Yuen, Ka Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 160-185). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.IV / ACKNOWLEDGEMENTS --- p.VI / LIST OF ABBREVIATIONS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XII / LIST OF FIGURES --- p.XIII / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- Pseudomalignant nature of the placenta --- p.2 / Chapter 1.1 --- Overview --- p.2 / Chapter 1.2 --- "Proliferation, migration and invasion behaviour" --- p.3 / Chapter 1.3 --- Gene expression --- p.4 / Chapter 1.3.1 --- Angiogenic factors --- p.5 / Chapter 1.3.2 --- Growth factors --- p.5 / Chapter 1.3.3 --- Proto-oncogenes --- p.6 / Chapter 1.3.4 --- Tumour suppressor genes --- p.8 / Chapter CHAPTER 2: --- Epigenetics --- p.10 / Chapter 2.1 --- Overview --- p.10 / Chapter 2.2 --- DNA methylation in mammals --- p.11 / Chapter 2.3 --- Regulation of DNA methylation machinery --- p.12 / Chapter 2.4 --- Role of DNA methylation --- p.13 / Chapter 2.5 --- Aberrant DNA methylation --- p.16 / Chapter 2.6 --- DNA methylation in normal cells --- p.17 / Chapter 2.6.1 --- X-chromosome inactivation --- p.17 / Chapter 2.6.2 --- Genomic imprinting --- p.18 / Chapter 2.6.3 --- Cell-type-specific methylation --- p.19 / Chapter 2.6.4 --- Placental-specific methylation --- p.20 / Chapter 2.7 --- Aim of Thesis --- p.21 / Chapter SECTION II: --- MATERIALS AND METHODOLOGY --- p.23 / Chapter CHAPTER 3: --- Materials and methods --- p.24 / Chapter 3.1 --- Preparation of samples --- p.24 / Chapter 3.1.1 --- Collection of placental tissues --- p.24 / Chapter 3.1.2 --- Preparation of blood cells --- p.25 / Chapter 3.1.3 --- Preparation of cell lines --- p.25 / Chapter 3.1.4 --- Treatment of JAR and JEG3 with 5-aza-2'-deoxycytidine (5-aza-CdR) and Trichostatin A (TSA) --- p.26 / Chapter 3.2 --- Nucleic acid extraction --- p.26 / Chapter 3.2.1 --- DNA extraction from tissue samples --- p.26 / Chapter 3.2.2 --- DNA extraction from blood cells --- p.29 / Chapter 3.2.3 --- RNA extraction from cell lines --- p.30 / Chapter 3.3 --- Methylation analysis --- p.31 / Chapter 3.3.1 --- Principles of bisulfite modification --- p.31 / Chapter 3.3.2 --- Bisulfite Conversion --- p.32 / Chapter 3.3.3 --- Primer design for methylation-specific polymerase chain reaction / Chapter 3.3.4 --- Methylation-specific polymerase chain reaction (MSP) --- p.33 / Chapter 3.3.5 --- Primer design for bisulfite sequencing --- p.34 / Chapter 3.3.6 --- Cloning and bisulfite genomic sequencing --- p.35 / Chapter 3.4 --- Quantitative measurements of nucleic acids --- p.39 / Chapter 3.4.1 --- Principles of real-time quantitative PCR --- p.39 / Chapter 3.4.2 --- Real-time quantitative MSP --- p.42 / Chapter 3.4.3 --- Real-time reverse transcriptase (RT)-PCR --- p.42 / Chapter 3.5 --- MALDI-TOF mass spectrometry (MS) --- p.43 / Chapter 3.5.1 --- Principle of homogeneous MassEXTEND assay and MALDI-TOF MS --- p.43 / Chapter 3.5.2 --- Methylation-sensitive restriction enzyme digestion and homogeneous MassEXTEND assay for APC and H19 --- p.46 / Chapter SECTION III: --- A SEARCH FOR HYPERMETHYLATED TUMOUR SUPPRESSOR GENES IN THE HUMAN PLACENTA --- p.48 / Chapter CHAPTER 4: --- Screening on TSGs and non TSGs --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.2 --- Materials and methods --- p.50 / Chapter 4.2.1 --- Sample collection --- p.50 / Chapter 4.2.2 --- Sample processing and DNA extraction --- p.50 / Chapter 4.2.3 --- Experimental Design --- p.51 / Chapter 4.3 --- Results --- p.63 / Chapter 4.3.1 --- Identification of hypermethylated TSGs by methylation-specific PCR screening --- p.63 / Chapter 4.3.2 --- Validation of hypermethylated TSGs by bisulfite sequencing --- p.69 / Chapter 4.4 --- Discussion --- p.77 / Chapter CHAPTER 5: --- Methylation status of TSGs in different tissues --- p.80 / Chapter 5.1 --- Introduction --- p.80 / Chapter 5.2 --- Materials and methods --- p.81 / Chapter 5.2.1 --- Sample collection --- p.81 / Chapter 5.2.2 --- Sample processing and DNA extraction --- p.81 / Chapter 5.2.3 --- Experimental design --- p.81 / Chapter 5.3 --- Results --- p.86 / Chapter 5.3.1 --- Methylation patterns of TSGs in non-placental fetal tissues --- p.86 / Chapter 5.4 --- Discussion --- p.90 / Chapter SECTION IV: --- FUNCTIONAL IMPLICATION OF HYPERMETHYLATED TUMOUR SUPPRESSOR GENES IN THE PLACENTA --- p.94 / Chapter CHAPTER 6: --- Imprinting checking --- p.95 / Chapter 6.1 --- Introduction --- p.95 / Chapter 6.2 --- Materials and methods --- p.96 / Chapter 6.2.1 --- Sample collection --- p.96 / Chapter 6.2.2 --- Sample processing and DNA extraction --- p.97 / Chapter 6.2.3 --- Experimental design --- p.97 / Chapter 6.3 --- Results --- p.100 / Chapter 6.3.1 --- Imprinting checking of H19 by enzyme digestion on placental tissues --- p.100 / Chapter 6.3.2 --- Imprinting checking of　APC by enzyme digestion on placental tissues --- p.101 / Chapter CHAPTER 7: --- CORRELATION OF HYPERMETHYLATION AND GENE EXPRESSION --- p.107 / Chapter 7.1 --- Introduction --- p.107 / Chapter 7.2 --- Materials and methods --- p.108 / Chapter 7.2.1 --- Sample preparation and processing --- p.108 / Chapter 7.2.2 --- DNA and RNA extraction from cell lines --- p.108 / Chapter 7.2.3 --- Experimental design --- p.108 / Chapter 7.3 --- Results --- p.111 / Chapter 7.3.1 --- Methylation status of APC in choriocarcinoma