structure in Western France: Loire River as genetic barrier Joanna Giemza1*, Floriane Simonet1, Matilde Karakachoff1, Karen Rouault2, Eric Charpentier1, Simon Lecointe1, Pierre Lindenbaum1, Jade Violleau1, Claude Férec2, Hervé Le Marec1, Stéphanie Chatel1, Serge Hercberg3, Pilar Galan3, Jean-Jacques Schott1, Emmanuelle Génin2, Richard Redon1, Christian Dina1 1. L'institut du thorax, Inserm UMR 1087 / CNRS UMR 6291, IRS - Université de Nantes, Nantes France 2. Inserm UMR 1078, CHRU Brest, University Bretagne Occidentale, EFS, Brest France 3. Université Paris 13, Equipe de Recherche en Epidémiologie Nutritionnelle, Centre de Recherche en Epidémiologie et Statistiques, Inserm (U1153), Inra (U1125), Cnam, COMUE Sorbonne Paris Cité, F-93017, Bobigny, France Background The genetic structure of human populations varies throughout the world, being influenced by migration, admixture, natural selection and genetic drift. Human population structure has first been investigated at broad scales, between and within continents. Currently researchers focus on finer scales, examining genetic structure within countries or regions. Characterising such genetic variation is of interest as it provides insight into demographical history and informs research on disease association studies, especially the ones focusing on rare variants. We here explored the genetic structure of a population living on the whole French territory and then on Western part where interesting stratification was identified. Methods and Results We genotyped 456 individuals from Western France Atlantic Coast, from Finistère to Vendée, with at least three of their grandparents born within a 15 kilometres distance using Axiom CEU Chip. Principal Components analysis revealed that individuals from the same departments form clusters and we observed a high correlation between geographical position and components (p-value < 2e-16). The main geographical barrier in the region is Loire River. Many independent methods support the hypothesis that Loire River is also a genetic barrier. The two groups of individuals, from north or south of Loire, are well differentiated along PC1 axis. ADMIXTURE estimated different ancestry proportions for the two groups. The first split of hierarchical clustering returned by fineSTRUCTURE, and the one based on normalized counts of identity-by-descent segments is between north and south of Loire. Conclusion We here report stratification at the level of continental French territory. The migration pattern is following the geographical structure. A specific pattern is noticed around the Loire River. We confirm both evidence for isolation by distance and existence of a genetic barrier, the Loire River. The discovered fine-scale population structure may have consequences in association analyses, especially for rare variants which tend to be geographically clustered. keywords: population structure, rare variants, PCA GLUAIS MAUDE 2nd year PhD student - Laboratoire d’Ingénierie Ostéo-articulaire et Dentaire LIOAD Design and evaluation of electrospun structured biomaterial for intervertebral disc regeneration Maude Gluais1,2,*, Johann Clouet1,2, Jérôme Guicheux1,2, Catherine Le Visage1,2. 1INSERM,UMR_S791, LIOAD «Skeletal Tissue Engineering and Physiopathology» (STEP) Group, Center for Osteoarticular and Dental Tissue Engineering, Nantes, France. 2 University of Nantes, France Intervertebral disc degeneration and injuries are common causes of low back pain affecting a large percentage of the global population. Current surgical strategies are limited to treating the symptoms and mainly focused on the repair of the gelatinous central part of the disc, the nucleus pulposus (NP), at the expense of the structural integrity of the external circumferential fibrous structure of the disc, the annulus fibrosus (AF). Unrepaired defects in the AF are often associated with postoperative reherniation and further tissue damage and degeneration. Thus, the aim of this study is to design a cell-seeded biomaterial composed of polycaprolactone (PCL) that mimics the oriented fibrous structure of the native AF. PCL biomaterials were produced using electrospinning technology and characterized by scanning electron microscopy (SEM) and uniaxial tensile mechanical analysis. They were then seeded with human adipose stem cells (ASCs) and observed with SEM and confocal microscopy to evaluate cell attachment and proliferation. Oriented and non-oriented scaffolds with tunable fibers diameters and mechanical properties were successfully obtained and promoted cell attachment and proliferation. Moreover, the orientation of fibers seemed to enhance cell alignment in a parallel manner to the underlying scaffold. In this study, we validated the feasibility of using the electrospinning technology in our laboratory to synthesize aligned nano- and micro-sized fibrous scaffold mimicking AF native structure and their use for hASCs cell culture. Their potential application will be further investigated with longer in vitro study and in vivo implantation in a rabbit animal model to assess cell differentiation, extracellular deposition and the regeneration of the annulus fibrosus. Keywords: Intervertebral disc, Annulus Fibrosus, Regeneration, Biomaterial, Electrospinning, Polycaprolactone. HERIVAUX ANAIS 2nd year PhD student – GEIHP, UPRES EA 3142 Hybrid histidine kinases: role in stress adaptation and pathogenicity in Scedosporium apiospermum Anaïs Hérivaux1*, Amandine Gastebois1, Patrick Vandeputte1, Jean-Philippe Bouchara1,2, Jean-Paul Latgé3, Nicolas Papon1 1 Université d'Angers, Groupe d'Etude des Interactions Hôte-Pathogène, EA 3142, Angers, France. 2 Laboratoire de Parasitologie Mycologie, Centre Hospitalier Universitaire, Angers, France. 3Unité des Aspergillus, Institut Pasteur, Paris, France. Scedosporium apiospermum ranks second among filamentous fungi colonizing the respiratory tract of patients with cystic fibrosis (CF) (1). In the airways of these patients, the fungus must face specific environmental conditions (hypoxia, increased carbon dioxide partial pressure, alkaline pH, low osmotic pressure and increased lactate concentration) in the mucus layer. Thus, to enable lung colonization, S. apiospermum must be able to perceive environmental signals and to cope with these constraints. In this project, we will study the role that hybrid histidine kinases (HHK) may play in these adaptive and pathogenic mechanisms (2). Indeed, HHK have been described in bacteria (3, 4, 5) and among some fungi (6) as major proteins implicated in stress adaptation and may constitute excellent targets in the perspective of developing new antimicrobials (7). Thanks to the recent S. apiospermum genome sequencing, six HHK genes have been identified. First, each of these genes will be independently disrupted thanks to genetic tools which we will have to develop beforehand. Then, phenotypic characterization will be carried out in order to determine their potential role in the adaptation to stress or virulence in S. apiospermum Keywords: hybrid histidine kinases, genetic tools, Scedosporium apiospermum References: 1. Cimon B, Carrère J, Vinatier JF, Chazalette JP, Chabasse D, Bouchara JP. Clinical significance of Scedosporium apiospermum in patients with cystic fibrosis. Eur J Clin Microbiol Infect Dis. 2000 Jan;19(1):53-6. PubMed PMID: 10706182 2. Mascher T, Helmann JD, Unden G. Stimulus perception in bacterial signal-transducing histidine kinases. Microbiol Mol Biol Rev. 2006 Dec;70(4):910‑38. Review. PubMed PMID: 17158704; PubMed Central PMCID: PMC1698512. 3. Mizuno T, Wurtzel ET, Inouye M. Osmoregulation of gene expression. II. DNA sequence of the EnvZ gene of the ompB operon of Escherichia coli and characterization of its gene product. J Biol Chem. 1982 Nov 25;257(22):13692‑8. PubMed PMID: 6292200. 4. Rudolph J, Oesterhelt D. Chemotaxis and phototaxis require a CheA histidine kinase in the archaeon Halobacterium salinarium. EMBO J. 1995 Feb 15;14(4):667‑73. PubMed PMID: 7882970; PubMed Central PMCID: PMC398130. 5. Kehoe DM, Grossman AR. Similarity of a chromatic adaptation sensor to phytochrome and ethylene receptors. Science. 1996 Sep 6;273(5280):1409-12. PubMed PMID: 8703080. 6. Ota IM, Varshavsky A. A yeast protein similar to bacterial two-component regulators. Science. 1993 Oct 22;262(5133):566‑9. PubMed PMID: 8211183. 7. Shor E, Chauhan N. A case for two-component signaling systems as antifungal drug targets. PLoS Pathog. 2015 Feb;11(2):e1004632. doi: 10.1371/journal.ppat.1004632. eCollection 2015 Feb. PubMed PMID: 25723524; PubMed Central PMCID: PMC4344368. MARCHAND JEREMY 2nd year PhD student – LABERCA, Oniris, Nantes Combining NMR and MS fingerprinting for fine characterisation of lipid profiles. Application to chemical food safety issues Jérémy Marchand*1, 2, Gaud Dervilly-Pinel1, Yann Guitton1, Estelle Martineau2, 3, Patrick Giraudeau2, 4, Bruno Le Bizec1 1 LABERCA, Route de Gachet, 44307 Nantes, France 2 CEISAM, UMR6230, Université de Nantes, BP 92208, 2 rue de la Houssinière, 44322 Nantes Cedex 3, France 3 Spectromaitrise, CAPACITES SAS, 26 Bd Vincent Gâche, 44200 Nantes, France 4 Institut Universitaire de France, 1 rue Descartes, 75005 Paris Cedex 5, France As leaner meat is expected upon the use of growth promoting agents in livestock, it can be hypothesised that lipid profiles may be disrupted as a consequence of such practice. Although the link between steroids and lipid metabolism has been highlighted decades ago in farm animals, analytical strategies to investigate thoroughly these fine changes did not follow. Technological advances in liquid chromatography, mass spectrometry (MS), Nuclear Magnetic Resonance (NMR) and omics strategies are now well suited to address this issue (Marchand et al., 2017). Recent results demonstrated the relevance of MS-based lipidomics for highlighting biomarkers of anabolic practices in livestock (Kouassi Nzoughet et al., 2015). However, as the precise identification of those compounds remains a bottleneck, a deeper characterisation of the lipidome is needed. To resolve this issue we propose an original lipidomics strategy, combining MS and NMR, applied to the characterisation of the illegal use of a veterinary drug in livestock. This PhD-work involved the development of a suitable sample preparation tailored for the different requirements of NMR and MS, which has been assessed in terms of sensitivity and repeatability. Various NMR and MS experiments were optimised and evaluated for their use in the frame of the lipidomics study. The analytical strategy was subsequently applied to the lipidomic study of ractopamine in pigs, where the serum lipid profiles from two groups of animals treated and control were compared. A suitable multivariate statistical model was then developed. The first results showed the ability of the strategy to differentiate the two sample groups and revealed potential biomarkers of such a growth promoting treatment, that require further investigation in order to determine their precise structure and the metabolic pathways involved. Further work will also involve the full NMR analysis of the same samples and the combination of the data acquired from the different techniques, in order to create a fully combined MS/NMR lipidomics platform. Keywords: Lipidomics, Mass spectrometry, Nuclear Magnetic Resonance, Anabolics, Chemical Food safety. References: Marchand J, Martineau E, Guitton Y, Dervilly-Pinel G, Giraudeau P: Multidimensional NMR approaches towards highly resolved, sensitive and high-throughput quantitative metabolomics. Current Opinion in Biotechnology 2016, 43:49–55 Kouassi-Nzoughet J, Gallart-Ayala H, Biancotto G, Hennig K, Dervilly-Pinel G, Le Bizec B: Hydrophilic interaction (HILIC) and reverse phase liquid chromatography (RPLC)–high resolution MS for characterizing lipids profile disruption in serum of anabolic implanted bovines. Metabolomics 2015, 11(6):1884–1895 MARIN EROS 2nd year PhD student – CRTI, INSERM UMR 1064, Nantes Human autologous tolerogenic dendritic cells impair T-cell proliferation through contact-independent mechanisms Eros Marin1,2,3*, Maria-Cristina Cuturi1,2,3, Aurelie Moreau1,2,3 1.INSERM UMR1064, Center for Research in Transplantation and Immunology, Nantes, France 2.CHU de Nantes, Institut de Transplantation Urologie Nephrologie (ITUN), Nantes, France 3.Université de Nantes, Nantes, France Low dose GM-CSF generated-Autologous tolerogenic dendritic cell (ATDC) has been proved to increase the allograft survival in “in vivo” models in rodents. Insomuch as it has been demonstrated the lack of toxicity in non-human primates, ATDC therapy has been moved to clinic in a phase I/II clinical assay in kidney transplantation. However mechanisms underlying human-ATDCs (hATDC) remain unclear. This study aims to investigate the suppressive mechanisms of hATDC. We demonstrated that hATDCs present an immature phenotype and are maturation-resistant based on the low expression of HLA-DR and co-stimulatory molecules CD80/83/86. Furthermore hATDCs impair T-cell proliferation in mixed lymphocyte reaction (MLR) leading to a decrease of Th1 and Th17 populations and an increase of CD4+CD25+FoxP3+ Treg. In this study we demonstrated that the impairment of T-cell proliferation by hATDCs is driven by soluble factors and not by contact-dependent mechanisms. To support this data we showed that hATDCs display a low expression of inhibitory molecules such as PD-L1 and HLA-G and a high expression of the immunomodulatory cytokine TGF-. Otherwise we belittle the suppressive effect of IL-10 of hATDCs because the production is quite similar to monocyte derived-dendritic cells. In