During normal placental development, a primitive vascular network develops through vasculogenesis and angiogenesis, and is then remodelled through maturation and regression. The mechanism behind this regression is unknown, but data from other systems suggests that it could be due to an endothelial-mesenchymal transition (EndMT). If this is the case, then dysregulated EndMT could lead to increased vascular regression, which could result in placental hypovascularisation. As the placental vasculature is the area of exchange between maternal and fetal circulations, a reduction in its surface area could result in fetal growth restriction (FGR). The hypothesis of this thesis is that EndMT occurs during normal placental development, but is increased during FGR and contributes to placental hypovascularisation. A primary cell model consisting of endothelial and mesenchymal cells was isolated from human first trimester placental villous stroma. These cells were shown to lose CD31 mRNA (n = 1-3) and protein (n = 15) over 4 passages, with no loss of cell viability (n = 8). EndMT-associated transcription factors were also present in these cells at all 4 passages (n = 2-4). When cells were isolated from this mixed cell model based on their CD31-positivity and examined immediately after isolation, a small proportion also expressed αSMA (n = 5). Co-expression of endothelial and mesenchymal markers suggests that an EndMT was occurring. After 24 hours in culture, the proportion of these cells expressing αSMA increased (n = 5), and some cells co-expressed vWF and αSMA, while others lost their CD31-positivity, indicating that these cells had undergone EndMT. Cells isolated based on their CD31-positivity were treated with factors shown to inhibit EndMT in other systems. However, culture with 10µM SB431542 (TGFβ receptor inhibitor; n = 6), 10µM Dorsomorphin (BMP receptor inhibitor; n = 3), or 0.1µM PDGFR-β Tyrosine Kinase Inhibitor IV (n = 3) did not inhibit gain of αSMA by these cells. Culture on Matrigel in endothelial growth medium containing VEGF and FGF also failed to stabilise the endothelial phenotype (n = 3). The possibility that EndMT occurs in placenta in vivo was examined; genes associated with EndMT were shown to be present in placenta (n = 5), and there was limited evidence of CD31 or vWF co-expression with αSMA in tissue. Preliminary evidence was obtained to suggest that expression of EndMT-associated genes was altered in FGR placentas compared to normal. In summary, the data presented in this thesis demonstrate that an EndMT occurs in primary placental microvascular endothelial cells in vitro. Furthermore, these studies provide evidence to suggest that this transition also occurs in vivo and could be altered in placentas from pregnancies complicated by FGR.

<p>PUFAs serve many important biological and physiological functions within the body and are key for the structure and function of the brain. Omega-6 and omega-3 PUFAs are found in abundance in phospholipids of neuronal membranes that impart structure and function of neurons. Omega-6 PUFAs are instrumental for neurotransmission, neuronal elongation, and neuritogenesis; whereas, omega-3 PUFAs promote neuronal maturation through synaptogenesis. The types of PUFAs incorporated into neuronal membranes is especially important in determining the progression of development. The processes of neurogenesis, neuritogenesis and elongation require large amounts of PUFAs to be incorporated into the membrane phospholipids. To accommodate for the high PUFA needs, maternal dietary PUFA, especially EPA and DHA, recommendations, mobilization of fatty acids into maternal circulation increases, and the accretion rate of PUFA are increased. If maternal nutritional inadequacy of PUFAs occurs during gestation, this can result in impaired cognition, behavioral abnormalities, reduced number of neurons, decreased dendric arborization, altered myelin sheath, and a reduction in brain size. </p> <p> Even though the essentiality of PUFAs in neuronal development is widely accepted, the mechanism is not well understood. There is a lack of consensus in the current literature on the effects of individual PUFAs on each stage of neuronal development and the molecular pathways involved. Despite the inconsistent evidence, the results of numerous studies have consistently suggested that neuronal membrane PUFA composition is associated with neuronal development outcomes, such as number of neurons and neurites, neurite length, and neurotransmitter release. The varying results may be the result of methodological discrepancies with PUFA composition and concentrations, as well as the models used for neuronal development. Additionally, very few studies have taken into consideration the competitive relationship of omega-6 and omega-3 PUFAs in the body when assessing neurodevelopment. </p> <p> This thesis was focused on addressing the role of PUFAs in neuronal development and to