Éditeur(s) : HAL CCSD
Résumé : Spermatogenesis in mammals is a complex biological function including cellular processes such as proliferation, meiosis and differentiation, aiming to the production of male gametes in the testis. If the seminiferous epithelium is well described in terms of organization and cellular morphology of cells that compose it, the processes by which undifferentiated diploid germ cells enter meiosis and give haploid cells that undergo many morphological transformations, are not fully decrypted. These processes rely on the coordinated and sequential expression of genes, including specific products for each stage of germ cell development These gene products are essential at each key stage of spermatogenesis. Transcriptomics since the 1990s, and proteomics since the 2000s have contributed to the improved. understanding of these mechanisms. A long term proteomic study aiming at characterizing the proteomes of Sertoli cells and germ cells, and a recent study that characterized and quantified the transcriptome of isolated rat testicular cells at high resolution using de novo sequencing of transcripts (RNA-Seq), have been the basis of my thesis work. The latter study showed the accumulation of long non-Coding RNAs (lncRNAs) and testicular unannotated transcripts (TUTs) at meiotic and post-Meiotic stages of spermatogenesis in the rat. In this context, my thesis work aimed at validating the coding potential of many genes expressed in germ cells using RNA-Seq combined with shotgun proteomics, a so-Called PIT (Proteomics Informed by transcriptomics) approach. In this approach, the protein sequences translated from the transcripts assembled by RNA-Seq in the different testicular cell types are integrated into a custom database of protein sequences used to query mass spectrometry data obtained from proteins of meiotic and post-Meiotic cells. The PIT approach showed that 69 TUTs or lncRNA (corresponding to 44 loci) code for proteins in meiotic cells and post meiotic cells, and we confirmed experimentally the meiotic and post-Meiotic expression for two new transcripts encoding for VAMP9, a protein of the SNARE family, and a new testicular enolase T-ENOL. The post-Meiotic expression of T-ENOL protein was confirmed by immunohistochemistry using a polyclonal antibody raised against the recombinant protein. This approach also allowed us to identify new isoforms of known proteins, specific to each stage of spermatogenesis. Germ cells and Sertoli cells maintain a dialogue which is necessary to the success of spermatogenesis and spermiogenesis. Another part of my work aimed at identifying membrane proteins, in germ cells and residual bodies, that may be involved in the dialogue between Sertoli cells and germ cells, using a ICPL relative quantification proteomic approach. The ICPL analysis enabled us to establish a list of 166 proteins whose expression is differential between pachytene spermatocytes, round spermatids and residual bodies. Their differential expression suggests that these proteins may play a role in spermiogenesis. Thanks to the Gene Ontology annotations, a list of 8 proteins with a putative role in signal transduction, cell recognition or differentiation, thus potentially involved in the dialogue between Sertoli and germ cells was drawn. In addition, I provided a first proteome of rat Sertoli cells, germ cells and residual bodies obtained by shotgun proteomics.
La spermatogenèse chez les mammifères est une fonction biologique complexe incluant des processus de prolifération cellulaire, de méiose et de différenciation uniques visant à la production des gamètes mâles au sein du testicule. Si l’épithélium séminifère est bien décrit sur le plan de son organisation et de la morphologie des cellules qui le composent, les processus par lesquels les cellules germinales diploïdes indifférenciées entrent en méiose pour donner ensuite des cellules haploïdes subissant par la suite de nombreuses transformations morphologiques, ne sont pas totalement décryptés. Ils reposent sur l’expression coordonnée et séquentielle de gènes dont les produits spécifiques de chaque stade de développement des cellules germinales sont essentiels aux étapes clés de la spermatogenèse. La transcriptomique depuis les années 1990 et la protéomique depuis les années 2000 ont contribué à l’amélioration de la connaissance de ces mécanismes. Une étude protéomique visant à caractériser par des approches systématiques et différentielles les protéomes des cellules de Sertoli et de la lignée germinale, et d’autre part une étude récente, réalisée dans notre unité, qui a permis de caractériser et de quantifier le transcriptome des cellules testiculaires isolées de rat en utilisant le séquençage de novo des transcrits (RNA-Seq), ont été à la base de mes travaux de thèse. Cette dernière étude a mis en évidence l’accumulation de longs ARNs non codants (lncRNAs) et de transcrits testiculaires non annotés (TUTs) aux stades méiotique et post- méiotique de la spermatogenèse chez le rat. Dans ce contexte, mon travail a consisté à valider le potentiel codant de nombreux gènes exprimés dans les cellules germinales par une approche dite PIT (Proteomics Informed by Transcriptomics) couplant protéomique Shotgun et RNA-Seq. Dans ce type d’approche, les séquences protéiques déduites des transcrits des différents types cellulaires, assemblés par RNA-Seq, sont intégrées dans une base personnalisée de séquences protéiques utilisée pour interroger les données de spectrométrie de masse obtenues à partir de protéines de cellules méiotiques et post-Méiotiques. L’approche PIT a permis de montrer que 69 TUTs ou lncRNA (correspondant à 44 loci) codent pour des protéines dans les cellules méiotiques et post méiotiques. L’expression post-Méiotique de deux nouveaux transcrits, l’un codant pour la protéine VAMP9, une protéine de la famille SNARE, et l’autre pour une nouvelle énolase T-ENOL a pu être confirmée. L’expression post-Méiotique de T-ENOL a été confirmée par immunohistochimie à l’aide d’un anticorps polyclonal produit contre la protéine recombinante. Cette approche nous a également permis d’identifier de nouvelles isoformes de protéines connues spécifiques de chaque stade de la spermatogenèse. Les cellules germinales et les cellules de Sertoli entretiennent le dialogue nécessaire au bon déroulement de la spermatogenèse. Une autre partie de mon travail a consisté à identifier des protéines membranaires des cellules germinales et des corps résiduels, susceptibles d’intervenir dans le dialogue entre les cellules de Sertoli et les cellules germinales, par une approche protéomique de quantification relative ICPL. Cette approche a permis d’établir une liste de 166 protéines différentiellement exprimées entre les spermatocytes pachytène, les spermatides rondes et les corps résiduels, qui sont susceptibles de jouer un rôle dans la spermiogénèse. Grâce aux annotations de le Gene Ontology, j’ai pu établir une liste de 8 protéines ayant un rôle supposé dans la transduction du signal, la reconnaissance cellulaire ou bien la différenciation. Par ailleurs, j’ai pu établir par protéomique Shotgun un premier protéome des cellules de Sertoli, des cellules germinales et des corps résiduels chez le rat.
https://tel.archives-ouvertes.fr/tel-01127389