summary, hATDCs impair T-cell proliferation through contact-independent mechanisms. These data suggest an approach to several mechanisms that can explain the tolerogenic role of hATDCs in transplantation. Keywords: Autologous tolerogenic dendritic cells, Transplantation, Tolerance, Cell therapy ORVAIN CORENTIN 2nd year PhD student – Plateforme STE, Angers - INSERM892/CNRS6299 GATA2 Expression Level in Chronic Myeloid Leukemia (CML) Patients Correlates with Their Prognostic Scores and Is Associated with Disease Stage at Diagnosis Corentin Orvain,1,2,3*Nick C.P. Cross,4 Martine Gardembas,3 Marc Spentchian,5 Estelle Pedrono,1 Diane Lambert,1 Anne Coutolleau,1 Anne Bouvier,6 Odile Blanchet,7 Nicole Piard,8 Norbert Ifrah,2,3 Frank Giles,9 Andreas Hochhaus,10 Jerald P. Radich,11 Philippe Rousselot,12 and Philippe Guardiola.1,2,3 1Plateforme STE, University Hospital, Angers, France; 2INSERM892/CNRS6299, University Angers, Angers, France; 3Maladies du Sang, University Hospital, Angers, France; 4Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, United Kingdom;5Service de Biologie Médicale, Hôpital André Mignot, Versailles, France; 6Laboratoire d’Hématologie, University Hospital, Angers,France; 7Centre de Ressources Biologiques, University Hospital, Angers, France; 8EFS Pays de la Loire, Angers, France;9NMDTI, Northwestern University, Chicago, IL; 10Universitätsklinikum Jena, Jena, Germany; 11Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 12Hématologie Clinique, Hôpital André Mignot, Versailles, France. The Sokal, Hasford, and EUTOS scoring systems have been successively proposed to predict the outcome of CML patients in chronic phase (CP-CML), both in terms of disease response and survival. No clear link has been identified between the covariates used to calculate those scores and CML molecular pathophysiology. A gene expression profiling study was performed on 48 CP-CML patients included in the Novartis ENEST1st study. The comparison of patients with low and high-risk Sokal scores lead to the identification of 14 genes differentially expressed between those groups. GATA2, which encodes a critical hematopoietic transcription factor, was among significantly overexpressed in high Sokal score CP-CMLs. Using a quantitative RT-PCR assay, GATA2 expression was subsequently assessed on an independent set of 80 CP-CML patients followed at Angers University Hospital from 2005 to 2014. This validation set confirmed that GATA2 expression level was significantly correlated to the 3 prognostic scores. Among the covariates used in those scores, basophil percentage, platelet count, and peripheral blood blast percentage were significantly correlated to GATA2 expression level. The ENEST1st gene expression dataset and a linear regression approach were then used to better define the role of GATA2 in CP-CMLs. This analysis identified 36 genes significantly correlated to GATA2 in terms of expression levels with half of those being basophil-related genes. To extend this analysis, the gene expression dataset from Radich et al (Proc Natl Acad Sci USA 2006; GEO Microarray, GSE4170), based on 108 peripheral blood samples from patients with CP-CML, accelerated phase (AP)CML, or blast crisis (BC)CML, and confirmed that GATA2 expression level was significantly increased from CP-CMLs to myeloid BC-CMLs whereas it was expressed at the lowest levels in lymphoblastic BC-CMLs. GATA2 is a key gene affecting the outcome of CP-CMLs and the evolution into myeloid BC-CMLs. Its association with a basophil-related gene expression signature might explain the prognostic influence of the basophil percentage within the EUTOS score. Keywords: chronic myeloid leukemia; GATA2. PARCOURET SIMON 2nd year PhD student – Laboratoire de Thérapie Génique (UMR1089) A Rapid and Robust Identity and Homogeneity Assay for AAV Preparations Simon Pacouret*, Mohammed Bouzelha, Rajani Shelke, Eva Andres-Mateos, Ru Xiao, Anna Maurer, Mathieu Mevel, Heikki Turunen, Trisha Barungi, Magalie Penaud-Budloo, Frederic Broucque, Veronique Blouin, Philippe Moullier, Eduard Ayuso and Luk H Vandenberghe Adeno-associated virus (AAV) vectors have emerged as key clinical candidates for gene therapy. Yet, the efficiency and safety of these 20-25 nm biological nanoparticles remain difficult to harmonize across pre-clinical studies due to the limitations of current analytical tools. The presence of residual DNA, protein contaminants, empty particles and VP subunits resulting from incomplete capsid assembly are variables that can strongly modulate the reliability of in vivo data and that, therefore, need to be closely monitored in AAV research laboratories. In this work, 70 AAV preparations, obtained with various production (baculo/Sf9 and triple transfection system) and purification (iodixanol gradient and double cesium-chloride gradient) techniques were analyzed using a thermal shift assay based on the fluorescent dye Sypro® Orange. The fluorescence fingerprint obtained did not only allow to discriminate various AAV serotypes based on their capsid melting temperatures, but also enabled to probe the homogeneity and purity of AAV vector preparations, investigated in parallel using dynamic light scattering (DLS) and polyacrylamide gel electrophoresis. In particular, a double fluorescence transition indicated the presence of capsid-associated protein contaminants whereas a high initial fluorescence background correlated with the presence of free protein contaminants and capsid subunits, possibly resulting from capsid degradation during vector purification or storage. The variability, sensitivity and precision of this assay were further investigated in two different AAV research laboratories. This simple, fast (analysis of 94 preps in ~6 hrs) and low-cost assay emerges as a relevant tool for characterization of AAV vector preparations and will help to increase the reliability of in vivo gene transfer studies. Keywords: AAV vectors, thermostability, idensity assay, quality control PRASANNA MARUTHI 2nd year PhD student - Unité Fonctionnalité et Ingénierie des Protéines, Nantes Designing the semisynthetic glyco-vaccines for effective immunization against pneumonia Maruthi Prasanna*1, Emilie Camberlein1, Noemi Csaba2, Cyrille Grandjean1 1) UFIP, University of Nantes, France 2) CIMUS, University of Santiago de Compostela, Spain Streptococcus pneumoniae is the leading cause of morbidity and mortality, by causing pneumonia, otitis media, meningitis and bacteremia, in both children and adults. Currently available vaccines 23-valent pneumococcal polysaccharide vaccine (PPSV23), the 7-valent pneumococcal conjugate vaccine (PCV7) and the 13-valent pneumococcal conjugate vaccine (PCV13) are not very effective in immunocompromised patients. We aim in preparing semisynthetic conjugate vaccine consisting of a synthetic tetrasaccharide linked to pneumococcal immunogen (Pneumococcal surface adhesion –A (PsaA)). PsaA is a membrane associated metal binding lipoprotein, which can act as both immunogenic and antigen carrier; it is present in all serotypes of pneumococcal bacteria. To our knowledge, PsaA has never been used as an antigen carrier. The selected tetrasaccharide is a synthetic mimic to the capsular polysaccharide specific to Streptococcus pneumoniae serotype-14. Moreover, it is the smallest tetrasaccharide that is capable of inducing opsonophagocytic adhesion. We hypothesize that the combination of the tetrasaccharide and the carrier molecule that is common in all the serotypes may produce synergistic effect, and may give rise to vaccine with improved immunity. Initially the tetrasaccharide was synthesized in the lab. Followed by the expression of PsaA in E coli BL21 strain and purified. Later, the tetrasaccharide was linked to the carrier protein using the linkers S-acetyl thioacetate. The obtained semisynthetic glyco-vaccine will be encapsulated into the suitable mucoadhesive nanocarrier which may enhance the stability of vaccine and can be easily internalized by the antigen presenting cells. Further, the formulations will be evaluated for their ability to evoke the immune response in the mice by administering them intra-nasally. 
Keywords: Glyco-vaccines, Pneumonia, Conjugate vaccines, PsaA POIZAT AXELLE 2nd year PhD student – BIOEPAR Special feature of the French young bulls’ value chain and associated sanitary issue to control bovine respiratory diseases A. Poizat1*, F. Beaugrand1, A. Rault1, C. Fourichon1, N. Bareille1 1 BIOEPAR, INRA, Oniris, La Chantrerie, 44307, Nantes, France Mail address corresponding author: axelle.poizat@oniris-nantes.fr In France, the young bulls sector has a very complex organization where beef calves are raised by a cow-calf producer and then sold to a fattener through numerous middle men. This organization is in favour of an increased incidence of respiratory diseases in fattening units, and thus increased use of antibiotics. No study allows a holistic understanding of the young bulls’ value chain, in France or in other countries. This would lead to a better apprehension of the influence of stakeholders to control bovine respiratory diseases (BRD) and of the possible lever to improve the existing situation. In this research, we aimed at (i) describing the functioning of the French young bulls value chain and the different roles of stakeholders and institutions, (ii) understanding how the present organization influences the risk to develop BRD in fattening units, (iii) observing the existing strategies of stakeholders to control the risk of BRD and discussing some possible alternative methods. We collected information from cow-calf producers, middle men, and fatteners relying on qualitative semi-structured interviews and on observations on field conducted in major beef cattle regions in France. The main findings first confirmed the expected complexity of different existing commercial paths in the value chain and the role of this vertical organization in increasing risks factors for BRD. Second, there was a preventive and quasi systematic use of antibiotics which sharply reduced (i) the potential effects of technical and sanitary interdependency between the cow-calf producer and the fattener, (ii) the risk to develop BRD, when there were an increased number of middle men before fattening. Finally, main opportunities to improve sanitary health status of animals would be to (i) reduce the number of intermediaries and (ii) the number of farms of origin in weanling batches, (iii) increase the use of vaccination by cow-calf producers. Key words : Antibiotic use, sanitary issues, cow-calf producer, fattener, middle man, vertical coordination. RMAIDI ASSIA 2nd year PhD student UMR INSERM 1066, MINT, Angers - UMR CNRS 6283, IMMM, Le Mans Synthetic vectors and mechanical-biological approach to optimize the use of stem cells in neurodegenerative medicine Rmaidi A. 1, 2 *; Boury F. 1; Delorme N. 2; Montero-Menei C,1 1 INSERM UMR-1066, Micro et Nano-Médecines Biomimétiques (MINT), Angers F-49933, France. 2 Institut des Molécules et Matériaux du Mans (IMMM), F-72085 Le Mans, France. A neurodegenerative disease, corresponds to any pathological condition primarily affecting the brain and more generally the central nervous system (CNS), it induces the progressive loss of neurons. Cell therapy is the transplantation of human or animal cells to replace or repair damaged tissue, and a lot off study confirmed that pharmacologically active microcarriers (PAMs) may serve as a support for cells transplanted and used for cells culture because this microcarriers (3D) presenting a controlled delivery of growth factors encapsulated inside for tissue engineering. PAMs providing a biomimetic surface of different extracellular matrix (ECM) molecules enhance cells implanted and stimulate cell survival and differentiation. Many studies confirm that laminin (LM) affect stem cell differentiation toward a neuronal. Our aims in this thesis is to study the effect of the surface properties of the microparticles formed with two types of biodegradable and biocompatible polymers poly(lactic-co-glycolic acid) (PLGA) and PLGA-P188-PLGA prepared by emulsion process (O/W), and functionalized by molecules presented in the extracellular matrix (Laminin (LM) and Poly-D-Lysine (PDL)) on the adhesion and differentiation of MIAMI cells on the surface of PAMs. Chemical and physical particle surface properties (topography, hydrophobicity, charge and mechanical properties) and protein adsorption are therefore important points to study. The particles were characterized in size, charge, topography and morphology. In order to understand and to study the influence of these parameters on the surface/cells interaction it may be interesting to prepare a model 2D similar to microparticules (3D). Results indicate that PLGA and PLGA-P188-PLGA are two polymers completely different. AFM results represent that PLGA-188Poloxamer-PLGA surface is rougher than PLGA for the MS (coated and uncoated) and films. The cells are easily adhered on the PLGA surface and not on PLGA-P188-PLGA these results confirmed by the confocal microscopy because the distribution of LM molecules on PLGA surface is homogenous and not on PLGA-188Poloxamer-PLGA surface. We can confirm that the roughness, distribution of the coating and hydrophobicity of the surface are the major parameters influence the adhesion of cells on the particles surfaces. POSTERS COMMUNICATIONS LEON ALEXIS PhD student 1st year - Ifremer LBCO/LABERCA Screening halogenated contaminants in marine environment based on isotopic pattern and mass defect provided by high resolution mass spectrometry profiling LEON Alexis*, 44311 