address some of the inconsistencies in the literature. attempt to elucidate the individual roles of ALA, ARA, and EPA on neuronal membrane composition and neuronal development. The aim of the thesis research project was to assess the impact of individual PUFAs on neuronal membrane PUFA composition, the membrane n-6:n-3 ratio, and the morphology of SH-SY5Y cells during differentiation. The results of this study demonstrated that supplementation of individual PUFAs alters membrane PUFA composition and the n-6:n-3 ratio. However, there wasn’t a significant effect on neurite number with ALA, ARA, and EPA treatment. Lastly, ARA treatment decreased cell viability compared to the other treatments and the BSA control. Furthermore, additional research needs to be conducted to address other morphological measures and functional outcomes, such as neurotransmitter production and release.</p>

Orientadores: Ricardo Barini, Lourenço Sbragia Neto / Tese (doutorado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas / Made available in DSpace on 2018-11-27T12:29:22Z (GMT). No. of bitstreams: 1 Bueno_MarciaPereira_D.pdf: 2898828 bytes, checksum: 2c6dcaa4a20ec185bea4b47114c30a0e (MD5) Previous issue date: 2010 / Resumo: A restrição do crescimento intrauterino (RCIU) limita o desenvolvimento fetal adequado aumentando a morbidade e mortalidades perinatais. Os mecanismos fetais adaptativos na RCIU podem desencadear alterações endócrinas e metabólicas que explicariam a ocorrência de doenças na idade adulta. O objetivo do estudo foi avaliar na RCIU experimental pela ligadura da artéria uterina se existem alterações na morfometria e histologia do fígado, intesti no e rins e se existem diferenças na expressão dos receptores de insulina, IR-(3, IRS-1, IRS-2, IGF-IR(3 no grupo de fetos submetidos à RCIU. O presente experimento foi submetido ao Comitê de Ética e Experimentação Animal da Universidade Estadual de Campinas (CEEA-UNICAMP) e aprovado como projeto de pesquisa N° 1644-1. Para realização do estudo utilizamos fetos de ratas Sprague Dawley divididos em 3 grupos. Grupo I (RCIU) - 40 fetos submetidos à ligadura da artéria uterina unilateral com 18,5 dias de gestação, Grupo II (Controle-RCIU) - 40 fetos do corno oposto ao da ligadura da artéria uterina e Grupo III (Controle Externo) - 40 fetos sem procedimento cirúrgico ou alimentar. Os resultados mostraram no modelo experimental de RCIU uma diminuição do peso corporal (PC), hepático (PH) e intestinal (PI) (p<0,01) no grupo RCIU, as relações entre PH/PC, PI/PC, PR/PC foram mantidas, fetos RC IU tem diminuição das camadas submucosas e mucosas intestinais (p<0,05); diminuição da camada cortical renal e do número de glomérulos, com aumento do volume glomerular (p<0,05). Na RCIU encontramos menor expressão hepática do IR-(3, IRS-1 e IRS-2, menor expressão do IRS-2 no intestino e rins e maior expressão do IGF-IR(3 em todos os tecidos. O modelo experimental estudado causou uma RCIU simétrica com alterações morfométricas e do metabolismo da glicose que poderiam justificar no futuro um maior risco de doenças metabólicas / Abstract: Intrauterine growth restriction (IUGR) limits appropriate fetal development increasing morbidity and perinatal mortality. Adaptive mechanisms in fetal IUGR may leave to endocrine and metabolic alterations that could explain the occurrence of diseases in adulthood. The aim of this study was to evaluate whether experimental IUGR by uterine artery ligation causes changes in morphology and histology of the liver, intestines and kidneys. We also evaluated if there were differences in the expression of insulin receptors, IR-(3, IRS-1, IRS-2, IGF-IR(3 of fetuses subjected to IUGR. This experiment was submitted to the Ethics and Animal Experimentation of the Campinas State University (UNICAMP CEEA) and was approved as a research project No. 1644-1. The study used fetuses Sprague-Dawley rats divided into 3 groups. Group I (IUGR) - 40 fetuses who underwent uterine artery ligation sided with 18.5 days of pregnancy Group II (Control-IUGR) - 40 fetuses of the horn opposite to the uterine artery ligation, and Group III (External Control) - 40 fetuses without surgery or food The results showed the experimental model of IUGR, a reduction in body weight (BW), liver (PH) and intestine (PI) (p <0.01) in IUGR, the relationship between PH/PC, PI/PC, PR/PC have been retai ned, IUGR fetuses have reduced layers of the intestinal mucosa and submucosa (p<0,05), decreased renal cortical layer and the glomerular number and increased volume rate (p<0,05). In IUGR found lower hepatic expression of IR-(3, IRS-1 and IRS-2, reduced expression of IRS-2 in the intestine and kidney and increased expression of IGF-IR(3 in all tissues. The experimental model studied caused a symmetrical IUGR with histological changes and glucose metabolism that could justify a greater risk of metabolic diseasesin the future / Doutorado / Ciencias Biomedicas / Doutor em Tocoginecologia