No description available.

Novel techniques, mainly three-dimensional (3D) reconstructions from image stacks and finite element analysis (FEA), were combined to study the oxygen transport mechanism in the human placenta and its relationship to placental structure. Initial research relates to the development of a platform suitable for realistic computational simulations. The work shows how the 3D architecture of a terminal villus can be accurately reconstructed from confocal laser scanning microscopic (CLSM) images. By combining the resultant 3D structures of terminal villi with finite element analysis, the diffusion of oxygen from the maternal bloodstream towards the fetal blood across the villous membrane is assessed. The results correlate with theoretical studies demonstrating that image-based computational modelling is a robust platform to explore the structure-function relationship in the placenta. Following work deals with the study of blood flow through the fetal capillary network, with particular interest in its role on the oxygen transport capacity of the terminal villi. The computational models are corroborated by a particle image velocimetry (PIV) experiment. The study shows that the variation in capillary diameter is key for effective oxygen uptake by the fetus. The fetus invests minimum energy needed for the blood to travel fast enough in order to provide oxygenated blood, but at the same time slow enough to allow for good oxygenation. This is achieved by the combination of narrow and dilated segments. Additionally, the results demonstrate that there is no vortical flow or whirling. In the subsequent work, the effect of blood properties is investigated. The calculated oxygen flux is 75 times higher than in the previous study (blood flow models), highlighting the importance of haemoglobin molecules in transporting oxygen. Fetal blood affinity is shown to improve fetal oxygen uptake by 11.5%. However, when accounting for haemoglobin concentration the data suggest that the different villous structures have a constant oxygen transport capacity. The methodology developed herein helps to elucidate the structure-function relationship in the human placenta. Additionally, 3D image-based multi-physics computational modelling is demonstrated to be a powerful tool to investigate in detail the mechanics of transport in the human placenta. This technique has the potential to enlighten on the development of pregnancy complications and serve as an in vivo diagnostic tool.

No description available.

Em diferentes mopdelos, o aumento dos níveis séricos de glicose pode ativar cascatas de sinalização envolvendo receptores semelhantes a Toll, o fator de transcrição NF-kB, moléculas da familia NLRP, ASC e caspase-1, que culminam na produção, ativação e liberação de moléculas inflamatórias. Investigamos o perfil inflamatório sérico e placentário em gestantes com distúrbios glicêmicos e a possível participação da via do inflamassoma NLRP1 e NRLP3 para o estabelecimento desse perfil. Para avaliar o papel da hiperglicemia no estabelecimento desse perfil, a via do inflamassoma foi também estudada em explantes de vilos coriônicos tratados com 100, 200, 300 e 400 mg/dL de glicose. Nossos resultados mostraram aumento na ativação de NF-kB e da expressão de NLRP1, NLRP3, ASC e Caspase-1 nas placentas de gestantes hiperglicemicas e nas culturas tratadas com doses de 300 r 400 mg/dL de glicose. Esses dados sugerem que a ativação da via do inflamassoma na porção fetal da placenta contribui para o processo inflamatório nessas pacientes. / In different models, the increase in serum glucose levels can activate signaling cascades involving Toll-like receptors, the transcription factor NF-kB, NLRP proteins, ASC and caspase-1, which culminate in the production, activation and release of inflammatory molecules . We investigated the serum and placental inflammatory profile in pregnant women with glucose disorders and the possible participation of via the NLRP1 and NRLP3 inflammasome to establish this profile. To evaluate the role of hyperglycemia, molecules of the inflammasome pathway was also studied in explants of chorionic villi treated with 100, 200, 300 and 400 mg / dL glucose. Our results showed an increase in NF-kB activation and expression of NLRP1, NLRP3, ASC and Caspase-1 in placentas of pregnant hyperglycemic women and in cultures treated with 300 and 400 mg / dL glucose. These data suggest that the inflammasome activation of the fetal portion of the placenta contributes to the inflammatory process in these patients.