Cedex 03, Rue de l'Île d'Yeu, 44980 Nantes The Stockholm Convention is a global treaty aiming at protecting human health and the environment from the Persistent Organic Pollutants (POPs). POPs meet 4 criteria: (i) remain intact for long periods of time, (ii) become widely distributed throughout the environment, (iii) accumulate in the fatty tissue of living organisms, and are found at higher concentrations at the top of the food chain and (iv) are toxic to both humans and wildlife. To date, 26 chemicals substances are listed under the Convention and 4 others are under review. A common feature is that all POPs are halogenated compounds (Br, Cl, F). Besides, the increasing regulatory pressure against POPs has led to the emergence of numerous alternative compounds. Research is currently focused on some of these compounds but it is anticipated that many other ones remain unidentified. In order to discover or inventory the increasing number of compounds to monitor, a trend is to use untargeted approaches based on fingerprints obtained by High Resolution Mass Spectrometry. Thus, post-acquisition data treatment became challenging to highlight useful information from the large datasets. The LABERCA has been interested in such approach since 2014 and has laid the foundation of an analytical method by developing automated operating data bioinformatics tools managed via a friendly-user application to identify chromatographic signals based on precise mass and isotopic pattern both relying on halogens specificities (Cariou et al., 2016). The present PhD project (2016-2019) is aiming to further develop this innovative analytical strategy to trace and identify new persistent halogenated contaminants in the marine environment. Passive samplers and sentinel species (fish, mollusk) collected with the IFREMER will be used to investigate halogenated compounds present in related environments. JACQUEMONT LOLA 2nd year PhD student - INSERM UMR 1064, CRTI, Nantes In vivo blockade of IL-7Rα in non-human primates does not alter the T cell response to polyclonal stimulation. Jacquemont L.1,2*, Belarif L.1, Tilly G.1, Poirier N.1, Mai H.1, Vanhove B.1, Blancho G.1,2, Brouard S.1,2, Degauque N.1. 1 Inserm UMR 1064, 30 boulevard Jean Monnet, 44093 Nantes Cedex 1, France 2 CHU de Nantes, ITUN, 30 boulevard Jean Monnet, 44093 Nantes Cedex 1, France Objective. IL-7 is a type 1 cytokine that plays a key role in thymopoiesis and homeostasis of B and T lymphocytes and is critical for generating memory T cells after antigen stimulation in murine models. However, the role of IL-7 is not well defined in humans. In this work, we investigate the consequences of IL-7Rα blockade on T cells in a model of delayed type hypersensitivity in non-human primates (NHP). Methodology. NHP vaccinated with BCG and with a positive tuberculin skin test received a treatment with an antibody anti-IL-7Rα (CD127) at 10mg/kg at day 0. They were also challenged monthly with an intradermal injection of tuberculin. PBMC were collected at days -30, 0, 7, 14, 30 and 60 and the phenotype of T cells subsets was analyzed according to the expression of CD45RA and CCR7. Expression of activation markers (CD25 and CD69) by T cells was measured after 48h of polyclonal stimulation. Oxygen Consumption Rate and ExtraCellular Acidification Rate of T cells were monitored with a Seahorse XF24 Analyzer. Finally, glucose uptake by T cells was analyzed using a fluorescent form of glucose (2NBDG). Results. Single injection of IL-7Rα blockade induces a long lasting decrease in erythema after intradermal injection of tuberculin in the absence of lymphopenia or any modification of T cells subsets. Furthermore, the immunometabolic properties of T cells are not changed after treatment with anti-CD127 both at steady state and after polyclonal stimulation. Finally, the blockade of IL-7 pathway does not modify the expression of activation markers such as CD25 and CD69 neither at steady state nor after ex vivo stimulation with aCD3 alone or with IL-2 or IL-7. Collectively, we show that a single injection of anti-IL-7Rα mAb in NHP blunts the tuberculin recall memory response without lymphopenia and preserves the immuno-metabolic properties of T cell to polyclonal stimulation. FARAJ SEBASTIEN 2nd year PhD student - CRCNA, UMR INSERM U. 892 – CNRS 6299 Immunotherapy for neuroblastic tumors targeting O-acetylated GD2 Sébastien Faraj*, Marc-David Leclair, Stéphane Birklé 7 quai Moncousu - 44000 Nantes Cedex1 Despite recent advances in the treatment of high risk neuroblastic tumors, patients prognosis still remain pejorative. Treatment modalities are aggressive and many children suffer from side effects, degrading their quality of life. Anti-GD2 immunotherapy offers in this context an alternative therapy, which improves the prognosis of these tumors, but its use is nevertheless limited by the presence of a toxicity of the existing molecules. The O-acetylated GD2 is not expressed in the surface of peripheral nerve fibers. Choosing it as a target therapy can be effective while reducing toxicity immunotherapy. Here we describe the different mechanisms of specific antibodies targeting the O-acetylated GD2 leading to destruction of neuroblastic tumor cells. Keywords : Immunotherapy, neuroblastic tumors, O-acetylated GD2 LERUEZ STEPHANIE 2nd year PhD student - Biologie Neurovasculaire et Mitochondrie Integré UMR INSERM 1083 Mitochondrial haplotypes as biomarkers in primary open angle glaucoma Stéphanie Leruez* (1,2), Pascal Reynier (2), Dan Miléa (1,3), Vincent Procaccio (2) (1) Département de Neurosciences, service d’Ophtalmologie, CHU Angers, Angers, France (2) Institut MITOVASC, CNRS UMR 6214, Université d’Angers, Angers, France (3) Singapore Eye Research Insitute, Singapore National Eye Centre and Duke-NUS Primary open angle glaucoma (POAG) is one of the leading causes of irreversible blindness worldwide. Its pathophysiology is still not well understood. The only major risk factor associated with optic nerve damage is an elevated intraocular eye pressure in POAG. Recent findings have suggested that genetic factors including the mitochondrial genetic background may play a role in POAG in the pathogenesis of glaucoma. Mitochondria have their own DNA (mtDNA) which can be subdivided into distinct mtDNA haplogroups, based on geographic origins. Given the clinical specificities of glaucoma in various populations, we hypothesize that mtDNA haplogroups could be involved in POAG pathogenesis. Our primary aim is to evaluate the prevalence of various mtDNA haplogroups in POAG patients compared to a large cohort of age and sex- matched controls. The secondary aim is to determine if certain haplogroups could be associated with severity of the disease. This observational, cross-sectional study, which has already obtained an ethical committee agreement, will involve two university hospital centers (CHU Angers/Nantes), including 400 POAG patients and 400 controls, with clinically detailed ophthalmic phenotypes. New sequencing technologies will be used to determine mitochondrial haplogroups in all included subjects, followed by mitochondrial bioinformatics data processing. We hypothesize that mitochondrial haplogroups could be valid biomarkers in glaucoma, predictive of disease severity. If proven true, this hypothesis could translate into a therapeutic paradigm shift in glaucoma, by including medications which improve mitochondrial metabolism. Keywords : Glaucoma, Mitochondria, mtDNA, Mitochondrial Haplogroups, Aging ESPITA OLIVIER 2nd year PhD student - UMR 975-LPRO Influence of cardiovascular risk factors and arterial bed on peripheral arterial atherosclerotic plaque calcification, clinicopathological study on 272 cases Olivier Espitia1,2*, Mathias Chatelais2, Béatrice Guyomarc’h3 , Blandine Maurel2,4, Marc-Antoine Pistorius1, Yann Gouëffic2,4, Thibaut Quillard2. 1 Department of Internal Medicine, University Hospital of Nantes, France 2 INSERM UMR957 – LPRO, University of Nantes, France 3 Department of Vascular Surgery, University Hospital of Nantes, France 4L’institut du thorax – INSERM-UMR 1087 IRT – University of Nantes, France Keywords : peripheral arterial disease, calcification, lower limbs ischemia, stroke. Introduction Vascular calcification is associated with a worse prognosis after lower limb arteries stenting. Clinical imaging approach is based on the quantification of vascular calcification on CT but it can evaluate the “quality “of calcification. Previous study showed an arterial heterogenicity in plaques composition with more lipid in carotid plaques and more calcification in femoral arteries. Moreover, histological types of calcification were different in carotid and femoral, with osteoid metaplasia in feral and micro nodular (clear center) calcification in lipid plaque in carotid. Influence of cardiovascular risk factor on atherosclerotic localization is well known. Hypertension promotes intra cranial atherosclerosis, tobacco is more frequent in critical limb ischemia than in stroke. The aim of this study was to analyze the relationship of cardiovascular risk factors, arterial territories and type of plaques calcifications. Materials and methods Patients From February 2008 to December 2015, atheromatous plaques were harvested from patients undergoing carotid or femoral endarterectomy at the department of vascular surgery of the Nantes University Hospital, we used our ECLA and ECLAGEN biocollections of human atherosclerotic lesions from different arterial locations (carotid, femoral, and infrapopliteal arteries). Patients suffering from non-atherosclerotic peripheral arterial disease, thrombosis or restenosis, and patients who could not give their written consent were excluded. Demographic and clinical data were collected, including age, gender, treatment, cardiovascular risk factors (high blood pressure, diabetis mellitus, dyslipidémia, obesity BMI ≥30Kg/m2), Cokroft creatinime clearance. Prior to surgery, blood specimens were collected for lipid balance and phospho-calcic metabolism. Sample collection and handling was performed in accordance with the guidelines of the Medical and Ethical Committee in Nantes, France, and a written informed consent was requested for each patient. Tissue sampling Endarteriectomies were performed on a consecutive series of patients using conventional surgical techniques. The plaque was removed at the bifurcation from within the lumen as a single specimen. All plaques were divided into several equal parallel sections; a part was processed for histological analysis, Histology processing Atherosclerotic plaques were immediately fixed in 10% formalin overnight and decalcified in Sakura TDE 30 fluid during 24 h. They were embedded in paraffin. Sections (4µm thickness) were stained with hematoxylin eosin (HE) added with safran. Whole sections were imaged with the NanoZoomer digital slide scanner (Hamamatsu Photonics, Hamamatsu, Japan). Histological classification of atherosclerotic plaque’s calcification The sections were graded according to previous calcification description. Atherosclerotic plaque’s calcification classification is based on 5 categories, no calcification, microcalcification clear-center, sheet-like calcifications, osteoid metaplasia, nodular calcifications. Four exclusive sub-types of calcifications can be observed: microcalcification clear-center calcification consisted of vesicle like structure outlined with calcium deposits; sheet-like calcification defined as a calcification front within fibrosis, surrounded by numerous calcified micronodules; nodular calcification characterized by numerous stratified deposits of calcification with multinodular edges, consistent with an aggregation phenomenon, and the presence of very few cells and osteoid metaplasia consisting of mature bone with typical lamellar structure and often bone marrow. Each section was classified by 2 independent investigators. In case of discordant classification, a third investigator was consulted. Results 272 artery were evaluated, 153 carotid arteries (91 asymptomatics, 62 symptomatics), 94 femoral arteries and 25 infrapoliteal arteries. Femoral arteries were very calcified ; calcification in femoral vs carotid arteries, and in femoral vs infra-politeal arteries were significantly differente respectively p=0,003 and p=0,003. Osteoïd metaplasia is siginficantly more frequent in femoral than in carotid arteries (p<0,0001) and more present in femoral tan infra-popliteal arteries ( p<0,0001). Micro-calcifications (clear center) were more present in carotid than femoral arteries (p<0,0001). Discussion Cardiovascular risk factors have little influence on the type of calcification of atheroma plaques. The embryological origin of the different territories seems to be at the origin of these processes because Preliminary data from RNA chip classified the different territories based on the level of expression of various genes involved in the calcification process. Conclusion Arterial calcifications are little influenced by the cardiovascular risk factors. The embryological origin of the different arteries may contribute to variations in expression of genes involved in bone metabolism, thus explaining the differences in type calcifications in different arterial beds. MEIGNAN THOMAS 2nd year PhD student – UMR INRA ONIRIS BIOEPAR Effects of feeding extruded linseed on production performance and milk fatty acid profile in dairy cows: a meta-analysis T. Meignan*(1)(2), C. Lechartier(3), G.Chesneau(2) and N.Bareille(1) (1)BIOEPAR, INRA, Oniris, La Chantrerie, F-44307 Nantes, France (2)VALOREX, La Messayais, F-35210 Combourtillé, France (3)Unité de Recherche