A protocol adapted for the propagation of monolayer cultures from human villous placenta is described. Keratin immunofluorescent localization was utilized to characterize trophoblast cells in sub-cultivated cultures derived from first trimester placentae. The appearance of autofluorescent particles in long-term first trimester placental cultures is reported. The monolayer culture system was employed to study living mitochondria. Using the vital fluorescent dye rhodamine 123, fluorescence microscopy of living first trimester human placental cells and choriocarcinoma cells was carried out. The length and intracellular distribution of mitochondria are described for normal placental cells, somatic hybrids of normal cells, and choriocarcinoma cells. In normal first trimester placental cells, quantitative relationships between the projected cell surface areas and numbers/lengths of their mitochondria are described by regression equations. From the equations, it is deduced that the total (aggregate) length of mitochondria per cell is directly proportional to the 0.86 +/- 0.07 power of the cell's projected surface area. The number of mitochondria per cell is also a direct function of the cellular surface area, with an exponent of 0.82 +/- 0.07; whilst the total length of mitochondria per unit projected cell surface area is an inverse function (exponent = -0.14 + 0.07) of the cell surface area. The reaction of living mitochondria in cultured first trimester placental cells to a number of chemical agents including colchicine was investigated. In the presence of colchicine, it appears that the distribution of mitochondria becomes more restricted to the perinuclear cytoplasm and that the mean length of the organelles is reduced. However, the total length of all mitochondria in colchicine-treated cells was not significantly different from that in similar cells which were not exposed to the drug. Experiments investigating the recovery of cells from colchicine-mediated changes in their mitochondrial orientation and length is presented.

Um experimento foi conduzido com os objetivos de avaliar a influência de diferentes tempos de aquecimento antes do armazenamento e diferentes tempos de armazenamento de ovos férteis de matrizes Cobb 500 com 44 semanas antes da incubação, sobre a eclodibilidade e características de pintos de um dia. Utilizou-se o delineamento inteiramente ao acaso em arranjo fatorial 3x3, com os fatores: aquecimento antes do armazenamento (37ºC por 0, 6 e 12h) e armazenamento antes da incubação (12ºC por 4, 9 e 14 dias), totalizando nove tratamentos com 22 repetições de 10 ovos cada. O procedimento de incubação foi o convencional adotado pela avicultura industrial. Avaliaram-se as características de eclosão (480 e 498h), peso do pintainho, perda de peso dos ovos durante o aquecimento e armazenamento, temperatura de superfície da casca, embriodiagnóstico, peso relativo de órgãos e características morfométricas e histológicas intestinais. O aquecimento por 6h não prejudicou a eclodibilidade em comparação ao não aquecimento dos ovos. O aquecimento por 12h resultou em menores taxas de eclosão. O peso dos pintainhos não foi influenciado pelo tempo de armazenamento dos ovos, no entanto, os ovos aquecidos por 0 e 6h produziram pintainhos com pesos superiores quando comparados com aqueles ovos aquecidos por 12h. As características de vilosidades intestinais não foram influenciadas pelos tratamentos, com exceção da profundidade de cripta. Conclui-se que o aquecimento pré-incubação de ovos férteis a 37ºC por 6h é adequado em manter as taxas normais de eclosão sem prejudicar as características de pintos de um dia, quando os ovos são armazenados por até quatro dias. / An experiment was carried out to evaluate the influence of different heating times before storage and different storage times of the fertile eggs from Cobb 500 broiler breeders, aged 44 weeks, before incubation on hatchability and day-old chicks characteristics. The experimental design was randomly assigned in a 3x3 factorial scheme: egg heating before storage (37ºC for 0, 6 and 12h) and egg storage before incubation (12ºC for 4, 9 and 14 days), totalizing nine treatments with 22 replicates of ten eggs each. The incubation procedure was the same used by the poultry industry. It was evaluated the hatchability characteristics (480 and 498h), day-old chick weight, egg weight loss during heating and storage, superficial eggshell temperature, and intestine morphologic and histological characteristics. The heating time of 6h did not impair hatchability of the eggs when compared to the control treatment (no heating 0h). On the other hand, heating eggs for 12h was observed significantly reduction of the hatchability. Day-old chicks weight was not influenced by the storage time, however, the eggs heated for 0 and 6h provided heavier day-old chicks than eggs heated for 12h. There were not influences of the treatments on intestine villous characteristics, except for crypt height. It was concluded that the pre-incubation fertile eggs heating at 37ºC for 6h is adequate to maintain normal rates of hatchability without impair day-old chicks characteristics when eggs are stored up to four days.

