Memoria para optar al Título Profesional de Médico Veterinario / Durante décadas, la estructura de las membranas biológicas fue caracterizada como un Mosaico Fluido, pero posteriormente surgió evidencia de la presencia de Lipid Rafts o Microdominios Lipídicos, que son estructuras de membrana enriquecidas en esfingolípidos y colesterol que contienen diversas proteínas y cumplen roles específicos dentro de la membrana. Recientemente, se ha reportado la presencia de estas estructuras, además de una serie de canales iónicos, en las membranas del Sinciciotrofoblasto placentario humano (hSTB), epitelio sincicial responsable del intercambio materno-fetal en la gestación. Este tejido posee membrana basal y membrana apical, la cual se subdivide en dos subdominios; uno pesado o MVM y uno liviano o LMVM. Por otra parte, existen antecedentes de que la asociación de un canal de potasio a microdominios de membrana tiene implicancias en el funcionamiento del canal, por lo que, el objetivo del presente estudio es describir la segregación de los canales de potasio previamente identificado en placenta KV 2.1, KIR 2.1, TREK-1 y TASK-1 en dominios Rafts de MVM y LMVM del hSTB. A partir de placentas de término, se obtuvieron la membrana basal y los dos sub-dominios de membrana apical del hSTB. Luego, MVM y LMVM se solubilizaron con detergente Tritón-X-100 y se centrifugaron en una gradiente discontinua de sacarosa, la cual se alicuotó en once fracciones, donde las fracciones insolubles en detergente (1-5) fueron Rafts y el resto no-Rafts. Mediante Western Blot y Dot Blot, se probaron marcadores Rafts (Fosfatasa Alcalina Placentaria o PLAP y gangliósido GM1) y no-Rafts (Receptor de Transferrina Humano o hTf-R), como también los canales de potasio KV 2.1, KIR 2.1, TREK-1 y TASK-1 en las once fracciones. Los resultados indicaron que las fracciones Rafts son ricas en PLAP y GM1, mientras que las no-Rafts ricas en hTf-R, lo cual permite inferir que se lograron aislar los Lipid Rafts. En el caso de los canales de potasio, Kv 2.1 se ubicó en fracciones no-Rafts y KIR 2.1 en fracciones Rafts. La presencia de KIR 2.1 en Rafts además se confirma mediante un experimento con extracción de colesterol. Este es la primera evidencia de segregación de canales de potasio en dominios Rafts y no-Rafts realizado en el hSTB. Sabiendo que KV 2.1 se ubica en sitios no-Rafts y KIR 2.1 en sitios Rafts, resta investigar la implicancia funcional de dicha asociación en la membrana apical, ya sea en condiciones normales como en patologías de la gestación

