Le syndrome d’embolie de liquide amniotique (SELA) est une complication rare et souvent catastrophique de l’accouchement chez la femme caractérisée classiquement par une hypotension sévère, un arrêt cardiorespiratoire et une coagulation intra-vasculaire disséminée. Malheureusement, sa physiopathologie est encore mal connue. Le rôle des kinines n’a notamment pas été étudié. L’objectif de notre projet était de développer un modèle animal de SELA et d’étudier le rôle éventuel des kinines dans ce syndrome. Douze lapines en fin de gestation ont été incluses dans l’étude. Pour chacune d’entre-elles, le liquide amniotique était aspiré de chaque sac amniotique après une laparotomie. Six lapines recevaient un bolus de liquide amniotique injecté via la veine auriculaire alors que les six autres recevaient un bolus de saline. Parallèlement, les effets in vitro de liquide amniotique sur la coagulation étaient évalués par thrombelastographie (TEG) et comparés aux effets de la saline. L’injection de liquide amniotique n’a pas permis de reproduire les signes cliniques de SELA, n’a pas entrainé la génération de bradykinine, et n’a pas eu d’effet sur le temps de prothrombine, le temps de thromboplastine partielle activée, et l’activité du facteur VIII de. Une thrombocytopénie sévère et transitoire a cependant été notée 5 minutes après l’injection de liquide amniotique. De plus, en additionnant in vitro de liquide amniotique au sang on a observé un tracé de TEG hypercoagulable comparé à celui obtenu avec la saline. Le modèle n’ayant pas pu reproduire le SELA, le rôle des kinines dans ce syndrome reste à déterminer. / Amniotic fluid embolism (AFE) is a rare but catastrophic complication of parturition characterized by severe hypotension, cardiovascular collapse, and massive consumptive coagulopathy. Its pathophysiology remains obscure. In particular, the potential role of bradykinin in hypotension is unknown. The objective of this study was to develop a suitable animal model of AFE and to study the effects of amniotic fluid injection on bradykinin release in this model. Twelve rabbits in late gestation (25 days) were used. For each rabbit, amniotic fluid was collected from foetal amniotic sacs by laparotomy. For six rabbits, the amniotic fluid was then injected as a bolus via the left auricular vein, whereas the six other rabbits received saline (control group). In parallel, the in vitro effects of amniotic fluid on coagulation was assessed by thrombelastography (TEG) and compared to the effects of saline. Injection of amniotic fluid did not reproduce clinical signs of AFE, did not provoke bradykinin generation and had no effect on prothrombin time, the activated partial thromboplastin time, nor Factor VIII activity. However, a significant thrombocytopenia was observed five minutes after amniotic fluid administration. This thrombocytopenia resolved within 60 minutes. In vitro addition of amniotic fluid to blood resulted in accelerated clotting on TEG tracings as compared to the effect of saline. As we were not able to reproduce AFE with our model, the role of kinins in this syndrome remains to be determined.

O objetivo do presente trabalho foi o desenvolvimento de um teste não invasivo para a trissomia do cromossomo 21 através da análise de ácidos nucleicos fetais livres no plasma materno por sequenciamento de última geração realizado no sequenciador automático Ion Torrent. A metodologia proposta para o teste é a análise de SNPs com alta taxa de heterozigosidade na população brasileira localizados em dois genes presentes no cromossomo 21 (PLAC4 e C21orf105), e a detecção da cópia extra do cromossomo 21 é feita pela razão dos alelos desses SNPs, sendo que a razão de 1:1 indica que o feto é normal, e a razão de 2:1 indica que o feto tem uma cópia extra do cromossomo 21. Para a validação da metodologia foram utilizadas 50 amostras de DNA livre extraídas de líquido amniótico, previamente caracterizadas por análise citogenética consideradas como o padrão ouro pois contém apenas material genético fetal abundante. A metodologia foi validada com sucesso nessas amostras, sendo que as 24 amostras de fetos com trissomia foram claramente distinguidas das 26 amostras de fetos normais. A metodologia validada foi aplicada a 44 amostras de DNA livre extraídas de plasma de gestantes (21 amostras de fetos com trissomia do 21 e 23 de fetos normais), porém não foi possível fazer a distinção entre fetos normais e fetos com trissomia