sur les Systèmes d’Elevage, Univ Bretagne Loire, Ecole Supérieure d’Agricultures, 55 rue Rabelais, F-49007 Angers, France. The objectives of this study were to quantify the effects on production performance and milk fatty acid (FA) composition of feeding dairy cows extruded linseed (EL), a feed rich in α-linolenic acid, and to assess the variability of the responses related to the dose of EL and the basal diet composition. To process our meta-analysis, we selected after the literature search only trials with a control diet without fat supplementation. The dependent variables were defined by the mean differences between values from EL supplemented groups and values from control groups. The data were processed by regression testing the dose effect, multivariable regression testing the effect of each potential interfering factor associated with the dose effect, and then stepwise regression with backward elimination procedure with all potential interfering factors retained in previous steps. The whole dataset consisted of 17 publications representing 21 control diets and 29 EL supplemented diets. The daily intake of fat from EL supplementation ranged from 87 to 1194 g/cow/d. The dry matter intake was reduced in high-fat diets. Milk fat content decreased when EL was supplemented to diets with high proportion of corn silage in the forage (-2.8 g/kg between low and high corn silage-based diets in the restricted dataset) but did not decrease when the diet contained alfalfa hay. The sum of pairs 4:0 to 14:0 (FA synthesized de novo by the udder), palmitic acid and the sum of saturated FA decreased linearly whereas oleic acid, trans-10 18:1, vaccenic acid, rumenic acid, α-linolenic acid and the sums of monounsaturated and polyunsaturated FA increased linearly when the daily intake of fat from EL was increased. Milk trans-10 18:1 proportion increased after EL supplementation by increasing the proportion of corn silage in the forage. In experimental conditions, EL supplementation modified linearly milk FA proportions to a profile favorable for human health but should be used cautiously in corn silage-based diets. Keywords: dairy cow, extruded linseed, milk yield, milk fatty acid, meta-analysis BARBE LAURE 2nd year PhD student - Unité Mixte de Recherche 892 Inserm - 6299 CNRS Glycan attachlent specificity, toward rotavirus vaccine improvement. Laure Barbé1*, Béatrice Le Moullac-Vaidye1, Nathalie Ruvoën-Clouet1,2, Jacques Le Pendu1 1INSERM, UMR 892; CNRS, UMR 6299; University of Nantes, France 2Oniris, Ecole Nationale Vétérinaire, Agroalimentaire et de l’Alimentation, Nantes, France Human strains of rotavirus A (RVAs) recognize fucosylated glycans of the histo-blood group family (HBGAs) as well as gangliosides through the VP8* protein of their capsid. Interaction with gangliosides is essential for cell entry and lack of fucosylated ligands due to HBGAs genetic polymorphism is associated with resistance to severe RVA gastroenteritis. However, the contribution of each of these ligands on infection remains unclear. Our goals are to delineate the contribution of HBGAs and gangliosides in the infection process and to explore the consequences of HBGAs polymorphisms on the virus transmission and efficacy of the available live vaccines. Binding to glycans of GST-VP8* proteins from various strains was studied using glycan microarrays and saliva mucins of individuals of the major HBGA subgroups. We observed that the VP8* from recent clinical strains of the most frequently isolated genotype in France (P8) showed binding to difucosylated structures as well as to saliva mucins from individuals expressing both FUT2 and FUT3 fucosyltransferases, with little effect of the ABO phenotype. The lack of either one or both enzymes led to an absence of binding to mucins. VP8* from cell culture-adapted Wa and vaccine strains showed the same binding profile, demonstrating conservation within the P8 genotype. Infection of MA-104 and HT-29 cells by cell culture-adapted and vaccine strains in the presence of an inhibitor of fucosylation demonstrated that fucosylated structures were dispensable for infection by cell culture-adapted P8 strains. The concordance between the glycan specificity of P8 strains VP8* and the HBGA-dependant susceptibility to severe RVA gastroenteritis indicates that HBGA-binding of human RVA strains is essential to the infection. Yet, our results suggest that HBGA binding corresponds to an early event since it is not required for infection of poorly differentiated cells. Besides, the fact that HBGA recognition is conserved between recent and older (Wa and vaccine strains) P8 strains suggest that HBGA polymorphism may contribute to explain the low efficacy of vaccines in areas where the frequency of FUT2(-) or FUT3(-) individuals is high. Keywords : Rotavirus, Histo-Blood Group Antigens FONTENAU ANDREA 2nd year PhD student - l’institut du thorax, Inserm UMR 1087 / CNRS UMR 6291 Scn5a gene, expressed in myocardium but not only: functional involvements of Nav1.5 channel in lungs. Fonteneau A1,2*, Jagu B1,2, Amossé E1,2, Carcouët A1, Dilasser F1,2, Cheminant MA1, Sauzeau V1, Magnan A1,2, Le Tourneau T1,2, Charpentier F1, Toumaniantz G1,2. 1 INSERM UMR 1087 / CNRS UMR 6291 l’institut du thorax, Nantes, France, 2 Université de Nantes, Nantes, France Cardiac Nav1.5 channel, encoded by Scn5a gene is responsible for the cardiac action potential initiation and propagation. In heart failure following myocardial infarction, which remain the major causes of mortality and morbidity in cardiovascular field, transcriptional and post-translational modifications of ionic channels, among them Nav1.5, are described and associated with lethal arrhythmias. It has been described in various animal models that these cardiac alterations in this pathological context may also have impacts on pulmonary function, following increased blood pressure upstream to the left ventricle. During our investigations, the Scn5a predominantly expression in heart was confirmed but significant expression was recorded in lungs, suggesting possible Nav1.5 inter-organ functional implications. The aim of this study was to confirm and characterize the Nav1.5 expression in lungs, and try to explain its potential functional role using two transgenic mouse models, a heterozygous knock-out Scn5a+/- and a human « loss of function » mutation T220I. We have shown Nav1.5 expression in lungs, localized a minima in airways smooth muscle cells. Then, transgenic mice pulmonary function tests have shown respiratory hypo responsiveness. Two explanations were considered. First, heart failure consequences were sought and overturned. Secondly, an intrinsic pulmonary remodeling was investigated. Modest fibrotic and muscular remodeling are suggested for T220I+/+ model, and appears not to be concomitant to inflammatory response. An adjusted protocol for airways smooth muscle primary cells culture will allow us to examine bronchomotility signaling pathway and its potential modifications in our transgenic mouse models. Then it would be possible to study in case of loss of Nav1.5 expression and/or function the cardiopulmonary impacts in heart failure following myocardial infarction. Keywords : Nav1.5, lungs, heart, remodeling, fibrosis. SOUALAH ZINEB 2nd year PhD student - CNRS UMR 6214, Inserm U1083 Drugs, alcohol and benzodiazepines abuse in fatal poisonings Zineb SOUALAH* , Habib BELMAH, César Mattei, Christian Legros CNRS UMR 6214, Inserm U1083, Univ, Angers Faculty of medicine, Angers-France Toxicology Service CHU Constantine Benzodiazepines (BZDs) are the most prescribed drugs for treating anxiety, which affects millions of people worldwide, but they generate undesired effects, including withdrawal symptoms, sedation, amnesia, cognitive impairments, aggressiveness, and also risk of Alzheimer’s disease (Rudolph and Knoflach, 2011; Billioti de Gage et al., 2014). However, BZDs are also frequently used by people who consume alcohol and drugs. Here, we reviewed 1945 patients who deceased by poisoning in the Toxicology Service CHU Constantine/ post-mortem forensic toxicology. Cases recorded between 01/01/2004, and 31/12/2015, circumstances and clinical signs of accidental oral poisoning of humans by benzodiazepines). Our results showed that, among the numerous potential drugs available, that the poisoning by BZD reports to other drug intoxication is 47%, two molecules are particulary appraisals at the drug addicts in Algeria, Diazepam, Valzépam, Valium®, and Clonazépam (Rivotril®, called "Roche")knowing that most consumers are women.the death by overdose also inceased among the million Algerians using of benzodiazepines. HER CHITDAVONE 2nd year PhD student – UMR-S INSERM1066, MINT, Angers Formulation of curcumin lipid nanocapsule-loaded gels for inflammatory bowel disease application Chithdavone HER*, Marie-Claire VENIER-JULIENNE, Emilie ROGER INSERM U1066, Micro et Nanomédecines Biomimétiques, IBS, Univeristy of Angers, France Inflammatory bowels diseases (IBD) are chronic inflammatory disorder of the gastrointestinal tract, there are some conventional drugs for IBD treatment to induce remission, prevent and treat symptom but these conventional therapies present side effects that lead to the disease complications. Curcumin, a natural polyphenol found in the rhizomes of Curcuma longa L, is well known for its beneficial effects in various illnesses without significant adverse effect. Curcumin have anti-inflammatory properties that are interesting for IBD remission. Nevertheless, administration is limited due to it presents a low aqueous solubility, instability at pH neutral to basic, poor absorption, rapid metabolism and low bioavailability. And additionally, drug delivery to intestinal inflame tissue via oral administration to improve therapeutic efficacy is challenge for IBD treatment due to the present of mucus layer which is an obstacle for drug deliver through the gastro-intestinal tract. The aim of this study is, to prepare and characterize curcumin lipid nanocapsule (cur-LNC) loaded mucoadhesive gels for oral administration for IBD application. The use of mucoadhesive polymer has been proposed as a strategy to increase the interaction of the nanoparticle with the mucus in order to prolong the residence time and LNCs allow to improved curcumin bioavailability. Method: Firstly, curcumin solubility was screened in different excipient. Then, Curcumin was encapsulated in lipid nanoparticle by phase inversion method. Finally, mucoadhesive gel was prepared by ionotropic gelation. In the first step we were attempted to formulate a mucoadhesive gel and looking for a system to produce a cylindrical gel and curcumin was screened for its solubility in oil phase before encapsulation. Preliminary results, we found interesting curcumin solubility in Transcutol® HP oil (≥ 80mg/g). Moreover, we obtained a mucoadhesive gel formulation in the cylindrical form with a system of Tanswell® polycarbonate with 5µm pore size 11.8 mm high and 4.9 mm in diameter. Conclusion and perspectives: Transcutol was selected as a solubilizer and the system of Tanswell® is suitable for formulating cylindrical gel that could allow to delivered nanoparticle into gastro-intestinal tract inflame tissue. Therefore, further studies mucoadhesive gels and cur-LNC will characterize for their formulations before incorporation each other. ARBEZ BAPTISTE 2nd year PhD student – GEROM Groupe d'Etudes sur le Remodelage Osseux et les bioMatériaux Synthesis and characterization of planar β-TCP blocs. Baptiste Arbez1*, Romain Mallet2, Daniel Chappard1,2 1GEROM – Groupe d'Etudes sur le Remodelage Osseux et les bioMatériaux – IRIS IBS, France 2SCIAM – Service Commun d'Imagerie et Analyses Microscopiques de l'Université d'Angers – IRIS IBS, France. Beta-tricalcium phosphate (β-TCP) is a synthetic ceramic that belongs to the calcium orthophosphate family. It crystallizes in a rhombohedral system which corresponds to 2 Bravais lattices: rhombohedral and hexagonal. Its chemical composition (β-Ca3(PO4)2) allows it to be used as a bone substitute for filling defects in bone surgery. β-TCP is resorbable, osteoconductive and biocompatible. It gradually degrades over time as new bone grows: it plays the role of a scaffold before being almost entirely replaced by bone. In this study, we have used a slurry of elementary β-TCP grains sintered at high temperature (>1000°C) to obtain planar blocs (Ra < 500 nm). The chemical composition of the synthesized material was verified by Raman spectroscopy. The material has been subsequently characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM) to obtain information on its surface morphology. A comparison before and after sintering has also been established. SEM and AFM analyses have shown that planar blocs of β-TCP present a tessellated surface whose elementary components present defects identified as shear bands. These shear bands form during sintering when lacunae and dislocation occur with the motion of atoms at high temperature. Dislocations create defect lines (called dislocation lines) that have the ability to move through the material when submitted to shear stress. Dislocation lines move along the denser crystallographic planes which have been identified as the family {1¯1 00} for β-TCP (in the hexagonal lattice). Knowing the lattice parameters, the minimum distance between two dislocation lines has been estimated at 15.449 Å. The distance between two observed dislocation lines can therefore be estimated as n x 15.449 Å with n being an integer. Keywords: Biomaterials; Beta-tricalcium phosphate; Crystal structure; Shear band; Surface defects