<p>Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants (BFRs) that have been heavily used in consumer products such as furniture foams, plastics, and textiles since the mid-1970’s. BFRs are added to products in order to meet state flammability standards intended to increase indoor safety in the event of a fire. The three commercial PBDE mixtures, Penta-, Octa-, and DecaBDE, have all been banned in the United States, however, limited use of DecaBDE is still permitted. PBDEs were phased out of production and added to the Stockholm Convention due to concerns over their environmental persistence and toxicity. Human exposure to PBDEs occurs primarily through the inadvertent ingestion of contaminated house dust, as well as though dietary sources. Despite the phase-out and discontinued use of PBDEs, human exposure to this class of chemicals is likely to continue for decades due to the continued use of treated products and existing environmental reservoirs of PBDEs. Extensive research over the years has shown that PBDEs disrupt thyroid hormone (TH) levels and neurodevelopmental endpoints in rodent and fish models. Additionally, there is growing epidemiological evidence linking PBDE exposure in humans to altered TH homeostasis and neurodevelopmental impairments in children. Due to the importance of THs throughout gestation, there is a great need to understand the effects of BFRs on the developing fetus. Specifically, the placenta plays a critical role in the transport, metabolism, and delivery of THs to the fetal compartment during pregnancy and is a likely target for BFR bioaccumulation and endocrine disruption. The central hypothesis of this dissertation research is that BFRs disrupt the activity of TH sulfotransferase (SULT) enzymes, thereby altering TH concentrations in the placenta.</p><p>In the first aim of this dissertation research, the concentrations of PBDEs and 2,4,6-TBP were measured in a cohort of 102 placenta tissue samples from an ongoing pregnancy cohort in Durham, NC. Methods were developed for the extraction and analysis of the BFR analytes. It was found that 2,4,6-TBP was significantly correlated with all PBDE analytes, indicating that 2,4,6-TBP may share common product applications with PBDEs or that 2,4,6-TBP is a metabolite of PBDE compounds. Additionally, this was the first study to measure 2,4,6-TBP in human placenta tissues.</p><p>In the second aim of this dissertation research, the placenta tissue concentrations of THs, as well as the endogenous activity of deiodinase (DI) and TH SULT enzymes were quantified using the same cohort of 102 placenta tissue samples. Enzyme activity was detected in all samples and this was the first study to measure TH DI and SULT activity in human placenta tissues. Enzyme activities and TH concentrations were compared with BFR concentrations measured in Aim 1. There were few statistically significant associations observed for the combined data, however, upon stratifying the data set based on infant sex, additional significant associations were observed. For example, among males, those with the highest concentrations of BDE-99 in placenta had T3 levels 0.80 times those with the lowest concentration of BDE-99 (95% confidence interval (CI): 0.59, 1.07). Whereas females with the highest concentrations of BDE-99 in placenta had T3 levels 1.50 times those with the lowest concentration of BDE-99 (95% CI: 1.10, 2.04). Additionally, all BFR analyte concentrations were higher in the placenta of males versus females and they were significantly higher for 2,4,6-TBP and BDE-209. 3,3’-T2 SULT activity was significantly higher in female placenta tissues, while type 3 DI activity was significantly higher in male placenta tissues. This research is the first to show sex-specific differences in the bioaccumulation of BFRs in human placenta tissue, as well as differences in TH concentrations and endogenous DI and SULT activity. The underlying mechanisms of these observed sex differences warrant further investigation. </p><p>In the third aim of this dissertation research, the effects of BFRs were examined in a human choriocarcinoma placenta cell line, BeWo. Michaelis-Menten parameters and inhibition curves were calculated for 2,4,6-TBP, 3-OH BDE-47, and 6-OH BDE-47. 2,4,6-TBP was shown to be the most potent inhibitor of 3,3’-T2 SULT activity with a calculated IC50 value of 11.6 nM. It was also shown that 2,4,6-TBP and 3-OH BDE-47 exhibit mixed inhibition of 3,3’-T2 sulfation in BeWo cell homogenates. Next, a series of cell culture exposure experiments were performed using 1, 6, 12, and 24 hour exposure durations. Once again, 2,4,6-TBP was shown to be the most potent inhibitor of basal 3,3’-T2 SULT activity by significantly decreasing activity at the high and medium dose (1 M and 0.5 M, respectively) at all measured time points. Interestingly, BDE-99 was also shown to inhibit basal 3,3’-T2 SULT activity in BeWo cells following the 24 hour exposure, despite exhibiting no inhibitory effects in the BeWo cell homogenate experiments. This indicates that BDE-99 must act through a pathway other than direct enzyme inhibition. Following exposures, the TH concentrations in the cell culture growth media and mRNA expression of TH-related genes were also examined. There was no observed effect of BFR treatment on these endpoints. Future work should focus on determining the downstream biological effects of TH SULT disruption in placental cells, as well as the underlying mechanisms of action responsible for reductions in basal TH SULT activity following BFR exposure. </p><p>This was one of the first studies to measure BFRs in a cohort of placenta tissue samples from the United States and the first study to measure THs, DI activity, and SULT activity in human placenta tissues. This research provides a novel contribution to our growing understanding of the effects of BFRs on TH homeostasis within the human placenta, and provides further evidence for sex-specific differences within this important organ. Future research should continue to investigate the effects of environmental contaminants on TH homeostasis within the placenta, as this represents the most critical and vulnerable stage of human development.</p> / Dissertation

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Eliška Marková Suprevisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Study of the effect of novel antiretroviral drugs on carnitine transport in the placenta Nowadays, the antiretroviral treatment of HIV-positive pregnant women is the standard approach for restriction of transmission of HIV infection from mother to the fetus. In spite of necessity of this pharmacotherapy, it is important to know its safety and risks. For the correct fetal development and function of placenta it is (besides others) essential to ensure the optimal supply of L-carnitine, the key factor for oxidation of fatty acids from mother's blood to the placenta and fetal blood circulation. The deficiency of L-carnitine generally leads to significant metabolic changes in the cells and in it usually demonstrated with cardiomyopathies and myopaties. Published studies indicate higher incidence of cardiovascular diseases and cardiomyopathies in children born to mothers treated with antiretroviral therapy during pregnancy. Optimal transport of carnitine into the placental cells, is ensured due to the presence of functional transport protein OCTN2 in the apical membrane of trophoblast. The aim of this study was...

