The aim of the research was to find new potential biomarkers of preterm premature rupture of membranes. The amniotic fluid and fetal membranes peptidic composition was analyzed using a fully automated 2D liquid chromatographic system coupled to mass spectrometry. A comparison of peptidomes of amniotic fluid and amniochorionic membranes with preterm premature rupture and term intact membranes was performed. Ten proteins from amniotic fluid were identified as potential biomarkers for PPROM. The created map of amniotic fluid peptides and proteins depending on the gestational age is important for proteomics-based identification of biomarkers for fetal abnormalities and other pregnancy complications. / Mokslinio darbo metu siekta nustatyti potencialius priešlaikinio neišnešioto vaisiaus dangalų plyšimo biožymenis. Panaudojant dvidimensinę skysčių chromatografiją bei masių spektrometriją išanalizuota vaisiaus vandenų ir dangalų peptidinė sudėtis. Ištirti ir palyginti amniochorioninės membranos ir vaisiaus vandenų peptidai bei su jais siejami baltymai, kai prieš laiką plyšta neišnešioto vaisiaus dangalai arba vaisius išnešiojamas iki numatyto gimdymo termino ir dangalai išlieka sveiki. Išanalizavus skirtumus, nustatyti nauji galimi priešlaikinio neišnešioto vaisiaus dangalų plyšimo biožymenys.

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Made available in DSpace on 2014-06-11T19:29:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-27Bitstream added on 2014-06-13T20:19:19Z : No. of bitstreams: 1 moya_cf_dr_botfmvz.pdf: 348880 bytes, checksum: dfb1789c8b1c607bbc3d25ebe0e02063 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Tendo em vista, a incidência de problemas fetais observados no momento do parto de animais produzidos in vitro (PIV) e a escassez de literatura nesta área, o presente estudo teve por objetivo determinar os níveis de fator de necrose tumoral-alfa (TNF-), interferon-gama (IFN-), interleucina-6 (IL-6) e IL-8 no fluido amniótico de bezerros oriundos de PIV comparando com aqueles obtidos pela transferência de embrião convencional e inseminação artificial no momento do parto, na tentativa de auxiliar na resolução de problemas ainda pouco conhecidos que são observados a campo durante emprego comercial da PIV. Foram utilizados sessenta animais divididos em grupo 01: vinte vacas gestando bezerros da raça Nelore PO de inovulações de embriões oriundos de doadoras superovuladas pelo método convencional (TE); grupo 02: vinte vacas gestando bezerros Nelore PO de inovulações de embriões oriundos de PIV; grupo 03: vinte vacas Nelore PO gestando bezerros oriundos de inseminação artificial (IA) - controle. Todos os partos dos animais do experimento foram observados. Durante a fase de expulsão e após a ruptura do alantocórion, foi realizada a punção do âmnion para a colheita de 15mL de líquido amniótico utilizando-se agulhas e seringas descartáveis compatíveis. O material foi depositado em tubo plástico e congelado em freezer para posterior análise laboratorial. O acompanhamento do padrão de desenvolvimento dos bezerros no periparto foi realizado. Após a descongelação das amostras foi realizado ensaio imunoenzimático (ELISA) para a dosagem de TNF-, com emprego do Kit comercial: ESS0011 - Bovine TNF Alpha, para IFN- foi Kit comercial: ESS0026 - Bovine IFN-, para IL-6 foi Kit comercial: ESS0029 - Bovine IL-6 da Pierce Biotechnology e para IL-8 Kit... / In view, the incidence of fetal problems seen at the time of birth of animals in vitro production and the be short of the literature in this area, this study aimed to determine the levels of tumor necrosis factor (TNF-), interferon-gamma (IFN-), interleukin-6 (IL-6) and IL-8 in amniotic fluid of calves from in vitro production (IVP) compared with those obtained by conventional embryo transfer and artificial insemination at the delivery, in an attempt to assist in solving problems that are still little known observed in simulations and the experimental field of commercial employment during IVP. Sixty animals used were divided into groups: 1 - Twenty Nelore cross-breed cows pregnant with Nelore calves of embryos transfer conventional method (ET); 2 - Twenty Nelore cross-breed cows pregnant with Nelore calves obtained by in vitro production after follicular aspiration; 3 - Twenty pregnant Nelore cows bearing calves obtained by artificial insemination (AI). All births of the animals of the experiment were observed. During the expulsion phase the amnion was punctured and 15mL of fluid were collected using a needle and syringes compatible. The material was deposited on plastic tube and frozen in freezer for later analysis. Monitoring the pattern of development of the calves in peripartum was performed. The cytokines levels were measured by immunoenzymatic assay (ELISA). The commercial kit used was: ESS0011 - Bovine TNF Alpha, ESS0026 - Bovine IFN- gamma, ESS0029 - Bovine IL-6 Pierce Biotechnology and comercial Kit: D8000C - Human IL-8 of the R & D Systems. The protocol was performed according to the manufacturer´s recommendations. The densities were evaluated in automatic optical reader ELISA. The well plates were read at 450nm. The Kruskal-Wallis Test was used for statistical analysis... (Complete abstract click electronic access below)

