Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Proefschrift Universiteit van Amsterdam. / Met samenvatting in het Nederlands.

Thesis (doctoral)--Liège, 1979.

Thesis (M.S.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: Edward E. Schmidt. Includes bibliographical references (leaves 71-83).

Proefschrift Universiteit Maastricht. / Auteursnaam op omslag: Marijke Buckx-Sanders. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

The aim of this project was to study ATP binding cassette (ABC) transporters in the human placenta, in particular their regulation and role in trophoblast differentiation and survival. The presence and localisation of four major placental drug transporters, multidrug resistance gene product 1 and 3 (MDR1 and 3)/ABC subfamily B members 1 and 4 (ABCB1 and 4), multidrug resistance associated proteins 1 and 2 (MRP1 and 2)/ABCC1 and 2 and breast cancer resistance protein (BCRP)/ABCG2 was initially studied in term human placenta, cultured primary trophoblast and BeWo and Jar trophoblast-like cell lines. Jar cells were found to be more similar to nondifferentiated cytotrophoblast with respect to their ABC protein expression profile, whereas BeWo cells more closely reflected differentiated syncytiotrophoblast. Treatment of primary term trophoblasts in vitro with cytokines (TNF- or IL-1) decreased expression and activity of apical transporters ABCB1/MDR1 and ABCG2/BCRP. Growth factors, on the other hand, increased BCRP expression and activity, while estradiol stimulated BCRP, MDR1 and MDR3 expression MDR1/3 functional activity. The ability of BCRP/ABCG2 to abrogate the apoptotic effects of TNF- and ceramides was studied in primary trophoblast and BeWo cells using pharmacological and molecular (siRNA) approaches. The results suggest that BCRP/ABCG2 contributes to the resistance of trophoblast cells to cytokine-induced (extrinsic) apoptosis, whereas its effects on apoptosis activated via the intrinsic mitochondrial pathway is minimal. This altered resistance was associated with increased intracellular accumulation of ceramides and reduced ability to maintain phosphatidylserine in the inner leaflet of the plasma membrane. A role for BCRP/ABCG2 in cell protection from differentiation-induced stressors was also demonstrated during the process of cell fusion associated with transient loss of plasma membrane lipid asymmetry. Finally, expression of BCRP/ABCG2 (and 9 other genes) was studied in 50 placentas from normal pregnancy and pregnancies complicated with fetal growth restriction (FGR). A marked reduction of BCRP/ABCG2 and MDR1/ABCB1 expression was observed in FGR placentas, while other transporter genes were unaffected. Collectively these data suggest that BCRP/ABCG2 and probably other ABC transporters may play a hitherto unrecognised survival role in the placenta, conferring a “stress resistance” to trophoblast cells.

Resumo: Avaliar a influência do índice apoptótico e da imuno-expressão da survivina em tecido molar no prognóstico e tratamento de pacientes com mola hidatiforme completa (MHC). Estudo observacional, retrospectivo, incluindo 78 pacientes com MHC diagnosticadas, tratadas e acompanhadas no Centro de Doenças Trofoblásticas de Botucatu/SP, Brasil, entre 1995 e 2006. Baseado nas curvas de regressão da gonadotrofina coriônica, as pacientes foram divididas em dois grupos: remissão espontânea (MHC-RE - 59 pacientes) e evolução para NTG pós-molar (MHC-NTG - 19 pacientes). Avaliação imunohistoquímica do trofoblasto viloso foi realizada pela técnica da avidina-biotina-peroxidase, usando dois marcadores: anticorpo policlonal anti-caspase-3 (diluição 1:200; Cell Signaling Technology; TX, USA) e anticorpo monoclonal anti-survivina (clone 5E8; diluição 1:100; Neomarkers, TX, USA). O índice apoptótico foi expresso em porcentual (número de células caspase-3 positivas / número de células contadas x 100). A imuno-expressão da survivina foi determinada por um método semi-quantitativo. Foi significativo o efeito do índice apoptótico sobre a evolução de pacientes com MHC, de tal modo que, o aumento de 1 unidade no índice apoptótico reduziu, em média, 61% a chance de desenvolvimento de NTG pósmolar (OR = 0,61, 95% IC: 0,45-0,84). Nenhuma influência significativa da imunoexpressão da survivina foi observada no desenvolvimento de NTG pós-molar (p > 0,01; teste exato de Fisher). Não foi possível estabelecer correlações entre efeito do índice apoptótico e da imuno-expressão da survivina e variáveis do tratamento. Nesse estudo, o índice apoptótico foi bom preditor do desenvolvimento de NTG depois de MHC, com potencial para ser usado como biomarcador prognóstico dessa doença. Ao contrário, a imuno-expressão... (resumo completo, clicar acesso eletrônico abaixo) / Abstract: To assess the influence of the apoptotic index and survivin expression of molar tissue on the prognosis and treatment of patients with complete hydatidiform mole (CHM). This retrospective observational study included 78 patients with CHM, who were diagnosed, treated and followed up in the Center of Trophoblastic Diseases, Botucatu/SP, Brazil, between 1995 and 2006. Based on chorionic gonadotrophin regression curves, patients were divided into two groups: spontaneous remission (CHM-RE - 59 patients) and post-molar GTN (CHM-NTG - 19 patients). Immunohistochemical analysis of the villous trophoblast was perfomed by avidin-biotin-peroxidase, using anti-caspase-3 polyclonal antibodies (1:200; Cell Signaling Technology; TX, USA) and anti-survivin monoclonal antibodies (clone 5E8; 1:100; Neomarkers, TX, USA). The apoptotic index was expressed in percent (number of caspase-3-positive cells / number of cells counted x 100). Survivin immuno-expression was determined by a semiquantitative method. The influence of the apoptotic index on the prognosis of patients with CHM was significant. A 1-unit increase in the apoptotic index represented an average 61% reduction in the chance of developing post-molar GTN (OR = 0.61, 95% CI: 0.45-0.84). No significant influence of survivin immuno-expression was observed on the development of post-molar GTN (p > 0.01; Fisher's exact test). No correlations of treatment variables with apoptotic index or survivin immunoexpression were found. In this study, the apoptotic index was a good predictor of GTN development after CHM and may be a useful prognostic biomarker of this disease. On the other hand, survivin immuno-expression in the villous trophoblast had no influence on the development of post-molar GTN. / Orientador: Marilza Vieira da Cunha Rudge / Coorientador: Izildinha Maestá / Coorientador: Maria Aparecida Custódio Domingues / Banca: José Carlos Peraçoli / Banca: Odair Carlito Michelin / Banca: Jorge Fontes de Rezende Filho / Banca: Rafael Cortés Charry / Doutor