cell lines --- p.111 / Chapter 7.3.2 --- Demethylation of APC in choriocarcinoma cell lines --- p.114 / Chapter 7.4 --- Discussion --- p.115 / Chapter SECTION V: --- CONSERVATION OF METHYLATION IN PLACENTA ACROSS DIFFERENT SPECIES --- p.118 / Chapter CHAPTER 8: --- Methylation analysis of hypermethylated TSG homologues in the placentas of the mouse and rhesus monkey --- p.119 / Chapter 8.1 --- Introduction --- p.119 / Chapter 8.2 --- Materials and methods --- p.120 / Chapter 8.2.1 --- Sample collection --- p.120 / Chapter 8.2.2 --- Sample processing and DNA extraction --- p.120 / Chapter 8.2.3 --- Experimental design --- p.120 / Chapter 8.3 --- Results --- p.124 / Chapter 8.3.1 --- Methylation status of TSGs in rhesus monkey and murine placental tissues --- p.124 / Chapter 8.4 --- Discussion --- p.136 / Chapter SECTION VI: --- CONCLUDING REMARKS --- p.138 / Chapter CHAPTER 9: --- Conclusion and future perspectives --- p.139 / Chapter 9.1 --- Pseudomalignant nature of placenta at the epigenetic level --- p.139 / Chapter 9.2 --- Functional implication of TSG hypermethylation --- p.140 / Chapter 9.3 --- Significance of hypermethylated TSGs in the placental evolution --- p.142 / Chapter 9.4 --- Clinical implication of TSG hypermethylation --- p.143 / Chapter 9.5 --- Future perspectives --- p.145 / APPENDIX I COMPLETE BISULFITE SEQUENCING DATA FOR HYPERMETHYLATED TSGS --- p.147 / APPENDIX II BISULFITE SEQUENCING DATA FOR PTEN --- p.156 / APPENDIX III BISULFITE SEQUENCING DATA OF LOCI NOT SHOWING HYPERMETHYLATION --- p.158 / REFERENCES --- p.160

A interação materno fetal na espécie equina depende exclusivamente da complexidade anatômica e fisiológica da placenta e este órgão endócrino provisório é a peça chave do presente estudo, onde objetivou-se avaliar a influência do número de partos sob a eficiência placentária. Buscando, por meio da morfometria das macro e microrregiões, além da expressão do VEGF, VEGFR-1/Flt-1 e VEGFR-2/FLK-1 na membrana corioalantóide a termo, dados referentes à inter-relação da pluriparidade, contato materno fetal e peso dos neonatos da raça Puro Sangue Inglês. O delineamento experimental consistiu em um estudo analítico, observacional, transversal, prospectivo. Foram acompanhados cinquenta partos de éguas divididas em três grupos de acordo com a pluriparidade: primíparas (n=18), 2 a 5 partos (n=14) e 6 a 10 partos (n=18). Foram coletados e fixados em formol fragmentos de 3 cm2 das regiões do corpo do útero (Cut), corno uterino gestante (CG) e corno uterino contralateral (CnG) da membrana corioalantóide a termo pós delivramento. Após processamento foram corados pela técnica de Hematoxilina & Eosina e Picro-Sirus-Red. Os estudos imunohistoquímico (método avidina-biotina) e esterológico foram realizados ao microscópio óptico. Foram correlacionados número de partos, altura e perímetro torácico (PT) maternos; idade, altura e PT paternos; composição volumétrica dos compartimentos placentários juntamente com as áreas de superfície de contato materno-fetal e peso, altura e escore de vitalidade neonatal. A paridade, além de diretamente relacionada ao peso do neonato, demonstrou na avaliação morfofuncional ser determinante ao ganho de eficiência placentária. As membranas corioalantóides das éguas pluríparas tenderam a apresentar maior volume total no CG, aumento na porcentagem e volume total de vilos, além de maior densidade microcotiledonária e expressão do VEGF na região do CG. Entre as muitas correlações determinadas no grupo primíparas as mais relevantes foram peso da membrana com variáveis neonatais como peso (r=0,598), PT (r=0,775), tempo para mamar (r=0,553) e tempo de gestação (r=-0,527), além do PT da mãe com o tempo para ruptura do cordão umbilical (r=0,564), reflexo de sucção (r=0,625), para levantar (r=0,756) e decúbito esternal (r=-590). Da mesma forma no grupo 2 a 5 partos, neonatos que nasceram com maior peso, altura, e PT apresentaram menor tempo para levantar (r=-0,546; r=-0,869, r=-0,892), eliminação do mecônio (r=-0,535) e ruptura do cordão umbilical (r=-0,528; r=-0,881). Assim como no grupo 6 a 10 partos, o peso do potro apresentou correlação com o peso (r=0,793), PT (r=0,716) e altura (r=0,667) materna. O volume total do CG correlacionou com volume total do parênquima (r=-0,816) e epitélio nos vilos placentários do CG (r=-0,915), tempo de gestação (r=-0,483), tempo para reflexo de sucção (r=-0,672), para mamar (r=-0,525), e para eliminação do mecônio (r=-0,525). No que diz respeito às variáveis paternas, o peso paterno correlacionou com o volume total do parênquima nos vilos placentários do CnG (r=0,393) , além do PT do garanhão com o peso (r=0,316) e PT (r=0,425) do potro e volume total do CG (r=0,319). Desta forma, acredita-se que estes resultados possam fornecer ferramentas práticas para auxiliar na escolha das fêmeas e machos mais indicados para geração de potros neonatos com desenvolvimento adequado. / Fetomaternal interaction in equine species depends exclusively on the complexity of placental anatomy and physiology. This transient endocrine organ is the key of the present study, which aimed to evaluate the influence of parity on placental efficiency. Morphometric of macro and micro regions, expression of VEGF, VEGFR-1/Flt-1 and VEGFR-2/FLK-1 in term corioallantois were performed and data