Orientador: Nereu Carlos Prestes / Resumo: A proposta do presente estudo foi descrever as diferenças no padrão morfológico das células do fluido amniótico, no momento do parto. Comparando os resultados obtidos de bezerros nelores oriundos de produção in vitro, transferência de embriões e inseminação artificial. Utilizaram-se 60 animais, divididos em Grupo 01: vinte vacas inovuladas com embriões Nelore produzidos in vitro (PIV) a partir de colheita de oócitos por aspiração folicular de doadoras, Grupo 02: vinte vacas gestando bezerros nelores originados de inovulações de embriões oriundos de doadoras superovuladas pelo método convencional, e Grupo 03: vinte vacas nelores PO gestando bezerros oriundos de inseminação artificial (controle). Próximo ao parto as vacas foram transferidas para piquete maternidade com acompanhamento dos partos. Durante a fase de expulsão e após a ruptura do alantocórion realizou-se a punção do âmnion para colheita de 20mL de liquido, que foi depositado em tubo plástico e congelado em freezer. Para a classificação celular as amostras, pós-descongelação, foram centrifugadas na Citocentrifuga (Revan Centrifuga Ciclo Cito) a 4000rpm durante seis minutos e empregou-se a técnica de coloração Hematoxilina-Shorr. Ao final deste procedimento, as lâminas citológicas foram montadas com lamínulas empregando Bálsamo do Canadá e posteriormente (após o período de secagem) observadas em microscópio óptico com aumentos de 200x e 400x para visualização das características morfológicas e tintoriais das células epiteliais do feto. Para avaliação da maturidade epidérmica utilizou-se a técnica de coloração de Sulfato Azul do Nilo bem como a porcentagem de células queratinizadas (coloração Hematoxilina-Shorr). As células basais e parabasais não foram encontradas nas lâminas citológicas das amostras de líquido amniótico analisadas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of the present study was to describe the differences in the cytological pattern of the amniotic fluid, at the moment of delivery, comparing the data from Nelore calves obtained by in vitro production, embryo transfer and artificial insemination. Sixty (n=60) Nelore cows were divided in group 01: Twenty cows (n=20) pregnant with Nelore calves obtained by in vitro production (IVP) after follicular aspiration; group 02: Twenty cows (n=20) pregnant with Nelore calves obtained by superovulation of embryo donors (ET); group 03: Twenty Nelore cows (n=20) pregnant of calves obtained by artificial insemination (AI; control). Near to the labor, the cows were transferred to a maternal paddock, permitting delivery observation. During the expulsion phase, after the rupture of the alantochorion the amnion was punctured and 20ml of fluid were collected in a plastic device and frozen. The samples, after being thawed, were centrifuged at 4000 rpm, during 06 minutes the Hematoxilin-Shorr stain was applied, after this, the glass slides were covered with Canadian balsame and analyzed in optical microscopy. To the evaluation of the epidermal maturity were used the Blue Nile Sulfate stain, as well the percent of keratinizaded cells observed in the Hematoxilin-Shorr stain. The basal and parabasal cells were not found on the slides of the amniotic fluid samples analyzed. The small intermediate cells (CIP) had its shape as an oval or polygonal form with large cytoplasm and central nucleus. The large intermediate cell (CIG) showed a central nucleus and a bigger nucleus-cytoplasm relation compared to the superficial cells. These were the largest cells found. They showed keratinization with angular edges, having both a picnotic nucleus (CSN) and no nucleus at all. (CSA). The technique of Blue Nile Sulfate stain did not showed satisfactory for the evaluation of the fetal maturity, in spite of some records... (Complete abstract click electronic access below) / Mestre