Um experimento foi conduzido com os objetivos de avaliar a influência de diferentes tempos de aquecimento antes do armazenamento e diferentes tempos de armazenamento de ovos férteis de matrizes Cobb 500 com 44 semanas antes da incubação, sobre a eclodibilidade e características de pintos de um dia. Utilizou-se o delineamento inteiramente ao acaso em arranjo fatorial 3x3, com os fatores: aquecimento antes do armazenamento (37ºC por 0, 6 e 12h) e armazenamento antes da incubação (12ºC por 4, 9 e 14 dias), totalizando nove tratamentos com 22 repetições de 10 ovos cada. O procedimento de incubação foi o convencional adotado pela avicultura industrial. Avaliaram-se as características de eclosão (480 e 498h), peso do pintainho, perda de peso dos ovos durante o aquecimento e armazenamento, temperatura de superfície da casca, embriodiagnóstico, peso relativo de órgãos e características morfométricas e histológicas intestinais. O aquecimento por 6h não prejudicou a eclodibilidade em comparação ao não aquecimento dos ovos. O aquecimento por 12h resultou em menores taxas de eclosão. O peso dos pintainhos não foi influenciado pelo tempo de armazenamento dos ovos, no entanto, os ovos aquecidos por 0 e 6h produziram pintainhos com pesos superiores quando comparados com aqueles ovos aquecidos por 12h. As características de vilosidades intestinais não foram influenciadas pelos tratamentos, com exceção da profundidade de cripta. Conclui-se que o aquecimento pré-incubação de ovos férteis a 37ºC por 6h é adequado em manter as taxas normais de eclosão sem prejudicar as características de pintos de um dia, quando os ovos são armazenados por até quatro dias. / An experiment was carried out to evaluate the influence of different heating times before storage and different storage times of the fertile eggs from Cobb 500 broiler breeders, aged 44 weeks, before incubation on hatchability and day-old chicks characteristics. The experimental design was randomly assigned in a 3x3 factorial scheme: egg heating before storage (37ºC for 0, 6 and 12h) and egg storage before incubation (12ºC for 4, 9 and 14 days), totalizing nine treatments with 22 replicates of ten eggs each. The incubation procedure was the same used by the poultry industry. It was evaluated the hatchability characteristics (480 and 498h), day-old chick weight, egg weight loss during heating and storage, superficial eggshell temperature, and intestine morphologic and histological characteristics. The heating time of 6h did not impair hatchability of the eggs when compared to the control treatment (no heating 0h). On the other hand, heating eggs for 12h was observed significantly reduction of the hatchability. Day-old chicks weight was not influenced by the storage time, however, the eggs heated for 0 and 6h provided heavier day-old chicks than eggs heated for 12h. There were not influences of the treatments on intestine villous characteristics, except for crypt height. It was concluded that the pre-incubation fertile eggs heating at 37ºC for 6h is adequate to maintain normal rates of hatchability without impair day-old chicks characteristics when eggs are stored up to four days.

Objetivo: estudiar la frecuencia de positividad de las pruebas serológicas en pacientes con biopsias compatible con enfermedad celiaca. Material y métodos: estudio transversal. Se incluyeron pacientes con biopsia duodenal histológicamente compatible con enfermedad celiaca y determinación de anticuerpos antigliadina, antiendomisio y antitransglutaminasa IgA. Definimos como caso de enfermedad celiaca a quienes tuvieran biopsia positiva y anticuerpos antiendomisio y/o antitransglutaminasa positivos. Resultados: 31 pacientes fueron incluidos de los cuales 6 fueron antiendomisio positivo, 5 fueron antitransglutaminasa positivo y antigliadina fue positivo en 14. Por lo tanto de 31 pacientes con cambios histológicos compatibles con enfermedad celiaca sólo 10 tuvieron serología diagnóstica. Sólo uno de los pacientes tuvo positividad tanto para antitransglutaminasa como para antiendomisio. Conclusiones: a) encontramos que la mayoría de biopsias de duodeno con un cuadro histológico sugerente de enfermedad celiaca no se corresponden con serología diagnóstica de esta enfermedad; b) encontramos baja coincidencia en la positividad serológica entre antiendomisio y antitransglutaminasa. / Objective: to study the frequency of positive serology for celiac disease (CD) in patients with duodenal biopsies suggestive of this disease. Material and methods: cross sectional study. We included patients with duodenal biopsies histologically compatible with CD and antigliadin, antiendomysial and IgA antitransglutaminase antibodies. We defined a “case” of CD if there was a positive biopsy and either antiendomisial or antitransglutaminase positive antibodies. Results: thirty one patients were included in our study. Six were antiendomysial positive and 5 antitransglutaminase positive while the antigliadin was positive in 14 cases. Therefore, out of 31 patients only 10 had a serology compatible with CD and only one had positive both antibodies, antiendomysial and antitransglutaminase. Conclusions: a) we have found that most of the duodenal biopsies compatible with CD are not diagnosed with positive serology; and b) we found a low correlation between serological diagnostic tests.