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Biologia Celular e do Desenvolvimento, Florianópolis, 2014. / Made available in DSpace on 2015-02-05T20:34:40Z (GMT). No. of bitstreams: 1 332307.pdf: 4190550 bytes, checksum: 47afc464a32ec01695950d5da6b3dda6 (MD5) Previous issue date: 2014 / A placenta é um órgão essencial no desenvolvimento embrionário e fetal. Este órgão contém vários tipos de células, entre as quais diferentes populações de células-tronco ou progenitoras, tais como as células-tronco mesenquimais (CTM), células-tronco hematopoéticas (CTHs) e trofoblásticas. O objetivo deste trabalho foi estudar a organização do nicho molecular de células-tronco no tecido da placenta humana a termo. Para isto, foram utilizadas técnicas histológicas, histoquímicas, imuno-histoquímicas e de microscopia eletrônica de transmissão (MET). As técnicas de coloração histológica empregadas nas amostras do tecido tiveram como objetivo identificar, principalmente, as características estruturais das vilosidades coriônicas (VC) e da decídua basal (DB). A técnica de coloração de Hematoxilina e eosina proporcionou uma visão geral do tecido placentário, de modo a permitir a identificação dos elementos teciduais da placenta. A técnica de coloração azul brilhante de Coomassie foi utilizada para identificar a presença de proteínas totais neste tecido. Outro teste utilizado foi Azul de toluidina (ATO) para identificar a presença de polissacarídeos ácidos, através da reação de metacromasia. As amostras, contendo o tecido placentário, reagiram mais fortemente com o ATO nas regiões do sinciciotrofoblasto, nos núcleos das células que compõem o estroma e na parede dos vasos sanguíneos das vilosidades coriônicas. Na região da decídua basal, a reação também foi positiva ao ATO nas células deciduais, porém, na matriz extracelular esta reação foi menos intensa, quando comparada com as regiões do sinciciotrofoblasto e da parede dos vasos sanguíneos das vilosidades coriônicas. Testes realizados com o ácido periódico de Schiff mostraram que a reação foi positiva em praticamente todas as regiões das vilosidades, mas houve uma positividade maior na região do sinciciotrofoblasto. Com relação à decídua basal (DB), a presença de polissacarídeos neutros também foi confirmada em praticamente todas as regiões da decídua. A técnica de Mallory revelou que fibras de colágeno estão presentes em grande parte nas regiões tanto das vilosidades coriônicas, bem como, na decídua basal. Esta técnica é capaz de revelar a presença dos diferentes tipos colágenos que estão presentes tanto nas vilosidades coriônicas quanto na decídua basal, porém, sem a identificação de quais são os tipos presentes neste tecido. A técnica de imuno-histoquímica empregada no tecido da placenta teve como objetivo avaliar a expressão dos marcadores CD73, CD105 e CD34, bem como, o perfil de expressão do colágeno do tipo I, fibronectina e laminina na matriz extracelular (MEC). O perfil expresso10pelos marcadores CD73+, CD105+ e CD34+ na superfície da membrana das células, e pelas moléculas de MEC como o colágeno do tipo I, a fibronectina e a laminina, se mostrou positivo. Estes resultados nos mostram que as CTMs tem uma disposição na região das vilosidades coriônicas, em associação com moléculas de laminina e de colágeno. A fibronectina se expressa nas vilosidades coriônicas sempre com associação à lâmina basal e perivascularmente. No sinciciotrofoblasto as CTM se associam próximas às células CD34+ com característica de CTH. As análises ultraestruturais demonstraram plena atividade metabólica e de células que organizam a placenta, tanto bioquimicamente quanto metabolicamente, sugerindo que a placenta, mesmo a termo, é ainda capaz de produções celulares, tanto de CTs quanto na homeostase do tecido.<br> / Abstract : Placenta is an essential organ in the development of the embryo and/or fetus. It is a readily available material, easy to obtain, and there is no association between its use and risks for the mother or fetus health. The organ contains several types of cells, including different populations of stem cells or progenitor cells, such as mesenchymal stem cells (CTM), hematopoietic stem cells (CTHs) and trophoblast. The aim of this work was to study the molecular niche organization of stem cells in the tissue of human placenta at term. Therefore, it was possible to use histological, histochemical and immunohistochemical techniques, and also, transmission electron microscopy (TEM). Histological staining techniques employed on tissue samples aimed to identify, mainly, the structural characteristics of chorionic villi (VC) and decidua basalis (DB). Hematoxylin and eosin stain provided placental tissue overview in order to enable the identification of placenta tissue elements. Coomassie brilliant blue staining technique was used to determine the presence of total protein in the tissue. Results showed the placental tissue structures as a whole. Another test performed was toluidine blue (ATO) to identify the presence of acidic polysaccharides by metachromasia reaction. Samples with placental tissue reacted more strongly to the ATO in regions of syncytiotrophoblast, cells nuclei comprising the stroma and blood vessels wall of the chorionic villi. In the basal decidua region, there was also a positive reaction to ATO. However, it was less intense when compared to the regions of syncytiotrophoblast of chorionic villi of tissue. Tests performed with periodic acid of Schiff showed that the reaction was positive in virtually all regions of the villi, but there was a greater positivity in the syncytiotrophoblast region. With respect to basal decidua (DB), the presence of neutral polysaccharides was also confirmed in practically all regions of decidua. Mallory technique revealed that collagen fibers are present largely in the areas of both chorionic villi and basal decidua. That technique can reveal the presence of different collagen types that are present in both chorionic villi and basal decidua; however, without identifying which types are present in the tissue. The immunohistochemical technique employed in placental tissue aimed to evaluate the markers expression: CD73, CD105 and CD34, as well as the expression profile of collagen type I, fibronectin and laminin in the extracellular matrix (MEC). Profile expressed by markers CD73+, CD105+ and CD34+ on cells membrane surface and MEC molecules such as collagen type I, fibronectin and laminin showed in a positive12way. These results show that CTMs have a disposition in the region of chorionic villi in association with molecules of laminin and collagen. Fibronectin is expressed in chorionic villi always associated with basal lamina and perivascularly. In the Syncytiotrophoblast, CTM associate close to CD34+ cells with CTH characteristics. Ultrastructural analysis presented full metabolic activity and cells that organize the placenta both biochemically and metabolically, suggesting that the placenta even at term is still capable of cells production not only of CTs, but also for tissue homeostasis.

