In this thesis I evaluate the impact of an experiment with free sales of strong beer in two Swedish counties that took place in the 1960s. I do this by studying adult earnings of persons in utero during the experiment. My data includes date and place of birth and allows me to evaluate the impact of the experiment using a difference-in differences methodology, comparing earnings across cohorts and counties. Since the availability of alcohol increased most heavily for persons under the age of 21, and male fetuses are less physiologically robust than female fetuses, I choose to study persons born by mothers younger than 21 separately and also estimate the impact of the experiment separately for men and women. I find that persons born by mothers under the age of 21 during the experiment have lower average earnings than persons born before the experiment, and that the impact is larger on men. My results indicate that the experiment has led to adverse effects on adult earnings, probably caused by the prenatal alcohol exposure’s negative impact on fetal development. This means that alcohol consumption have long-term consequences that represent large costs to society. Since these costs are generally disregarded when evaluating the cost of alcohol consumption, society’s cost of alcohol is probably higher than usually estimated.

A Cannabis sativa, genericamente conhecida por maconha é uma das drogas ilícitas mais utilizadas no mundo. Por ser considerado inofensiva em alguns países seu uso recreativo é liberado, o que tem aumentado em grandes proporções do uso desta substância, incluindo mulheres gestantes. A gestação é um período em que ocorrem alterações dinâmicas fisiológicas consideráveis na mãe, placenta e feto e com isso, os processos de absorção, distribuição e eliminação de drogas utilizadas pode ser muito variável. O que torna a utilização de desta droga mais perigosa nesse período. Diversos desfechos gestacionais negativos estão associados a seu uso, porém pouco se sabe sobre os mecanismos envolvidos nesta associação. Assim, neste estudo avaliamos experimentalmente utilizando um modelo animal, se os desfechos gestacionais negativos podem estar associados a alterações placentárias. Resumidamente, fêmeas gestantes de camundongos foram expostas a fumaça proveniente da queima da Cannabis diariamente durante a gestação via inalação, mimetizando o uso por humanos. No 18,5° dia da gestação (a termo) foram eutanasiadas e as placentas coletadas apara avaliação estereológica. Os resultados mostram que a o consumo da cannabis em baixa dose pode provocar alterações na estrutura da placenta. Há um aumento significativo em seu volume total decorrente do aumento no volume do labirinto, além disto o volume de capilares fetais está aumentados e a barreira inter-hemal fica mais fina. Estas alterações apesar de sugerirem um mecanismo compensatório, o crescimento fetal normal não é mantido / Cannabis sativa, commonly known as marijuana or pot, is one of the most used ilicit drug in the world. In some countries its recreational use is legal because this drug is not considered harmful to health. Legalization of its use has increased the number of users of this drug including pregnant women. Gestation is a period of physiological dynamic changes in the mother, fetus and placenta, thus the process of absorption, metabolism and excretion is different. Association between negative gestational and fetal outcomes and gestational exposure to marijuana is described in the medical literature, however little is known about the mechanisms involved. Taking this into account, in this study we investigated experimentally, using in a animal model that mimics human use, if the negative gestational outcomes could be associated to placental structural and functional changes. Briefly, pregnant mice were exposed during gestation to either filtered or marijuana smoke by inhalation and at gestational Day 18 (at term) they were euthanized and placentas collected for stereological analysis. Our preliminary results indicate that the use of cannabis sativa during pregnancy is associated to structural changes in the placenta. Placental total volume is increased and is due to increases in the volume of the labyrinth. In the labyrinth the volume of fetal capillaries and their surface area are increased and the interhemal membrane thickness decreased. These changes suggest a compensatory mechanism however, they fail to support the fetal normal growth.