Em diferentes mopdelos, o aumento dos níveis séricos de glicose pode ativar cascatas de sinalização envolvendo receptores semelhantes a Toll, o fator de transcrição NF-kB, moléculas da familia NLRP, ASC e caspase-1, que culminam na produção, ativação e liberação de moléculas inflamatórias. Investigamos o perfil inflamatório sérico e placentário em gestantes com distúrbios glicêmicos e a possível participação da via do inflamassoma NLRP1 e NRLP3 para o estabelecimento desse perfil. Para avaliar o papel da hiperglicemia no estabelecimento desse perfil, a via do inflamassoma foi também estudada em explantes de vilos coriônicos tratados com 100, 200, 300 e 400 mg/dL de glicose. Nossos resultados mostraram aumento na ativação de NF-kB e da expressão de NLRP1, NLRP3, ASC e Caspase-1 nas placentas de gestantes hiperglicemicas e nas culturas tratadas com doses de 300 r 400 mg/dL de glicose. Esses dados sugerem que a ativação da via do inflamassoma na porção fetal da placenta contribui para o processo inflamatório nessas pacientes. / In different models, the increase in serum glucose levels can activate signaling cascades involving Toll-like receptors, the transcription factor NF-kB, NLRP proteins, ASC and caspase-1, which culminate in the production, activation and release of inflammatory molecules . We investigated the serum and placental inflammatory profile in pregnant women with glucose disorders and the possible participation of via the NLRP1 and NRLP3 inflammasome to establish this profile. To evaluate the role of hyperglycemia, molecules of the inflammasome pathway was also studied in explants of chorionic villi treated with 100, 200, 300 and 400 mg / dL glucose. Our results showed an increase in NF-kB activation and expression of NLRP1, NLRP3, ASC and Caspase-1 in placentas of pregnant hyperglycemic women and in cultures treated with 300 and 400 mg / dL glucose. These data suggest that the inflammasome activation of the fetal portion of the placenta contributes to the inflammatory process in these patients.

A novel molecular mechanism of autophosphorylation-dependent activation of the ser/thr S6/H4 kinase isolated from human placenta is described. Phosphopeptide mapping of the enzyme was used to determine the rate and extent of site-specific autophosphorylation. These data were correlated to phosphotransferase activity of the protein kinase. The results indicated that a sequential phosphorylation of two sites in the catalytic domain is required for maximum activation. Kinetic analysis determined that site 1 is modified by an intramolecular phosphorylation, and site 2 is modified by an intermolecular phosphorylation. On the basis of these data a model is proposed in which autophosphorylation of the pseudosubstrate domain and on a serine residue in subdomain VIII are both required for maximum activation of the S6/H4 kinase.