do 21, possivelmente devido à variações na fração fetal do DNA livre em relação à fração materna. Como nosso objetivo principal não foi alcançado, Propomos aqui que o teste realizado em líquido amniótico seja utilizado como uma alternativa mais simples, rápida e barata ao cariótipo convencional atualmente utilizado para fazer o diagnóstico da trissomia do cromossomo 21 em amostras coletadas por procedimentos invasivos, enquanto as deficiências do teste não-invasivo pelo plasma materno são aprimoradas / The purpose of this study was to develop a test for trisomy 21 by analyzing cell-free fetal nucleic acids in maternal plasma by next-generation sequencing performed on automated sequencer Ion Torrent. The proposed methodology for the test is based on analysis of SNPs with high heterozygosity rates in Brazilian population, located in two genes present on chromosome 21 (PLAC4 and C21orf105), and the detection of the extra copy of chromosome 21 is made by the allelic-ratio of these SNPs, where 1:1 ratio indicates a normal fetus, and the 2:1 ratio indicates that the fetus has an extra copy of chromosome 21. In order to validate the methodology 50 cell-free DNAs extracted from amniotic fluid were used representing a gold standard since it contains abundant genetic material exclusively from the fetus. The methodology has been successfully validated in these samples, all the 24 samples from fetuses with trisomy 21 were clearly distinguished from 26 samples of normal fetuses. The validated method was applied to 44 cell-free DNA samples extracted from plasma of pregnant women (21 samples from fetuses with trisomy 21 and 23 from normal fetuses), but unfortunately it was not possible to distinguish between normal and trisomy 21 fetuses, possibly due to variations on the fetal fraction of the cell-free DNA in relation to maternal fraction. As our main goal was not achieved, we propose here that the test performed on amniotic fluid sample could be used as a simpler, faster and cheaper alternative test to traditional karyotype, which is used nowadays to make the diagnosis of trisomy 21 in samples collected by invasive procedures. In parallel, minor improvements in the described method may enable its clinical use

O objetivo do presente trabalho foi o desenvolvimento de um teste não invasivo para a trissomia do cromossomo 21 através da análise de ácidos nucleicos fetais livres no plasma materno por sequenciamento de última geração realizado no sequenciador automático Ion Torrent. A metodologia proposta para o teste é a análise de SNPs com alta taxa de heterozigosidade na população brasileira localizados em dois genes presentes no cromossomo 21 (PLAC4 e C21orf105), e a detecção da cópia extra do cromossomo 21 é feita pela razão dos alelos desses SNPs, sendo que a razão de 1:1 indica que o feto é normal, e a razão de 2:1 indica que o feto tem uma cópia extra do cromossomo 21. Para a validação da metodologia foram utilizadas 50 amostras de DNA livre extraídas de líquido amniótico, previamente caracterizadas por análise citogenética consideradas como o padrão ouro pois contém apenas material genético fetal abundante. A metodologia foi validada com sucesso nessas amostras, sendo que as 24 amostras de fetos com trissomia foram claramente distinguidas das 26 amostras de fetos normais. A metodologia validada foi aplicada a 44 amostras de DNA livre extraídas de plasma de gestantes (21 amostras de fetos com trissomia do 21 e 23 de fetos normais), porém não foi possível fazer a distinção entre fetos normais e fetos com trissomia do 21, possivelmente devido à variações na fração fetal do DNA livre em relação à fração materna. Como nosso objetivo principal não foi alcançado, Propomos aqui que o teste realizado em líquido amniótico seja utilizado como uma alternativa mais simples, rápida e barata ao cariótipo convencional atualmente utilizado para fazer o diagnóstico da trissomia do cromossomo 21 em amostras coletadas por procedimentos invasivos, enquanto as deficiências do teste não-invasivo pelo plasma materno são aprimoradas / The purpose of this study was to develop a test for trisomy 21 by analyzing cell-free fetal nucleic acids in maternal plasma by next-generation sequencing performed on automated sequencer Ion Torrent. The proposed methodology for the test is based on analysis of SNPs with high heterozygosity rates in Brazilian population, located in two genes present on chromosome 21 (PLAC4 and C21orf105), and the