INTRODUÇÃO: O desenvolvimento das glândulas salivares envolve um processo coordenado de interações moleculares complexas, nas quais as integrinas têm papel fundamental. As integrinas são uma família de receptores transmembrânicos, heterodímeros, compostos por duas subunidades: alfa e beta que estão ligadas de forma não covalente e dependentes de cations bivalentes. Estes heterodímeros medeiam sinais intra e extracelulares envolvidos na organização das células em tecidos e órgãos durante seu desenvolvimento. Em particular a integrina beta1 está envolvida na proliferação e diferenciação de células no desenvolvimento dos tecidos epiteliais. OBJETIVOS: Para compreender o papel da integrina beta1 no desenvolvimento das glândulas salivares humanas, estudamos a sua expressão por meio da hibridização in situ em tecido fetal humano em desenvolvimento. MATERIAL E MÉTODOS: A hibridização in situ com o método chromogenic in situ hibridization (CISH) foi utilizada para investigar a expressão genética da integrina beta1 em espécimes de glândulas salivares em desenvolvimento derivados de 30 fetos humanos em vários estágios gestacionais (de 825 semanas de vida intrauterina). Os resultados obtidos com a hibridização in situ foram relacionados com os aspectos morfológicos das glândulas salivares em desenvolvimento em cada fase da evolução da morfogênese glandular, por meio de análise qualitativa. RESULTADOS: Expressão da integrina beta1 foi detectada em raras células na fase prebotão. A expressão dessa molécula foi detectada em mais estruturas com a evolução do desenvolvimento morfogenético das glândulas salivares. CONCLUSÕES: A expressão a integrina beta1 parece ser regulada de forma temporoespacial e é associada ao estabelecimento do fenótipo maduro das glândulas salivares / INTRODUCTION: Salivary gland development entails coordinated processes involving complex molecular interactions in which integrins have a fundamental role. The integrins are a family of heterodimeric transmembrane receptors comprising alpha and beta subunits that mediate intercellular and extracellular signals involved in the organisation of cells in tissues and organs during development. The beta1 integrin in particular have been implicated in proliferation and differentiation of cells involved in the development of epithelial tissues. OBJECTIVE: To understand the role of beta1 integrin in salivary gland development we have studied its expression in human foetal tissues. MATERIAL AND METHODS: In situ hybridisation CISH technique was used to compare the expression and localisation of integrin beta1 with differentiation markers in developing human salivary glands obtained from foetuses of 825 gestational weeks. RESULTS: Integrin beta1 first appeared during bud stage in a few cells and its distribution increased as salivary gland morphogenesis progressed. CONCLUSIONS: The developmentally regulated expression of integrin beta1 in association with the establishment of a mature phenotype of salivary glands is suggestive of its role in salivary gland morphogenesis

Conexinas são proteínas que compõem as junções comunicantes do tipo gap, e a diminuição na sua expressão tem sido relacionada com diversas alterações fisiológicas, entre elas algumas síndromes, malformações genéticas, o aumento da proliferação celular e a carcinogênese. Dentre as isoformas das conexinas presentes nos tecidos animais, a Cx43 é a mais abundante e a mais estudada, tendo a sua importância relatada in vivo em camundongos que tiveram um dos alelos de Cx43 deletado (Cx43+/-), devido a morte desses animais logo após o nascimento devido a malformações cardíacas. Considerando o fato de esse gene ser expresso em dezenas de tipos celulares, tivemos como objetivo avaliar os outros tecidos em busca de anomalias ocorridas durante o desenvolvimento, e a possível interferência do background gentético. Para isso acompanhamos dia-a-dia o último terço gestacional de camundongos de background C57BL/6 e CD1, avaliando histologica e morfologicamente os fetos em busca de anomalias nos animais Cx43+/- e Cx43-/- em relação aos animais Cx43+/+. Exceto pelo tecido ósseo, não encontramos alterações nos órgãos que expressam esse gene, bem como alterações causadas pelo refluxo de sangue causado pela malformação da válvula tricúspide. Durante a osteogênese, por meio da avaliação das costelas e tíbia, percebemos um retardo no desenvolvimento, que se agrava conforme a deficiência do gene Cx43. Percebemos nitidamente que o processo de diferenciação celular ocorre de maneira menos eficiente, atrasando processos como deposição de colágeno e de matriz óssea. Conclui-se que a Cx43 é importante para o desenvolvimento ósseo na fase fetal em camundongos. / Connexins are proteins that compose the gap junctions, and the reduction in its expression has been related with diverse physiological alterations, like some syndromes, malformations, the increase of the cellular proliferation and carcinogenesis. Among isoforms of the connexins in animal cells, the Cx43 is the most abundant and studied, having its importance been shown up in alive mice that had one allele of Cx43 (Cx43+/-) deleted. REAUME et al. related that Cx43-/- mice presented cardiac malformation and died immediately after birth. Considering the fact that this gene is expressed in many cell types, we evaluate the possibility of other tissues also to present alterations during the fetal development. Due to this, we studied the mouse development initiating in 12.5 to 19.5 DE (embryologic day) and evaluated the histology of C57BL/6 and CD1 mice searching for anomalies of Cx43+/- and Cx43-/- mice in relation to the Cx43+/+ animals. We did not find alterations in the main organs that express Cx43, nor alterations due to blood out flow related to cardiac malformations. We only found significant difference was the bones; through the evaluation of the ribs and tibia. It has been observed a delay in the development, that was more important in Cx43 knockout mice. We observed clearly that the process of cellular differentiation occurs in less efficient way, delaying processes as deposition of collagen and bone matrix. In conclusion, this study showed that Cx43 is important for bone development in mice.