Droits : info:eu-repo/semantics/OpenAccess
NNT : 2014REN1S064
tel-01127389
https://tel.archives-ouvertes.fr/tel-01127389
https://tel.archives-ouvertes.fr/tel-01127389/document
https://tel.archives-ouvertes.fr/tel-01127389/file/2014REN1S064.pdf

Éditeur(s) : HAL CCSD American Society for Microbiology
Résumé : International audience
A rapid and specific high-performance liquid chromatography method with UV detection (HPLC-UV) for the simultaneous determination of 12 beta-lactam antibiotics (amoxicillin, cefepime, cefotaxime, ceftazidime, ceftriaxone, cloxacillin, imipenem, meropenem, oxacillin, penicillin G, piperacillin, and ticarcillin) in small samples of human plasma is described. Extraction consisted of protein precipitation by acetonitrile. An Atlantis T3 analytical column with a linear gradient of acetonitrile and a pH 2 phosphoric acid solution was used for separation. Wavelength photodiode array detection was set either at 210 nm, 230 nm, or 298 nm according to the compound. This method is accurate and reproducible (coefficient of variation [CV] < 8%), allowing quantification of beta-lactam plasma levels from 5 to 250 μg/ml without interference with other common drugs. This technique is easy to use in routine therapeutic drug monitoring of beta-lactam antibiotics.
ISSN: 0066-4804

hal-00875271
https://hal.archives-ouvertes.fr/hal-00875271
https://hal.archives-ouvertes.fr/hal-00875271/document
https://hal.archives-ouvertes.fr/hal-00875271/file/Simultaneous_Determination-accepted.pdf
PUBMED : 21788467