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.

Made available in DSpace on 2014-06-11T19:29:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-21Bitstream added on 2014-06-13T19:39:22Z : No. of bitstreams: 1 moreli_jb_me_botfm.pdf: 522780 bytes, checksum: f823279b232bbcd45aa94cb502231c63 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo dessa revisão foi explorar a literatura sobre a atuação das citocinas IL-10 e TNF-α na gestação, destacando aspectos de interesse para a clínica obstétrica. A literatura disponível destaca várias funções da IL-10 e do TNF-α na gestação. Via de regra, estas citocinas desempenham papéis antagônicos e dependentes do balanço entre elas, condição que é orquestrada pela função imunomoduladora específica da IL-10. O TNF-α tem ação de característica inflamatória, sendo relacionado a perdas fetais recorrentes, síndromes hipertensivas, restrição do crescimento fetal e diabete melito gestacional. Entretanto, os resultados ainda são controversos e não completamente definidos. Tais conflitos são atribuídos à heterogeneidade dos estudos, principalmente relacionada à natureza, ao tamanho amostral, aos métodos de avaliação e à multiplicidade de fatores e condições que influenciam a produção das citocinas. Estas questões são fundamentais e devem ser consideradas em futuras investigações para que resultados mais consistentes possam nortear a prática obstétrica / The purpose of this work was to review the literature concerning the action of cytokines IL-10 and TNF-a in pregnancy, giving emphasis to aspects of interest to clinical obstetrics. The literature available highlights several IL-10 and TNF-a actions in pregnancy. These cytokines usually play balance-dependent opposing roles orchestrated by the IL-10-specific immunomodulatory function. TNF-a action is characteristically inflammatory and has been associated with recurrent fetal loss, hypertensive syndromes, fetal growth restriction and gestational diabetes mellitus. However, study results are controversial and remain not clearly defined. These conflicts are attributed to the heterogeneity of the studies, particularly regarding sample nature and size, evaluation methods, and the multiplicity of factors and conditions that influence cytokine production. These questions are fundamental, and should be addressed in future investigations so that more consistent results can be obtained and applied to obstetric practice