WALENCIK ALEXANDRE 2nd year PhD student - EFS Pays de la Loire Topological Data Analysis: an emerging analyze haplotype-based likelihoods of finding a 10/10 match donor for HLA known patients A Walencik1,2*, M. L. Balere3, C. Faucher3, P. Loiseau4, A. Dormoy5, E. Marry3 & F. Garnier3 Devi Ramanan 6, Anne Cesbron 2, PA Gourraud 1 1 UMR1064, CHU de Nantes, France 2 Laboratoire d’Histocompatibilité et d’Immunogénétique, EFS Pays de la Loire, Nantes, France 3 Agence de la Biomédecine, Registre France Greffe de Moelle, Paris, France 4 Laboratoire d’Histocompatibilité et d’immunologie, Hôpital Saint Louis, Paris, France 5 Laboratoire d’Immunogénétique, Etablissement Français du Sang Bourgogne-Franche-Comté, Besançon, France 6 Ayasdi, 4400 Bohannon Dr #200, Menlo Park, CA 94025, USA Topological data analysis (TDA) is an increasingly popular method applied in various data-intensive fields such as finances. Because the diversity of HLA phenotypes is central to determine the chance to find a suitable match for Hematopoietic Stem Cell Transplantation (HSCT) candidates, we revisited a published analysis of 2,126 transplanted and non-transplanted patients (Gourraud et al., Tissue Antigens, 2015) using available TDA software named Ayasdi in order to evaluate the ability of TDA to identify groups of patients with low vs high chance to find a 10/10 matched unrelated donor. Input data consist of the likelihood of estimations of HLA-A, -B, (-C), -DRB1, (-DQB1) phenotypes of the HSCT candidates. These likelihoods were computed from published haplotype frequencies for four genetic ancestries (Caucasian, African-American, Hispanic and Asian-Pacific Islanders) and provide a set of coordinates for each patient further analyzed in TDA software blind to the actual transplanted outcome (transplanted or not). TDA delivers a visual representation of the datasets in the form of weighted networks. Analysis parameters such as data reduction techniques, lenses and metrics were investigated for their ability to define subgroups of transplanted and not transplanted patients. Using Variance Normalized Euclidean with neighborhood lenses, TDA succeeds to define networks representing non-transplanted patients with rare phenotypes whatever their genetic ancestry. Despite the fact that self-declared ancestry is not known to the search coordinators for legal reasons; TDA successfully identifies subgroups of transplanted patients with frequent HLA phenotype in one or all ancestries. Transformations of input as logarithmic transformation influence the network display. When used with appropriate parameters, TDA delivers actionable visual representation of chance to find a HLA matched donor. Further analyses as inputting actual HLA polymorphism are required to evaluate the representation modalities that would accelerate the decisional process for HSCT candidates. KHOURY JOSEPH 3rd year PhD student - Laboratory of Integrated Neurovascular and Mitochondrial Biology, UMR CNRS 6214, INSERM U1083, Faculty of Medicine, University of Angers, France The aqueous buds extract of Eucalyptus neutralizes the main enzymatic activities of Montivipera bornmuelleri venom Joseph Khoury 1, 2£, Ranin Dabbousy 1£, Souad Hraoui-Bloquet3, Riyad Sadek4, Walid Hleihel5, Jean-Marc Sabatier6, Christian Legros2 and Ziad Fajloun1, 7 * £. Both authors contributed equally to this work 1-Laboratory of Applied Biotechnology, Azm Centre for Research in Biotechnology and its Application, Doctoral School for Sciences and Technology, Lebanese University, El Mittein Street, Tripoli, Lebanon; 2-Laboratory of Integrated Neurovascular and Mitochondrial Biology, UMR CNRS 6214, INSERM U1083, Faculty of Medicine, University of Angers, France; 3-Faculty of Sciences II, Lebanese University, Fanar El Metn, Lebanon; 4-American University of Beirut, Department of Marine Sciences, Beirut, Lebanon; 5-Department Biology, Department of biology, university of Balamand, Kourah, Lebanon; 6-INSERM UMR 639, Luminy Science and Technology Park, Marseille, France; 7-Department of Biology, Faculty of Science III, Lebanese University, Tripoli, Lebanon. In recent years, it has emerged the idea that plant extracts represent a novel alternative to neutralize the deleterious effects of snake bites. In this context, we investigated the aqueous Buds extract of Eucalyptus (ABEE) to counteract the enzymatic and the antibacterial activities of the venom of the vipera, Montivipera bornmuelleri. Proteolytic and PLA2 activities of M. bornmuelleri were evaluated using both milk and egg yolk in agar plates. AChE, anti-AChE and L-Amino Acid Oxidase (LAAO) activities were determined by spectrophotometric methods. Anti-bacterial activity was tested against Staphylococcus aureus using the disc-diffusion method. The antioxidant potential was evaluated by α,α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging method. We first showed that ABEE efficiently counteracts the proteolytic, PLA2 and the LAAO activities of M. bornmuelleri venom. In addition, ABEE was found to inhibit AChE and possess a potent antioxidant activity, but it displays no antibacterial activity against staphylococcus aureus. We also showed that of M. bornmuelleri venom lacks AChE, either anti-AChE activities. In conclusion, our data indicate that the aqueous buds extract of Eucalyptus efficiently neutralize the proteolytic, PLA2 and the LAAO activities of M. bornmuelleri venom, and also AChE in vitro. Thus, this plant extract represents a promising natural source of antivenomic compounds against the deleterious effects of M. bornmuelleri or other vipera species bites. Keywords : Montivipera bornmuelleri, Eucalyptus Plant, AChE, PLA2, protease activity, Antioxidant activity. STAERCK CINDY 3rd year PhD student, Groupe d’Etude des Interactions Hôte-Pathogène (GEIHP UPRES-EA 3142) Oxidative stress defense of Scedosporium apiospermum, a filamentous fungus associated with cystic fibrosis C. Staerck*1, A. Gastebois1, P. Vandeputte1.2, C. Godon1, J-P. Bouchara1,2, and M.J.J. Fleury1 1L’UNAM Université, Université d’Angers, Groupe d’Etude des Interactions Hôte-Pathogène, GEIHP UPRES EA 3142, Angers France ; 2Laboratoire de Parasitologie Mycologie, Centre Hospitalier Universitaire, Angers, France Species of the Scedosporium apiospermum complex are able to chronically colonize the airway of cystic fibrosis (CF) patients, at the second ranks after Aspergillus fumigatus. Establishment of a fungal colonization depends on the capacity of the fungus to evade killing by the host immune system, particularly the oxidative response of phagocytes, since macrophages and neutrophils release antimicrobial compounds, especially reactive oxygen species (ROS). Pathogens can evade the high levels of ROS produced during the oxidative burst response by the induction of several genes. The aim of this study was to investigate all the genes implicated in the defenses of S. apiospermum against the oxidative stress. Analysis of S. apiospermum sensu stricto (strain IHEM 14462) genome allowed to identify 34 genes coding for proteins potentially implicated in the defense against oxidative stress. Gene expression was evaluated by RT-PCR on 24-h-old hyphae stressed by conditions mimicking those encountered in the bronchial mucus of CF patients, with chemical compounds generating ROS or in presence of activated phagocytic cells. The results show that genes expression is not significantly modulated by CF conditions. Six genes are highly overexpressed in chemical oxidative stress conditions and 5 genes are overexpressed in co-culture with phagocytic cells. Interestingly, two genes coding for thioredoxin reductases are overexpressed in chemical oxidative stress and in co-culture with activated phagocytic cells. In others fungi, thioredoxin reductases are implicated in ROS degradation and in the virulence. They are also immunogenic marker for serodiagnosis and could be used as vaccine. To confirm their role as virulence factors in Scedosporium apiospermum, defective mutants are currently underway for these two genes and for their potential transcription factor SaYap1. Finally, the aim of this work is the identification of Scedosporium apiospermum virulence factors as potential new therapeutic targets. Keys words: oxidative stress, response, fungus NAVARRO LAURENT 2nd year PhD student - Centre de Recherche en Cancérologie Nantes - Angers (CRNCA) “Synthesis and evaluation of radioiodinated and astatinated prosthetic groups for bioorthogonal conjugation to antibodies for nuclear imaging and therapy” Laurent Navarro1*, Cyrille Alliot2, Michel Chérel1, Frédéric Pecorari1, Jean-François Gestin1, François Guérard1 1 Inserm U892/CNRS UMR6299/Université de Nantes, Nantes, France 2 Arronax GIP, Nantes 44817, France Heavy radiohalogen astatine and iodine are increasingly studied for therapeutic or diagnostic purpose in nuclear medecine.1 Particularly 211At (t1/2 = 7.2 h, α-emitter) is a promising isotope for the treatment of small clusters or isolated tumor cells by targeted α-particle therapy. To associate this type of radiohalogen on cancer targeting biomolecules (peptides, antibodies), the conventional method consists in performing the radiohalogenation of an arylstannane precursor bearing a N-Hydroxysuccinimide function (NHS), which is then reacted with lysines of biomolecule.2 However, although widely used, this approach requires basic aqueous conditions leading to competitive hydrolysis of the radiolabeled prosthetic group, resulting in suboptimal conjugation yields (< 50 %). To overcome this issue and to simplify the radiolabeling process, we have initiated the investigation of click bioorthogonal conjugation approaches. These highly chemoselective reactions are more inert and highly reactive in biological media due to the nature of the chemical functions employed. In this study, a set of arylstannane and diaryliodonium precursors bearing clickable functionalities (tetrazine, azide, alkyne) were synthesized and radiolabeled with 125I and 211At. Kinetics of the click conjugation were then conducted on a model peptide simulating antibodies reactivity and bearing a clickable function (trans-cyclooctene (TCO), bicyclononyne (BCN) or alkyne). All reactions tested produced quantitative conjugation yields from less than 1 minute to 9 hours with the following reactivity order: TCO-tetrazine > copper(I)-catalyzed alkyne-azide > BCN-tetrazine > BCN-azide, as expected from literature data.3 The results obtained highlight the higher efficiency of click chemistry in comparison with the conventional NHS-lysine coupling method. They open perspectives for quantitative radiolabelling with reduced conjugation time. Translation of this approach to the radioiodination and the astatination of antibodies is now in progress, with the aim of assessing the in vivo fate of such radiolabeled biomolecules. [1] M. J. Adam, D. S. Wilbur, Chem. Soc. Rev. 2005, 34, 153–163 [2] Zalutsky, M.R. & Narula, A.S. Int. J. Rad. Appl. Instrum. A. 1988, 39, 227-232 [3] Lang, K. & Chin, J. W. ACS Chem. Biol. 2014, 9, 16–20 PATIN JUSTINE 2nd year PhD student - l’institut du thorax, INSERM, CNRS, Université de Nantes Gap-134, a gap-junctional activator, decreases ventricular fibrosis in Scn5a+/- mice Justine Patin1*, Claire Castro1, Cynthia Ore Cerpa1, Arnaud Tessier2, Jacques Lebreton2, Agnès Carcouët1, Agnès Hivonnait1, Isabelle Baró1, Flavien Charpentier1 and Mickaël Derangeon1 1 : l’institut du thorax, INSERM, CNRS, UNIV Nantes, IRS-UN, 8 quai Moncousu, BP 70721, 44001 Nantes Cedex1, France ; 2 : CEISAM UMR CNRS 6230, UFR sciences et techniques, 2 rue de la Houssinière, BP 92208, 44322 Nantes cedex 03, France Progressive Cardiac Conduction Disease (PCCD) progressively alters cardiac conduction during ageing and has been associated with fibrosis. Genetic forms of PCCD have been linked to loss-of-function mutations of SCN5A gene product, the Nav1.5 cardiac voltage-gated Na+ channel. To understand the pathophysiology of PCCD, we studied a Scn5a heterozygous knockout (Scn5a+/-) mouse model, which exhibits age-related deterioration in conduction and progressive development of ventricular fibrosis between 45 and 60 weeks of age mediated by TGF-β pathway. Since abnormal expression of connexin 43 (Cx43) has been observed in models of cardiac fibrotic remodeling, we investigated Cx43 expression in wild-type (WT) and Scn5a+/- mice. During ageing of WT mice the ventricular expression and phosphorylation of Cx43 decreased between 45 and 60 weeks of age. In Scn5a+/- mice, expression and phosphorylation state of Cx43 were higher at 30 weeks and then decreased markedly to levels equivalent to WT mice at 60 weeks. We hypothesized that the decrease of Cx43 expression induced by ageing associated with the decrease of Nav1.5 participates to the age-dependent fibrotic remodeling observed in Scn5a+/- mice. To validate this hypothesis, Scn5a+/- mice have been treated with Gap-134 ([2S,4R]-1-[2-aminoacetyl]4-benzamidopyrrolidine-2-carboxylic acid), a gap-junction activator. Gap-134 (5mg/kg/d, p.o.) was chronically administered to Scn5a+/- mice from the age of 45 weeks to the age of 60 weeks. After 15 weeks of treatment, Gap-134 did not prevent the amplification of conduction defect but increased the expression and phosphorylation of Cx43 on serine368. Furthermore, Scn5a+/- mice treated with Gap-134 did not show any sign of ventricular areas of patchy fibrosis at the age of 60 weeks but a decreased of collagen I / collagen III ratio. Altogether, the results support the hypothesis that fibrotic remodeling in Scn5a+/- mice is correlated with the ageing-induced decrease of Cx43. CHAMPIN TRISTAN 2nd year PhD student - Laboratoire BNMI Angers Neutrophil and Thrombospondin-1 implications in flow-mediated remodeling of resistance arteries from mice. Tristan Champin*1 , Céline Grenier1, Olivier Blanc-Brude2, Daniel Henrion1, Laurent Loufrani1. 