Chan Wai-Hon. / "September 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 137-156). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

A maconha é a droga ilícita mais consumida entre gestantes. Entretanto, os efeitos do uso materno sobre a gestação e o desenvolvimento fetal não são bem esclarecidos. Estudos experimentais e epidemiológicos apresentam resultados conflitantes devido à via de administração, tempo de exposição, dose e como a toxicidade da Cannabis é testada. Neste estudo experimental foram investigados os efeitos da inalação materna da fumaça de Cannabis sativa, aproximando as reais condições de uso da droga por humanos. Camundongas gávidas (n=20) foram expostas diariamente durante 5 minutos à fumaça decorrente da queima de maconha ( 0,2 g de Cannabis), ou ao ar filtrado, a partir do 5,5° dia gestacional (DG) ao 17,5° DG. A ingestão de alimentos e o peso materno foram registrados. Análise por ultrassom foram realizadas entre o 10,5° e o 16,5° DG. No 18,5° DG metade das fêmeas foram eutanasiadas para a a avaliação dos fetos a termo, reabsorções e placentas. A duração da gestação, desfechos neonatais e comportamentais (reflexo, força muscular, ansiedade e memória) em neonatos e adultos foram avaliados na outra metade das fêmeas. A exposição diária de 5 min (dose baixa) durante a gestação resultou em redução do peso ao nascer mas o tamanho da prole não foi alterado; No entanto, o número de filhotes machos por prole foi maior. Além disso, o peso líquido da placenta foi aumentado e a proporção feto/placenta foi diminuída em machos, mostrando um efeito específico do sexo. A exposição também alterou a força muscular e reflexos em neonatos, além de causar prejuízos na memória e efeito ansiolítico na prole exposta. Em conclusão, os resultados indicam que o fumo da maconha durante a gestação, mesmo em doses baixas, pode ser embriotóxico, fetotóxico e pode alterar o comportamento em neonatos e adultos / Marijuana is the most used illegal drug among pregnant women. However, the effects of maternal use on pregnacy and fetal development are not well understood. Experimental and epidemiologicals studies have show conflicting results due to the route of administration, duration of exposure, dose, and a toxicity of Cannabis is tested. In this experimental study we investigated the effects of maternal inhalation of smoke Cannbis sativa , approaching the actual drug use conditons for humans. Pregnant mice (n=20) were exposed (only noise) daily for 5 min to smoke resulting from burning marijuana (0.2g Cannabis), or filtered air, from gestational day (GD) 5.5° to GD 17.5°. Food intake and maternal weight were recorded. Ultrasound analysis was performed on 10.5 and 16.5° GD. On 18.5° GD half the mice were euthanized for evaluation of term fetus, resorptions and placenta. The duration of pregnancy , neonatal outcomes and behavioral reponses (reflex,muscle strenght, anxiety and memory) were assessed in the other half of the females. The daily exposure of 5 min (low dose) during pregnancy resulted in reduced brth weiht but the size of the offspring was not changed; however, the number of males per offspring pups was higher. In addition, placental weight was increased and fetus/placenta ratio was decreased in males, showing a particular effect of sex.The exposure also altered muscle strength and reflexes in newborns and cause impars in memory and anxiolytic effects in exposed offspring. In conclusion, the results indicate that smoking marijuana during pregnancy, even in low doses, can be embryotoxic, foetotoxic and may change in the behavior in neonates and adults

In this 15 year follow up study in a Chinese population, we confirmed that maternal gestational diabetic status significantly increased women’s future cardiometabolic risk. Glycaemic levels below the current criteria for a positive screening test for gestational diabetes and for the diagnosis of gestational diabetes still significantly predict women’s future risk. In utero hyperinsulinaemia, which caused by an intrauterine hyperglycaemic environment, was found to predict children’s AGT and adolescents’ overweight and MetS. The results had important implication that the current diagnostic criteria for gestational diabetes may not be discriminative in predicting both the mothers and their children’s future cardiometabolic risk. Although recent research has re-visited and emphasised on the diagnostic criteria of gestational diabetes which best predicted adverse pregnancy outcome, future study should also scrutinise on the optimal glycaemic threshold, either in screening or diagnostic test, that relate to the mothers’ and children offspring’s long term cardiometabolic risk. / Tam, Wing Hung. / Thesis (M.