Le placenta assure les échanges materno-fœtaux et possède une fonction endocrine autonome. Les hormones placentaire lactogène (hPL) et chorionique gonadotrope (hCG) sont synthétisées par le syncytiotrophoblaste. A ce jour, les mécanismes impliqués dans le contrôle de la sécrétion de ces deux hormones ne sont pas connus. In vitro, l’influx d’ions Ca2+ entraîne une augmentation immédiate et soutenue de la libération d’hPL et d’hCG à partir d’explants de placentas à terme. En outre, l’élévation de la concentration extracellulaire en albumine, principale protéine maternelle circulante en contact direct avec le trophoblaste, stimule de manière immédiate et transitoire la libération d’hPL et d’hCG. L’objectif de nos travaux a été de vérifier la spécificité de l’activité sécrétagogue de l’albumine au niveau du placenta, de caractériser les messagers cellulaires potentiellement impliqués dans la libération d’hPL et d’hCG, et de définir l’interaction entre l’albumine et le trophoblaste, en utilisant des explants provenant de placentas humains à terme. Nos travaux démontrent que la riposte sécrétoire à l’albumine (5%, m/v) est largement mimée par d’autres agents colloïdaux (dextran et polygéline). Cette stimulation colloïdale de la libération d’hPL et d’hCG impliquerait une mobilisation de Ca2+ à partir de réserves intracellulaires. L’intervention de 3 messagers cellulaires a été envisagée: les IPs/DAG, l’AMPc, et le GMPc. Le fluorure de sodium, la forskoline, ou le nitroprussiate sodique, activateurs connus de la production respective des IPs, de l’AMPc, et du GMPc, augmentent de manière significative les taux placentaires de chacun de ces messagers, sans toutefois affecter la libération d’hPL ou d’hCG. De plus, l’élévation de la concentration extracellulaire en albumine (5%, m/v) ne modifie pas les taux des IPs, de l’AMPc et du GMPc dans les explants placentaires, tandis qu’elle stimule la sécrétion hormonale. Ces systèmes de signalisation, bien que fonctionnels au niveau du trophoblaste, ne joueraient donc pas un rôle majeur dans la régulation de la libération d’hPL et d’hCG. Nos résultats mettent en évidence une internalisation rapide d’albumine marquée, avec de l’125I ou de la fluorescéïne, dans le syncytiotrophoblaste. Une large fraction de cette albumine est recyclée, intacte, vers la circulation maternelle selon un processus sensible à l’abaissement de la température et indépendant du cytosquelette. L’albumine marquée restant dans les explants placentaires est partiellement dégradée. Trois mécanismes ont été envisagés pour expliquer ces mouvements d’entrée et de sortie de l’albumine au sein du placenta humain: l’endocytose médiée par l’albondine via les caveolae, le système des coated pits clathrine-dépendant, et l’endocytose médiée par la mégaline. Par immunohistochimie, nous avons montré que, dans le tissu placentaire, la caveoline-1, protéine caractéristique des caveolae, est localisée uniquement dans l’endothelium des capillaires fœtaux. La clathrine, au niveau des coated pits, et la mégaline se trouvent au contraire dans le syncytiotrophoblaste. La méthyl-b-cyclodextrine et l’hydrochlorure de chlorpromazine, inhibiteurs d’une endocytose dépendant de la clathrine, réduisent significativement l’internalisation placentaire de l’albumine marquée. Par contre, le DIDS ou le NPPB, susceptibles de perturber l’endocytose médiée par la mégaline, n’affectent pas la captation d’albumine marquée par les explants placentaires. L’albumine pénétrerait donc dans le syncytiotrophoblaste principalement par un processus clathrine-dépendant. La mégaline ne jouerait ici qu’un rôle mineur dans l’entrée de la protéine. Un tel processus de recyclage de l’albumine pourrait être similaire à celui décrit pour les immunoglobulines G au niveau du syncytiotrophoblaste. Ces mouvement d’entrée et de sortie de l’albumine ne semblent pas associés à la stimulation de la libération d’hPL et d’hCG par l’albumine. Ils pourraient par contre participer significativement, étant donné leur ampleur, à la nutrition fœtale. L’albumine est en effet un transporteur notoire d’ions et d’acides gras, molécules qui pourraient être acheminées au fœtus via le phénomène de recyclage placentaire de l’albumine mis en évidence par ce travail. / The human placenta is the site of all maternal-fetal exchanges, and is also an active endocrine organ. Placental lactogen (hPL) and chorionic gonadotrophin (hCG) hormones are synthesized by the syncytiotrophoblast. So far, the mechanisms involved in the regulation of both hormones secretion remain elusive. In vitro, calcium inflow causes an immediate and sustained rise in the hPL and hCG releases from human term placenta explants. Moreover, increasing the extracellular concentration of albumin, the major maternal plasma protein in direct contact with the human trophoblast, stimulates the hPL and hCG releases in an immediate and transient way. Our study have aimed to check the specificity of this secretory effect of albumin, to investigate the potential cellular messengers involved in the hPL and hCG releases, and to define the interaction between albumin and the throphoblast layer, using human term placenta explants. Our results indicate that the triggering effect of albumin (5%, w/v) is largely mimicked by two other colloidal agents (dextran and polygelin). This “colloidal” stimulation of the hPL and hCG releases would involve the mobilization of calcium from intracellular pools. Three cellular messengers have been considered to mediate this process: the IPs/DAG, the cAMP, and the cGMP. Sodium fluoride, forskolin, or sodium nitroprusside, known activators of respectively the IPs, cAMP, and cGMP production, significantly increase the placental content of each of those messengers, without modifying the hPL and hCG releases. In addition, raising the extracellular concentration of albumin does not cause any change in the placental level of IPs, cAMP, and cGMP, while stimulating the hormonal release. These three signaling pathways are thus functional in human term trophoblast but do not appear to significantly modulate the hPL and hCG secretions. Our findings show that albumin, labeled with 125I or with fluorescein, is rapidly internalized into the syncytiotrophoblast. Thereafter, the intact protein is largely recycled to the maternal circulation, through a temperature-sensitive and cytoskeleton-independent process. The labeled albumin remaining in placental explants is partially degraded. Three different mechanisms could participate to the albumin entry into the human placenta: the albondin-mediated endocytosis via the caveolae, the clathrin-dependent coated pits system, and the megalin-mediated endocytosis. Using immunohistochemistry, caveolin-1, marker of the caveolae, is localized in the endothelium of the fetal capillaries and not in the syncytiotrophoblast. By contrast, clathrin and megalin are observed only in the syncytiotrophoblast. Methyl-b-cyclodextrin, and chlorpromazine hydrochloride, known inhibitors of the clathrin-dependent endocytotic process, significantly reduce the placental uptake of labeled albumin. On the other hand, DIDS or NPPB, able to perturb the megalin-mediated endocytosis, do not affect the labeled albumin uptake. Thus, albumin seems to be internalized into the syncytiotrophoblast mainly through a clathrin-dependent mechanism. Megalin would only play a minor role in this process. Such movements of albumin in the human placenta may be similar to the recycling process reported for IgG at that site. The placental apical recycling of albumin is not associated to the albumin triggering effect on the hPL and hCG releases. This quantitatively significant internalization process may participate to the fetus’ nutrition. Indeed, Albumin carries ions and fatty acid, which could be brought to the fetus via the protein recycling evidenced by our study.