Brain injury, either surgically induced or as a result of trauma or stroke, is one of the leading causes of death and disability worldwide. Since transplantable stem cell sources are showing a great deal of promise and are actively being pursued to provide neuroprotection post-injury, in this body of work, we set out to characterize and examine the therapeutic potential of amniotic fluid derived (AF) cells as a potential cell source for cell-based therapies in mediating neuroprotection post-injury. Despite their heterogeneity, we found that AF cells are mainly epithelial in origin and express various genes involved in stem cell maintenance and neural commitment. A very small subset of AF cells also express pluripotency markers OCT4a, SOX2 and NANOG, which can be enriched for by single cell cloning. SOX2 positive clones have the capacity to give rise to a neuronal phenotype, in neural induction conditions, which can be used to examine the neural differentiation capabilities of AF cells. Subsequently, we examined the ability of AF cells to mediate a neuroprotective effect in a surgically induced brain injury model through gap junctional-mediated direct cell-cell communication and as a vehicle for GDNF delivery post-injury. AF cells express high levels of CX43 and are able to establish functional gap junctional intercellular communication (GJIC) with cortical astrocytes. We report an induction of Cx43 expression in astrocytes following injury and demonstrate, for the first time, CX43 expression at the interface between implanted AF cells and host astrocytes. In an effort to boost host endogenous neuroprotective mechanisms post-injury, via neurotrophic factor delivery, we engineered AF cells to secrete GDNF (AF-GDNF). GDNF pre-treatment significantly increased AF cell and cortical neuron survival rates following exposure to hydrogen peroxide. AF-GDNF cells, seeded on polyglycolic acid (PGA) scaffolds, survived longer in serum-free conditions and continued to secrete GDNF post-implantation activating the MAPK/ERK signaling pathway in host neural cells in the peri-lesion area. Despite some promising trends, we did not observe significant behavioural improvements following AF-GDNF/PGA implantation nor reduced lesion volume during the 7 day time-frame. In conclusion, through GJIC with cortical astrocytes and delivery of exogenous neurotrophic factors, AF cells hold great promise in mediating neuroprotection post-injury.

This study was designed to examine the hypothesis that the amount of amniotic fluid present during gestation is critical to normal lung growth and maturation. On day 16 of gestation the amniotic sacs of the right or left uterine horns of timed pregnant Sprague-Dawley rats were punctured with a 20 gauge needle. The fetuses of the opposite horn served as controls. On day 21 of gestation (one day prior to natural delivery) the fetuses were delivered by Cesarean section. An unbalanced, mixed model analysis of variance was performed on the data collected from each fetus. Probability values of less than 0.05 between control and experimental animals were considered significant. Amniotic sac puncture resulted in a significant loss of amniotic fluid as indicated by reduced amniotic fluid volume on day 21. Experimental body weight was significantly reduced indicating fetal growth retardation. Lung growth was also retarded as indicated by significantly reduced lung weight to body weight ratios and lung volume to body weight ratios following amniotic sac puncture. There was a reduction in the amount of fluid present within the experimental lungs. There appeared to be no significant effect on the structural units of the lung as indicated by no significant difference between control and experimental fetal lungs in terms of cell number, cell size, total protein to body weight ratio, maturation of type II cells, volume fraction of saccular air, saccular wall, conducting air and nonparenchyma, airspace size, saccular surface area to body weight ratio and surface to volume ratio. Thus, loss of amniotic fluid significantly affected lung growth, more than it affected overall body growth, without having an effect on lung maturation. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

The role that amniotic fluid (AF) may play in HIV vertical infection is unresolved. We used the FIV-infected cat model to study this question. We hypothesized that AF may be a source of fetal infection if the virus is present in the fluids. However, virus neutralizing (VN) antibodies in AF may limit vertical transfer. Fetuses were delivered from FIV-infected queens by cesarean section at early and late gestation. AFs were aspirated from intact fetal membranes and tested for viral antigen and RNA and for FIV-specific antibody. Randomlyselected samples were tested for VN activity using a syncytium reduction assay. Neither FIV antigen nor RNA was detected in any AFs. AFs and parallel serum samples from early and late pregnancy were positive for FIV-specific antibody. VN activity was detected in three early-term AFs and a parallel serum, but not late-term AFs. AF appears to play no appreciable role in FIV vertical transmission.