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 119-146). / Abstract also in Chinese. / LIST OF TABLES --- p.xxii / LIST OF FIGURES --- p.xxv / LIST OF ABBREVIATIONS --- p.xxvi / Chapter Chapter 1 --- Gestational diabetes & future cardiometabolic risk - an overview / Chapter 1.1 --- Historical background --- p.2 / Chapter 1.2 --- Pregnancy physiology vs. gestational diabetes --- p.5 / Chapter 1.3 --- Diabetes mellitus - a global epidemic --- p.6 / Chapter 1.4 --- History of gestational diabetes & progression to Type 2 DM --- p.7 / Chapter 1.5 --- History of gestational diabetes & cardiometabolic risk --- p.8 / Chapter 1.6 --- Type 2 DM among children and adolescents --- p.9 / Chapter 1.7 --- Type 2 DM among offspring of mothers with gestational diabetes --- p.10 / Chapter 1.8 --- Cardiometabolic risk in children exposed to maternal gestational diabetes --- p.12 / Chapter 1.9 --- Long term follow up on mothers & children cohort --- p.12 / Chapter Chapter 2 --- Research methodology / Chapter 2.1 --- Subjects --- p.16 / Chapter 2.2 --- Obstetric and neonatal information --- p.18 / Chapter 2.2.1 --- Maternal glycaemic indices at pregnancy --- p.18 / Chapter 2.2.2 --- Umbilical cord blood C-peptide & insulin levels --- p.18 / Chapter 2.2.3 --- Definition of antenatal variables --- p.19 / Chapter 2.3 --- Follow up assessment of the mothers --- p.19 / Chapter 2.4 --- Follow up assessment of the children and adolescents --- p.22 / Chapter 2.5 --- Definition of abnormal glucose tolerance and metabolic syndrome --- p.24 / Chapter 2.5.1 --- Definition of abnormal glucose tolerance --- p.24 / Chapter 2.5.2 --- Definition of metabolic syndrome in adult --- p.24 / Chapter 2.5.3 --- Definition of metabolic syndrome in adolescent --- p.25 / Chapter 2.6 --- Determination of insulin resistance and pancreatic beta cell function --- p.26 / Chapter 2.6.1 --- Definition of insulin resistance --- p.26 / Chapter 2.6.2 --- Definition of pancreatic beta cell function --- p.26 / Chapter 2.6.3 --- Measurement of insulin resistance and pancreatic β-cell function --- p.27 / Chapter 2.7 --- Statistical analysis --- p.31 / Chapter 2.7.1 --- Statistical programme --- p.31 / Chapter 2.7.2 --- Comparison between group differences --- p.31 / Chapter 2.7.3 --- General Linear Model --- p.32 / Chapter 2.7.4 --- Multivariate logistic regression --- p.33 / Chapter 2.7.5 --- Receiver operating characteristic analysis --- p.37 / Chapter 2.8 --- Ethics approval --- p.41 / Chapter 2.9 --- Funding --- p.42 / Chapter Chapter 3 --- History of gestational diabetes and women’s future cardiometabolic risk / Chapter 3.1 --- Maternal clinical parameters at the index pregnancy --- p.44 / Chapter 3.2 --- Maternal cardiometabolic status at 8 years post-delivery --- p.45 / Chapter 3.3 --- Maternal cardiometabolic status at 15 years post-delivery --- p.49 / Chapter 3.4 --- Prediction of cardiometabolic risk by maternal gestational diabetic status --- p.50 / Chapter 3.4.1 --- Abnormal glucose tolerance and metabolic syndrome at 8 years by maternal gestational diabetic status --- p.52 / Chapter 3.4.2 --- Abnormal glucose tolerance, DM, hypertension and metabolic syndrome at 15 years by maternal gestational diabetic status --- p.52 / Chapter 3.5 --- The role of insulin resistance in predicting women’s DM and metabolic syndrome --- p.55 / Chapter 3.6 --- Discussion --- p.57 / Chapter 3.7 --- Conclusion --- p.62 / Chapter Chapter 4 --- Glycaemic variables measured at mid-gestation of the index pregnancy predict women’s future cardiometabolic risk / Chapter 4.1 --- Glycaemic levels in pregnancy and perinatal outcome --- p.64 / Chapter 4.2 --- Glycaemic levels in pregnancy and women’s future cardiometabolic risk --- p.65 / Chapter 4.2.1 --- Prediction of women’s cardiometabolic risk at 8 and 15-year --- p.66 / Chapter 4.2.2 --- Optimal cut-off levels in predicting women’s future cardio- metabolic risk --- p.69 / Chapter 4.3 --- Discussion --- p.75 / Chapter 4.4 --- Conclusion --- p.78 / Chapter Chapter 5 --- Maternal