CD146 est une glycoprotéine appartenant à la superfamille des immunoglobulines, exprimée de manière ubiquitaire sur l'endothélium, principalement au niveau des jonctions. Une forme soluble de CD146 (CD146s) est générée par clivage de la forme membranaire par un mécanisme dépendant des métalloprotéases. CD146 et CD146s interviennent dans le maintien de l'intégrité de l'endothélium, la transmigration endothéliale des monocytes et possèdent des propriétés angiogéniques. Les concentrations sériques de CD146s varient au cours de pathologies liées à une atteinte vasculaire. Notre travail a pour but d'étudier l'implication de CD146s dans deux contextes indépendants: la physiopathologie obstétricale et la sclérodermie systémique. La première partie de notre travail a consisté à analyser le rôle de la molécule au cours de la grossesse. Dans le placenta normal, l'expression de CD146 est restreinte aux trophoblastes possédant des capacités de migration et d'invasion, les trophoblastes extra-villeux (EVT). Ces EVT remodèlent les artères utérines spiralées afin d'établir une circulation foeto-maternelle. Au cours de ce processus, appelé pseudo-vasculogenèse, les EVT acquièrent un phénotype endothélial. De plus, l'expression placentaire de CD146 et les concentrations de CD146s varient au cours de grossesses pathologiques. L'hypothèse de notre travail est que CD146s régulerait les capacités invasives des EVT et interviendrait dans le développement placentaire. / CD146 is a member of the immunoglobulin superfamily. This is a membrane glycoprotein localized at the endothelial junction. CD146 also exists as a soluble form in the plasma (sCD146), generated by proteolysis of membrane CD146 through a mechanism involving metalloproteinases. CD146 and sCD146 are involved in endothelium integrity, monocyte transmigration and display angiogenic properties. CD146s level variations are observed in vascular pathologies. The aim of our work was to investigate the role of sCD146 in obstetrical physiopathology and in systemic sclerosis. In a first part, the involvement of our molecule was analyzed during pregnancy. In normal placenta, CD146 is expressed in intermediate and extra-villous trophoblasts (EVT), presenting migratory and invasive properties. EVT invade the spiral arteries and convert from an epithelial to an endothelial phenotype during the pseudo-vasculogenesis process. Moreover, CD146 placental expression and sCD146 levels are modified during pathologic pregnancies. We hypothesized that sCD146 could regulate EVT invasiveness and placental development. Using placental villous explants, we demonstrated that sCD146 inhibits EVT outgrowth. Consistently, we showed that sCD146 inhibits the ability of EVT cells (HTR8/SVneo) to migrate, invade and form tubes in Matrigel®. The involvement of sCD146 in human pregnancy was investigated by evaluation of sCD146 levels in 50 pregnant women. We observed physiological down-regulation of sCD146 throughout pregnancy. These results prompted us to investigate the effect of prolonged sCD146 administration in a rat model of pregnancy.

Objectives: The study aim was to analyse the kinetics of stem and transit cells in the crypts of jejunal mucosa infected with HIV and Microsporidia. Design: The size of villi, depth of crypts and proliferative activity of transit and stem cells in jejunal mucosa were measured using morphometric techniques. Methods: The surface area/volume ratio (S/V) of jejunal biopsies was estimated under light microscopy using a Weibel graticule. Crypt length was measured by counting enterocytes along the crypt side from the base to the villus junction, and the mean crypt length was calculated. The S/V and crypt lengths of the jejunal mucosa of 21 HIV and Microsporidia-infected test cases were compared with 14 control cases. The labelling index in relation to the crypt cell position of 10 of the test cases was analysed compared with 13 control cases. Results: Differences were found in the S/V and crypt length, and there was a negative correlation between S/V and crypt length in test and control cases combined. Cell labelling indices fell into low and high proliferation groups. There were significant differences in labelling indices between low proliferation test cases and controls, between high proliferation test cases and controls, and between high and low proliferation test cases. Conclusion: Villous atrophy induced by HIV and Microsporidia is attributed to crypt cell hyperplasia and the encroachment of crypt cells onto villi. These infections induce crypt hypertrophy by stimulating cell mitosis predominantly in transit cells but also in stem cells. Increased stem cell proliferation occurs only in high proliferation cases.

