Éditeur(s) : Elsevier
Résumé : Because Vibrio aestuarianus is known to cause serious infections in Pacific oyster Crassostrea gigas, a real-time PCR assay was developed targeting the dnaJ gene of this bacterium. Only V. aestuarianus strains isolated from C. gigas mortality events in different geographic areas and the reference strain tested positive, whereas no amplification products was obtained with type strains belonging to 23 other species of Vibrio. Sensitivity and reproducibility of the method were assessed using either seawater or oyster homogenate samples spiked with one V aestuarianus strain. All these samples were stored at -20 degrees C in order to mimic retrospective or grouped natural sample analysis without quantification bias due to prolonged freezing. Analysis of standard curves revealed excellent correlation values between light microscopy cell enumerations and PCR Threshold Cycle (Ct) values, and acceptable PCR reaction efficiencies for all type of samples. Quantification curves of both sample types were equivalent, with a detection level as low as 1.6 V. aestuarianus cells in the PCR reaction tube, corresponding to 1.6.10(2) cells ml(-1) and 1.6.10(2) cells mg(-1) in seawater and entire oyster samples, respectively, taking into account the dilution factor used for appropriate template DNA preparation. Comparison of PCR assay reproducibility according to the complexity of samples revealed that seawater samples gave more reproducible quantification measures than samples from oyster homogenate, with precision of measured Ct values inferior to 0.4 and 0.6 respectively at 99% confidence. Use of the real-time PCR assay allowed us to monitor V. aestuarianus load in oysters naturally infected with this pathogen. Furthermore, we were able to detect V aestuarianus in samples of seawater in which oysters had been reared and in algal cultures used for feeding oysters. Because of the rapidity and reliability of the real-time PCR assay method used in this study, just a few hours are needed compared with the two days required using the classic culture method, this technique will be particularly valuable in mollusc pathology laboratories, for monitoring the source and course of infections by V. aestuarianus in pathogenesis and epidemiologic studies, as well as for designing appropriate prophylactic control measures. (C) 2009 Elsevier B.V. All rights reserved.
Journal of Microbiological Methods (0167-7012) (Elsevier), 2009-05 , Vol. 77 , N. 2 , P. 191-197

Droits : 2009 Elsevier B.V. All rights reserved.
http://archimer.ifremer.fr/doc/2009/publication-6447.pdf
DOI:10.1016/j.mimet.2009.01.021
http://archimer.ifremer.fr/doc/00000/6447/

Éditeur(s) : HAL CCSD
Résumé : International audience
This article describes a previously unreported mutation at position 210 (C210T) of the mitochondrial large subunit ribosomal RNA (mtLSUrRNA) gene of Pneumocystis jirovecii, which led to a false-negative result of a real-time polymerase chain reaction (PCR) assay. Since the aforementioned real-time PCR assay is widely used in France, a French multicenter study was conducted to estimate the mutation frequency and its potential impact on the routine diagnosis of Pneumocystis pneumonia (PCP). Through analysis of data obtained from eight centers, the mutation frequency was estimated at 0.28%. This low frequency should not call into question the routine use of this PCR assay. Nonetheless, the occurrence of the false-negative PCR result provides arguments for maintaining microscopic techniques combined to PCR assays to achieve PCP diagnosis.
Medical Mycology

hal-01477200
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01477200
DOI : 10.1093/mmy/myw051
PUBMED : 27489302

Éditeur(s) : HAL CCSD American Society for Microbiology
Résumé : International audience
Toxoplasmosis is a life-threatening infection in immunocompromised patients (ICPs). The definite diagnosis relies on parasite DNA detection, but the incidence and burden of disease are ill-known in HIV-negative patients. A three-year retrospective study was conducted in 15 reference laboratories from the network of the French National Reference Center for Toxoplasmosis, to record the frequency of Toxoplasma DNA detection in ICPs, to review the molecular methods used for diagnosis and the prevention measures implemented in transplant patients. During the study period, out of 31,640 PCR performed in ICPs, 610 were positive (323 patients). Blood (n=337), cerebrospinal fluid (n=101) and aqueous humor (n=100) samples were more frequently positive. Chemoprophylaxis schemes in transplant patients differed between centers. PCR follow-up of allogeneic hematopoietic stem-cell transplant (allo-HSCT) patients was implemented in 8/15 centers. Data from 180 patients (13 centers) were further analyzed regarding clinical setting and outcome. Only 68/180 (38%) patients were HIV+; the remaining 62% consisted in 72 HSCT, 14 solid-organ transplants, and 26 miscellaneous immunodeficiencies. Cerebral toxoplasmosis and disseminated toxoplasmosis were most frequently observed in HIV and transplant patients, respectively. Of 72 allo-HSCT patients with a positive PCR, 23 were asymptomatic; all were diagnosed in centers performing systematic blood PCR follow-up, and received specific treatment. Overall survival of allo-HSCT patients at two months was better in centers with PCR follow-up than in other centers (p<0.01). This study provides updated data on the frequency of toxoplasmosis in HIV-negative ICPs and suggests that regular PCR follow-up of allo-HSCT patients could guide pre-emptive treatment and improve outcome.
ISSN: 0095-1137