Although not obvious at first sight, several parallels can be drawn between pregnancy andcancer. Many proliferative, invasive and immune tolerance mechanisms that supportnormal pregnancy are also exploited by malignancies to establish a nutrient supply andevade or edit the immune response of the host. The human placenta, of crucial importancefor pregnancy success, and its main cells, the trophoblast, share several features withmalignant cells such as high cell proliferation rate, lack of cell-contact inhibition andinvasiveness. Both in cancer and in pregnancy, the immune defense mechanisms,potentially threatening the survival of the tumor or the fetus, are progressively blunted oreven turned into tumor- or pregnancy-promoting players. Amongst immune mechanisms that are meant to protect the host from cancer and can be apotential threat to the fetus, the NKG2D receptor-ligand system stands out as the mostpowerful, stress-inducible “danger detector” system that comprises the activating NK cellreceptor NKG2D and its ligands, the MIC (MHC class I Chain-related proteins A and B)and ULBP (UL-16 Binding Proteins) families. It is the major cytotoxic mechanism in thebody promoting surveillance and homeostasis. In the present thesis we investigate theNKG2D receptor-ligand system in human early normal pregnancy and in theleukemia/lymphoma cell lines Jurkat and Raji and ask the questions “How is the NKG2Dreceptor-ligand system functioning in pregnancy and tumor? How is the danger of cytotoxicattack of the fetus avoided? Why is the immunosurveillance function compromised incancer patients?” We developed a method to isolate and culture villous trophoblast from early human normalplacenta and used it to study the NKG2D receptor-ligand system. We discovered that theNKG2D ligand families of molecules MICA/B and ULBP1-5 are constitutively expressedby the syncytiotrophoblast of the chorionic villi. Using immnunoelectron microscopy, westudied the expression of these molecules at the subcellular level and could show for thefirst time that they are preferably expressed on microvesicles in multivesicular bodies(MVB) of the late endosomal compartment and are secreted as exosomes. Exosomes arenanometer sized microvesicles of endosomal origin, produced and secreted by a great7variety of normal and tumor cells. The exosomes are packages of proteins and ribonucleicacids that function as “mail” or “messengers” between cells conveying different biologicalinformation. We isolated and studied exosomes from placental explant cultures. We foundthat they carry NKG2D ligands on their surface and are able to bind and down-regulate thecognate receptor on NK-, CD8+ and <img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Cgamma" /><img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Cdelta" />T cells. The down-regulation selectively causedimpairment of the cytotoxic response of the cells but did not affect their lytic ability asmeasured by perforin content and gene transcription. Thus, the NKG2D ligand-bearingexosomes suppress the cytotoxic activity of the cells in the vicinity of the placenta, leavingtheir cytolytic machinery intact, ready to function when the cognate receptor isrestored/recycled. These findings highlight the role of placental exosomes in the fetalmaternalimmune escape and support the view of placenta as an unique immunomodulatoryorgan. Next, we studied the expression and exosomal release of NKG2D ligands by tumor cellsusing the leukemia cell lines Jurkat and Raji as a tumor model. We found that NKG2Dligand-bearing exosomes with similar immunosuppressive properties as placental exosomesare constitutively secreted by the tumor cells, as a mechanism to blunt the cytotoxicresponse of the immune cells and thus protect themselves from cytotoxic attack by the host.Interestingly, we found that thermal- and oxidative stress up-regulates the exosomesecretion and the amount of exosome-secreted NKG2D ligands. Our results imply thattumor therapies that cause stress-induced damage, such as thermotherapy and stripping ofoxygen supply to the tumor, might have a previously unrecognized side effect causingenhanced exosome production and secretion, which in turn suppresses the natural antitumorimmune response and thus should be taken into account when designing an optimaltherapy of cancer patients. In conclusion, we describe a novel stress-inducible mechanism shared by placenta andtumors as an immune escape strategy. We found that placenta- and tumor-derived NKG2Dligand-bearing exosomes can suppress immune responses to promote the survival and wellbeing of the fetus or the tumor. Our work comprises an important contribution to theelucidation of the NKG2D ligand-receptor system and its mode of operation in the humanbody and opens new perspectives for designing novel therapies for infertility and cancer.