detection of the extra copy of chromosome 21 is made by the allelic-ratio of these SNPs, where 1:1 ratio indicates a normal fetus, and the 2:1 ratio indicates that the fetus has an extra copy of chromosome 21. In order to validate the methodology 50 cell-free DNAs extracted from amniotic fluid were used representing a gold standard since it contains abundant genetic material exclusively from the fetus. The methodology has been successfully validated in these samples, all the 24 samples from fetuses with trisomy 21 were clearly distinguished from 26 samples of normal fetuses. The validated method was applied to 44 cell-free DNA samples extracted from plasma of pregnant women (21 samples from fetuses with trisomy 21 and 23 from normal fetuses), but unfortunately it was not possible to distinguish between normal and trisomy 21 fetuses, possibly due to variations on the fetal fraction of the cell-free DNA in relation to maternal fraction. As our main goal was not achieved, we propose here that the test performed on amniotic fluid sample could be used as a simpler, faster and cheaper alternative test to traditional karyotype, which is used nowadays to make the diagnosis of trisomy 21 in samples collected by invasive procedures. In parallel, minor improvements in the described method may enable its clinical use

Le syndrome d’embolie de liquide amniotique (SELA) est une complication rare et souvent catastrophique de l’accouchement chez la femme caractérisée classiquement par une hypotension sévère, un arrêt cardiorespiratoire et une coagulation intra-vasculaire disséminée. Malheureusement, sa physiopathologie est encore mal connue. Le rôle des kinines n’a notamment pas été étudié. L’objectif de notre projet était de développer un modèle animal de SELA et d’étudier le rôle éventuel des kinines dans ce syndrome. Douze lapines en fin de gestation ont été incluses dans l’étude. Pour chacune d’entre-elles, le liquide amniotique était aspiré de chaque sac amniotique après une laparotomie. Six lapines recevaient un bolus de liquide amniotique injecté via la veine auriculaire alors que les six autres recevaient un bolus de saline. Parallèlement, les effets in vitro de liquide amniotique sur la coagulation étaient évalués par thrombelastographie (TEG) et comparés aux effets de la saline. L’injection de liquide amniotique n’a pas permis de reproduire les signes cliniques de SELA, n’a pas entrainé la génération de bradykinine, et n’a pas eu d’effet sur le temps de prothrombine, le temps de thromboplastine partielle activée, et l’activité du facteur VIII de. Une thrombocytopénie sévère et transitoire a cependant été notée 5 minutes après l’injection de liquide amniotique. De plus, en additionnant in vitro de liquide amniotique au sang on a observé un tracé de TEG hypercoagulable comparé à celui obtenu avec la saline. Le modèle n’ayant pas pu reproduire le SELA, le rôle des kinines dans ce syndrome reste à déterminer. / Amniotic fluid embolism (AFE) is a rare but catastrophic complication of parturition characterized by severe hypotension, cardiovascular collapse, and massive consumptive coagulopathy. Its pathophysiology remains obscure. In particular, the potential role of bradykinin in hypotension is unknown. The objective of this study was to develop a suitable animal model of AFE and to study the effects of amniotic fluid injection on bradykinin release in this model. Twelve rabbits in late gestation (25 days) were used. For each rabbit, amniotic fluid was collected from foetal amniotic sacs by laparotomy. For six rabbits, the amniotic fluid was then injected as a bolus via the left auricular vein, whereas the six other rabbits received saline (control group). In parallel, the in vitro effects of amniotic fluid on coagulation was assessed by thrombelastography (TEG) and compared to the effects of saline. Injection of amniotic fluid did not reproduce clinical signs of AFE, did not provoke bradykinin generation and had no effect on prothrombin time, the activated partial thromboplastin time, nor Factor VIII activity. However, a significant thrombocytopenia was observed five minutes after amniotic fluid administration. This thrombocytopenia resolved within 60 minutes. In vitro addition of amniotic fluid to blood resulted in accelerated clotting on TEG tracings as compared to the effect of saline. As we were not able to reproduce AFE with our model, the role of kinins in this syndrome remains to be determined.