regarding parity, fetomaternal contact and neonatal weight were evaluated in Thoroughbred. The experimental study consisted of an analytical, observational, cross-sectional, prospective study. Fifty deliveries were monitored in mares divided into three groups according to parity: nulliparous (n=18), 2 to 5 deliveries (n=14) and 6 to 10 deliveries (n=18). Tissue sections measuring 3 cm 2 were collected and fixed in formol saline from term chorioallantois regions: uterine body (UtB), pregnant uterine horn (PH) and contralateral uterine horn (CtH). After processing, samples were stained using Hematoxylineosin and Picro-Sirus-Red. Imunnohistochemistry (avidin-biotin method) and stereology were performed using an optic microscope. Correlations between parity, maternal height and thoracic perimeter; paternal age, height and thoracic perimeter; volume composition of placental compartments, surface area of fetomaternal contact and weight, height and neonatal vitality score were performed. Parity was directly related to weight of the neonate and demonstrated in morphofunctional evaluation to be determinant for placental efficiency. Chorioallantoic membranes from pluriparous mares tended to have greater total PH volume, higher percentage and total villi volume, and greater microcotiledonary density and VEGF expression in PH region. There were several correlations found in nulliparous group, the most relevant were membrane weight and neonatal parameters such as weight (r=0,598), thoracic perimeter (r=0,775), time to nurse (r=0,553) and gestational length (r=-0,527) and also mares thoracic perimeter with time to rupture the umbilical cord (r=0,564), suckle reflex (r=0,625), time to stand (r=0,756) and to achieve sternal recumbency (r=-590). Likewise, for the 2 to 5 deliveries group, neonates that were born heavier, taller and with greater thoracic perimeter took less time to stand (r=-0,546; r=-0,869, r=-0,892), pass meconium (r=-0,535) and rupture the umbilical cord (r=-0,528; r=-0,881). In the group of mares between 6 and 10 deliveries, foals weight was correlated to maternal weight (r=0,793), thoracic perimeter (r=0,716) and height (r=0,667). Total PH volume correlated to total parenchyma (r=-0,816) and epithelium in placental villi in PH (r=-0,915), gestational lenght (r=-0,483), time to suckle reflex (r=- 0,672), to nurse (r=-0,525), and pass meconium (r=-0,525). Regarding Paternal variables, paternal weight correlated to total parenchyma volume in placental villi of the CtH (r=0,393). Also thoracic perimeter of the stallion correlated to neonatal weight (r=0,316) thoracic perimeter (r=0,425) and total volume of the PH (r=0,319). Therefore, these data provides rationale for an adequate choice of mares and stallions to generate well-developed neonates.

Perdas gestacionais, malformações, restrição de crescimento intrauterino (IUGR) são associadas a gestações diabéticas. Para ampliar o conhecimento nesse tema, nosso grupo desenvolveu um modelo de gestação complicada por diabetes tipo 1 em camundongos que, nessa tese, foi utilizado para analisar o ciclo estral, desenvolvimento fetal e organização placentária. O diabetes foi induzido por aloxana e estudado em dois períodos 30-50D (curto prazo) e 90-110D (longo prazo). Placentas e fetos foram coletados, pesados, e submetidos a técnicas moleculares, bioquímicas e morfológicas. Detectaram-se alterações no perfil temporal do ciclo estral. O grupo 30-50D apresentou altas taxas de perdas embrionárias e IUGR, e o 90-110D malformações, mortes fetais, IUGR e aumento no peso placentário. As placentas diabéticas apresentaram aumento e desorganização da zona juncional, redução do labirinto e vasodilatação. A expressão dos colágenos I e III aumentou e a do V diminuiu em 30-50D, porém, a deposição destes aumentou concomitante com a redução da atividade da MMP9. A deposição dos colágenos III e V e a atividade da MMP2 aumentaram em 90-110D. Nossos resultados reiteram a importância do fator temporal nas complicações do diabetes sobre a gestação. / Gestational loss, malformations and intrauterine growth restriction (IUGR) are often associated with pregnancies. To increase the knowledge about this topic, our group has developed a model of pregnancy complicated by type 1 diabetes in mice. In this study, was analyzed the estrous cycle and the fetal and placental development. For this, diabetes was induced by alloxan and studied in two time-periods 30-50D (short term) and 90-110D (long term). Placentas and fetuses were collected, weighed and analyzed by biochemical, morphological and molecular procedures. We detected changes in the temporal profile of the estrous cycle. The 30-50D group showed high rates of embryonic loss and IUGR whereas malformations, fetal death, IUGR and increased placental weight was detected in 90-110D. Increase and disorganization of junctional zone, reducing labirinth and vasodilation characterize diabetic placentas. The expression of collagen I and III was increased whereas collagen V decreased in the 30-50D. The deposition of this collagen, however increased concomitant with the reduction of MMP9 activity. In 90-110D deposition of collagen III and V and the MMP2 activity was increased. Together, our results reinforce the relevance of the time factor in the complications of diabetes on pregnancy.