gestational diabetes and offspring’s cardiometabolic risk / Chapter 5.1 --- Offspring’s cardiometabolic risk at 8 years age --- p.80 / Chapter 5.1.1 --- Baseline characteristics at pregnancy and delivery --- p.80 / Chapter 5.1.2 --- Children’s clinical and biochemical parameters at 8 years age --- p.82 / Chapter 5.2 --- Offspring’s cardiometabolic risk at 15 years age --- p.84 / Chapter 5.2.1 --- Adolescents’ clinical and biochemical parameters at 15 years age --- p.84 / Chapter 5.2.2 --- Clinical parameters of adolescents with abnormal glucose tolerance --- p.84 / Chapter 5.3 --- Discussion --- p.88 / Chapter 5.4 --- Conclusion --- p.90 / Chapter Chapter 6 --- In utero hyperinsulinaemia and offspring’s cardiometabolic risk / Chapter 6.1 --- Umbilical cord blood insulin and C-peptide --- p.92 / Chapter 6.1.1 --- Umbilical cord insulin and C-peptide concentrations in the original cohort --- p.92 / Chapter 6.1.2 --- Determination of in utero hyperinsulinaemia by umbilical cord insulin and C-peptide levels --- p.95 / Chapter 6.2 --- The effect of in utero hyperinsulinaemia on children’s abnormal glucose tolerance at 8 years of age --- p.98 / Chapter 6.2.1 --- Receiver operating characteristic analysis --- p.98 / Chapter 6.2.2 --- Logistic regression analysis --- p.98 / Chapter 6.3 --- The effect of in utero hyperinsulinaemia on adolescents’ cardio- metabolic risk at 15years of age --- p.102 / Chapter 6.3.1 --- Logistic regression analysis --- p.102 / Chapter 6.4 --- Discussion --- p.105 / Chapter 6.5 --- Conclusion --- p.108 / Chapter Chapter 7 --- Summary and conclusion / Chapter 7.1 --- Summary of the thesis --- p.110 / Chapter 7.1.1 --- Women’s long term cardiometabolic risk after a pregnancy with gestational diabetes --- p.110 / Chapter 7.1.2 --- The long term cardiometabolic risk of children born to mothers who had gestational diabetes --- p.111 / Chapter 7.1.3 --- New findings from the studies and their implications --- p.111 / Chapter 7.2 --- Strength and weakness in the study --- p.113 / Chapter 7.2.1 --- Unique cohort from universal screening --- p.113 / Chapter 7.2.2 --- Study design --- p.113 / Chapter 7.2.3 --- Response rate and loss to follow up --- p.114 / Chapter 7.2.4 --- Treatment effect of gestational diabetes --- p.115 / Chapter 7.3 --- Issues of future research --- p.115 / Chapter 7.3.1 --- Follow up study on the HAPO cohort --- p.115 / Chapter 7.3.2 --- Opportunity for international collaboration --- p.117 / Chapter 7.4 --- Conclusion --- p.118 / REFERENCES --- p.119

INTRODUÇÃO: O desenvolvimento das glândulas salivares envolve um processo coordenado de interações moleculares complexas, nas quais as integrinas têm papel fundamental. As integrinas são uma família de receptores transmembrânicos, heterodímeros, compostos por duas subunidades: alfa e beta que estão ligadas de forma não covalente e dependentes de cations bivalentes. Estes heterodímeros medeiam sinais intra e extracelulares envolvidos na organização das células em tecidos e órgãos durante seu desenvolvimento. Em particular a integrina beta1 está envolvida na proliferação e diferenciação de células no desenvolvimento dos tecidos epiteliais. OBJETIVOS: Para compreender o papel da integrina beta1 no desenvolvimento das glândulas salivares humanas, estudamos a sua expressão por meio da hibridização in situ em tecido fetal humano em desenvolvimento. MATERIAL E MÉTODOS: A hibridização in situ com o método chromogenic in situ hibridization (CISH) foi utilizada para investigar a expressão genética da integrina beta1 em espécimes de glândulas salivares em desenvolvimento derivados de 30 fetos humanos em vários estágios gestacionais (de 825 semanas de vida intrauterina). Os resultados obtidos com a hibridização in situ foram relacionados com os aspectos morfológicos das glândulas salivares em desenvolvimento em cada fase da evolução da morfogênese glandular, por meio de análise qualitativa. RESULTADOS: Expressão da integrina beta1 foi detectada em raras células na fase prebotão. A expressão dessa molécula foi detectada em mais estruturas com a evolução do desenvolvimento morfogenético das glândulas salivares. CONCLUSÕES: A expressão a integrina beta1 parece ser regulada de forma temporoespacial e é associada ao estabelecimento do fenótipo maduro das glândulas salivares / INTRODUCTION: Salivary gland development entails coordinated processes