hal-01134189
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01134189
DOI : 10.1128/JCM.03282-14
PUBMED : 25762774

Éditeur(s) : HAL CCSD American Society for Microbiology
Résumé : International audience
Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations (P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal.
ISSN: 0095-1137

hal-01526432
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526432
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526432/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526432/file/Evaluation%20of%20the%20Toxoplasma.pdf
DOI : 10.1128/JCM.02379-16

Éditeur(s) : HAL CCSD
Résumé : Molds are common contaminants in indoor environments. Health effects associated with these microscopic fungi are multiple, including irritations, allergic reactions, infections and toxicity. Depending on the exposure level and the vulnerability of the populations, fungi may represent different public health risks. For example, damp and moldy dwellings are associated with chronic, respiratory diseases such as asthma and allergic rhinitis. However, the lack of valid tools for accurately measuring the environmental fungal exposure is one of the main challenges to better understanding the role of fungal products and particles in human health. In this thesis, diverse sampling techniques and analytical methods were evaluated for measuring the fungal contamination in the air, on surfaces and in settled dust. A quantitative PCR-based index was notably tested and compared with other approaches to assess fungal exposure in dwellings. By collecting and analyzing settled floor dust, this index successfully distinguished between contaminated and non-contaminated homes in Brittany, France. Quantitative PCR and this index are promising techniques for fungal exposure assessment in indoor environments. Further validation of these methods may lead to disease prevention approaches, particularly for vulnerable populations.
Les moisissures sont des micro-organismes présents dans tous les environnements intérieurs. Les effets sur la santé associés à ces champignons microscopiques sont multiples : irritations, réactions immunoallergiques, infections et effets toxiques. Selon le niveau d'exposition et la vulnérabilité des populations, les moisissures peuvent représenter des risques plus ou moins importants pour la santé publique. Les logements humides et moisis ont par exemple été associés à des maladies chroniques respiratoires telles que l'asthme et les rhinites allergiques. Cependant, le manque d'outils valides permettant d'évaluer quantitativement l'exposition fongique environnementale constitue une des principales difficultés pour mieux appréhender le rôle des particules et des composés fongiques sur la santé humaine. Lors de ce travail de thèse, diverses techniques d'échantillonnage et méthodes d'analyses ont été évaluées afin de mesurer la contamination fongique de l'air, des surfaces et des poussières. Un indice moléculaire basé sur la technique de PCR quantitative a notamment été testé et comparé à d'autres approches pour mesurer l'exposition fongique dans des logements. Après analyse des poussières domestiques collectées au sol, cet indice a permis de discriminer les habitats avec ou sans moisissures visibles. La quantification de groupes fongiques par PCR quantitative s'avère une technique prometteuse pour la mesure de l'exposition aux moisissures dans les environnements intérieurs. Outre le développement et la validation d'outils de mesure, une démarche globale de prévention du risque fongique semble nécessaire, particulièrement chez les populations vulnérables.
https://tel.archives-ouvertes.fr/tel-00768444

Droits : info:eu-repo/semantics/OpenAccess
tel-00768444
https://tel.archives-ouvertes.fr/tel-00768444
https://tel.archives-ouvertes.fr/tel-00768444/document
https://tel.archives-ouvertes.fr/tel-00768444/file/MA_heust_Delphine.pdf

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
PCR is the most widely applied technique for large scale screening of bacterial clones, mouse genotypes, virus genomes etc. A drawback of large PCR screening is that amplicon analysis is usually performed using gel electrophoresis, a step that is very labor intensive, tedious and chemical waste generating. Single genome amplification (SGA) is used to characterize the diversity and evolutionary dynamics of virus populations within infected hosts. SGA is based on the isolation of single template molecule using limiting dilution followed by nested PCR amplification and requires the analysis of hundreds of reactions per sample, making large scale SGA studies very challenging. Here we present a novel approach entitled Long Amplicon Melt Profiling (LAMP) based on the analysis of the melting profile of the PCR reactions using SYBR Green and/or EvaGreen fluorescent dyes. The LAMP method represents an attractive alternative to gel electrophoresis and enables the quick discrimination of positive reactions. We validate LAMP for SIV and HIV env-SGA, in 96- and 384-well plate formats. Because the melt profiling allows the screening of several thousands of PCR reactions in a cost-effective, rapid and robust way, we believe it will greatly facilitate any large scale PCR screening
ISSN: 1932-6203

hal-01163756
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01163756
DOI : 10.1371/journal.pone.0128188
PUBMED : 26053379