La naissance est l’une des étapes les plus délicates à laquelle les nouveau-nés mammifères doivent faire face. Le nouveau-né doit opérer des changements physiologiques et comportementaux pour s’adapter à l’environnement aérien, et l’un des premiers défis est d’ingérer du colostrum et du lait. Il est surprenant que la nature des stimuli et les mécanismes impliqués dans le déclenchement de la tétée soient encore mal connus, alors que la survie du nouveau-né est conditionnée par le succès de la première tétée. Par conséquent, cette étude a pour but de comprendre comment un nouveau-né immature et inexpérimenté réussit à s’orienter vers une tétine, à la saisir et à la téter de façon efficace? Cette question générale est posée chez la souris en focalisant sur : i) la nature des substrats chimiques utilisés par les souriceaux pour atteindre les tétines maternelles ; ii) la variation de la puissance attractive de ces substrats au cours du développement ; et iii) l’implication des effets de l’expérience dans l’établissement des réponses adaptatives précoces. Premièrement, nos résultats mettent en lumière que les odeurs mammaires de femelles allaitantes induisent plus d’approches et de saisies de la tétine chez les souriceaux que celles émanant de femelles non allaitantes. Deuxièmement, les odeurs de liquide amniotique et de lait déclenchent la première saisie orale de la tétine chez des souriceaux à la naissance, alors que les odeurs de salives maternelle et infantile n’induisent ce comportement qu’après une brève expérience de tétée. Troisièmement, les souriceaux âgés de 0, 2 et 6 jours postnatals (P), ayant eu une expérience de tétée, affichent une attraction sélective envers des odeurs de laits collectés en début de lactation plutôt qu’en fin de lactation, alors que les souriceaux plus âgés P15 ne montrent aucune réponse sélective envers ces odeurs. En résumé, certains substrats biologiques présents sur les tétines de femelles allaitantes sont immédiatement attractifs après la naissance, tandis que d’autres ont besoin d’être appris pour être réactogènes. Par conséquent, la réponse initiale de recherche de la tétine chez le souriceau est contrôlée par des processus d’apprentissage postnatal. A ce stade, l’implication de l’apprentissage prénatal et de processus prédisposés n’a pu être prouvée, bien qu’elle ne soit pas exclue. Ces résultats montrent des capacités d’apprentissage sophistiquées chez le souriceau nouveau-né / Birth is one of the most delicate periods mammalian infants have to deal with. Newborns have then to adapt physiologically and behaviorally to the aerial environment, and one of their first challenges is to ingest colostrum and milk. It is paradoxical that the survival of pups is conditioned by the success of this first suckling, and that we have so little understanding of the stimuli that underlie and promote it. Thus, the present work aims to contribute to answer how immature and naïve newborns do manage to orient to a nipple, to grasp it, and to suckle efficiently? This general issue will be addressed in the mouse in focusing on: i) the nature of the chemical substrates that newborn mice use to reach nipples; ii) whether the attractive potency of these substrates changes as a function of development; and iii) whether exposure effects underly the establishment of early adaptive responses? The results highlight that mammary odors of lactating females are more behaviorally active for newly born pups than those of non-lactating females. Secondly, amniotic and milk odors provoke the first nipple grasping in newborns right at birth, while maternal and pup salivary odors induce this behavior after short sucking experience. Thirdly, younger pups on postnatal day (P) 0, 2 and 6 (with sucking experience), display a selective orientation toward the odor of milk collected during early-lactation rather than to the odor of late-lactation milk, whereas older pups P15 do not exhibit any selective attraction to these odors. To summarize, some of the biological substrates which are present on a nursing mouse nipples are attractive immediately after birth, while some others need to be postnatally learned to be active. Thus, the initial nipple search response of mouse pups is controlled by processes involving postnatal learning. At this stage, the involvement of prenatal learning and of predisposed processes that do not depend on previous exposure effect are not conclusive, although not excluded. These results show highly sophisticated learning abilities in a newborn mammal

The aim of this thesis was to establish methods for selected PAPP-A (Pregnancy- Associated Plasma Protein A) gene polymorphisms analysis and to study genetic background of PAPP-A and biochemical background of PAPP-A and PlGF (Placental Growth Factor) in relation to risk pregnancy. Secondly, the aim was to establish method for two-dimensional (2D) electrophoresis of amniotic fluid. Methods for analysis of ten PAPP-A gene polymorphisms were established. These polymorphisms, PAPP-A and PlGF levels were studied in together 165 women in third trimester pregnancies complicated with threatening preterm labor (n=98), preeclampsia (n=35), IUGR (Intrauterine Growth Restriction) (n=34) and ICP (Intrahepatic Cholestasis of Pregnancy) (n=15). 114 healthy pregnant women served as controls. The method for 2D electrophoresis of amniotic fluid was established. Preeclamptic patients had significantly higher frequency of TT genotype of Cys327Cys (C/T) PAPP-A gene polymorphism compared to controls. Patients with ICP had increased serum levels of PAPP-A compared to controls, in patients with threatening preterm labor PAPP-A levels were rather decreased. PlGF levels did not differ from control group in patients with ICP and threatening preterm labor. Positive correlation was found between PAPP-A and PlGF in group of...

