Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A Atresia de esÃfago (AE) à uma anomalia estrutural que acontece em fetos quando nÃo ocorre a septaÃÃo completa do septo esofagotraqueal. Estudou-se a repercussÃo da AE induzida por Doxorrubicina (Doxo) na contratilidade in vitro do esÃfago distal e fundo de estÃmago. 26 Ratas wistar (267 g), com acasalamento controlado, nos dias 8 e 9 de gestaÃÃo receberam 2,2 mg/Kg de Doxo intraperitonealmente, enquanto 13 ratas controle receberam o mesmo volume de NaCl 0,9%. No dia 21,5 as ratas foram submetidas a cesariana, anÃlise dos fetos para confirmaÃÃo da AE e divisÃo em 3 grupos: controle, cujas mÃes receberam apenas NaCl 0,9%; Doxo sem AE, cujas mÃes receberam Doxo, mas nÃo desenvolveram AE; e Doxo com AE, os que desenvolveram AE. Foram montados em sistema para banho de ÃrgÃo isolado os anÃis de fundo de estÃmago dos fetos e em sistema miÃgrafo de agulha os segmentos de esÃfago distal, ambos contendo soluÃÃo fisiolÃgica Tyrode a 37 ÂC, pH 7,4 e oxigenaÃÃo constante, tensÃo basal de 1 g para estÃmago e 8 mN para esÃfago. Realizou-se curva concentraÃÃo-efeito ao agonista colinÃrgico Carbacol (CCh) (0,01 â 300 &#956;M) em ambos os tecidos nos 3 grupos. Em seguida, agora apenas nos segmentos de esÃfago isolado, foi construÃda uma curva concentraÃÃo-efeito ao KCl (10 â 100 mM), em que a contraÃÃo se deve prioritariamente à entrada de cÃlcio do meio extracelular por meio de canais operados por voltagem (VOC). A anÃlise estatÃstica foi determinada utilizando two-way anÃlise de variÃncia (ANOVA) e a significÃncia foi testada pelo Student-Newman-Keuls test. No fundo de estÃmago nÃo houve diferenÃa estatÃstica entre os grupos na resposta contrÃtil ao CCh (p>0,05, ANOVA), os valores da CE50 dos animais controle foram 2,17 [1,03 â 4,58] &#956;M e Emax 0,084  0,016 g/mg de tecido (n=7); Doxo sem AE 1,47 [0,83 â 2,61] &#956;M e 0,068  0,006 g/mg de tecido (n=12); Doxo com AE 3,26 [1,90 â 5,60] &#956;M e 0,070  0,022 g/mg de tecido (n=6). No esÃfago, animais controle com Emax 5,97  0,58 mN (n=11) foram estatisticamente diferentes (p<0,05, ANOVA) dos grupos Doxo sem AE 4,48  0,34 mN (n=11) e Doxo com AE 4,42  0,68 mN (n=8), enquanto a CE50 nÃo apresentou diferenÃa estatÃstica significativa entre os grupos (p>0,05, ANOVA) controle 190 [96 â 379] nM, Doxo sem AE 228 [125 â 418] nM e Doxo com AE 439 [206 â 936] nM, quanto a resposta contrÃtil ao CCh. Na resposta ao KCl houve incremento de tensÃo inferior ao observado com CCh sem diferenÃa entre os trÃs grupos (p>0,05, ANOVA), 8 valores de Emax foram no controle 1,31  0,14 mN (n=5), Doxo sem AE 1,27  0,42 mN (n=7) e Doxo com AE 1,21  0,20 mN (n=7). Concluiu-se que o tratamento de ratas com Doxo durante o perÃodo gestacional leva a uma diminuiÃÃo da contratilidade de esÃfago isolado de seus fetos, independente do desenvolvimento de AE. Aparentemente, essa diminuiÃÃo nÃo se deve a uma menor funcionalidade dos canais VOC. O fundo de estÃmago isolado nÃo apresentou alteraÃÃes da resposta contrÃtil. / Esophageal atresia (EA) is a structural anomaly that results from an incomplete esophago-traqueal septation in the fetus during intrauterine development. The in vitro contractility of the distal esophagus and gastric fundus of fetuses with esophageal atresia induced by Doxorubicin (Doxo) was studied. 