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Whilst it used to affect mostly intravenous drug users and patients who underwent valvular surgery with suboptimal infection control procedures, fungal endocarditis is now mostly observed in patients with severe immunodeficiency (onco-haematology), in association with chronic central venous access and broad-spectrum antibiotic use. The incidence of fungal endocarditis has probably decreased in most developed countries with access to harm-reduction policies (i.e. needle exchange programmes) and with improved infection control procedures during cardiac surgery. Use of specific blood culture bottles for diagnosis of fungal endocarditis has decreased due to optimisation of media and automated culture systems. Meanwhile, the advent of rapid techniques, including fungal antigen detection (galactomannan, mannan/anti-mannan antibodies and β-1,3-d-glucans) and PCR (e.g. universal fungal PCR targeting 18S rRNA genes), shall improve sensitivity and reduce diagnostics delays, although limited data are available on their use for the diagnosis of fungal endocarditis. New antifungal agents available since the early 2000s may represent dramatic improvement for fungal endocarditis: (i) a new class, the echinocandins, has the potential to improve the management of Candida endocarditis owing to its fungicidal effect on yeasts as well as tolerability of increased dosages; and (ii) improved survival in patients with invasive aspergillosis with voriconazole compared with amphotericin B, and this may apply to Aspergillus sp. endocarditis as well, although its prognosis remains dismal. These achievements may allow selected patients to be cured with prolonged medical treatment alone when surgery is considered too risky.
ISSN: 0924-8579

hal-01118441
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01118441
DOI : 10.1016/j.ijantimicag.2014.07.003
PUBMED : 25178919

Éditeur(s) : HAL CCSD Elsevier Masson
Résumé : International audience
The diagnosis of diabetic foot infections is difficult due to limitations of conventional culture-based techniques. The objective of this study was to evaluate the contribution of denaturing gradient gel electrophoresis (DGGE) in the microbiological diagnosis of diabetic foot ulcers in comparison to conventional techniques, and also to evaluate the need to perform a biopsy sample for this diagnosis. Twenty diabetic patients (types 1 and 2) with foot ulcers (grades 1-4) were included. After debridement of their wounds, samples were taken in duplicate by surface swabbing and deep-tissue biopsy. The samples were analyzed by conventional culture and by a new molecular biology tool, DGGE technology. Polymerase chain reaction (PCR)-DGGE led to the identification of more bacteria than did conventional cultures (mean: 2.35 vs 0.80, respectively). In 11 cases, the technology detected pathogenic species not isolated by classical cultures. PCR-DGGE also identified significantly more pathogenic species at deep levels compared with species detected at superficial levels (87% vs 58%, respectively; P = 0.03). In 9/20 cases, pathogenic bacteria were detected only in deep samples, revealing the need to perform tissue biopsy sampling. DGGE, achievable in 48h, could be a useful technique for the bacteriological diagnosis of diabetic foot infections. It may help to identify pathogenic bacteria in deeply infected ulcers, thereby contributing to a more appropriate use of antibiotics.
ISSN: 1262-3636

hal-01122240
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01122240
DOI : 10.1016/j.diabet.2014.03.002
PUBMED : 24751989

Éditeur(s) : HAL CCSD Lippincott, Williams & Wilkins
Résumé : International audience
Purpose of review Toxoplasmosis in immunocompromised patients is associated with a high mortality rate. Molecular techniques are important tools to diagnose acute disease in immunocompromised patients, but there are various methods with variable efficiency. Some of them have been validated for the diagnosis of congenital toxoplasmosis, but the impact of their use has not been evaluated in immunocompromised patients. Recent findings Toxoplasmosis is of increasing importance in non-HIV immunocompromised patients. In addition, the picture of disease shows greater severity in South America, both in immunocompetent study participants and in congenitally infected infants. These epidemiological differences could influence the sensitivity of diagnostic methods. This review analyzes recent data on molecular diagnosis and compares them with older ones, in light of progress gained in molecular techniques and of recent epidemiological findings. Most recent studies were conducted in South America and used PCR targeting the B1 gene. PCR on blood could allow diagnosing a significant proportion of patients with ocular toxoplasmosis in Brazil. Summary Quantitative PCR methods with specific probes should be used to improve sensitivity and warrant specificity. Performance of quantitative PCR targeting the repeated 529 bp sequence for the diagnosis of toxoplasmosis in immunocompromised patients needs evaluation in field studies in South America and in western countries.
ISSN: 0951-7375

hal-01390970
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01390970
DOI : 10.1097/QCO.0000000000000275
PUBMED : 27191201