Crohn's disease, a chronic inflammation of the bowel, is a multi-factorial condition where uncontrolled immune responses to luminal bacteria occur in genetically predisposed individuals. The first observable clinical signs are small ulcers that form at a specialised form of epithelium, follicle-associated epithelium (FAB). The FAB covers immune inductive sites, Peyer's patches, which function primarily as sensory areas that sample the externaI gut environment. Dendritic cells are one of the key cells that are involved in sensing luminal contents and orchestrating the gut immune system. The main aim of this thesis was to determine whether the barrier of the FAB is breached in Crohn's disease and if dysfunctional immune regulators, namely dendritic cells, playaroIe in initiating and/or maintaining the chronic intestinal inflammation. Using biopsies and surgical specimens, we were able to show that in Crohn's disease, there was an increased transmucosaI transport of Escherichia coli compared to specimens from ulcerative colitis and non-inflammatory bowel disease (IBD) controIs. Dendritic cells internalised a higher percentage of bacteria that had translocated across the FAB in the Crohn's samples. Furthermore, significantly higher concentrations of TNF-u was released upon bacterial stimulation by tissues from patients with Crohn's disease than in controIs. We went on to characterise the dendritic cells present in the Peyer's patches of patients with Crohn's disease. We found an accumulation of both immature and mature dendritic cells beneath the FAB, in the sub-epithelial dome (SED). Normally, mature dendritic cells migrate towards T cell-rich areas. However, we observed mature dendritic cells accumulating in the SED because they lacked the CCR7 migratory receptor. Furthermore, they were more prone to take-up bacteria, and produced TNF-α. To study the function of mucosal dendritic cells, we performed isolation experiments and mixed Iymphocyte reactions. Dendritic cells from both the ileum and blood of patients with active Crohn's had reduced capacity for inducing T cell proliferation than non-IBD controIs. Blood dendritic cells of patients in remission had normalised function that was similar to dendritic cells from healthy controls. The SAMPl/YitFc mice, considered an appropriate murine model for Crohn's disease, had an inherent permeability defect that increased with the chronicity of intestinaI inflammation. However unlike in human Crohn's disease, dendritic cells did not seem to playaroIe in murine ileitis. This thesis highlights the accumulation of the actively surveying dendritic cells that are prone to bacterial internalisation, and points to their possible different functional roles in active versus in-active disease; thereby confirming dendritic cells as one ofthe key components in the pathogenesis ofCrohn's disease.

Coeliac disease (CD) is a clinically heterogeneous autoimmune disease with variable presentation and progression triggered by gluten intake. Molecular or genetic factors contribute to disease heterogeneity, but the reasons for different outcomes are poorly understood. Transcriptome studies of tissue biopsies from CD patients are scarce. Here, we present a high-resolution analysis of the transcriptomes extracted from duodenal biopsies of 24 children and adolescents with active CD and 21 individuals without CD but with intestinal afflictions as controls. The transcriptomes of CD patients divide into three groups—a mixed group presenting the control cases, and CD-low and CD-high groups referring to lower and higher levels of CD severity. Persistence of symptoms was weakly associated with subgroup, but the highest marsh stages were present in subgroup CD-high, together with the highest cell cycle rates as an indicator of virtually complete villous atrophy. Considerable variation in inflammation-level between subgroups was further deciphered into immune cell types using cell type de-convolution. Self-organizing maps portrayal was applied to provide high-resolution landscapes of the CD-transcriptome. We find asymmetric patterns of miRNA and long non-coding RNA and discuss the effect of epigenetic regulation. Expression of genes involved in interferon gamma signaling represent suitable markers to distinguish CD from non-CD cases. Multiple pathways overlay in CD biopsies in different ways, giving rise to heterogeneous transcriptional patterns, which potentially provide information about etiology and the course of the disease.