A placenta dos mamíferos é um órgão transitório formado pela justaposição das membranas fetais e tecidos maternos, sendo responsável pelas trocas fisiológicas entre o feto e a mãe e pela síntese de hormônios fundamentais para o sucesso da gestação. O crescimento placentário e a nutrição fetal requerem altas taxas de crescimento e diferenciação celular, sendo fundamental um balanço adequado entre proliferação e morte celular das células trofoblásticas placentárias. Estas células possuem propriedades particulares no que diz respeito a suas funções metabólicas, endócrinas e angiogênicas, porém a atividade proliferativa destas células em diferentes regiões da placenta bovina é desconhecida. Neste estudo, levantamos a hipótese de que as células de diferentes regiões placentárias de bovinos devem apresentar padrões distintos de atividade proliferativa e morte celular, podendo ser algumas regiões mais ou menos responsáveis pelo desenvolvimento fetal ao longo da prenhez. Para testar esta hipótese, analisamos as células de diferentes regiões da placenta e em diferentes estágios gestacionais através de citometria de fluxo e coloração com marcador histoquímico, além de observações macroscópicas e histológicas para avaliar a morfologia da placenta e suas regiões. Foram analisadas as regiões: central e marginal dos placentônios, área interplacentomal, microplacentônios (≤1,0cm) e fusões placentomais. Por meio da citometria de fluxo realizamos uma distribuição das células placentárias nas diferentes fases do ciclo celular, observando a porcentagem de células em proliferação e apoptose. Em nossos resultados, encontramos particularidades macroscópicas, histológicas e metabólicas nas células de cada região placentária. A margem do placentônio apresentou um aumento prematuro de células em apoptose, com um aumento crescente a partir de 170 dias até o final da gestação, sugerindo que nesta região se inicie a desconexão placentomal. As áreas interplacentomais e fusões de placentônios apresentaram um equilíbrio na atividade proliferativa ao longo da gestação, sugerindo que a proliferação celular possua uma menor importância para a homeostase tecidual nestas regiões. Os microplacentônios apresentaram uma atividade proliferativa mais lenta, porém crescente ao longo da gestação, sugerindo pouca contribuição destas estruturas para a liberação placentária. Estes achados estão de acordo com nossa hipótese de que cada região placentária contribui de forma distinta para o crescimento da placenta bovina, cada uma delas participando de maneira particular no processo de maturação e desconexão placentária e no crescimento e nutrição fetal ao longo da gestação. O entendimento das taxas proliferativas e apoptóticas é importante para a compreensão da patogênese das perdas embrionárias e anormalidades no desenvolvimento placentário. A descrição dos processos de proliferação e morte celular em diferentes regiões da placenta de gestações bovinas poderá ajudar a elucidar processos fisiológicos desconhecidos relacionados ao desenvolvimento do concepto e a falhas gestacionais. Espera-se que este estudo forneça elementos essenciais à melhor compreensão do desenvolvimento placentário, estabelecendo padrões de normalidade para cada região da placenta bovina e auxiliando na compreensão dos mecanismos de falhas gestacionais que levam a anormalidades fetais e placentárias, muito comuns em técnicas avançadas de manipulação embrionárias, como a fertilização in vitro, a produção de animais transgênicos e a clonagem animal. / The mammalian placenta is a transitory organ consisting of maternal-fetal tissues union, responsible for nutrient exchange and synthesis of many hormones which are essential for success of the gestation. Placental growth and fetal nutrition require high rates of cellular turnover and differentiation, and a balance between proliferation and apoptosis in the trophoblastic cells are essential to the placental development. The trophoblastic cells, that form the fetal portion of placenta, has unique properties and a wide range of metabolic, endocrine and angiogenic functions, but the proliferative profile of those cells. In this study we hypothesed that cells from different placental regions on bovine placenta must reveal distinct patterns of proliferative activity and apoptosis, and some regions may be potentially responsible to distinct modulation patterns in fetal growth and nutrition throughout pregnancy. We analyzed cells form different regions of normal bovine placenta, namely the central region of the placentome, the placentomes edges, interplacentomal areas, placentomal fusion, and microplacentomes (≤1.0cm). The cells of those regions were analyzed by flow cytometry and with a histochemical marker, as well as macroscopical and histological analysis. Using flow cytometry we performed the percentage of cells in each cell cycle phase, including apoptosis. Our findings indicated macroscopical, histological and metabolic particularities in different placental region. The edges of the placentome revealed a premature increase in apoptotic cells, with a significantly higher number of apoptotic cells from 170 days of gestation. That suggests this region to be the point of initiation of placentomal detachment in cattle. Interplacentomal areas and placentomal fusions showed no significant variations during pregnancy, suggesting that proliferation is minor importance for tissue homeostasis in those regions. Microplacentomes revealed a lower but an ascending proliferative activity, that suggests the relatively small role of those structures, with microplacentomes not contributing significantly to normal of placental functions and conceptus development during pregnancy. Taken together, the complete understanding of placental apoptotic and proliferative profiles at distinct placental tissue regions during gestation will shed light to unknown physiological processes related to conceptus development, as some regions may be potentially responsible, at different degrees of influence, to distinct modulation patterns in fetal growth and nutrition throughout pregnancy. In that regard, results from the present study using non-manipulated bovine gestations indicated that proliferation and apoptosis exhibit patterns that are specific for the individual placental areas during gestation and at term in normal, non-manipulated pregnancies in cattle. Variations involving proliferation and apoptotic rates may influence placental maturation and detachment, compromising placental functions and leading to fetal stress, abnormalities in development and abortion, as frequently seen in bovine pregnancies from in vitro fertilization and cloning procedures. Our findings describing the pattern of cell proliferation and apoptosis in normal bovine pregnancies may be useful for unraveling some of the developmental deviations frequently seen in nature and after in vitro embryo manipulations.