Éditeur(s) : HAL CCSD
Résumé :   Diplôme : DEA
il s'agit d'un type de produit dont les métadonnées ne correspondent pas aux métadonnées attendues dans les autres types de produit : DISSERTATION
L'étude de la répartition géographique de la diversité génétique résultant de l'histoire des populations apparaît comme fondamentale pour la conservation des espèces. Ainsi, la mise en évidence des relations phylo-géographiques au court du Quaternaire récent, au sein d'une espèce modèle Vouacapoua americana (Caesalpiniaceae), en Guyane française apporte des informations importantes sur la dynamique des populations naturelles. Dans cette étude les loci trn Q-trn S et trn Sa-trn fM sont utilisés afin de déterminer l'histoire évolutive des populations issues des refuges forestiers tropicaux. Le but principal est l'obtention des séquences après amplification PCR. L'analyse de ces séquences chez cinq populations testées a permis de mettre en évidence leur caractère hypervariable, ainsi que des régions présentant des motifs conservés répétés plusieurs fois. Les constructions phylogéographiques obtenues à partir de celles-ci accentuent la régionalisation des différents chlorotypes, existant en Guyane pour cette espèce, mise en évidence au cours de travaux précédents. Ainsi, les informations apportées par cette approche apparaissent comme complémentaires aux résultats obtenus par d'autres techniques d'analyse moléculaire.
https://hal.archives-ouvertes.fr/hal-01189224

hal-01189224
https://hal.archives-ouvertes.fr/hal-01189224
PRODINRA : 16622

Éditeur(s) : Elsevier
Résumé : Shrimp farming is a small but growing industry in New Caledonia. Since 1993, "Syndrome 93" has been affecting New Caledonian shrimp farming industry every cold season, causing severe epizootic mortalities in grow-out ponds and significant losses. Highly pathogenic strains of Vibrio penaeicida are considered the etiological agent of the disease in Litopenaeus stylirostris. On one hand, studies demonstrated that healthy shrimp may carry V penaeicida for weeks with a high overall prevalence, regardless of any seasonal pattern or temperature conditions. On the other hand, larvae are free of V penaeicida and are also resistant to experimental infection. V penaeicida is frequently detected in incoming water pumped from the bays, which was shown, by a molecular typing study, to be the infectious source. This particular epidemiological pattern highlights the major role of the factors that trigger and aggravate the disease in grow-out ponds, where shrimp populations carry the pathogen all year round. In order to gain a better understanding of "Syndrome 93" epidemiology, quantification of V penaeicida both in shrimp and the shrimp farm ecosystem is necessary. This article describes the steps in the successful development of a real-time PCR quantification assay of V penaeicida in shrimp haemolymph, seawater (from ponds or bays) and sediment pore water, including the choice of an accurate extraction technique. The entire detection method; including sample processing, DNA extraction and real-time PCR amplification, can be completed within 4 h. (c) 2006 Elsevier B.V All rights reserved.
Journal of Microbiological Methods (0167-7012) (Elsevier), 2006-10 , Vol. 67 , N. 1 , P. 27-35

Droits : 2006 Elsevier
http://archimer.ifremer.fr/doc/2006/publication-1903.pdf
DOI:10.1016/j.mimet.2006.02.013
http://archimer.ifremer.fr/doc/00000/1903/

Éditeur(s) : Elsevier
Résumé : A collection of 57 isolates of Vibrio nigripulchritudo from either diseased or healthy shrimp and from shrimp farms environment was studied in order to gain a better understanding of the epidemiology of this pathogen, notably isolated from two distinct shrimp disease complexes. Molecular typing using two different techniques, arbitrarily primed PCR (AP-PCR) and multi-locus sequence typing (MLST), studied together with experimental pathology data allowed a relevant epidemiological insight into this possibly emerging pathogen. Additionally, results obtained with the two molecular typing techniques were congruent and allowed discriminating the strains associated with the "Summer Syndrome" from strains isolated from other contexts, especially the other shrimp vibriosis "Syndrome 93". These results highlight that the "Summer Syndrome" is most probably caused by an emergent clonal pathogen that therefore deserves surveillance and that AP-PCR can satisfactorily be used for that purpose
Systematic and Applied Microbiology (0723-2020) (Elsevier), 2006-11 , Vol. 29 , N. 7 , P. 570-580