Estudos de placentação foram desenvolvidos em quatorze fêmeas de mocós em diferentes fases de gestação. As fêmeas foram pré-anestesiadas associando-se cloridrato de quetamina (15mg/kg) e midazolan (1mg/kg). Em seguida anestesiadas com isoflurano em associação com oxigênio com 100% de saturação. Após a anestesia realizou-se a cirurgia para a exposição das estruturas fetais e a coleta de dados. Macroscopicamente, identificou-se uma placenta discoidal, o saco vitelínico e o âmnio de aspecto transparente e avascular. Microscopicamente, o cordão umbilical apresentou duas artérias, uma veia e o ducto alantoideano, além de uma artéria e uma veia vitelínicas. A placenta mostrou uma relação mesometrial com o útero e apresentou-se constituída por lóbulos delimitados por regiões de interlóbulo e, perifericamente, uma região de sincício marginal contendo locais com espongiotrofoblasto e células trofoblásticas gigantes. A subplacenta esteve composta por lóbulos e por trofoblasto de natureza sincicial e celular. O saco vitelínico apresentou uma porção parietal sustentada pela membrana de Reichert´s e uma porção visceral muito vascularizada. Os estudos de placentação em mocós indicaram a presença de um útero bicórneo, uma placenta corioalantoídea discoidal e labiríntica, com barreira placentária hemocorial de subtipo hemomonocorial separando um fluxo sangüíneo materno-fetal do tipo contracorrente. / Placentation studies of fourteen rock cavy females in different gestation phases were conducted. Females were pre-anesthetized associating ketamine chloridrate (15mg/kg) and midazolan (1mg/kg). Soon afterwards, they were anesthetized by isoflurane inhalation in association with oxygen at 100% saturation. After anesthesia, the surgery allowed to exhibit fetal structures and then data collection was performed. Macroscopically, a discoidal placenta, vitelline sack and the amnion of a transparent aspect and avascular, were identified. Microscopically, the umbilical cord presented two arteries, a vein and the allantoid duct, besides an artery and a vitelline vein. The placenta showed a relationship between the mesometrium and the uterus and was constituted by lobes delimited by interlobular areas and, peripherically, by an area of marginal syncytium containing places with spongiotrophoblast and gigantic trophoblastic cells. The subplacenta was composed by lobules and by a trophoblast of syncytium and cellular nature. The vitelline sack showed a parietal portion sustained by the Reichert´s membrane and a well-vascularized visceral portion. The placentation studies in rock cavies indicated the presence of a bicornuate uterus, a chorioallantoid discoidal and labirynthic placenta, with a hemochorial placental barrier of hemomonochorial subtype separating the maternal-fetal countercurrent sanguine flow.

by Pui-Kwong Chan. / Thesis -- The Chinese University of Hong Kong. / Bibliography: leaves 139-145.

Kwok-pui Fung. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1975. / Bibliography: leaves 122-131.

Thesis (M.Ph.)--Chinese University of Hong Kong. / Bibliography: leaves 87-96.