1 UMR Inserm 1083 UMR CNRS 6214 Laboratoire de Biologie Neurovasculaire et Mitochondriale Intégrée, Angers, France. 2 UMR Inserm 970 Paris Centre de recherche Cardiovasculaire à l’HEGP, Paris, France On vascular endothelium, shear stress produces vasorelaxation and outward arterial remodeling. A chronic increase in blood flow induces arterial diameter expansion (normalizing shear stress), and is associated with a compensatory hypertrophy to normalize wall strain. This remodeling is due to inflammation and nitric oxide (NO) production in resistance arteries. A large number of studies have demonstrated that Thrombospondin-1 (TSP1), a homotrimeric glycoprotein, can have an inhibitory action on the NO pathway and a positive effect on inflammation in vitro. Nevertheless, the role of TSP1 on remodeling is poorly understood. We hypothesized that TSP1 is able to participate in this outward arterial remodeling and hypertrophy by immune cells recruitment. We investigated this remodeling in mesenteric resistance arteries of WT or TSP1-KO mice aged to 12-16 weeks. An IgG2a isotype control mAb 2A3 or Ly6G-specific mAb 1A8 are used for neutrophil depletion by in vivo administration. Ligation of mesenteric resistance arteries of WT/TSP1-KO and IgG2a/neutrophil-depleted mice allowed modifying blood flow in vivo, thus exposing arteries to low (LF), normal (NF), or high flow (HF). After 7 days, arteries were isolated for in vitro study. Phenylephrine-mediated contraction and Acetylcholine-mediated relaxation were stronger in HF than in NF vessels on WT and IgG2a control mice. Any difference between HF or NF in TSP1-KO and neutrophil-depleted mice were found. Furthermore, passive diameter analysis revealed a higher diameter in HF than NF in WT and IgG2a mice. These differences are lost in TSP1-KO and neutrophil-depleted mice. These results suggest that TSP1 are essential for remodeling in mesenteric arteries by promoting neutrophils recruitment. PERROTEAU JEANNE 2nd year PhD student – CRCNA UMR 892 Inserm 6299 CNRS Identification of human self-reactive iNKT cell subset Jeanne PERROTEAU*1, Leslie HESNARD1, Marie-Claire DEVILDER1, Laurent GAPIN3, Emmanuel SCOTET1, Xavier SAULQUIN1,2, Laetitia GAUTREAU-ROLLAND1,2 1 CRCNA, UMR 892 Inserm 6299 CNRS, France 2 Université de Nantes, France 3 National Jewish Health and University of Colorado, Denver, USA iNKT cells are unconventional T lymphocytes that express both a semi-invariant αβ T Cell Receptor (TCR) and Natural Killer (NK) cell receptors. These cells are able to recognize glycolipids presented by a monomorphic and non-classical MHC-I molecule called CD1d. iNKT cells play an important role in the anti-infectious immunity by recognizing glycolipids from pathogens or self-derived glycolipids expressed by immune cells in an inflammatory context. Thus, iNKT lymphocytes have the ability to recognize both exogenous and endogenous glycolipids, suggesting that these cells present an autoreactive potential. However, mechanisms of this autoreactivity are still poorly defined, especially in humans. In particular, it is still unknown if all iNKT lymphocytes present an autoreactive potential or if only iNKT sub-populations have this feature. In this context, the characterization of human self-reactive iNKT cells from peripheral blood is the main objective of this study. The understanding of iNKT cell autoreactivity has been hampered, at least in part, by the difficulty to identify and isolate potential autoreactive iNKT cells from an immune repertoire and by their low frequency in PBMCs (less than 0,5%). In this study, we used a very sensitive tetramer-based cell sorting strategy (threshold frequency 10-7) to identify iNKT cells stained by CD1d molecule loaded with undefined self-derived glycolipids. Tetramer-specific iNKT cells stained by self-glycolipid/CD1d tetramers were enriched, purified and amplified in vitro upon unspecific activation. Only a part of cells, recognizing self-glycolipids, showed a strong autoreactivity against various target cells expressing endogenous glycolipids/CD1d complexes, in terms of degranulation, cytokine production or cytotoxicity, unlike iNKT cells not stained by self-glycolipid/CD1d tetramers. Moreover, this recognition was dependent on CD1d expression. Thanks to our highly sensitive immunomagnetic cell sorting approach, we were able to isolate and then characterize a human self-reactive iNKT cell subset. MANIANGOU BERCELIN 2nd year PhD student - EFS pays de la Loire, Université de Nantes Broad KIR allelic polymorphism investigated by Next Generation Sequencing technology Bercelin Maniangou1, Nolwenn Legrand1, Catherine Willem1, Gaëlle David1, Raphaelle Riou1, Eric Charpentier2, Mehdi Alizadeh3, Alexandre Walencik4, Christelle Retière1 and Katia Gagne1, 4, 5 1Etablissement Français du Sang and Université de Nantes, Immunovirologie et Polymorphisme Génétique, EA4271, Nantes, France; 2Plateforme BIRD, Institut du Thorax, Nantes, France; 3Laboratoire d’Histocompatibilité, EFS Rennes, France; 4Laboratoire d’Histocompatibilité et d’Immunogénétique, EFS Nantes, France, 5LabEx Transplantex, Université de Strasbourg, France. The impact of KIR+ NK cell alloreactivity on Hematopoietic Stem Cell Transplantation (HSCT) is still debated due to the heterogeneity of graft parameters, HLA environment, nature of KIR/KIR ligand genetic combinations studied and KIR+ NK cell repertoire size. KIR are known to be polymorphic in terms of gene content, copy number variation and number of alleles (n=753). This allele polymorphism may impact on the phenotype and function of KIR+ NK cells as we and other groups having reported for KIR3DL1. We speculate that KIR allele polymorphism may alter donor KIR+ NK cell phenotype/function thus modulating KIR+ NK cell alloreactivity post-HSCT. However, the impact of KIR allele polymorphism on HSCT outcome remains today difficult to assess due to the lack of suitable KIR allele typing methods. In this study, we developed a Next Generation Sequencing (NGS) technology for KIR allele typing from 30 reference DNA samples of the 10th International Histocompatibility Workshop with known KIR allele typing. Intergenic KIR primers enabled amplification of each gene from 5’ UTR to 3’ UTR region. Clonal amplification and sequencing were performed from KIR libraries on an Illumina MiSeq. The alignment of raw data to the Human genome reference (hg19) demonstrated that all KIR genes were amplified. Although the assignment of KIR alleles showed a high rate of concordance, some discordance with the reference typing were highlighted probably due to the limitations of KIR typing methods used in the past. Overall, our results showed a reliable NGS.KIR method to investigate the broad KIR allelic polymorphism to improve our knowledge, not only on KIR+ NK cell alloreactivity in HSCT, but also on KIR+ NK cell implication in control of viral infections and diseases. Key words : NK cell, receptors and signaling, Clinical applications of NK cells POIROUX LAURENT 2nd year PhD student - Laboratoire BNMI Prospective randomized study to assess the effect of humidification on the comfort of patient receiving oxygen inhalation therapy Laurent Poiroux*(1), Lise Piquilloud-Imboden (2), Cyril Le Roy (1), François Beloncle (1), Alain Mercat (1) (1) Département de Réanimation Médicale et de Médecine Hyperbare – CHU d'Angers, 4 rue Larrey, 49933 Angers cedex 9 - France (2) Service de Médecine Intensive CHUV, rue du Bugnon 46, 1011 Lausanne - Suisse Introduction Hypoxemia is frequent in acutely ill patients and often requires oxygen administration. This therapy is often associated with discomfort. Humidifying delivered gas could potentially reduce discomfort associated to the oxygen inhalation therapy but data available on this question are sparse and previous study results are controversial. Patients, Material and Method This study was a randomized prospective multicentric non-inferiority open trial designed to demonstrate that using non humidified oxygen was not associated with a significant increase in reported discomfort scores compared to using humidified oxygen. After inclusion, patients were randomized either in the humidified or non-humidified oxygen groups and humidification was started as soon as possible if indicated. The randomization was stratified on the initial prescribed oxygen flow rate (≤ 4L/min and > 4L / min). A specific score of comfort was then assessed between the 6th and 8th hour after inclusion. The global score was calculated by adding the points obtained (between 0 and 10) for each of the fifteen assessed items. Results The main finding of this study is that, 6 to 8 hours after oxygen therapy was started, oxygen humidification did not improve global patient comfort. At 24 hours after oxygen administration initiation, the global comfort scores were also similar in patients receiving humidified and non-humidified oxygen. Interestingly, the results were similar in the subgroups of patients receiving oxygen at a flow of ≤ 4L/min and > 4 L / min. Conclusion The results of this randomized controlled trial, including a significant number of patients from 9 intensive care units, give a real credibility to the results. They appear as a key element to update the current recommendations for practice. MAXIME LORENZINI 2nd year PhD student - l'institut du thorax Inserm UMR 1087 / CNRS UMR 6291 Regulation of Nav1.5 Channels by Ca2+/Calmodulin-dependent protein Kinase II in heart failure Maxime Lorenzini*, Sophie Burel, Fabien C. Coyan, Matthew R. Meyer, Cheryl F. Lichti, Joan H. Brown, Flavien Charpentier, Jeanne M. Nerbonne, R. Reid Townsend, Lars S. Maier and Céline Marionneau IRS-Université de Nantes, 8 Quai Moncousu, BP 70721, 44007 Nantes Cedex 1 Voltage-gated Na+ (Nav) channels are key regulators of myocardial excitability, determining depolarization, duration, waveform and refractoriness of action potentials. In heart failure, action potentials are prolonged while conduction velocity is slowed, and several evidences suggest that these arrhythmogenic abnormalities are associated with Ca2+/Calmodulin-dependent protein Kinase II (CaMKII) and altered phosphorylation of the main ventricular Nav channel subunit, Nav1.5. However, the phosphorylation sites implicated in these defects are still unknown. Phosphoproteomic analyses were therefore undertaken in the laboratory to identify and quantify in situ the phosphorylation sites on Nav1.5 proteins purified from adult wild-type (WT) and failing CaMKII c-overexpressing (CaMKII c-Tg) mouse ventricles. Among the nineteen native Nav1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in CaMKII c-Tg, compared with WT, ventricles. Because these two phosphoserines are located in close proximity to the binding sites for Fibroblast Growth Factor 13 (FGF13) and Calmodulin (CaM), two key regulators of channel inactivation, we tested the hypothesis that phosphorylation at these sites impairs the FGF13/CaM-mediated regulation of channel inactivation and recapitulate the arrhythmogenic abnormalities associated with CaMKII in heart failure. Preliminary experiments in HEK293 cells showed that phosphomimetic mutations on serines-1938 and -1989 reduce the interaction of Nav1.5 with FGF13 and CaM, resulting in increased late INa and reduced Nav1.5 channel availability. The aim of my thesis is to validate these findings in a more integrative, cardiac cellular model, the rat neonatal cardiomyocytes, and to confirm the role of these two phosphorylation sites in the CaMKII-dependent dysregulation of Nav1.5 channels in heart failure. Key words : Heart failure - Nav1.5 channels – CaMKI - INa late BIRONNEAU VANESSA 2nd year PhD student, INSERM U1063, Stress Oxydant et pathologie Métaboliques (SOPAM) Impact of obstructive sleep apnea on the endothelial function in patients with type 2 diabetes V Bironneau*1, MC Martinez1, M Le Vaillant3, S Dubois4, PH Ducluzeau5, F Goupil6, A Paris6, P Priou1,2, N Meslier1,2, C Sanguin7, W Trzépizur1,2, R Andriantsitohaina1, P Abraham8, F Gagnadoux1,2 1 INSERM U1063, Stress Oxydant et Pathologies Métaboliques, Université d’Angers, Angers 2 Département de Pneumologie, CHU d’Angers, Angers 3 CERMES, CNRS UMR8211- INSERM U988-EHESS, Villejuif 4 Service d’Endocrinologie, de Diabétologie, et Nutrition, CHU d’Angers, Angers 5 Service de Médecine Interne Nutrition, CHRU de Tours, Tours 6 Service de Pneumologie, CH du Mans, Le Mans 7 Service d’Endocrinologie, diabétologie, CH Du Mans, Le Mans 8 Service d’Exploration Fonctionnelles Vasculaires, CHU d’Angers, Angers Introduction: The endothelial dysfunction is a predictive infra-clinical marker of the cardiovascular risk that is largely described in the type 2 diabetes (DT2) and in the obstructive sleep apnea (OSA), two conditions frequently associated. Objective: The objective was to estimate if the association of two pathologies increases the endothelial dysfunction compared with two isolated conditions. Methods: We included 175 patients without cardiovascular disease except hypertension for clinical suspicion of OSA. The patients were distributed in 3 groups: OSA (AHI ≥ 15/hour, n=30), DT2 (IAH < 15/h, n=39), and DT2+OSA (n=97). In-vivo endothelium function was estimated by the hyperemic reactivity measured by digital plethysmography (RH-PAT) (n=146) and by laser-Doppler (RH-LD) (n=70). In-vitro endothelial function was evaluated by assessing endothelial NO production (DAF2-DA