Droits : 2005 Elsevier
http://archimer.ifremer.fr/doc/2006/publication-1913.pdf
DOI:10.1016/j.syapm.2005.12.005
http://archimer.ifremer.fr/doc/00000/1913/

Résumé : Une étude de typage moléculaire par AP-PCR de souches de Vibrio impliqués dans des épisodes de mortalité d'élevages de crevettes en Nouvelle-Calédonie (Syndrome 93) et au Japon a été réalisée fin 1996 avec le concours de l' Institut Pasteur de Nouvelle-Calédonie. Elle nous a permis une identification rapide des isolats en terme de génospecies ainsi que des discriminations d' intérêt épidémiologique entre les isolats du génospecies majoritaire. Ainsi, des sous-groupes ont été identifiés au sein de l' espéce majoritaire : V. penaeicida. Certains de ces sous-groupes correspondent à des topotypes et permettent de discriminer les isolats du Japon de ceux de Nouvelle-Calédonie, ainsi que ceux de deux baies différentes de Nouvelle Calédonie, distinctes de 50 km seulement. D'autres sous-groupes ont pu être identifiés au sein de l' un des topotypes calédoniens, sans qu'il soit possible de l'expliquer par des éléments épidémiologiques. L' analyse des géotypes et des données zootechniques permettent de qualifier cette vibriose de maladie hydrique, c'est à dire dont le pathogène est véhiculé par l'eau. Des recommandations d' ordre zoosanitaires en sont déduites, qui tendent principalement à limiter les transferts d'animaux entre les différents sites d'élevage. Cette technique d'AP-PCR est utilisée pour la première fois dans l'étude épidémiologique d' une vibriose de crevettes. Sa rapidité, sa fiabilité et les informations d'ordre épidémiologique qu' elle fournit en font une technique de choix pour ce type d'étude

Droits : 1997 Ifremer
http://archimer.ifremer.fr/doc/00274/38541/37061.pdf
http://archimer.ifremer.fr/doc/00274/38541/

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
BACKGROUND: Estrogens are thought to play a critical role in prostate carcinogenesis. It has been suggested that polymorphisms of genes encoding enzymes involved in estrogen metabolism are risk factors for prostate cancer. However, few studies have been performed on populations of African ancestry, which are known to have a high risk of prostate cancer. OBJECTIVE: We investigated whether functional polymorphisms of CYP17, CYP19, CYP1B1, COMT and UGT1A1 affected the risk of prostate cancer in two different populations of African ancestry. METHODS: In Guadeloupe (French West Indies), we compared 498 prostate cancer patients and 565 control subjects. In Kinshasa (Democratic Republic of Congo), 162 prostate cancer patients were compared with 144 controls. Gene polymorphisms were determined by the SNaPshot technique or short tandem repeat PCR analysis. Logistic regression was used to estimate adjusted odds ratios (OR) and 95% confidence intervals (CI). RESULTS: The AA genotype and the A allele of rs4680 (COMT) appeared to be inversely associated with the risk of prostate cancer in adjusted models for both Afro-Caribbean and native African men. For the A allele, a significant inverse association was observed among cases with low-grade Gleason scores and localized clinical stage, in both populations. CONCLUSIONS: These preliminary results support the hypothesis that polymorphisms of genes encoding enzymes involved in estrogen metabolism may modulate the risk of prostate cancer in populations of African ancestry
ISSN: 1932-6203

hal-01305477
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01305477
DOI : 10.1371/journal.pone.0153609
PUBMED : 27074016