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Glioblastoma (GB), the most frequent primary tumor of the central nervous system, remains one of the most lethal human cancers despite intensive researches. Current paradigm in the study of GB has been focused on inter-patient variability and on trying to isolate new classification elements or prognostic factors. Here, using ICPL, a technique for protein relative quantification by mass spectrometry, we investigated protein expression between the four regions of GB on clinically relevant biopsies from 5 patients. We identified 584 non-redundant proteins and 31 proteins were found to be up-regulated in the tumor region compared to the peri-tumoral brain tissue, among which, 24 proteins belong to an interaction network linked to 4 biological processes. The core of this network is mainly constituted of interactions between beta-actin (ACTB) with heat shock proteins (HSP90AA1, HSPA8) and 14-3-3 proteins (YWHAZ, YWHAG, YWHAB). A cluster of three isoforms of the sodium pump α-subunit (ATP1A1, ATP1A2, ATP1A3) was also identified outside this network. The differential expression observed for ACTB and 14-3-3γ was further validated by western blot and/or immunohistochemistry. Our study confirms the identity of previously proposed molecular targets, highlights several functional processes altered in GB such as energy metabolism and synaptic transmission and could thus provide added value to new therapeutic trails.
ISSN: 1874-3919

hal-00877756
https://hal.archives-ouvertes.fr/hal-00877756
DOI : 10.1016/j.jprot.2012.04.034
PUBMED : 22575386