A família dos VEGFs é a principal responsável pela angiogênese aumentando a neovascularização e a permeabilidade da interface endometrial/placentária. O estudo comparativo da distribuição temporal do sistema VEGF na placenta de cães e de bovinos, que apresentam diferenças tanto na sua estrutura como na sua função, pode contribuir para esclarecer o papel deste sistema ao longo da gestação. Amostras da cinta placentária de cadelas (nos dias 20, 40 e 60 de gestação) foram coletadas assim como amostras de placentomas bovinos (dias 90, 150, 210 e 270 da gestação). As amostras foram congeladas em nitrogênio líquido e armazenadas em -80ºC até serem homogeneizadas, ou fixadas em solução de formol tamponado e embebidas em paraplast usando os procedimentos convencionais. Foi realizado Western Blotting para a quantificação do sistema VEGF utilizando-se anticorpos específicos combinados com um sistema amplificador de quimio-luminescência: ECL®. A imuno-histoquímica para a localização do Flt-4 canino foi realizada de acordo com o método ABC, utilizando o Nova Red® como cromógeno. Na placenta bovina, o VEGF apresentou uma alta expressão no início da gestação seguida por queda que se manteve deste até o termo. O Flt-1, o KDR e o Flt-4 demonstraram uma alta expressão no início e no meio da gestação, e um declínio em sua expressão ao final da mesma. Na placenta canina, a proteína do VEGF e do Flt-1 apresentaram uma elevada expressão no início da gestação, a qual diminuiu na metade e assim se manteve até o final da gestação, enquanto o KDR demonstrou uma alta expressão no início e no meio da gestação, e um decréscimo em sua expressão próximo ao termo. A proteína do Flt-4 foi localizada nas regiões do labirinto placentário, do corion frouxo e do alantocorion e sua expressão variou nos diferentes compartimentos ao longo da gestação. A expressão similar do sistema VEGF e do Flt-4 em ambas as espécies estudadas sugere que estas proteínas apresentem funções similares tanto na placenta bovina como canina. / The VEGF family is known to be responsible for the angiogenesis increasing blood vessels number and permeability in the endometrial/placentary interface. A comparative study of VEGF system temporal distribution in canine and bovine placentas, which present very well described differences in structure and function, will contribute to better define the role of this system along pregnancy. Samples from placental belt of pregnant bitches (on days 20, 40 and 60) were collected as well as bovine placentomes samples (from days 90, 150, 210, 270 of gestation). All samples were frozen in liquid nitrogen and stored at -80º C until processed, or fixed in buffered formalin solution and embedded in paraplast using conventional procedures. Western Blotting for VEGF system quantification was carried out using specific antibodies combined to an amplification quimio-luminescence system: ECL®. Immunohistochemistry for canine Flt-4 demonstration was performed according to the ABC method using NovaRed® as chromogen. In the bovine placenta, VEGF demonstrate a higher expression in early gestation, which decreased at mid-gestation and remained so until term. Flt-1, KDR and Flt-4 demonstrated a constant expression in early and mid-gestation, and a decrease in the end of pregnancy. In the dog placenta, VEGF and Flt-1 proteins show an increased expression in early gestation, and a decreased expression at mid and late gestation; while KDR demonstrated an increased expression at early and mid-gestation, and a decreased expression near term. Flt-4 protein is localized in placental labyrinth, chorion and allantochorion. Similar expressions of these proteins in both species, that present considerable differences in placenta structure and function, suggest that VEGF system and Flt-4 play similar roles in the bovine and canine placenta.

Biofilme subgengival, conteúdo vaginal, âmnio e parênquima placentários foram obtidos de 93 puérperas. Os periodontopatógenos (BPPG) P.g., A.a, F.n e T.f foram identificadas por PCR, corioamnionite e vilosite por histopatologia e analisados estatisticamente. Roturas de membranas (22,2%), cesáreas (65,6%), pretermo (36,6%), baixo peso (34,4%), morte perinatal (5,4%), CAM (34,4%) e vilosite (5,5%) foram observados. Periodontite agressiva (62,4%) e cáries (83,9%). Periodontite ou BPPG em qualquer sitio não se associou com maior freqüência ou com maior risco de corioamnionite ou de desfecho reprodutivo ruim. No biofilme observou-se Aa em 2 (2,1%), Fn em 16 (17,20%), Pg em 30 (32,30%) e Tf em 29 (31.2%); na vagina, Aa em 3 (3,2%), Fn em 2 (2,1%), Pg em 16 (17,2%) e Tf em 3 (3,2%); no âmnio, Fn em 4 (4,2%) e Pg em 9 (9,7%); na placenta, Fn em 1 (1,07%), Pg em 4 (4,3%) e Tf em 1 (1,1%). P.g e F.n foram observados simultaneamente: 6/30 casos de Pg na boca estavam na vagina, 3 no âmnio e 1 na placenta; dos 16 Fn na boca, 1 foi encontrado na placenta. Esta taxa de disseminação sugere que as BPPG na vagina, âmnio ou placenta não se originaram na boca das puérperas. / Subgingival biofilm and buccal samples, vaginal contents, amnion and placental parenchyma were obtained from 93 pregnant. The periodontopathogens (PPG) Pg, Aa, Fn, and Tf were identified by PCR, the diagnosis of chorioamnionitis (CAM) and villitis by histopathology methods and its relationships with adverse perinatal outcome (APO) statically analyzed. Rupture of membranes (22.2%), cesarean (65.6%), preterm (36.6%), low fetal weight (34.4%), perinatal death (5.4%), CAM (34.4%), and villitis (5.5%) were observed. Aggressive periodontitis (62,4%), and 83.9% had caries. Periodontitis or PPG (Aa, Fn, Pg, and Tf) in any site not associated with greater frequency or at greater risk of CAM or APO. In the subgingival biofilm Aa was observed in 2 (2.1%), Fn in 16 (17.20%), Pg in 30 (32.30%) and Tf in 29 (31.2%); in the vagina, 3 in Aa (3.2%), Fn 2 (2.1), Pg 16 (17.2%) and Tf 3 (3.2%); amnion, Fn in 4 (4.2%) and Pg 9 (9.7%); in the placenta, Fn 1 (1.07%), Pg 4 (4.3%) and Tf 1 (1.1%). Only Fn and Pg were observed simultaneously: 30 mothers with Pg in the mouth, 6 were detected in the vagina, 3 in amnion and 1 in the placenta; Fn 16 with the mouth, 1 was found in the placenta. This low rate of spread suggests that most of periodontopathogens present in vagina, amnion or placenta did not belong to the pregnant mouth.