Éditeur(s) : HAL CCSD
Résumé : Anti-Müllerian hormone (AMH) is a member of the TGF-ß superfamily. AMH is well known for its role in Müllerian duct regression in male fetuses. Postnatally, AMH is secreted by granulosa cells (GCs) of small growing follicles (preantral and small antral). However, despite the increasing interest of ovarian AMH in clinics, little is known on its mechanism of action and its role in female reproductive tract. My PhD project focuses on the identification of AMH function in the female reproductive tract.AMH signals through a type II transmembrane serine/threonine kinase receptor (AMHR-II) which forms a complex with a type I serine/threonine kinase receptor (ActR-IA, BMPR-IA, BMPR-IB). The type II receptor phosphorylates serine and threonine residues of type I receptor. Once activated, the type I receptor phosphorylates the receptor-regulated Smads (R-Smad1/5/8) which interact with a common partner Smad4. The Smad complex accumulates into the nucleus and regulates target gene expression. This canonical signalling pathway is regulated at different levels, in particular by co-receptors which amplify or antagonize TGF-ß family members action. The type I receptors and R-Smads involved in AMH effects on post-natal GCs remain unknown. In addition, to date, no co-receptor has been found for AMH. To define the involvement of the different type I receptors, we used siRNA technology to inactivate Acvr1, Bmpr1a and Bmpr1b in GC. In parallele, we analysed GC extracted from conditional mutant mice for Acvr1 and Bmpr1a. We found that BMPR-IA is the most important type I receptor for AMH to transduce its signal in GC. A Smad-Gal4/UAS-luciferase reporter gene technology allowed us to show that Smad1 and 5 are involved in AMH signaling pathway. Recently, new BMPs coreceptors were found, RGMs for Repulsive Guidance Molecules. There are three RGMs : RGMa, b and c. Because AMH shares with BMPs its type I receptors and R-Smad proteins, we hypothesized that they also share the same co-receptors, the RGM. We showed that RGMb was the only one expressed in GC and after siRNA transfection we demonstrated that this coreceptor is not essential for AMH to transduce its signal.To date, only few AMH target genes have been identified. Aromatase (Cyp19a1) and LH receptor (Lhcgr) are down-regulated by AMH in rat and porcine GCs. We used micro-array technology (Affymetrix) by comparing Wt and knockout immature ovaries to find new AMH target genes. This experiment evidenced that Ovgp1 and Kcnj2 are two new potential AMH target genes in the ovary.The last part of my project was to define a potential role of AMH in murine uterus. Only one study showed that AMHR-II is expressed in the mouse myometrium. We showed that Amh gene is slightly expressed in uterus but the results are not confirmed at the protein level. Using PCR-array, we found a lot of differentially expressed genes between Wt and Amh KO uterus. Therefore, AMH could regulate uterine function through the modulation of different genes located in the myometrium.
L’hormone anti-Müllerienne (AMH) est un membre de la famille TGF-β impliquée dans la différenciation du tractus reproductif mâle. Elle est aussi exprimée par les cellules de la granulosa de l’ovaire adulte. Cependant, son rôle physiologique chez la femelle n’a pas encore été entièrement établi. Mon projet de thèse a pour objectif d’élucider le(s) rôle(s) de l’AMH dans le tractus reproductif femelle. L’AMH transduit ses effets par l’intermédiaire de deux récepteurs transmembranaires sérine/thréonine kinase : un récepteur de type II qui lui est spécifique (AMHR-II) et un récepteur de type I (ActR-IA, BMPR-IA, BMPR-IB) qu’elle partage avec les BMPs. Après fixation de l’hormone sur le récepteur de type II, celui-ci recrute et phosphoryle le récepteur de type I. Ce dernier phosphoryle à son tour les Smads spécifiques (Smad1, 5 et 8) qui s’associent à la Smad commune, Smad4. L’ensemble transloque dans le noyau et en association avec des facteurs de transcription régule les gènes cibles de l’hormone. L'utilisation de souris KO conditionnelles pour les récepteurs Acvr1 et Bmpr1a et d'une technique de siRNA dirigés contre chacun des trois récepteurs de type I a permis de mettre en évidence que le récepteur BMPR-IA est un acteur essentiel de la voie de signalisation de l'AMH dans les cellules de la granulosa. Pour déterminer la ou les Smad(s) impliquées, une technique de gènes rapporteurs, Smad-Gal4/UAS-luciférase, a été utilisée. Nous avons pu montrer que les Smad1 et 5 sont importantes pour la transduction du signal de l'AMH dans les cellules de la granulosa. Récemment des corécepteurs aux BMPs, les Repulsive Guidance Molecule (RGMs), ont été mis en évidence. L’AMH partageant sa voie de signalisation avec les BMPs, nous avons cherché à déterminer si ces corécepteurs pouvaient également intervenir dans la voie de signalisation de l’AMH. Il existe 3 types de RGMs: RGMa, RGMb et RGMc. Nous avons montré en q-PCR que seul RGMb est exprimé dans les cellules de la granulosa alors que les 3 RGMs sont exprimés dans l’ovaire. L'utilisation de siRNA dirigés contre RGMb a permis de montrer que ce récepteur n'est pas nécessaire à la transduction du signal de l'AMH. Actuellement, seuls deux gènes cibles de l’AMH sont connus dans les cellules de la granulosa : l’aromatase et le récepteur LH. Nous avons réalisé des analyses de puces à ADN (ou micro-array) pour décrire de nouveaux gènes cibles de l'AMH. L'analyse des puces a permis de décrire de nouveaux gènes régulés par cette hormone tels qu’Ovgp1 ou Kcnj2. La dernière partie de mon projet visait à déterminer un rôle potentiel de l'AMH dans l'utérus. En effet, le récepteur de cette hormone est exprimé dans le myomètre utérin de souris permettant de supposer qu’elle peut agir sur cet organe. Nous avons pu mettre en évidence une expression faible du gène de l’Amh dans l’utérus. En revanche, l’expression et la localisation de la protéine restent encore à définir. Une expérience de PCR-array a permis de montrer que de nombreux gènes sont différentiellement exprimés entre l’utérus Wt et l’utérus KO Amh. Ceci indique que l’AMH jouerait un rôle sur la régulation de la fonction utérine qu’elle soit exprimée ou non dans cet organe.
https://tel.archives-ouvertes.fr/tel-00989662