Résumé : Bacteriological study of abyssal sediments: During the Demeraby mission of the N/O Jean Charcot in the western Atlantic Ocean, a study of the microflora of the digestive tracts of echinoderms (Holothuroidea of various genera, Asteroidea, Echinoidea) was compared with that of the surrounding sediment. The bacterial concentration observed in the digestive tracts of all the animals studied is higher than that in the surrounding sediments. The largest numbers were always found in the most anterior part of the digestive tracts of the holothurians. In some cases, the increase is considerable (7.10 versus 5.2. 10 cells per millilitre): It indicates a proliferation of bacterial cells in the anterior part of the digestive tract. As the sediment moves through the digestive tract, the bacterial level progressively decreases. In the most posterior part, the concentration is, however, always clearly greater than that in the surrounding sediment. In the case of the holothurian of the genus Deima, analysis according to the Adamsonian principle of comparison shows that the microflora isolated from the various parts of the digestive tract is composed of taxonomic groups showing little resemblance to those isolated from the sediment. In the case of the holothurian of the genus Pseudostichopus, no specialisation of the microflora from digestive tract contents appears with respect to that of the ingested sediment. Examination of metabolic activities suggests a juxtaposition of two types of communities: Free sediment - sediment in transit that complement one other in their enzymatic and nutritional behaviour Indeed, the strains of free sediment seem to be rather suitable for hydrolysing and using carbohydrates, while those of enteric communities prefer the hydrolysis of lipids and proteins and the assimilation of fatty acids, dicarboxylic acids, hydroxylic acids and certain amino acids. Moreover, the enteric microflorae possess a greater nutritional versatility that, linked with its total numbers, has a sizeable catabolic potential. .../... -2- Measurements of heterotrophic activity done by means of 14 C glucose tracers make it possible to show that the metabolic activities of the bacterial communities of superficial abyssal sediments vary according to zones. In the Amazon Cone, a clear glucose mineralization gradient over a distance of a few kilometres can be observed. In the abyssal plain of Demerara heterotrophic activities are slowed in relation to those of the superficial sediments of the Cone of the Amazon, even though the bacterial numbers and the depths are of the same order of magnitude in the two zones. The abundant microflorae associated with the various divisions of digestive tract contents of the Deima abyssal holothurian manifest little metabolic activity as regards the glucose. However, the bacterial communities of Pseudostichopus and Psychropotes manifest very strong heterotrophic activities, increasing from the oesophagus towards the cloaca. Current results are too fragmentary to allow us to observe possible physiological specialisation of enteric bacterial communities enabling the facilitation of digestion processes in the host. Conclusion: In the abyssal environment, characterised by poorly developed bacterial communities, microflorae associated with the digestive tract contents of echinoderms have considerably greater cellular concentrations. By means of their enzymatic equipment, these enteric communities, which complement those of the free microflorae of superficial sediments, can participate in the transformation of organic matter in the abyssal area. Study of the dynamics of microbial communities and their heterotrophic activities in shellfish farms: From experiments done on the water in the Arcachon basin, trials to quantify and describe the reactivity potential of bacterial communities were performed. Bacterial communities were defined by direct counts, counts after setting them up for cultivation, structure and diversity of these communities, dosage of AMP, ADP, ATP. Two opposite ecological conditions were considered: Autumn conditions (high temperature) and a winter conditions (low temperature). Tubs of sea water of 6 and 60 litres were enriched either with ammonium chloride or a mixture of amino acids, at about 500 uAtg N.L. The experiments lasted 24 hours, with sampling every 3 hours. The autumn community reacted rapidly (6 hours), the response to the enrichment in organic nitrogen being the highest. The structure of the community has unstable immature stages. On the contrary, during the winter experiment, no response could be perceived. This may mean that the latency phase is greater than 24 hours. The structure of this community has mature stages. In conclusion, bacterial communities react in the same way as macroscopic communities. Stable communities are diversified and correspond to mature stages of the ecosystem; the immature stages are unstable and less diversified. This type of experiment has made it possible to bring to light the rapidity of response of bacterial populations, and can be used to define the stability of a bacterial community in a precise biotope.
ETUDE BACTERIOLOGIQUE DES SEDIMENTS ABYSSAUX RESUME Au cours de la mission DEMERABY du n/o Jean CHARCOT en atlantique occidental , une étude de la microflore des tractus digestifs d'Echinodermes (Holothurides de différents genres, Astérides, Echinides) a été comparée à celle du sédiment environnant. La concentration bactérienne observée dans les tractus digestifs de tous les animaux étudiés est supérieure à celle des sédiments environnants. Les effectifs les plus importants sont toujours observés dans la partie la plus antérieure des tractus digestifs des holothuries. Dans certains cas, l'accroissement est considérable (7.10 contre 5,2. 10 cellules/ml) : il indique une prolifération des cellules bactériennes dans la partie antérieure du tractus digestif. Au.cours de la progression du sédiment dans le tractus digestif, la charge bactérienne décroit progressivement. Dans la partie la plus postérieure, la concentration est cependant toujours nettement supérieure à celle du sédiment environnant-. Dans le cas d'une holothurie du genre Deima, l'analyse selon le principe adansonien de comparaison montre que la microflore isolée des différentes parties du tractus digestif est constituée de groupes taxonomiques présentant peu de ressemblance avec ceux isolés du sédiment. Dans le cas d'une holothurie du genre Pseudostichopus il n'apparait pas de spécialisation de la microflore du contenu du tractus digestif par rapport à celle du sédiment ingéré. ¿ ¿ . L'étude des activités métaboliques suggère une juxtaposition de deux types de communautés : sédiment libre-sédiment en transit qui se complètent dans leur comportement enzymatique et nutritionnel. En effet les souches du sédiment libre semblent être plutôt aptes à hydrolyser et à utiliser les glucides, tandis que celles des communautés entériques portent leur préférence sur l'hydrolyse des lipides et protéines et 1'assimilation des acides gras, acides dicarboxyl iques, hydro'xy-acides et certains acides aminés. D'autre part les microflores entériques possèdent une versatilité nutritionnelle plus importante qui, liée à leurs effectifs, offre une potentialité catabolique non négligeable. .../... -2- Les mesures d'activité hétérotrophe effectuées au moyen de traceurs glucose 14 C permettent de montrer que les activités métaboliques des communautés bactériennes des sédiments abyssaux superficiels varient selon les zones. Dans le Cone de l'Amazone on observe un net gradient de minéralisation du glucose sur une distance de quelques kilomètres. Dans la plaine abyssale de Demerara les activités hétérotrophessont ralenties par rapport à celles des sédiments superficiels du Cone de l'Amazone, bien que les effectifs bactériens et les profondeurs soient du même ordre de grandeur dans ces deux zones. Les abondantes microflores associées aux différentes fractions du contenu du tractus digestif de l'holothurie abyssale Deima ne manifestent que peu d'activités métaboliques vis à vis du glucose. Par contre les communautés . bactériennes de Pseudostichopus et P syahropotes manifestent des activités hétérotrophes très fortes, croissant de 1'oesophage vers le cloaque. Les résultats actuels sont trop fragmentaires pour permettre d'observer une éventuelle spécialisation physiologique des communautés bactériennes entëriques permettant de faciliter les processus de digestion chez l'hôte. Conclusion: dans le milieu abyssal, caractérisé par des communautés bactériennes faiblement développées, les microflores associées aux contenus de tractus digestif des Echinodermes présentent des concentrations cellulaires considérablement plus importantes. Par leurs équipements enzymâtiques ces communautés entëriques complémentaires de ceux des microflores libres des sédiments superficiels, peuvent participer à la transformation des matières organiques du domaine abyssal. ETUDE DE LA DYNAMIQUE DES COMMUNAUTES MICROBIENNES ET DE LEURS ACTIVITES HETEROTROPHES DANS LES PARCS CONCHYLICOLES RESUME A partir d'expérimentations effectuées sur l'eau du bassin d'Arcachon, des essais de quantification et qualification du potentiel de réactivité des communautés bactériennes ont été effectuées. Les communautés bactériennes ont été définies par comptage directs, comptages après mise en culture, structure et diversité de ces communautés, dosage des AMP, ADP, ATP. Deux situations écologiques opposées ont étées considérées . Une situation automnale (température élevée) et une situation hivernale (température faible). Des bacs d'eau de mer de 6 et 60 litres ont été enrichis , soit avec du chlorure d'ammonium, soit avec un mélange d'acides aminés, à environ 500 uAtg N.l" . Les expériences ont duré 24 heures, avec des prélèvements toutes les 3 heures. La communauté automnale réagit rapidement (6 heures), la ' réponse à l'enrichissement en azote organique étant la plus importante. La structure de la communauté présente des stades immatures instables. Au contraire, au cours de l'expérimentation hivernale, aucune réponse n'a pu être perçue. Ceci peut signifier que la phase de latence est supérieure à 24 heures. La structure . de cette communauté présente des stades matures. En Conclusion, les communautés bactériennes réagissent de la même façon que les communautés macroscopiques. Les communautés stables sont diversifiées et correspondent à des stades matures de l'écosystème, les stades immatures sont instables et moins diversifiés. Ce type d'expérience a permi de mettre en évidence la rapidité de réponse des populations bactériennes, et peut être utilisé pour définir la stabilité d'une communauté bactérienne dans un biotope précis.