A função da eritrofagocitose observada após o extravasamento de sangue na interface materno-fetal é indefinida em várias espécies, incluindo o búfalo. Na ovelha, este processo foi muito estudado, e ocorre na zona arcada do placentônio (topos dos septos maternos e base dos vilos fetais), região onde o processo é realizado pelo trofoblasto. É possível que o ferro seja transferido para o feto mediante a eritrofagocitose trofoblástica nesta área hemófaga da placenta e nas glândulas endometriais. Para este estudo foram utilizadas placentas de búfalas entre 2-3, 4-5, 6-7, 8-9 e 10 meses de prenhez, fixadas por perfusão com solução aquosa de formaldeído a 10% e paraformaldeído a 4%, para microscopia de luz, e glutaraldeído a 2,5%, para microscopia eletrônica de transmissão, processadas e coradas para microscopia de luz (HE, azul de Toluidina, tricromo de Gomori, Hematoxilina-floxina, Azul de metileno - fucsina básica), histoquímica (reação de Perls, PAS e fosfatase ácida) além de microscopia eletrônica de transmissão. A metodologia utilizada permitiu-nos observar que as áreas hemófagas estavam presentes em determinadas regiões do placentônio, nas quais se identificavam áreas hemorrágicas entre o epitélio uterino e o trofoblástico, nas placentas de 4 a 10 meses de prenhez. Eritrócitos foram encontrados nas células trofoblásticas, elucidando, deste modo, a eritrofagocitose. A reação de PAS foi positiva, marcando substância mucóide, principalmente na base dos vilos fetais, células trofoblásticas binucleadas e nas glândulas endometriais da região interplacentomal. A reação de Perls foi negativa nos placentônios e positiva nas glândulas endometriais. A reação de fosfatase ácida foi positiva tanto nos placentônios, quanto na região interplacentomal. A ultraestrutura da região das áreas hemófagas revelou eritrócitos ingeridos dentro das células trofoblásticas epiteliais em diferentes fases de digestão e eletrondensidades, várias vesículas endocíticas, cavéola, muitas gotículas lipídicas, retículo endoplasmático rugoso bem desenvolvido e a presença de grande quantidade de mitocôndrias. O epitélio das glândulas endometriais da região interplacentomal é do tipo colunar com a presença de microvilos em seu ápice, e núcleos basais. / The function of erytrophagocytosis observed after blood extravasation in the maternal-fetal interface is indefinite various species, including the water buffalo. In ewe, this process had been hardly studied and it occurs in the placentome arcade zone (top of maternal septa), region where the process is performed by the trophoblast. It is possible that iron is being transferred to the fetus through the trophoblastic erytrophagocytosis in the hemophagous areas of the placenta and in the endometrial glands. In our research we have been using placentomes of buffaloes between 2-3, 4-5, 6-7, 8-9 and 10 months of pregnancy, fixed by perfusion with 10% formaldehyde aqueous solution, 4% paraformoldehyde for light microscopy and 2,5% glutaraldehyde for transmission electron microscopy, processed and stained for light microscopy (HE, Toluidine blue, Gomori trichrome, hematoxilin - floxin, methilen blue ? basic fucsin), histochemistry (Perls, PAS and acid phosfatase reaction) and transmission electron microscopy. The methodology used allowed us to observe that the haemophagous areas were present in determined regions of the placentome, in which there were showing haemorragic areas between the trophoblastic and uterine epithelium, in 4-10 months pregnant placentae. Erythrocytes had been found inside trophoblastic cells, thus contributing to explain the erytrophagocytosis. The PAS reaction was positive, staining mucoid substance, mainly in the basis of the fetal villi, trophoblastic binucleate cells and in an endometrial glands in the interplacentomal region. The Perls reaction was negative in the placenton as well as in the endometrial glands. The acid phosphatase reaction was positive in placenton as well as in the interplacentomal region. The ultrastructure of the haemophagous areas revealed ingested erythrocytes inside the epithelial cells of trophoblast in different phases of digestion and electrondensities, various endocitic vesicles, caveolae, many lipid droplets, well-developed rough endoplasmic reticulum and large number of mitochondrias. The endometrial glands epithelium of interplacentomal region is columnar type with microvilli and basal nuclei.

A busca por técnicas alternativas para suprir a escassez de tecidos e órgãos danificados levou ao surgimento da engenharia de tecidos. Scaffolds biológicos criados a partir da matriz extracelular (MEC) de órgãos e tecidos tem sido uma promissora ferramenta aplicada para suprir esta necessidade. A matriz extracelular placentária descelularizada surge como uma potencial ferramenta para a produção de scaffolds biológicos para recelularização e implantação em áreas lesionadas. Para ser classificado como um scaffolds biológico ideal, a matriz extracelular deve ser acelular e ter preservada suas proteínas e características físicas para viabilizar a adesão celular. Neste contexto, desenvolvemos o scaffolds biológico descelularizado a partir de placentas caninas com 35 e 40 dias de gestação. A eficiência da descelularização foi confirmada pela ausência de conteúdo celular e quantidade de DNA remanescente. A arquitetura vascular e as proteínas da matriz extracelular, tais como, colágenos tipo I, III e IV, laminina e fibronectina, foram preservadas. Para o processo de recelularização, utilizamos células-tronco progenitoras endoteliais derivadas do saco vitelino canino (SVC) e células tronco mesenquimais (CTMs) derivadas de medula óssea canina (CMOC) e de polpa de dente canina (CPDC). O processo de recelularização em placas não aderentes por 7 e 14 dias, na presença do scaffolds placentário secos em ponto crítico auxiliou na eficiência da recelularização, comprovada por imunofluorescência e microscopia eletrônica de varredura, evidenciando a adesão das células no scaffolds e comprovando ser um promissor biomaterial para utilização na medicina regenerativa tecidual. / The search for alternative techniques to address the scarcity of damaged tissues and organs has led to the emergence of tissue engineering. Biological scaffolds created from the extracellular matrix (ECM) of organs and tissues have been a promising applied tool to meet this need. The decellularized placental extracellular matrix appears as a potential tool for the production of biological scaffolds for recellularization and implantation in injured areas. To be classified as an ideal biological scaffold, the extracellular matrix must be acellular and have preserved its proteins and physical characteristics to enable cell adhesion. In this context, we developed the biological scaffold decellularized from canine placentas with 35 and 40 days of gestation. The efficiency of the decellularization was confirmed by the absence of cellular content and amount of DNA remaining. Vascular architecture and extracellular matrix proteins, such as collagens type I, III and IV, laminin and fibronectin, have been preserved. For the process of recellularization, we used stem cells derived from the canine yolk sac (CYSC) and mesenchymal stem cells (MSCs) derived from canine bone marrow (CBMC) and canine dental pulp (CDPC). The process of recellularization in non-adherent plaques for 7 and 14 days in the presence of placental scaffold dried at a critical point assisted in the efficiency of the recellularization, evidenced by immunocytochemistry and scanning electron microscopy, evidencing the adhesion of the cells in the scaffold and proving to be a promising biomaterial for use in tissue regenerative medicine.