La transplantation rénale, thérapie de choix de l'insuffisance rénale chronique terminale, est limitée par une pénurie d'organes. Les greffons issus de donneurs décédés par arrêt cardiaque (DDAC) peuvent contribuer à pallier à cette pénurie au prix de stratégies thérapeutiques visant à améliorer l'issue de la transplantation. Les cellules souches mésenchymateuses (MSC) de l'organisme adulte ont des propriétés de sécrétion, d'immunomodulation et de différenciation intéressantes dans ce contexte.L'objectif de ce travail est d'évaluer, dans un modèle pré-clinique porcin de DDAC, le potentiel thérapeutique de deux populations cellulaires d'intérêt : les MSC issues du liquide amniotique (AFSC) et les cellules de la fraction vasculaire stromale du tissu adipeux (SVF). Les AFSC porcines injectées dans l'artère rénale 7 jours post-greffe, en raison de leur sensibilité à une séquence d'hypoxie réoxygénation (HR) in vitro, accélèrent la reprise de fonction et réduisent l'extension des lésions chroniques du greffon et sont détectées dans le rein 24h après injection. La SVF porcine, phénotypiquement proche de celle de l'Homme, est moins sensible à cette séquence d'HR et peut être injectée dans l'artère du greffon à sa reperfusion sans perturbation du flux sanguin rénal, avec une rétention des cellules dans le rein 24h post injection.Ce travail met en évidence le rôle bénéfique des AFSC dans la réparation des lésions ischémie-reperfusion des greffons issus des DDAC, ainsi que la faisabilité de l'injection de la SVF dans l'artère rénale après transplantation, et ouvre des pistes pour l'optimisation les protocoles d'administration de produits de thérapie cellulaire en transplantation. / Kidney transplantation is the best therapeutic option for end stage chronic kidney failure, but is limited by transplant shortage. Use of transplants from deceased after cardiac death donors (DCD) could represent an additional graft source, but there is a need for developing new therapeutic approaches like cell therapy to increase their recovery. Mesenchymal stem cells (MSC) potentially extracted from many adult tissues have interesting paracrine, immune-modulating, and differentiation properties in this context. This work aims to assess, in a preclinical porcine model DCD donor, the therapeutic potential of two cell populations of interest: amniotic fluid derived MSC (AFSC) and cells from stromal vascular fraction of adipose tissue (SVF). Delayed injection of AFSC 7 days following kidney transplantation because of their sensitivity to a specific Hypoxia Reoxygenation (HR) sequence in vitro, accelerates graft function recovery and limits chronic injuries to the transplanted organ. Cells are detectable into the transplanted kidney 24h after injection. Porcine SVF is phenotypically similar to human. Injected in renal artery simultaneously with organ reperfusion because of its resistance to the HR sequence, porcine SVF does not disturb renal blood flow and allow cell-retention within the organ 24h after injection. This work highlights the protective effect of AFSC against ischemia reperfusion lesions in grafts from DCD donors and the feasibility of SVF injection directly into the renal artery of the graft following kidney transplantation in DCD conditions. Moreover it opens new lines for optimizing injection protocols of cellular products in kidney transplantation.

The aim of this thesis was to establish methods for selected PAPP-A (Pregnancy- Associated Plasma Protein A) gene polymorphisms analysis and to study genetic background of PAPP-A and biochemical background of PAPP-A and PlGF (Placental Growth Factor) in relation to risk pregnancy. Secondly, the aim was to establish method for two-dimensional (2D) electrophoresis of amniotic fluid. Methods for analysis of ten PAPP-A gene polymorphisms were established. These polymorphisms, PAPP-A and PlGF levels were studied in together 165 women in third trimester pregnancies complicated with threatening preterm labor (n=98), preeclampsia (n=35), IUGR (Intrauterine Growth Restriction) (n=34) and ICP (Intrahepatic Cholestasis of Pregnancy) (n=15). 114 healthy pregnant women served as controls. The method for 2D electrophoresis of amniotic fluid was established. Preeclamptic patients had significantly higher frequency of TT genotype of Cys327Cys (C/T) PAPP-A gene polymorphism compared to controls. Patients with ICP had increased serum levels of PAPP-A compared to controls, in patients with threatening preterm labor PAPP-A levels were rather decreased. PlGF levels did not differ from control group in patients with ICP and threatening preterm labor. Positive correlation was found between PAPP-A and PlGF in group of...