involving complex molecular interactions in which integrins have a fundamental role. The integrins are a family of heterodimeric transmembrane receptors comprising alpha and beta subunits that mediate intercellular and extracellular signals involved in the organisation of cells in tissues and organs during development. The beta1 integrin in particular have been implicated in proliferation and differentiation of cells involved in the development of epithelial tissues. OBJECTIVE: To understand the role of beta1 integrin in salivary gland development we have studied its expression in human foetal tissues. MATERIAL AND METHODS: In situ hybridisation CISH technique was used to compare the expression and localisation of integrin beta1 with differentiation markers in developing human salivary glands obtained from foetuses of 825 gestational weeks. RESULTS: Integrin beta1 first appeared during bud stage in a few cells and its distribution increased as salivary gland morphogenesis progressed. CONCLUSIONS: The developmentally regulated expression of integrin beta1 in association with the establishment of a mature phenotype of salivary glands is suggestive of its role in salivary gland morphogenesis

Conexinas são proteínas que compõem as junções comunicantes do tipo gap, e a diminuição na sua expressão tem sido relacionada com diversas alterações fisiológicas, entre elas algumas síndromes, malformações genéticas, o aumento da proliferação celular e a carcinogênese. Dentre as isoformas das conexinas presentes nos tecidos animais, a Cx43 é a mais abundante e a mais estudada, tendo a sua importância relatada in vivo em camundongos que tiveram um dos alelos de Cx43 deletado (Cx43+/-), devido a morte desses animais logo após o nascimento devido a malformações cardíacas. Considerando o fato de esse gene ser expresso em dezenas de tipos celulares, tivemos como objetivo avaliar os outros tecidos em busca de anomalias ocorridas durante o desenvolvimento, e a possível interferência do background gentético. Para isso acompanhamos dia-a-dia o último terço gestacional de camundongos de background C57BL/6 e CD1, avaliando histologica e morfologicamente os fetos em busca de anomalias nos animais Cx43+/- e Cx43-/- em relação aos animais Cx43+/+. Exceto pelo tecido ósseo, não encontramos alterações nos órgãos que expressam esse gene, bem como alterações causadas pelo refluxo de sangue causado pela malformação da válvula tricúspide. Durante a osteogênese, por meio da avaliação das costelas e tíbia, percebemos um retardo no desenvolvimento, que se agrava conforme a deficiência do gene Cx43. Percebemos nitidamente que o processo de diferenciação celular ocorre de maneira menos eficiente, atrasando processos como deposição de colágeno e de matriz óssea. Conclui-se que a Cx43 é importante para o desenvolvimento ósseo na fase fetal em camundongos. / Connexins are proteins that compose the gap junctions, and the reduction in its expression has been related with diverse physiological alterations, like some syndromes, malformations, the increase of the cellular proliferation and carcinogenesis. Among isoforms of the connexins in animal cells, the Cx43 is the most abundant and studied, having its importance been shown up in alive mice that had one allele of Cx43 (Cx43+/-) deleted. REAUME et al. related that Cx43-/- mice presented cardiac malformation and died immediately after birth. Considering the fact that this gene is expressed in many cell types, we evaluate the possibility of other tissues also to present alterations during the fetal development. Due to this, we studied the mouse development initiating in 12.5 to 19.5 DE (embryologic day) and evaluated the histology of C57BL/6 and CD1 mice searching for anomalies of Cx43+/- and Cx43-/- mice in relation to the Cx43+/+ animals. We did not find alterations in the main organs that express Cx43, nor alterations due to blood out flow related to cardiac malformations. We only found significant difference was the bones; through the evaluation of the ribs and tibia. It has been observed a delay in the development, that was more important in Cx43 knockout mice. We observed clearly that the process of cellular differentiation occurs in less efficient way, delaying processes as deposition of collagen and bone matrix. In conclusion, this study showed that Cx43 is important for bone development in mice.