Le placenta humain est un organe indispensable au maintien de la grossesse et au développement foetal. Son unité structurale et fonctionnelle est la villosité choriale constituée principalement de trophoblastes qui se différencient selon la voie villeuse endocrine ou extravilleuse invasive. Ces deux populations trophoblastiques sécrètent de l'hormone chorionique gonadotrope humaine (hCG), hormone indispensable à la grossesse. C'est une glycoprotéine constituée de deux sous-unités: la sous-unité alpha commune avec la LH, FSH et la TSH et la sous-unité beta, spécifique à chaque hormone, codée par un cluster de gênes regroupés en type I (gêne beta 7) et type II (gênes beta 3, 5 et 8). L'hCG est sécrétée dans le compartiment maternel où elle joue un rôle endocrine essentiel au maintien de la grossesse en stimulant la production de progestérone par l'ovaire. L'hCG joue également un rôle localement en stimulant la différenciation de chaque type de trophoblaste. Elle présente, dans le sang maternel, un pic de sécrétion à 10-12 semaines d'aménorrhée (SA), période ou le statut oxydatif placentaire change. En effet, les bouchons trophoblastiques obstruant la lumière des artères spiralées utérines se délitent à cette période, permettant l'entrée progressive du sang maternel dans la chambre intervilleuse. La pression en oxygène augmente de 18 mm/Hg (8-9 SA, 1er trimestre précoce) à 60 mm/Hg (12-14 SA, 1er trimestre tardif). Dans mon travail de thèse, j'ai cherché à mettre en évidence in situ et in vitro l'impact de ce changement de statut oxydatif sur la différenciation des trophoblastes villeux du 1er trimestre, et plus particulièrement sur l'expression des hCG beta de type I et de type II. J'ai ainsi mis en évidence que les trophoblastes villeux mononucléés du 1er trimestre précoce sécrétaient plus d'hCG beta de type I et II, fusionnaient plus rapidement et exprimaient un panel de facteurs de transcription différents par rapport aux trophoblastes villeux du 1er trimestre tardif. Dans un deuxième temps, j'ai comparé in vitro l'expression et la régulation des deux types d'hCG beta entre les trophoblastes villeux et extravilleux. J'ai montré que: 1) les trophoblastes villeux expriment plus d'hCG beta de type I et II que les trophoblastes extravilleux, 2) dans les deux cas l'hCG beta de type II est majoritaire et 3) PPAR gamma régule de façon opposée ces deux types d'hCG entre les trophoblastes villeux et extravilleux. Enfin j'ai mis en évidence que l'expression de ces deux types d'hCG était dérégulée dans la pré-éclampsie et le RCIU. L'étude des mécanismes impliqués dans la régulation des gênes codants pour l'hCG représente un enjeu important pour la compréhension de la différenciation du trophoblaste humain, du développement précoce du placenta et des pathologies de la grossesse. / The human placenta is an essential organ to maintain pregnancy and for foetal growth. Its structural and functional unit is the chorionic villous, which is mainly composed of cytotrophoblasts that follow two differentiation pathways: the endocrine villous and the invasive extravillous trophoblasts. These two trophoblastic subtypes secrete the human chorionic gonadotropin hormone (hCG), an essential hormone for trophoblast differentiation, placental development and pregnancy. hCG is a glycoprotein composed of two subunits: the alpha subunit, which is common to LH, FSH and TSH, and the beta subunit that confers hormone specificity. A gene cluster encodes the beta subunit, type I (CGB7) and type II (CGB3, 5 and 8), that code for two different proteins. hCG is detected in the maternal blood from the first week of pregnancy, with a peak level at 10-12 weeks of gestation (WG). During the first trimester the oxygen concentration in the intervillous space changes from about 2% (prior to 10 WG) to approximately 6-8% (after 12 WG) due to development of blood flow to the placenta. During my PhD work, I studied in situ and in vitro the impact of these different environments during the first trimester on villous cytotrophoblast differentiation, and more specifically on the type I and type II beta hCG gene expression. I showed that type I and type II beta hCG are more expressed in early first trimester cytotrophoblasts and that these cells exibit more fusion features and express a different panel of transcription factors compare to cells from late first trimester. In the second part of my work, I compared the expression and the regulation in vitro of the two types of beta hCG between villous and extravillous cytotrophoblasts. I demonstrated: 1) villous trophoblast express more type I and type II beta hCG compared to the extravillous trophoblast, 2) in both case type II hCG beta is the major form of beta hCG and 3) PPAR gamma differentially regulates type I and type II beta hCG expression in villous and extravillous trophoblasts. Lastly I showed that the expression of type I and type II beta hCG is deregulated in pre-eclampsia and FGR. The study of the mechanisms involved in hCG regulation represents an important issue for the understanding of human trophoblast differenciation and pregnancy pathophysiology.