The amniotic membrane and the amniotic fluid are one of life's most complex and delicate tissues and fluids, respectively. What was known about this tissue and fluid prior to the 20th century was extremely limited scientifically, but was significantly defined by beliefs entrenched in mysticism, folklore, and superstitions. A comprehensive literature review of the amniotic membrane tissue and amniotic fluid reveals the many unique and complex characteristics and biological properties that been heavily investigated since the turn of the 20th century and continues to surge into the 21st century. The historical perspectives, evolution, derivation, histology, structure, and composition of the amniotic membrane; and historical perspectives, volume and regulation, and cellular and non-cellular composition of the amniotic fluid are discussed here and are coalesced for an easy and comprehensible resource. Lastly, future perspectives regarding research and application of the amniotic membrane and amniotic fluid, including stem cells are discussed.

O objetivo do presente estudo foi o de determinar os efeitos da exposição prolongada ao KCN e ao KSCN em ratos, no período perinatal. Inicialmente, realizou-se o estudo toxicocinético do tiocianato em ratas, em três estados fisiológicos diferentes: fase estrogênica, gestacional e de lactação. Para tal, estes animais receberam, por via oral, na água de bebida ou gavage, a dose única de 3 mg/kg de KCN. Os resultados obtidos mostraram aumento significante nos níveis séricos, lácteos e no líquido amniótico, de tiocianato, após a administração do KCN. A partir destes dados, foram calculados os parâmetros toxicocinéticos. Na segunda etapa, foram utilizadas ratas, as quais foram divididas em 3 grupos controles e 18 experimentais, que receberam as diferentes doses de KCN (1; 3 e 30 mg/kg) ou KSCN (0,8; 2,4 e 24 mg/kg), na água de bebida, do 6º ao 20º dia de gestação e submetidas à eutanásia no 20º dia da gestação ou no 22º dia pós-parto; e do 1º ao 19º dia da lactação, e submetidas à eutanásia no 19º dia deste período. Ao final de cada experimento, os animais foram submetidos à eutanásia, no período apropriado, bem como os fetos e filhotes, coletando-se soro para análise dos níveis de tiocianato, glicose, colesterol, uréia, creatinina, alanina aminotransferase, aspartato aminotransferase, fosfatase alcalina, bem como foram retirados fragmentos do sistema nervoso central, do rim, do pâncreas, da tireóide, do fígado, do pulmão e do baço para estudo histopatológico. Ainda, realizou-se a dosagem de tiocianato no líquido amniótico e no leite, bem como a análise óssea e visceral dos fetos. Dos parâmetros bioquímicos analisados, em relação às mães, verificou-se que os níveis séricos de tiocianato estiveram aumentados, significantemente, em diversos grupos experimentais, no entanto, poucas foram as alterações nas enzimas e outras substâncias avaliadas. O estudo histológico revelou, tanto naquelas gestantes quanto nas lactantes, nefrose, hemorragia e hemossiderose renal; congestão, neuronofagia e gliose no SNC; congestão, vacuolização e proliferação dos ductos biliares e aumento no número de vacúolos de reabsorção no colóide dos folículos tireoidianos, de forma dose-dependente, nos diferentes grupos experimentais. Ratas gestantes, submetidas à eutanásia no 20º dia da gestação apresentaram, ainda, depleção de células das ilhotas de Langerhans. Em relação aos filhotes, tanto aqueles provenientes de fêmeas tratadas durante a gestação quanto da lactação, verificou-se que, embora a avaliação bioquímica tenha revelado alteração apenas no tiocianato sérico de filhotes de mães provenientes dos grupos experimentais, o estudo histológico, nestas proles, mostrou várias lesões, a saber: congestão, neuronofagia e gliose no SNC, congestão renal e hepática, vacuolização e proliferação dos ductos biliares. Por outro lado, não foram detectadas alterações na performance reprodutiva bem como na análise visceral e óssea dos fetos. Portanto, pode-se sugerir que o cianeto e/ou seu metabólito promovam efeito tóxico diretamente sobre o feto, no entanto estas alterações são passíveis de detecção apenas na fase pós-natal. Além disto, verificou-se, que o cianeto e/ou o tiocianato são carreados para o leite, podendo comprometer, também, a saúde do neonato. / The aim of the present study was to determinate the toxic effects of long-term exposure to KCN and KSCN in rats, during perinatal period. Initially, it was achieved the toxicokinetics study of the thiocyanate in female rats, in three different physiological states: estrogenic, gestation and lactation. These animals received, per os, in the drinking water or by gavage, the unique dose of 3.0 mg KCN/kg. The results showed significantly increased in the thiocyanate