Multidrug resistance phosphoglycoprotein (MDR1/P-gp) and breast cancer resistance protein (BCRP) were first isolated in chemoresistant cancer cells and have since been found in a variety of normal tissue, including the placenta. The potential function of MDR1/P-gp and BCRP in the human placenta is to protect the fetus from maternally circulating endogenous steroids and hormones, therapeutic drugs and toxins. The objective of this study was to examine the role of maternal steroids in the regulation of MDR1/P-gp and BCRP in the human placenta. Trophoblast cells were isolated from term placenta tissues and immunohistochemistry, western blot analysis and transport studies were used to determine the effect of maternal steroids on MDR1/P-gp and BCRP regulation. Maternal steroids, present at high concentrations in maternal serum, did not have an effect on BCRP in human syncytiotrophoblast. Estrogen and progesterone did not alter MDR1/P-gp levels in human syncytiotrophoblast, but cortisol significantly decreased MDR1/P-gp levels.

Preeclampsia and molar pregnancy are two devastating placental pathologies characterized by an immature proliferative trophoblast phenotype accompanied by excessive cell death. It is therefore of paramount importance to study the regulation of cell fate in the placenta, to gain a further understanding of the mechanisms that contribute to these diseases. In this dissertation we report that during normal placental development and in preeclampsia, Matador (Mtd), a pro-apoptotic member of the Bcl-2 family, has a dual function in regulating trophoblast cell proliferation and death. Importantly, we reveal a novel role of Mtd-L in promoting cyclin E1 expression and cell cycle progression. Of clinical importance, we also identify that both cyclin E1 and the CDK inhibitor p27, are increased in severe early onset preeclampsia. However, the inhibitory function of p27 in this pathology may be hampered due to its increased phosphorylation at Ser10, resulting in its nuclear export. Of equal importance, data presented demonstrate that placentae from severe early onset preeclampsia display a molecular profile distinct from late onset preeclampsia or intrauterine growth restricted pregnancies. In the final data chapter we demonstrate that Mtd is highly expressed in molar tissue, where it localizes to both apoptotic and proliferative cells. Our data suggests that an abundance of Mtd and cyclin E1 in conjunction with the low level of p27 may contribute to the hyperproliferative nature of the disorder. The body of work in this dissertation uncovers novel insights into the regulation of trophoblast cell fate. Importantly, the impact of Mtd on cyclin E1 to promote G1-S transition is a novel mechanism found to regulate trophoblast cell proliferation in normal and pathological placentation. Equally important is our identification of molecular differences between placental pathologies that may help to differentiate early and late onset preeclampsia, IUGR and molar pregnancy.