labeling) and examining capillary network formation on Matrigel®. For this, Human Aortic Endothelial cells (HAoEC) were treated for 24 hours by patients’ microparticles (MP) after characterization. Results: The groups of patients were comparable in terms age, smoking, frequency of the hypertension and the waist measurement. The BMI was higher in the DT2 and DT2+OSA groups (32±6.41 kg/m2 and 33.85±6.04 kg/m2) when compared with OSA group (30.8±3.4 kg/m2, p=0.03). The endothelial function measured by RH-PAT and RH-LD was better in the OSA group than in the other groups (p=0.03 vs DT2 and 0.005 vs DT2+OSA) without difference between DT2 and DT2+OSA groups (1.83±0.53 vs 1.84±0.47).The MP characterization only show a difference for the MP CD162+ (PSGL-1 carriers), increased for the diabetic patients. In-vitro, diabetes patients MP cause a decrease of NO, all the more important that OSAHS is severe. They also inhibit the action of ATP. Angiogenesis is more important when cells are treated with patients MP without significant difference between diabetic patients with or without apnea. Conclusion: These results are not in favor of a cumulative effect of the OSA on the endothelial dysfunction associated with the DT2. Keywords: obstructive sleep apnea, type 2 diabetes, endothelial dysfunction, microparticles JOALLAND NOEMIE 2nd year PhD student –Inserm UMR 892, Nantes Identification and characterization of human γδ T cells sup-populations with pro/anti- tumoral functions Noémie JOALLAND*, Ulrich JARRY, Emmanuel SCOTET (Inserm U892 – 8 quai Moncousu – 44000 Nantes) γδ T cells, which are at the interface between innate and adaptative immunity, represent an interesting population among cells that play key roles in immune responses in cancer. γδ T cells are characterize by gamma paired to delta chains TCR but also by NK and Toll-Like receptors allowing to recognize stressed cells. Although, γδ T cells implication during inflammatory response in infections is well described, their real implication during anti- tumoral response remain unclear. Then, the presence of γδ T cells inside the tumoral microenvironnement has been associated with poor prognosis in some cancers, suggesting the existence of sub-populations with pro- tumoral effects. This study aimed at identifying new sub-populations of human γδ T cells with suppressive/regulatory functions and investigate mechanisms which could be implicated in their establishment and functions in physopathological context. First we investigate the phenotype of γδ T cells isolate from blood of healthy donors by characterized their TCR based on the delta chain. The three main populations of γδ T cells (δ1, δ2 and δ3) were detected in the circulation and could be amplified in vitro. Second, the functions of the different sub-populations will be evaluated by analysis of the cytokine profil and the reactivity against tumoral cells. This study will allow to better identified γδ T cells with pro-tumoral functions and lead to the designing of new therapeutic strategies promoting anti-tumoral functions. Key words : γδ T cells – plasticity – cancer MELANIE LE DUFF 2nd year PhD student – Inserm U892, Angers Role of the EZH2 methyltransferase during escape to chemotherapy-mediated senescence. Mélanie Le Duff*, Julien Gouju, Barbara Jonchère, Eric Lelièvre, Catherine Guette and Olivier Coqueret Institut de Cancérologie de l’Ouest Paul Papin, Inserm U892 Equipe 12, Angers, France Chemotherapy and DNA damage activate signaling pathways that lead to the induction of senescence. Senescence is a definitive arrest induced by the p53-p21 and Rb-p16 pathways. Resistance mechanisms allow tumor cells to survive and as a consequence to avoid the establishment of senescence. In the laboratory, we analyzed cell escape in response to irinotecan, a first line treatment used in colorectal cancer that induced senescence. We have shown that specific subpopulations (called PLD) can escape and resume proliferation, while other cells remain arrested and senescent (called PLS). This heterogeneous population of persistent cells is resistant to anoikis and it depends on the expression of the Mcl-1 survival protein. To better understand this escape, we investigated the impact of the EZH2 methyltransferase, a protein overexpressed in cancers and involved in the methylation of the p16INK4 locus. We observed that the expression of the methylase is inhibited following genotoxic treatment but EZH2 remains expressed in the PLD dividing subpopulation. In addition, the inhibition of EZH2 via GSK343 or RNA interference reduces the number of emerging cells whereas this was not the case of HDAC inhibitors. We concluded that EZH2 is involved in cell emergence following chemotherapy treatment. We performed a proteomic analysis after EZH2 inhibition to characterize the targets that could be involved in senescence escape. This analysis identifies genes involved in self renewal and maintenance of a stem cell phenotype. We are actually validating these preliminary results. Keywords : Chemotherapy – Senescence – EZH2 – Escape – Resistance – Colorectal Cancer HENNI SAMIR 2nd year PhD student – University Hospital, Angers Impact of nilotinib treatment on subclinical cardiovascular biomakers Henni S (1), Gardembas M (2), Corby A (1), Omarjee L (2), Ifrah N (1), Lefthériotis G (2) 1) Vascular Exploration Department, University Hospital Center of Angers 2) Hematology Department, University Hospital Center of Angers Contact: samir.henni@chu-angers.fr Background: Nilotinib is a tyrosine kinase inhibitor targeting the BCR-ABL1 oncoprotein. It has been approved for the treatment of chronic myeloid leukemia-CP. Recently, a higher rate of cardiovascular events (CVE) have been reported as part of the non hematological nilotinib related adverse events. Although the CVE events may be influenced by several factors including pro-atherogenic and anti-angiogenic properties of nilotinib, the contribution in the atherosclerotic process still remains unclear. The purpose of this cross-sectional study was to determine the impact of nilotinib treatment on subclinical atherosclerotic biomarkers. Methods: 15 patients (mean 44 yrs ±14 yrs, 8 men) treated with nilotinib (Nilo) were compared to 30 age- and gender-matched patients with metabolic syndrom (MS) and 30 healthy controls. Subclinical atherosclerosis was assessed by the presence of carotid, aortic and femoral plaques, intima media thickness (IMT), arterial stiffness (Beta index), ankle brachial pressure index (ABI), and brachial arterial pressure (BAP). The overall cardiovascular risk (CVR) was determined according the 10 years-Framingham CV score. Statistical differences (p < 0.05) between the groups were determined by Chi2 and Kruskal-Wallis or ANOVA statistics as appropriate (Stata 12.1). Results: Median duration exposure to nilotinib was 1299 days (range 252-2196). Plaques were significantly more frequent (p = 0.007) in the Nilo group (53%) than in healthy controls (13 %,) but no difference between Nilo and MS group (63%, p = 0.371). There were no statistically significant differences in IMT (p = 0.271), Beta stiffness index (p = 0.428), ABI and BAP. The CVR score in Nilo group was similar than in the control group ( p = 0,136). Conclusions: Patients treated with nilotinib exhibited a higher amount of plaques although at a lower CVR score. These data would suggest that patients treated with nilotinib, likely develop focal lesions rather than a systemic arteriosclerotic process. Subclinical cardiovascular biomarkers could provide more targeting options for the personalized follow-up of patients treated with nilotinib. However, these preliminary data need to be confirmed in a larger longitudinal study. PENSEC CINDY 2nd year PhD student – Biofortis Mérieux NutriSciences Chemotherapy Impact on the Gut Microbiome of Patient-Derived Tumor Xenograft Models C. Pensec1*, A. De Martino1, IMODI Consortium2, S. Le Fres,e1, A. Groh3, Y. Amouzou1, S. Leuillet1, D. Guenit3, M. Campone4, T. Carton1, F. Le Vacon1 1 Biofortis Mérieux NutriSciences, Saint-Herblain, France, 2 http://www.imodi-cancer.org/, 3 EA 3430, University of Strasbourg, France, 4 UMR892, CRCNA, University of Nantes-Angers, France The French consortium IMODI (Innovative MODels Initiative) including 18 partners, aims to develop predictive preclinical models for new chemotherapeutic treatments discovery, to progress toward personalized medicine. These mouse models, named Patient-Derived Tumor Xenograft (PDX), are well characterized and concern 9 types of human cancer. In the last decade, the gut microbiota has been recognized as a powerful environmental factor that could influence the cancer development and therapeutic responses. In IMODI project, Biofortis Mérieux NutriSciences explores the chemotherapy impact on the gut microbiota of PDX models. Having established PDX models, mice were treated with chemotherapeutic agents. Stool analyses were assessed from 4 sampling times: Before treatment/ End of treatment/ One week post-treatment/ End of experiment. The gut microbiota composition during chemotherapy was studied by 16S rRNA genes metasequencing. Body weight and tumor volume were measured, in all groups, during the experiment. We will present our involvement in IMODI project and our first result about the impact of some chemotherapeutic agents on the gut microbiota composition. Microbiota represents an important additional “indicator” for evaluating the efficacy and the toxicity of pharmacological treatment that should be taken into account in the development of new therapeutic molecules. Keywords: Microbiota, Chemotherapy, PDX, Metasequencing DRUT AMANDINE 2nd year PhD student – Unité de Nutrition et d’Endocrinologie, Oniris Vitamin B12 (cobalamin) status in cats with chronic kidney disease Drut Amandine*, Unité de Pathologie Médicale des Carnivores Domestiques, Oniris, Nantes, France Siliart Brigitte, Unité de Nutrition et d’Endocrinologie, Oniris, Nantes, France Mallem Yassine, Unité de Pathophysiologie Animale et Pharmacologie Fonctionnelle, Oniris, Nantes, France Nguyen Patrick, Unité de Nutrition et d’Endocrinologie, Oniris, Nantes, France Simard Gilles, Département de Biochimie et Génétique, Centre Hospitalier Universitaire, Angers, France Soetart Nicolas, Unité de Pathologie Médicale des Carnivores Domestiques, Oniris, Nantes, France The kidneys are involved in the metabolism and possibly in the storage of cobalamin (vitamin B12). In humans, vitamin B12 deficiency is encountered in renal patients and may be detected before measurable hypocobalaminemia, by demonstrating hyperhomocysteinemia and methylmalonic acidemia. A previous study showed that homocystein is significantly higher in cats with naturally occurring chronic kidney disease compared with controls. We hypothesized that cobalamin disturbances would be encountered in cats with chronic kidney disease, and would be partly involved in the gastrointestinal signs and hematological abnormalities (nonregenerative anemia) seen in those patients. The first part of our study will aim at determining the pre-analytical conditions for the measurement of plasma homocysteine and methylmalonic acid in eight healthy laboratory cats. The second part of our work will be designed to establish the reference values for the measurement of plasma homocysteine and methylmalonic acid in 120 privately-owned healthy adult cats. The effect of age, breed, sex, body condition score and vitamin B12 content of the food on plasma homocysteine and methylmalonic acid in cats will also be determined. The third part of our study will assess the status of vitamin B12 in cats with naturally-occurring chronic kidney disease and will seek to establish a link with clinical and biological abnormalities. Keywords : cobalamin, cat, homocysteine, methylmalonic acid, chronic kidney disease TESSOULIN BENOIT 2nd year PhD student – CRCNA UMR 892, Nantes Oxidative metabolism targeting in Multiple Myeloma deficient for p53 pathway. Multiple Myeloma (MM) remains an incurable plasma cell malignancy, and patients harboring abnormalities of TP53 (deletion and/or mutation) have a dismal prognosis, whatever the treatment intensity. PRIMA-1Met is a compound selected by phenotypic screening, assumed to reactivate p53 proteins in SAOS2-His-273 by binding p53. We recently demonstrated that the sensitivity of human Myeloma Cell Lines (HMCLs) did not correlate with myeloma genomic heterogeneity or TP53 status, and PRIMA-1Met did not induce or increase expression of the p53 target genes (e.g. CDKN1A). However, PRIMA-1Met increased the expression of NOXA in a p53-independent manner, and NOXA silencing decreased PRIMA1Met-induced cell death. The PRIMA-1Met toxicity was associated with a burst of reactive oxygen species (ROS) and a decrease in glutathione cellular content (GSH). HMCLs with low GSH synthetase levels, measured by Gene Expression Profiling, had a significantly lower lethal dose (LD50) of PRIMA-1Met. Thus, inhibiting the endogenous GSH generation, either by silencing GSH synthetase or by inhibiting the γ-glutamyl cystein-synthase (Buthionin-Sulfoximin [BSO]), we strongly enhanced the PRIMA-1Met efficacy in HMCLs. The PRIMA-1Met resistance was overcome in most samples when associated with BSO in HMCLs and primary cells. This synergy was further demonstrated in TP53KO tumors subcutaneously xenografted in SCID-beige mice without overwhelming or unexpected toxicity. We’ll determine whether cell death is dependent on specific mitochondrial depolarization (i.e. BAX/BAK dependent cell death). Underlying metabolism regulation in our HMCLs will be investigated by SeaHorse® and sequencing of