Droits : info:eu-repo/semantics/OpenAccess
NNT : 2014PA11T014
tel-00989662
https://tel.archives-ouvertes.fr/tel-00989662
https://tel.archives-ouvertes.fr/tel-00989662/document
https://tel.archives-ouvertes.fr/tel-00989662/file/VA_SEDES_LAURIANE_03042014.pdf

Éditeur(s) : Academic Press Ltd- Elsevier Science Ltd
Résumé : The understanding of reciprocal interactions between Crassostrea gigas and Vibrio sp., whether these be virulent or avirulent, is vital for the development of methods to improve the health status of cultured oysters. We describe an original non-invasive experimental infection technique using cohabitation, designed to explore these interactions. Using real-time PCR techniques we examined the dynamics of virulent and avirulent Vibrio sp. in oyster hemolymph and tank seawater, and made a parallel study of the expression of four genes involved in oyster immune defense: Cg-BPI, Cg-EcSOD, Cg-IκB, Cg-TIMP. No mortality occurred in control animals, but oysters put in cohabitation for 2–48 h with animals previously infected by two Vibrio pathogens suffered mortalities from 2 to 16 days post-cohabitation. Our results show that virulent Vibrio infect healthy individuals after only 2 h of cohabitation, with values ranging from 4.5×102 to 2×104 cells ml−1 hemolymph. Simultaneously, an approximate ten-fold increase of the total Vibrio population was observed in control animals, with a 6.6–78.5-fold up-expression of targeted genes. In contrast, oysters exposed to harmful bacteria had mean expression levels strongly down-regulated by a factor of 9.2–29 (depending on the gene) compared with control animals. Although oysters were still found to be infected by virulent Vibrio after 6–48 h of cohabitation, no significant differences were noted when comparing levels of each transcript in control and infected oysters at the same sampling times during this period: the important differences were noted before 6 h cohabitation. Taken together, our data support (1) the hypothesis that virulent Vibrio disturbs the immune response of this invertebrate host both rapidly and significantly, although this occurs specifically during an early and transient period during the first 6 h of cohabitation challenge, and that (2) expression of targeted genes is not correlated with vibriosis resistance.
Fish & Shellfish Immunology (1050-4648) (Academic Press Ltd- Elsevier Science Ltd), 2011-02 , Vol. 30 , N. 2 , P. 691-699

Droits : 2011 Elsevier Ltd All rights reserved.
http://archimer.ifremer.fr/doc/00025/13604/10681.pdf
DOI:10.1016/j.fsi.2010.12.017
http://archimer.ifremer.fr/doc/00025/13604/

Éditeur(s) : HAL CCSD BioMed Central
Résumé : International audience
UNLABELLED: ABSTRACT: BACKGROUND: Genomics, transcriptomics and proteomics of glioblastoma multiforme (GBM) have recently emerged as possible tools to discover therapeutic targets and biomarkers for new therapies including immunotherapy. It is well known that macroscopically complete surgical excision, radiotherapy and chemotherapy have therapeutic limitations to improve survival in these patients. In this study, we used a differential proteomic-based technique (2D-Difference Gel Electrophoresis) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify proteins that may serve as brain tumor antigens in new therapeutic assays. Five samples of patients presenting a GBM and five samples of microscopically normal brain tissues derived from brain epileptic surgery specimen were labeled and run in 2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis) with an internal pool sample on each gel. Five gels were matched and compared with DIA (Difference In-gel Analysis) software. Differential spots were picked, in-gel digested and peptide mass fingerprints were obtained. RESULTS: From 51 protein-spots significantly up-regulated in GBM samples, mass spectrometry (MS) identified twenty-two proteins. The differential expression of a selected protein set was first validated by western-blotting, then tested on large cohorts of GBM specimens and non-tumor tissues, using immunohistochemistry and real-time RT-PCR. CONCLUSIONS: Our results confirmed the importance of previously described proteins in glioma pathology and their potential usefulness as biological markers but also revealed some new interesting targets for future therapies.
ISSN: 1477-5956