Pang, Weng I. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 116-148). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / PUBLICATIONS --- p.vii / TABLE OF CONTENTS --- p.viii / LIST OF TABLES --- p.xiii / LIST OF FIGURES --- p.xv / LIST OF ABBREVIATIONS --- p.xvi / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- CIRCULATING NUCLEIC ACIDS IN PRENATAL DIAGNOSIS --- p.2 / Chapter 1.1 --- Prenatal diagnosis --- p.2 / Chapter 1.2 --- Circulating fetal DNA in maternal plasma --- p.2 / Chapter 1.2.1 --- Biology of circulating fetal DNA --- p.2 / Chapter 1.2.2 --- Clinical applications of circulating fetal DNA --- p.3 / Chapter 1.2.2.1 --- Qualitative fetal-specific sequence detection --- p.4 / Chapter 1.2.2.2 --- Quantitative aberration detection --- p.4 / Chapter 1.2.3 --- Circulating fetal epigenetic markers --- p.5 / Chapter 1.3 --- Circulating fetal RNA in maternal plasma --- p.6 / Chapter 1.3.1 --- Biology of circulating fetal RNA --- p.6 / Chapter 1.3.2 --- Clinical applications of circulating fetal RNA --- p.8 / Chapter 1.3.2.1 --- Quantitative aberration detection --- p.8 / Chapter 1.3.2.2 --- Chromosomal aneuploidy detection --- p.9 / Chapter 1.3.3 --- Enrichment of fetal RNA --- p.10 / Chapter 1.4 --- Circulating microRNA in maternal plasma --- p.10 / Chapter CHAPTER 2: --- FETAL GROWTH AND WELL-BEING --- p.12 / Chapter 2.1 --- Normal fetal growth --- p.12 / Chapter 2.1.1 --- Role of the mother --- p.12 / Chapter 2.1.2 --- Role of the placenta --- p.12 / Chapter 2.1.3 --- Role of the fetus --- p.13 / Chapter 2.1.4 --- Role of the somatotrophic axis --- p.15 / Chapter 2.2 --- Abnormal fetal growth --- p.15 / Chapter 2.2.1 --- Intrauterine growth restriction --- p.16 / Chapter 2.1.2 --- Definition of IUGR --- p.16 / Chapter 2.2.3 --- Risk factors of IUGR --- p.17 / Chapter 2.2.4 --- Diagnosis of IUGR --- p.20 / Chapter 2.2.4.1 --- Biometric tests --- p.20 / Chapter 2.2.4.2 --- Biophysical tests --- p.21 / Chapter 2.2.4.3 --- Biochemical tests --- p.22 / Chapter 2.2.4.4 --- Others --- p.22 / Chapter 2.3 --- Limitations of current modalities in fetal growth assessment --- p.23 / Chapter 2.4 --- Aims of this thesis --- p.24 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.26 / Chapter CHAPTER 3: --- QUANTITATIVE ANALYSIS OF CIRCULATING RNA --- p.27 / Chapter 3.1 --- Sample collection and processing --- p.27 / Chapter 3.1.1 --- Preparation of plasm a --- p.27 / Chapter 3.1.2 --- Preparation of blood cells --- p.27 / Chapter 3.1.3 --- Preparation of placental tissues --- p.27 / Chapter 3.2 --- Total RNA extraction --- p.28 / Chapter 3.2.1 --- Plasma and blood cells --- p.28 / Chapter 3.2.2 --- Placental tissues --- p.32 / Chapter 3.3 --- Quantitative measurements of nucleic acids --- p.32 / Chapter 3.3.1 --- Principles of real-time quantitative PCR --- p.33 / Chapter 3.3.2 --- One-step QR T-PCR assays for placental mRNA quantification --- p.3 7 / Chapter 3.3.3 --- QPCR assays for checking genomic DNA contamination --- p.43 / Chapter 3.4 --- Statistical analysis --- p.45 / Chapter SECTION III: --- EVALUATION OF PLACENTA-DERIVED MRNA AS POSSIBLE MARKERS FOR FETAL GROWTH ASSESSMENT --- p.46 / Chapter CHAPTER 4: --- SELECTION OF POTENTIAL FETAL GROWTH MRNA MARKERS FOR MATERNAL PLASMA DETECTION --- p.47 / Chapter 4.1 --- Introduction --- p.47 / Chapter 4.2 --- Materials and methods --- p.49 / Chapter 4.2.1 --- Sample collection and processing --- p.49 / Chapter 4.2.2 --- Experimental design --- p.49 / Chapter 4.2.3 --- RNA extraction and quantification --- p.51 / Chapter 4.2.4 --- Statistical analysis --- p.51 / Chapter 4.3 --- Results --- p.52 / Chapter 4.3.1 --- Identification of potential fetal growth mRNA markers in maternal plasma --- p.52 / Chapter 4.3.2 --- Development of real-time QR T-PCR assays --- p.56 / Chapter 4.3.3 --- Validation of maternal plasma detectability and pregnancy-specificity --- p.58 / Chapter 4.3.4 --- Assessment of the gestational trend in maternal plasma --- p.64 / Chapter 4.4 --- Discussion --- p.68 / Chapter CHAPTER 5: --- RELATIONSHIP BETWEEN CIRCULATING PLACENTAL MRNA AND FETAL GROWTH --- p.72 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Materials and methods --- p.73 / Chapter 5.2.1 --- Sample collection and processing --- p.73 / Chapter 5.2.2 --- "Ultrasound measurement, placental weight and birth weight.…" --- p.74 / Chapter 5.2.3 --- Experimental design --- p.74 / Chapter 5.2.4 --- RNA extraction and quantification --- p.75 / Chapter 5.2.5 --- Statistical analysis --- p.75 / Chapter 5.3 --- Results --- p.75 / Chapter 5.3.1 --- Expression of potential growth markers in placental tissues --- p.76 / Chapter 5.3.2 --- Relationship between circulating placental mRNA and birth measurements --- p.76 / Chapter 5.3.3 --- Relationship between circulating placental mRNA and fetal biometric measurements --- p.77 / Chapter 5.4 --- Discussion --- p.85 / Chapter SECTION IV: --- CLINICAL APPLICATION OF POTENTIAL FETAL GROWTH MARKERS IN THE ASSESSMENT OF IUGR --- p.93 / Chapter CHAPTER 6: --- QUANTITATIVE ANALYSIS OF PLACENTAL MRNA IN IUGR WITH OR WITHOUT PET --- p.94 / Chapter 6.1 --- Introduction --- p.94 / Chapter 6.2 --- Materials and methods --- p.95 / Chapter 6.2.1 --- Sample collection and processing --- p.95 / Chapter 6.2.2 --- Experimental design --- p.96 / Chapter 6.2.3 --- RNA extraction and quantification --- p.96 / Chapter 6.2.4 --- Statistical analysis --- p.97 / Chapter 6.3 --- Results --- p.97 / Chapter 6.3.1 --- Cross-sectional comparison of placental mRNA concentrations --- p.97 / Chapter 6.3.2 --- Longitudinal comparison of placental mRNA concentrations --- p.102 / Chapter 6.4 --- Discussion --- p.103 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.107 / Chapter CHAPTER 7: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.108 / Chapter 7.1 --- A strategy for identifying circulating placental MRNA markers for fetal growth assessment --- p.108 / Chapter 7.2 --- Implications of mRNA marker development strategy --- p.111 / Chapter 7.3 --- Prospects for future work --- p.112 / REFERENCES --- p.116