Made available in DSpace on 2014-06-11T19:30:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-02Bitstream added on 2014-06-13T20:21:28Z : No. of bitstreams: 1 lourenco_ncv_dr_botib.pdf: 289471 bytes, checksum: 1e3de2164fad680893ad0ff7aece51c7 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A pré-eclâmpsia é uma síndrome específica da gestação humana, caracterizada por hipertensão arterial e proteinúria após a 20ª. semana de gestação. É aceito que esta patologia tem origem na placenta. O fator de crescimento placentário (PlGF) tem importante papel no desenvolvimento vascular durante a invasão trofoblástica e, citocinas de perfil Th2 podem estar envolvidas na manutenção da gestação durante a interação materno-fetal.O presente trabalho avaliou a concentração de citocinas e de fatores pró e anti-angiogênicos em homogenato de placenta, obtido de gestantes normais e com pré-eclâmpsia, correlacionando esses parâmetros com a gravidade da doença e com a idade gestacional.Foram estudadas 70 gestantes, sendo 30 normotensas e 40 portadoras de pré-eclâmpsia. As gestantes com pré-eclâmpsia foram classificadas de acordo com o aparecimento das manifestações clínicas em pré-eclâmpsia precoce (< 34 semanas de gestação, n=13) e pré-eclâmpsia tardia (≥ 34 semanas de gestação, n=27). Um fragmento de placenta foi obtido imediatamente após o parto, homogeneizado em solução salina tamponada com fosfatos e submetido à centrifugação. O sobrenadante obtido foi empregado para determinação de fator de necrose tumoral-alfa (TNF-a), fator estimulador de colônias de granulócitos e macrófagos (GM-CSF), interleucina-10 (IL-10), fator de crescimento e transformação beta (TGF-b1), fator de crescimento placentário (PlGF) e Endostatina por ensaio imunoenzimático (ELISA). Outro fragmento placentário foi obtido para análise histopatológica. As diferenças entre os grupos de gestantes normais e com préeclâmpsia foram avaliadas por teste t de Student, com nível de significância de 5%.Os níveis de TNF-a, Endostatina e GM-CSF detectados na placenta de gestantes com pré-eclâmpsia foram significativamente maiores em relação às gestantes... / Preeclampsia is a human pregnancy-specific syndrome characterized by the onset of hypertension and proteinuria after the 20th gestational week. It is widely accepted that this disorder is placental in origin. Placental growth factor (PlGF) plays an important role in the vascular development during trophoblast invasion and cytokines with Th2 profile may be involved in gestation maintenance and promoting a balance in maternal– fetal interaction. This study investigated the concentration of cytokines, angiogenic and anti-angiogenic factors in placental homogenate obtained from normotensive and preeclamptic pregnant women, and correlated these parameters with disease severity and gestational age.The subjects were 70 pregnant women of whom 30 were normotensive pregnant, and 40 were pregnant women with preeclampsia. The preeclamptic pregnant women were classified according to the onset of clinical manifestations in early-onset (< 34 weeks of gestation, n=13) and late-onset (≥ 34 weeks of gestation, n=27) preeclampsia. A tissue fragment of the placenta was obtained immediately after delivery, homogenized with phosphate-buffered saline solution and centrifuged. Separated supernatant was employed for determination of tumor necrosis factor alpha (TNF-a), granulocyte-macrophage colony-stimulating factor (GM-CSF) Interleukin-10 (IL-10), transforming growth factor beta (TGF-b1), placental growth factor (PlGF), and Endostatin, by enzyme-linked immunosorbent assay (ELISA). Another placental tissue segment was prepared for histopathological analysis. Differences between groups were evaluated by Student t unpaired test with significance set at p < 0.05.The levels of TNF-a, Endostatin and GM-CSF detected in placenta of preeclamptic women were significantly increased when compared with the normotensive pregnant women. On the other hand, the concentration of IL-10, TGF-b1 and PlGF have shown... (Complete abstract click electronic access below)

Background: Exercise in pregnancy is associated with optimized fetal growth; however, the implicated mechanisms remain unknown. We hypothesize that exercise-induced myokines may be acting on the placenta to optimize fetal growth across gestation. Methodology: 1) Circulating profiles of 11 myokines were analyzed in 2nd trimester plasma of women characterized as active (N=14) or non-active (N=16) during pregnancy. 2) First trimester human placental explants (N=5) were treated with SPARC in a dose-dependent manner (0-150ng/ml). Metrics of placental health/function, including GLUT-4 expression/regulation, were assessed. Results: 1) Active women demonstrated an elevation in circulating SPARC compared to non-active women (86±19pg/ml vs. 52±18pg/ml, p=0.0001). 2) Explants treated with SPARC at 100ng/ml demonstrated improved invasion, with improved maximum outgrowth distance (N=3; p=0.0219). Conclusion: SPARC is a myokine that is elevated in the circulation of active pregnant women and is associated with improved placental invasion, suggesting a possible role of SPARC in placentation.

Las HSMG son causa importante de morbimortalidad materna y perinatal especialmente en países en desarrollo y se presentan con una frecuencia entre 2 y 6 % y sus causas primarias son obstétricas, el desprendimiento prematuro de la placenta y la placenta previa. La etiología es multifactorial, pero determinadas condiciones se asocian a mayor incidencia de esta complicación como son alteraciones endometriales ó miometriales los que se dan en la edad madura, multiparidad, antecedente de cesárea, legrados uterino, miomas uterino, y cuando hay aumento relativo de la masa placentaria como en los gemelares, fetos de altura, y tabaquismo y en casos de Abruptio, también la hipertensión inducida por el embarazo, ruptura prematura de membranas y traumas. El objetivo general del trabajo es identificar los factores de riesgo que se asocian a las causas de hemorragia en esa etapa del embarazo, y la incidencia acumulada. Los resultados del estudio son que la incidencia general de las HSMG: 1.26%, la incidencia acumulada del Desprendimiento Prematuro de Placenta: 0.55% y de la Placenta Previa: 0.69%. Son factores de riesgo para placenta previa, la edad gestacional menor de 36 semanas, situación transversa, la anemia severa, peso menor de 2500gr de los recién nacidos y el número de transfusiones; y para desprendimiento prematuro de la placenta, además de la anteriores, la hipertensión inducida por el embarazo y la muerte fetal intraútero. En ambos casos el factor de riesgo protector destacable es el control prenatal. / Tesis