L’obésité, problème majeur de santé publique, est en augmentation constante. Elle est responsable d’une altération de la sécrétion des adipokines, telles que l’apeline. L’apeline est impliquée dans diverses fonctions de l’organisme et notamment dans la régulation du métabolisme énergétique. Au cours de la grossesse, ce système semble avoir un rôle crucial dans le développement foeto-placentaire. Le système apelinergique chez la femme enceinte obèse n’a encore jamais été étudié. L’objectif de cette thèse était de vérifier si le système apelinergique est modifié en cas d’obésité chez la femme enceinte, tout d’abord par une approche expérimentale sur un modèle de souris obèse et insulino-résistante, puis par une approche translationnelle vers l’humain (étude OB-APE). Matériel et méthode : Modèle murin : 40 souris femelles ont été réparties en 2 groupes : Témoin (T, n=20) et High Fat (HF, n=20). Après 3 mois de régime, les souris étaient mises en reproduction. Des prélèvements étaient réalisés à E6.5, E12.5 et E18.5. Lors du sacrifice à E18.5, la glycémie à jeun, l’insulinémie, l’apelinémie maternelle et foetale étaient dosées, les souriceaux et les placentas étaient pesés. La moitié de chaque placenta était mis dans du RNA later et l’autre moitié était mis dans de l’azote liquide. Les prélèvements étaient conservés à -80°C. Etude chez l’humain (étude OB-APE) : Etude prospective et comparative menée au sein de la maternité Jeanne de Flandre (Lille, France) entre mai 2016 et juillet 2017. Les patientes inclues étaient réparties en 3 groupes: N (normal, n= 30), O (obèses, n = 30) et ODG (obèse avec diabète gestationnel, n = 30). Trois prélèvements plasmatiques maternels d’apeline étaient réalisés : entre 35 et 40 SA), à l’accouchement et au 2ème jour du post-partum, ainsi qu’un prélèvement néonatal au cordon ombilical. Des fragments placentaires étaient prélevés à l’accouchement et du colostrum était récupéré à J2 en post-partum. Les dosages de l’apeline dans le plasma et dans le colostrum étaient réalisés par ELISA. L’expression placentaire de l’apeline et d’APJ était étudiée par RT-PCR quantitative. La sécrétion placentaire était étudiée dans un milieu nutritif standard (DMEM) ainsi qu’en présence d’insuline (50nM) ou d’angiotensine II (AT2, 1nM). Résultats:Modèle murin : Après 3 mois de régime, les souris HF étaient obèses et intolérantes aux hydrates de carbone. Il n’y avait pas de différence d’apelinémie à jeun entre les souris T et HF hors gestation. Les placentas des souriceaux HF avaient un poids supérieur à ceux des portées T (P=0.006). Il y avait au cours de la gestation une diminution de l’apelinémie dans les 2 groupes en fin de gestation, de façon plus importante dans le groupe HF que dans le groupe Témoin (P =0.01). Chez les souris obèses, il y avait une augmentation de l’insulinorésistance en fin de gestation par rapport au groupe T (P<0.05). L’expression placentaire de l’apeline et d’APJ était augmentée dans les placentas de souris obèses. Etude chez l’humain (étude OB-APE) : L’apelinémie maternelle était diminuée dans les groupes O et ODG en comparaison avec le groupe N aux 3 temps de l’étude. L’apelinémie néonatale était également diminuée dans ces mêmes groupes. Les concentrations en apeline dans le colostrum étaient à l’inverse plus élevées dans les groupes O et ODG que dans le groupe N (P = 0,007 et P = 0,05 respectivement). Ex-vivo, la sécrétion placentaire était diminuée dans les groupes O et ODG en comparaison avec le groupe N. L’ajout d’insuline dans le milieu entrainait une augmentation de la sécrétion d’apeline, alors que l’ajout d’AT2 aboutissait à une diminution de cette sécrétion. L’expression placentaire de l’ARNm d’APJ était plus importante dans les placentas de femmes obèses que chez patientes d’IMC normal (N) [...] / Obesity is a major public health problem and is constantly increasing. Obesity alters the adipokines’ secretion, such as apeline. Apeline is involved in various functions, such as energy metabolism regulating. During pregnancy, the apelinergic system seems to be crucial for fetal development. The apelinergic system in obese pregnant women has never been studied before. The aim of this thesis was to verify whether the apelinergic system is modified in case of obesity in pregnant women, first by an experimental approach on an obese and insulin-resistant mouse model and then by a translational approach to the human (OB-APE study). Material and methods : Murine model: 40 female mice were divided into 2 groups: Control (C, n = 20) and High Fat (HF, n = 20). After 10 weeks of diet, mice were mated. Samples were taken at E6.5, E12.5 and E18.5. Mice were sacrificed by decapitation at E18.5 and blood samples were collected. Fetuses and placentas were collected after cesarean section and weighed. Fasting blood glucose, insulinaemia, maternal and fetal apelinemia were measured. At E18.5, blood samples of fetuses were collected and placentas were frozen and stored at -80°C. Human model (OB-APE study) : Prospective and comparative study conducted in Jeanne de Flandre maternity (Lille, France) between May 2016 and July 2017. Patients were divided into 3 groups: group N (normal, n=30), group O (obese, n=30) and group ODG (obese with diabetes mellitus, n=30). The maternal plasma samples were obtained at 3 different times: at the end of pregnancy (35-40 weeks of gestation), at delivery and at day 2 in postpartum, as well as a neonatal umbilical cord sampling. Placental fragments were collected at delivery and colostrum was recovered on day 2 in postpartum. The measures of apelin concentrations in plasma and colostrum were performed by ELISA. The placental expression of apelin and APJ was studied by quantitative RT-PCR. Placental secretion was studied in a standard nutrient medium (DMEM) as well as in presence of insulin (50nM) or angiotensin II (AT2, 1nM). Results: Murine model: After 3 months of diet, HF mice were obese and intolerant to carbohydrates. There was no significant difference in fasted apelinemia between non-pregnant T and HF mice. The placentas of HF mice were heavier than controls (P=0.006). There was an increase in apelinemia at E12.5 in the 2 groups (P<0.05), higher in the group HF (4.89 ± 1.18 ng/mL, vs 2.44 ± 0.42 ng/mL, P<0.001). In obese mice, there was an increase in insulin-resistance at the end of pregnancy compared to the group T (P<0.05). The placental expression of apelin and APJ was increased in obese mice. Human model (OB-APE study) : Maternal apelinemia was decreased in the O and ODG groups in comparison with the N group at the 3 times of the study. Neonatal apelinemia was also decreased in these groups. The apelin concentrations in colostrum were higher in the groups O and ODG than in the group N (P=0.007 and P=0.05 respectively). Ex-vivo, placental secretion of apelin was decreased in the groups O and ODG compared to the group N. The addition of insulin in the medium led to an increase in apelin secretion, whereas the addition of AT2 led to a diminution of this secretion. Placental expression of APJ mRNA was greater in placentas of obese women than in normal (N) patients [...]