Droits : info:eu-repo/semantics/openAccess
http://archimer.ifremer.fr/doc/1981/rapport-1926.pdf
http://archimer.ifremer.fr/doc/00000/1926/

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Rapid cold hardening (RCH) is a form of thermal acclimation that allows ectotherms to fine-tune their physiological state to match rapid changes in thermal environment. Despite progress in recent years, there is still a considerable uncertainty regarding the physiological basis of RCH in insects. Here we investigated the physiological response of adult Drosophila melanogaster to a gradual reduction of temperature from 25 to 0°C followed by 1h at 0°C. As expected, this RCH treatment promoted cold tolerance, and so we hypothesized that this change could be detected at the proteomic level. Using 2D-DIGE, we found that only a few proteins significantly changed in abundance, and of these, we identified a set of four proteins of particular interest. These were identified as two different variants of glycogen phosphorylase (GlyP) of which three spots were up-regulated and another was down regulated. In subsequent experiments, we quantified upstream events by measuring the GlyP mRNA amount, but we found no marked effect of RCH. We also examined downstream events by measuring GlyP activity and the level of free sugars. We found no effect of RCH on GlyP activity. On the other hand, screening of whole animal sugar contents revealed a small increase in glucose levels following RCH while trehalose content was unaltered. This study highlights a complex regulation of GlyP in relation to RCH where we found associations between the cold tolerance, the protein abundance and the metabolite concentrations but no changes in mRNA expression and enzyme activity. These data stress the necessity of combining the hypothesis-generating power of an 'Omics' approach with subsequent targeted validations across several levels of the biological organization. We discuss reasons why different biological linked levels do not necessarily change stoichiometrically.
ISSN: 0022-1910

hal-00991032
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00991032
DOI : 10.1016/j.jinsphys.2014.01.009
PUBMED : 24508557