metabolic key regulators, to determine whether it’s predictive of PRIMA-1Met sensitivity. Our work will further investigate associations with PRIMA-1Met, with commonly used MM drugs. From a translational point of view, regarding the unmet need of treatment for patients with p53 abnormalities in MM, the best combinations will be tested in mice, especially in 5T mouse model of MM (Pr vanderkerken). PAOLINI LEA 3rd year PhD student - CRCNA UMR INSERM 892 – CNRS 6299 – UA Identification of a vaccine carrier protein to induce protective antitumor immune responses PAOLINI Léa * , DELNESTE Yves , BEAUVILLAIN Céline et JEANNIN Pascale In addition to conventional cancer therapies (surgery, chemotherapy, radiotherapy), immunotherapies, based on manipulating the immune system, have emerged as potent therapeutic alternatives with long lasting protection and limited side effects. Among other strategies, active immunotherapy (or therapeutic vaccination) aims to induce tumor-specific cytotoxic T lymphocyte (CTL). However, to induce CTL remains a challenge in vaccinology. We have identified a microbe-derived carrier protein (named MC; patenting in progress) that binds to endocytic receptors expressed by dendritic cells and involved in antigen presentation. We have produced a chimeric protein consisting in MC fused to ovalbumin (Ova), used as a model antigen. Results showed that recombinant MC-Ova protein (i) favours in vitro Ova presentation to CD8+ T cells, (ii) induces in vivo CTLs in wild type mice (iii) and delays the development of Ova-expressing tumor cells. Finally, we have observed that MC-Ova induces CTL in neuOT-I/OTII transgenic mice that express Ova in mammary epithelium and are tolerant to Ova, when combined with immune checkpoint blockade. Actually, we are evaluating the potential of MC-Ova in a model of therapeutic vaccination. These results identify MC as a promising vaccine for CTL-based vaccines, especially as it could be tagged with personalized immunogenic molecules. Keywords: cancer vaccine, immunotherapy, cross-presentation ADAM CLEMENT 2nd year PhD student – CRCNA – UMR Inserm 892 – CNRS 6299 – UA Lactic acid controls the differentiation of human macrophages ADAM Clément*,DELNESTE Yves, JEANNIN Pascale Bat IBS 4 rue Larrey CHU d’Angers 49933 Angers In case of infections or tissue injury, peripheral blood monocytes are recruited into tissues where they differentiate into macrophages (M). Macrophages are plastic cells, meaning that their differentiation is tightly controlled by environmental factors such as nutrients, cytokines or ligation of pattern recognition receptors. As a consequence of the diversity of differentiating molecules, a plethora of M subsets, with distinct phenotypes and functions, exist with pro-inflammatory M (M1-type) and immunoregulatory M (M2-type) representing the extremes of a spectrum of phenotypes. The generation and biology of M1-type and M2-type M can be analysed in vitro by culturing monocytes with GM-CSF and M-CSF, respectively. In solid tumours, infiltrating tumour-associated M (TAM) are M2 polarized cells which accumulation is correlated with a poor prognosis. To understand the mechanisms involved in their local polarization is thus crucial to propose strategies targeting TAM. In this study, we focus on the impact of lactic acid (LA), a glycolysis by-product which concentration is elevated in the tumour environment, on M polarization. Results showed that M generated in the presence of the pro-M1 cytokine GM-CSF acquire a non-conventional phenotype when cultured in the presence of LA. More precisely, LA favours autocrine M-CSF consumption, thereby enhancing the expression of some M2 markers, and inhibits the production of IL-12. However, and surprisingly, LA increases the production of the pro-inflammatory cytokines IL-1β and TNF. These results suggest that LA induces the differentiation of monocytes into a new M subset, exhibiting a mixed polarization profile. Keywords: Macrophages, tumor, polarization LAROCHETTE VINCENT 2nd year PhD student – INSERM UMR 892, Université d’Angers IL-26 reduces HCV replication by interfering with viral RNA replication Vincent Larochette*, Élodie Beaumont, Laurence Preisser, Simon Blanchard, Jonathan Dauvé, Minna Poranen, Alain Morel, Helmut Fickenscher, Yves Delneste, Philippe Roingeard, Pascale Jeannin University of Angers, France; Inserm, unit 892, Angers, France Interleukin-26 (IL-26), a recently discovered cytokine, has been described as a pro-inflammatory mediator mainly produced by lymphoid cells. Studies have reported that IL-26 is overexpressed during chronic inflammatory disorders. We recently reported that (i) the levels of circulating IL-26 were increased in patients with chronic hepatitis C infection and, (ii) that IL-26 enhances the cytotoxic activity of natural killer cells against HCV-infected cells. Our results also suggested a direct antiviral role of IL-26 against HCV infection. We observed, in an in vitro model of HCV infection using the hepatocarcinoma cell line Huh7.5 and the JFH-1 optimized HCV strain, that IL-26 protects hepatocytes against HCV. We demonstrated that this protection relies on the capacity of IL-26 to interact with viral RNA and to inhibit its replication. In parallel, we excluded that the protective role of IL-26 may result from an inhibition of virus entry or in the induction of antiviral genes by hepatocytes. Collectively, this study demonstrates that IL-26 may protect against HCV infection in an immune-independent mechanism and suggests that IL-26 may exhibit non-conventional properties. At this time, we are exploring the consequences of the interaction of IL-26 with viral nucleic acids on the activation of innate immune cells. Keywords: Immunology, Interleukin-26, Hepatitis C, dsRNA REDA AL SAYED-ZEINA 2nd year PhD student – l’institut du Thorax, INSERM UMR 1087 CNRS 691 Elucidate the role of IRX5 in human ventricular electrical conduction using cardiomyocytes derived from human induced pluripotent stem cells Zeina Reda Al Sayed*1, Mariam Jouni1, Guillaume Lamirault1, Patricia Lemarchand1, Carine Bonnard2, Bruno Reversade2, Nathalie Gaborit1. 1. Inserm UMR1087, CNRS UMR6291, l'institut du thorax, Nantes, France 2. A*Star, Institute of Medical Biology, Singapore IRX5 is one of the Iroquois transcription factors which are known to control embryonic and post embryonic developmental tissue pattering and function. Studies on murine heart have revealed a role for IRX5 in regulating ion channel gene expression throughout ventricular wall. However due to limitations inherent to current models including animal and human cell lines transfected with DNA constructs or transgenic animal models, the role of IRX5 transcription factor in the human heart has not been investigated. Hamamy syndrome affected patients, having a single mutation in IRX5 gene, present with cardiac electrical defects suggesting a role for IRX5 in modulating ion channel gene expression in the human heart. Taking advantage of these patient somatic cells reprogrammed into human induced pluripotent stem cells and differentiated into cardiomyocytes (hiPS-CM), we will search for IRX5 potential ion channel gene targets by revealing mRNA and protein expression variation in the mutated iPS-CM. Also, we will investigate whether IRX5 directly impact its target gene promoters by chromatin immunoprecipitation and we will identify the cofactors that are interacting with IRX5 by co-immunoprecipitation. Finally, we will analyze the impact of IRX5 mutation on the global electrophysiological activity by patch clamp. The work we performed during my first year PhD suggest that IRX5 regulates the expression of the ion channel SCN5A in human cardiomyocytes through a direct interaction to its promoter and that IRX5 binds to IRX3 and GATA4 to regulate gene expression. Keywords: Transcription factors; ion channels; human induced pluripotent stem cells; cardiomyocytes. SMITH AUDREY 2nd year PhD student - LIOAD INSERM U791 Human MSCs encapsulation in cytoprotective hydrogel: new prospect in osteoarthritis treatment? Smith A.(1)*, Hached F.(1), Vinatier C.(1), Marquis M.(2), Billon-Chabaud A.(1), Grimandi G.(1,3), Renard D.(2), Guicheux J.(1,4), Le Visage C.(1) (1) INSERM U791, LIOAD, Université de Nantes, UFR odontologie, Nantes F-44042, France ; (2) UR1268 BIA (Biopolymères Interactions Assemblages), INRA, 44300 Nantes, France ; (3) CHU de Nantes, PHU11 Pharmacie, Pharmacie Centrale F-44042, France ; (4) CHU Nantes, PHU 4 OTONN, Nantes F-44042, France Osteoarthritis is a degenerative joint disease in which cartilage degeneration goes along with synovium inflammation. Mesenchymal Stromal Cells (MSCs) ability to secrete anti-inflammatory and immuno-modulatory factors represents an attractive tool in the treatment of diseases with inflammatory components. In this context, MSCs could offer a great support to manage synovium inflammation. Considering the risk of cell leakage and the massive cell death upon intra-articular injection, cell encapsulation therefore could (i) allow limitation of cell death, (ii) avoid cell effusion outside the articular space, and (iii) supply a suitable micro-environment supporting the biological activity of MSCs. Here we have selected biocompatible biomaterials (alginate and silanized hydroxypropylmethylcellulose (Si- HPMC)) and encapsulated human MSCs into these hydrogels. We have compared several methods (dripping, emulsification, soft lithography) to prepare hydrogel-based particles with a controlled size, ranging from 80 microns to more than 1.5 mm, and demonstrated cell viability after encapsulation. Future work will focus on the bioactivity of the encapsulated MSCs, i.e their capacity to modulate the immune response through the secretion of factors that have immune-modulatory, pro-angiogenic, anti-apoptotic, anti-fibrotic, and anti-inflammatory effects. ABDEL MALAK INAS 2nd year PhD student – LABERCA Identification of Dechloranes by gas chromatography coupled to tandem mass spectrometry with atmospheric pressure chemical ionisation (GC-APCI-MS/MS) Abdel Malak I1,2*, Cariou R1, Dervilly-Pinel G1, Jaber F2, Le Bizec B1 1LUNAM Université, Oniris, Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), F-44307, Nantes, France; 2Lebanese University, Faculty of Sciences I, Laboratory of Analysis of Organic Compounds (LACO) - 508 - Hadath, Beirut, Lebanon. Flame retardants (FRs) are chemicals added to a wide range of consumer and industrial products in order to limit fire initiation and propagation in a context of fire safety. Some halogenated FRs has been registered to the United Nations Stockholm Convention annexes, due to persistence in the environment, long-range transport, bioaccumulation and toxicity (1). Yet, Dechlorane (syn- and anti-Dechlorane plus, Dechlorane 601, 602, 603, 604, 604CB and Chlordene Plus) are halogenated flame retardants (FRs) still used but raising concern (2). They have been detected in various environmental matrices and in a different aquatic and terrestrial biota (3), thus showing their bioaccumulation and biomagnifications potential. Dechlorane Plus (DP) was produced for the first time by OxyChem in North America more than 40 years ago. The technical product consists of the syn and anti isomers in a ratio of ~1:3. DP has a very high production volume (500-5000 t.y 1) and is used worldwide (4). As regard to analytical aspects, most of the authors use gas chromatography coupled to mass spectrometry (sometimes with tandem mass analysers) with electron impact (EI) or negative chemical ionisation (NCI). Such ionisation modes exhibit important (NCI) or extensive (EI) fragmentation, thus limiting specificity of monitored diagnostic ions. We have investigated the fragmentation patterns of a range of Dechloranes by Atmospheric Pressure Chemical Ionisation (APCI) to compare the results with Electron Impact (EI) (5). Thus, highly specific precursor ions were selected and transitions were optimised (selection of fragment, collision energy) on a triple quadruple mass spectrometer. Finally, a Selected Reaction Monitoring (SRM) method was defined. Thus, on-going work relies to the development and optimisation of sample preparation method dedicated to feed and foodstuffs in order to eventually produce occurrence data useful for human dietary exposure assessment. Keywords: Flame Retardants, Dechloranes, Mass Spectrometry, Atmospheric Pressure Chemical Ionisation, Selected Reaction Monitoring. References 1. Chen K, Zheng J, Yan X, Yu L, Luo X, Peng X, Yu Y, Yang Z, Mai B. Dechlorane Plus in paired hair and serum samples from e-waste workers: Correlation and differences. Chemosphere, 2015: 43–47.- 2. Barón E, Eljarrat E, Barceló D. Analytical method for the determination of halogenated norbornene flame retardants in environmental and biota matrices by gas chromatography coupled to tandem mass spectrometry. Journal of Chromatography A, 2012: 154-160. 3. Sühring R, Busch F, Fricke N. Distribution of brominated flame retardants and dechloranes between sediments and benthic fish — A comparison of a freshwater and marine habitat. Science of the Total Environment, 2016: 578–585. 4. Guerra P, Fernie K, Jiménez B, Pacepavicius G, Shen L, Reiner E, Eljarrat, Barceló, Alaee. Dechlorane Plus and Related Compounds in Peregrine Falcon (Falco peregrinus) Eggs from Canada and Spain. Environmental Science and Technology, 2011: 1284–1290 5. L’Homme B, Calaprice C, Damiana Calvano C, Zambonin C, Leardi R, Focant J-F. Ultra-trace measurement of Dechloranes to investigate food as a route of human exposure. Chemosphere, 2015: 525–533. 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