A imunidade inata na interface materno-fetal é um dos mecanismos de proteção essenciais na resposta antiviral, sobretudo, na infecção por HIV-1. Neste trabalho, avaliamos a influência da infecção materna por HIV-1 na expressão de fatores antivirais, nas moléculas do complexo inflamassoma e de retrovírus endógenos (HERV), em células mononucleares (CMN) maternas, células do cordão umbilical (recém-natos, RN), colostro e no tecido placentário. Os resultados mostraram que no vilo das placentas de mães infectadas há um aumento na expressão de mRNA dos fatores antivirais, como IFN tipo I e III, DAMPs damage-associated molecular patterns e HERVs. Entretanto, na análise proteica, essas diferenças não se confirmam, indicando que o sistema imune inato é capaz de reconhecer o vírus, ou ainda, o dano causado pela infecção, mas controla a produção exacerbada da proteína. Sob estímulo de agonista de TLRs Toll-like receptor, em CMNs a expressão de HERVs não se altera entre RNs. Além disto, avaliando os níveis de &#223-quimiocinas, CCL5 e CCL3, e de IFN-&#967 nos sobrenadantes das culturas de CMNs, a ativação por LPS foi capaz de diminuir a produção de CCL3 e CCL5 nas CMNs de mães infectadas por HIV em relação às mães controles, contudo o CL097 promoveu níveis similares às mães do grupo controle. Nos RNs, enquanto os níveis de CCL5 são inferiores aos de adultos, os níveis de CCL3 são semelhantes. Já o ligante TLR7/8 foi capaz de restaurar a secreção de IFN-&#945 no grupo infectado por HIV-1. Além disso, no vilo das placentas das mães infectadas por HIV, há intensa modificação na expressão de mRNA dos fatores analisados, sejam antivirais, como IFN tipo I (IFN-&#945), tipo III (IFN-&#955), fatores relacionados ao dano celular (DAMPs e seus receptores). Entre os DAMPs, um aumento da expressão de S100A9 e HMGB1 e seus receptores RAGE, TLR4 e TLR9 foi observado nos vilos de placentas de mães infectadas por HIV-1, contudo, os níveis séricos de HMGB1 estão diminuídos em mães infectadas e RN expostos. Quanto aos níveis séricos de citocinas, foram observados níveis reduzidos de HMGB1, IL-6 e IL-1&#223 nos RNs expostos, o que evidencia um controle do estado inflamatório na exposição ao HIV-1. Também observamos presença de níveis séricos de HERV-W, livre ou em exossomas, em ambos os grupos analisados. Já no colostro, não encontramos diferenças nas análises de fatores inflamatórios e HERVs indicando que, nesse compartimento, a infecção não altera os padrões de expressão desses alvos. A vigência do estado antiviral e a supressão do ambiente inflamatório podem equilibrar a resposta imune placentária, promovendo a homeostase para o desenvolvimento do feto e de proteção à infecção por HIV-1 nos neonatos. / Innate immunity at the maternal-fetal interface is one of the main protection mechanisms in the antiviral response, especially in HIV-1 infection. In this work, we present the influence of maternal HIV-1 infection on the expression of antiviral factors, inflammasome molecular complex and human endogenous retroviruses (HERV), in maternal mononuclear cells (MNCs), umbilical cord cells (newborns, NB), colostrum and placental tissue. The results show that in infected mothers cells has an increase in mRNA expression of antiviral factors, such as IFN type I and III, DAMPs (damage-associated molecular patterns) and HERVs. However, in protein analysis, these differences are not confirmed, indicating that the immune system is able to detect the virus, or even the damage caused by the infection, but controls the exacerbated protein production. Under stimulation of the TLR (Toll-like receptor) agonist, CMNs do not change among the RNs. In addition, by evaluating the levels of &#223-chemokines, CCL5 and CCL3, and of IFN-&#945 in the supernatants of CMNs cultures, LPS activation was able to decrease the production of CCL3 and CCL5 in CMNs of HIV-infected mothers compared to control mothers, nevertheless CL097 promoted similar levels between HIV-infected mothers and control group. In RNs, while CCL5 levels are lower than in adults, CCL3 levels are similar. TLR7/8 agonist was able to restore IFN- secretion in the HIV-infected group. In contrast, the TLR7/8 agonist was able to restore IFN- secretion in HIV-infected group. In addition, in placental villi, there is intense modification in the mRNA expression of the analyzed factors, whether they are antiviral, such as IFN type I (IFN-&#945), type III (IFN-&#955), related to cell damage (DAMPs and their receptors). Among DAMPs, increased expression of S100A9 and HMGB1 and their receptors RAGE, TLR4 and TLR9 was observed in placental villi of HIV-infected mothers, however, serum HMGB1 levels are decreased in infected-mothers and exposed-newborns. About the cytokines serum levels, reduced levels of HMGB1, IL-6 and IL-1&#223 were observed in the exposed-NBs, which evidences an inflammatory status control in HIV-1 exposure. We also observed the presence of free HERV-W or exosomes levels in serum in both groups analyzed. In colostrum, we did not find differences in inflammatory factors and HERVs analysis indicating that, in this compartment, the infection does not alter the expression patterns of these targets. The effectiveness of antiviral status and suppression of the inflammatory environment can balance the placental immune response, promoting homeostasis for fetal development and protection of HIV-1 infection in neonates.