Éditeur(s) : HAL CCSD
Résumé : Benzophenone derivatives (BPs) are used as UV filters in many different products. These lipophilic compounds were recently described as ubiquitous contaminant of different compartiments of aquatic systems, including fish. In this study, we evaluated estrogenic activity of ten BPs in a model fish species, the zebrafish (Danio rerio), by combining newly developed in vitro and in vivo models. In vitro, three specific zebrafish reporter gene models expressing zebrafish estrogen receptors (ZfERs) in zebrafish hepatic cell line (ZFL) were used.Estrogenic activity of BPs was quantify in vivo by using a newly developed transgenic zebrafish line that expresses green fluorescent protein (GFP) under the control of the cyp19a1b promoter. The cyp19a1b gene codes for the brain aromatase B that is under the control of estrogens. Vitellogenin (Vtg), a marker of estrogenic compound, was used to assess the potential estrogenic effect of certain BPs in male fish. Five of 10 BPs induced an estrogenic activity on the three models: BP2, THB, BP1, 4BP, while BP and 234 BP were non active. We noted diffrence in BPs ranking depending on the zfER subtypes suggesting ?/? receptor selectivity by certain BPs. By quantifying GFP expression in live zebrafish, we confirm the estrogenic activity of some BP, i.e., BP1, THB, 4BP and 44'BP, as shown by their ability to induce GFP in a concentration-dependent manner. However, the ranking of BPs as a function of their estrogenic activity assay is different between models. For instance, 4BP was the most estrogenic compound in vivo but not in vitro. Differences were also noticed regarding the estrogenic activity of BP2. In vitro, BP2 has strong activity but had no effect on GFP in cyp19a1b-GFP embryo. However, BP2 induced strongly Vtg in male. The lack of estrogenic activity of BP2 in embryo may be related to difference of biodisponibility and/or metabolisation of this compound between embryo and adult fish. Our approach allows to characterize the estrogenic potency of emerging pollutants, using innovative in vitro and in vivo assays within a unique model fish species. The differences observed between assays deserve further studies to characterize the behavior of compounds in the different biological models in term of biodisponibility and metabolism.
1. European Conference on the Replacement, Reduction and Refinement of Animal Experiments in Ecotoxicology
Dübendorf, Switzerland

ineris-00971019
https://hal-ineris.archives-ouvertes.fr/ineris-00971019
INERIS : EN-2012-243

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Among persisting questions on bone calcification, a major one is the link between protein expression and mineral deposition. A cell culture system is here proposed opening new integrative studies on biomineralization, improving our knowledge on the role played by non-collagenous proteins in bone. This experimental in vitro model consisted in human primary osteoblasts cultured for 60 days at the surface of a 3D collagen scaffold mimicking an osteoid matrix. Various techniques were used to analyze the results at the cellular and molecular level (adhesion and viability tests, histology and electron microscopy, RT-and qPCR) and to characterize the mineral phase (histological staining, EDX, ATG, SAED and RMN). On long term cultures human bone cells seeded on the osteoid-like matrix displayed a clear osteoblast phenotype as revealed by the osteoblast-like morphology, expression of specific protein such as alkaline phosphatase and expression of eight genes classically considered as osteoblast markers, including BGLAP, COL1A1, and BMP2. Von Kossa and alizarine red allowed us to identify divalent calcium ions at the surface of the matrix, EDX revealed the correct Ca/P ratio, and SAED showed the apatite crystal diffraction pattern. In addition RMN led to the conclusion that contaminant phases were absent and that the hydration state of the mineral was similar to fresh bone. A temporal correlation was established between quantified gene expression of DMP1 and IBSP, and the presence of hydroxyapatite, confirming the contribution of these proteins to the mineralization process. In parallel a difference was observed in the expression pattern of SPP1 and BGLAP, which questioned their attributed role in the literature. The present model opens new experimental possibilities to study spatio-temporal relations between bone cells, dense collagen scaffolds, NCPs and hydroxyapatite mineral deposition. It also emphasizes the importance of high collagen density environment in bone cell physiology.
ISSN: 1932-6203