inserm-00585020
http://www.hal.inserm.fr/inserm-00585020
http://www.hal.inserm.fr/inserm-00585020/document
http://www.hal.inserm.fr/inserm-00585020/file/1477-5956-9-16.pdf
DOI : 10.1186/1477-5956-9-16

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
BACKGROUND: Estrogens are thought to play a critical role in prostate carcinogenesis. It has been suggested that polymorphisms of genes encoding enzymes involved in estrogen metabolism are risk factors for prostate cancer. However, few studies have been performed on populations of African ancestry, which are known to have a high risk of prostate cancer.OBJECTIVE: We investigated whether functional polymorphisms of CYP17, CYP19, CYP1B1, COMT and UGT1A1 affected the risk of prostate cancer in two different populations of African ancestry.METHODS: In Guadeloupe (French West Indies), we compared 498 prostate cancer patients and 565 control subjects. In Kinshasa (Democratic Republic of Congo), 162 prostate cancer patients were compared with 144 controls. Gene polymorphisms were determined by the SNaPshot technique or short tandem repeat PCR analysis. Logistic regression was used to estimate adjusted odds ratios (OR) and 95% confidence intervals (CI).RESULTS: The AA genotype and the A allele of rs4680 (COMT) appeared to be inversely associated with the risk of prostate cancer in adjusted models for both Afro-Caribbean and native African men. For the A allele, a significant inverse association was observed among cases with low-grade Gleason scores and localized clinical stage, in both populations.CONCLUSIONS: These preliminary results support the hypothesis that polymorphisms of genes encoding enzymes involved in estrogen metabolism may modulate the risk of prostate cancer in populations of African ancestry.
ISSN: 1932-6203

hal-01668333
https://hal.univ-antilles.fr/hal-01668333
DOI : 10.1371/journal.pone.0153609
PUBMED : 27074016

Éditeur(s) : HAL CCSD Instituto Oswaldo Cruz, Ministério da Saúde
Résumé : International audience
A cross section of a human population (501 individuals) selected at random, and living in a Bolivian community, highly endemic for Chagas disease, was investigated combining together clinical, parasitological and molecular approaches. Conventional serology and polymerase chain reaction (PCR) indicated an active transmission of the infection, a high seroprevalence (43.3%) ranging from around 12% in < 5 years to 94.7% in > 45 years, and a high sensitivity (83.8%) and specificity of PCR. Abnormal ECG tracing was predominant in chagasic patients and was already present among individuals younger than 13 years. SAPA (shed acute phase antigen) recombinant protein and the synthetic peptide R-13 were used as antigens in ELISA tests. The reactivity of SAPA was strongly associated to Trypanosoma cruzi infection and independent of the age of the patients but was not suitable neither for universal serodiagnosis nor for discrimination of specific phases of Chagas infection. Anti-R-13 response was observed in 27.5% only in chagasic patients. Moreover, anti-R13 reactivity was associated with early infection and not to cardiac pathology. This result questioned previous studies, which considered the anti-R-13 response as a marker of chronic Chagas heart disease. The major clonets 20 and 39 (belonging to Trypanosoma cruzi I and T. cruzi II respectively) which circulate in equal proportions in vectors of the studied area, were identified in patients' blood by PCR. Clonet 39 was selected over clonet 20 in the circulation whatever the age of the patient. The only factor related to strain detected in patients' blood, was the anti-R-13 reactivity: 37% of the patients infected by clonet 39 (94 cases) had anti-R13 antibodies contrasting with only 6% of the patients without clonet 39 (16 cases).
ISSN: 0074-0276

hal-01256421
https://hal.archives-ouvertes.fr/hal-01256421
DOI : 10.1590/S0074-02762002000300002
IRD : fdi:010029219
PUBMED : 12048553

Éditeur(s) : HAL CCSD
Résumé : Diplôme : DEA
il s'agit d'un type de produit dont les métadonnées ne correspondent pas aux métadonnées attendues dans les autres types de produit : DISSERTATION
absent
https://hal.archives-ouvertes.fr/hal-01189541

hal-01189541
https://hal.archives-ouvertes.fr/hal-01189541
PRODINRA : 46419