levels in the serum, milk and amniotic fluid, after the administration of the KCN. The toxicokinetics parameters were calculated based on these data. In a second part, female rats were distributed in 18 experimental and 3 control groups. The experimental groups were dosed with 1, 3 and 30 mg KCN/kg or 0.8; 2.4 and 24 mg KSCN/kg, daily, in the drinking water, from days 6 to 20 of gestation and euthanized on day 20 of gestation or on day 22 postpartum; or received from days 1 to 19 of lactation and euthanized on day 19 of lactation. At the end of each experiment, the animals were euthanized, in the appropriated period, as well as the fetuses and pups. The serum samples were obtained in order to determine the thiocyanate, glucose, cholesterol, urea, creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase levels as well as samples of the following organs for the histopathological study: central nervous system, kidney, pancreas, thyroid, liver, lung and spleen. Yet, the levels of thiocyanate were evaluated in the amniotic fluid and milk, from the mothers, as well as the skeletal and visceral analysis of the fetuses. From the biochemical parameters, in relation to the dams, it was verified that the thiocyanate levels were significantly increased, in several experimental groups; otherwise, too few alterations were observed in the enzymes and others substances analyzed. The histopathological study revealed, in the pregnant and lactating rats, nephrosis, hemorrhage and hemosiderosis in the kidneys; congestion, neuronophagia and gliosis in the CNS; vacuolization and proliferation of the biliar ducts in the liver and increase in the number of reabsorption vacuoles in follicular colloid, in a dose-dependent manner, in some experimental groups. At the pregnant rats that were euthanized on day 20 of gestation, it was verified depletion of cells from the islets of Langerhans. In relation to the pups, from mothers exposed during the gestation and lactation, it was verified alteration in the thiocyanate levels from experimental groups and the histopathological study revealed: congestion, neuronophagia and gliosis in the CNS, renal and hepatic congestion, vacuolization and proliferation of biliar ducts. On the other hand, it was verified no alterations in the reproductive performance nor skeletal and visceral analysis of the fetuses. Thus, it was suggested that the cyanide and/or its metabolite promoted toxic effect straight to the fetuses; however, these alterations are susceptible of detection only at postnatal phase. Furthermore, the cyanide and/or thiocyanate are transferred to the breast milk of rats and can also compromise the health of the offspring.

Orientador: Márcia Guimarães da Silva / Banca: Rodrigo Paupéro de Camargo Soares / Banca: Vera Lúcia Mores Rall / Não disponível / Abstract: Microbial invasion of the amniotic cavity has been described in term deliveries and its role on the immune modulation is of interest to the better understanding of the underlying labor processes. The aim of this study was to determine the prevalence of Mycoplasma hominis and Ureaplasma urealyticum in the amniotic fluid of term pregnancies and to evaluate its influence on cytokines production at the end of pregnancy. A cross sectional study was conducted with fifty five pregnant women out of labor with intact membranes and gestational age between 37 and 41 weeks seen at the Bom Jesus hospital in Ariquemes, Rondônia, between June 2009 and May 2010. Amniotic fluid samples and fragments of chorioamniotic membranes were collected at cesarean section. M. hominis and U. urealyticum detection was performed by PCR and Interleukin (IL)-1β, IL-6, IL-8, IL-10 and Tumor Necrosis Factor (TNF)- levels were determined by ELISA. Chorioamniotic membranes were submitted to histopatological analyses. Presence of M. hominis was detected in 36.4% of amniotic fluid samples and any of them was positive for U. urealyticum. Regarding cytokines levels, 63.6% and 90.9% of samples have not shown detectable concentrations of TNF- and IL-1β. The median concentration of IL-6 and IL-8 were 107.9 pg/mL (0-517.1) and 208.1 pg/mL (0-1897.4), respectively. Interleukin-1, IL-6, IL-8 and TNF- concentrations were not associated with the presence of M. hominis in amniotic fluid, regardless the gestational age. No sample had detectable IL-10 levels. The histopatological analyses have shown no chorioamnionitis in any of the membranes, only a discreet mononuclear infiltration in the decidua could be observed in 40.4% of the samples. Presence of M. hominis was detected in 36.4% of amniotic fluid samples and any of them was positive for U. urealyticum. Regarding cytokines levels, 63.6% and 90.9% of samples have not... (Complete abstact click electronic access below) / Mestre