hal-01142641
http://hal.upmc.fr/hal-01142641
http://hal.upmc.fr/hal-01142641/document
http://hal.upmc.fr/hal-01142641/file/Silvent_2013_Collagen.pdf
DOI : 10.1371/journal.pone.0057344

Éditeur(s) : HAL CCSD
Résumé : Food-grade dairy propionibacteria are nutraceutical producers. They release short chain fatty acids (SCFAs), folic acid and cobalamin, which play a role in intestinal physiology. SCFAs induce apoptosis via the mitochondrial death pathway. We investigated the potential of dairy propionibacteria-fermented products to synergize with drugs used in gastric and colorectal cancer.Fermented milk contained high and viable populations of propionibacteria. This product, as well as its cell-free supernatant, were tested in vitro on gastric HGT-1 and colic HT-29 cells with respect to apoptosis and synergy with known anti-cancer molecules, camptothecin and the human cytokine TRAIL (TNF Related Apoptosis Inducing Ligand). Viability was monitored and apoptosis quantified by Hoescht staining, ROS production, mitochondrial membrane potential perturbation and caspases activity assay. A transcriptomic study on genome-wide microarray was performed in collaboration with the company Miltenyi.Fermented milk induced cell death in both gastric and colon cancer cells. Characterization of cells revealed cell cycle arrest, drop in intracellular ATP, depolarization of mitochondria, translocation of key apoptosis proteins, processing of caspases and fragmentation of the nucleus. Interestingly, the fermented milk was shown to potentiate the cytotoxic effect of molecules used in cancer chemotherapy, camptothecin and TRAIL. The observed synergistic pro-apoptotic effect between TRAIL and propionibacterial SCFAs depended on death receptors and on caspases, was more lethal on cancer cells than on normal human intestinal epithelial cells (HIEC), and was inhibited by Bcl-2 expression. Transcriptomics revealed that propionibacterial metabolites boosted TRAIL-induced modulation of gene expression in human colon cancer cells.These results open new perspectives in the field of colon cancer cells prevention and/or treatment. The synergy with pro-apoptotic chemotherapy molecules suggests that such a fermented product, or the propionibacterium alone, may be proposed as a food supplement to enhance the effects of anticancer treatments.
The 2nd International conference on Cell Death In Cancer (CDIC)
La Baule, France

hal-01209622
https://hal.archives-ouvertes.fr/hal-01209622
PRODINRA : 261291

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Risk assessment related to the exposure of humans to chemicals released into the environment is a major concern of our modern societies. In this context, toxicology plays a crucial role to characterize the effects of this exposure on health and identify the targets of these molecules. MALDI imaging mass spectrometry (IMS) is an enabling technology for biodistribution studies of chemicals. Although the majority of published studies are presented in a pharmacological context, the concepts discussed in this review can be applied to the toxicological evaluation of chemicals released into the environment. The major asset of IMS is the simultaneous localization and identification of a parent molecule and its metabolites without labeling and without any prior knowledge. Quantification methods developed in IMS are presented with application to an environmental pollutant. IMS is effective in the localization of chemicals and endogenous species. This opens unique perspectives for the discovery of molecular alterations in metabolites and protein biomarkers that could help for a better understanding of toxicity mechanisms. Distribution studies of agrochemicals in plants by IMS can contribute to a better understanding of their mode of action and to a more effective use of these chemicals, avoiding the current concern of environmental damage.
ISSN: 1874-3919

hal-01367141
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01367141
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01367141/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01367141/file/Potential%20of%20MALDI%20imaging.pdf
DOI : 10.1016/j.jprot.2016.05.008