26 Female Wistar rats (267 g), were subjected to date-controlled mating, subsequently receiving 2.2 mg/kg Doxo intraperitoneally on days 8 and 9 of pregnancy, while a controlled group of 13 rats received the same volume of 0.9% NaCl. On day 21.5 the pregnant rats were submitted to a cesarean surgery, with the fetuses analysed to confirm EA and thereafter divided into 3 groups: control, whose mothers received only 0.9% NaCl; Doxo without EA, whose mothers received Doxo but not developed EA; Doxo with EA, who developed EA. After being sacrificed, ring-strips of the gastric fundus were obtained from the fetuses and mounted in isolated organ bath, while the distal esophageal strips were mounted in wire myograph system; both strips contained a standard Tyrode solution maintained at 37 ÂC, pH 7.4, in addition to constant oxygenation and a basal tension of 1 g for the fundic strips and 8 mN for the esophagus. For each set up, we carried out a cholinergic-agonist concentration- effect curve with Carbachol (CCh) (0.01 â 300 &#956;M) in both tissue in the three groups. The participation of voltage-operated channels (VOCs) was studied; a KCl- concentration-effect curve (10 â 100 mM) was conducted on isolated esophageal strips. Collected data was subjected to two-way analysis of variance (ANOVA) and the significance was tested using Student-Newman-Keuls test. There was not significant statistical difference in fundic stripsâ contractility in response to CCh (p>0.05, ANOVA), the EC50 values of the control animals were 2.17 [1.03 â 4.58] &#956;M and Emax 0.084  0.016 g/mg tissue (n=7); Doxo without EA 1.47 [0.83 â 2.61] &#956;M and 0.068  0.006 g/mg tissue (n=12); Doxo with EA 3.26 [1.90 â 5.60] &#956;M and 0.070  0.022 g/mg tissue (n=6). However, significant statistical difference was noted (p<0.05, ANOVA), in esophageal stripsâ contractility in response to CCh in the Emax value of control 5.97  0.58 mN (n=11), vs Doxo without EA 4.48  0.34 mN (n=11) and Doxo with EA 4.42  0.68 mN (n=8), while there was not significant statistical difference (p>0.05, ANOVA) in the EC50 value of control 190 [96 â 379] nM, Doxo without EA 228 [125 â 418] nM and Doxo with EA 439 [206 â 936] nM. Tensional response to KCl were present in all groups, though lower than that seen in response &#65532;to CCh, however not statistically different when comparing all the three groups (p>0.05, ANOVA), Emax of control was 1.31  0.14 mN (n=5), Doxo without EA 1.27  0.42 mN (n=7) and Doxo with EA 1.21  0.20 mN (n=7). It is possible to conclude that the treatment of rats with Doxo during pregnancy leads to decrease contractility of isolated esophagus of their fetuses, independent of the development of EA. Apparently, such a decrease is not due to a lower functionality of VOC channels. The isolated gastric fundus strips showed no change in contractile response.

Thesis (doctoral)--University of Lund, 1981. / Includes bibliographical references.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-05-18T18:52:58Z No. of bitstreams: 2 Dissertação - Lívia Roberta Rodrigues Conceição - 2017.pdf: 1928394 bytes, checksum: 6893367c65e0cfe9d78998113c7466a9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-19T10:40:36Z (GMT) No. of bitstreams: 2 Dissertação - Lívia Roberta Rodrigues Conceição - 2017.pdf: 1928394 bytes, checksum: 6893367c65e0cfe9d78998113c7466a9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-19T10:40:36Z (GMT). No. of bitstreams: 2 Dissertação - Lívia Roberta Rodrigues Conceição - 2017.pdf: 1928394 bytes, checksum: 6893367c65e0cfe9d78998113c7466a9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-04-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Esophageal atresia is a congenital malformation characterized by lack of continuity of the esophagus, which may or may not be associated with communication with the trachea. Newborns with esophageal atresia have a good survival rate in developed countries, but there is still a high mortality rate in developing countries. The objective was to analyze the neonatal and maternal characteristics related to the occurrence of death in newborns with esophageal atresia. This is a descriptive, cross-sectional study with a quantitative approach. The data used are composed of the units' entry books and the charts of the newborn infants with diagnosis of esophageal atresia of a large public hospital of the Unified Health System (SUS) network of the central-western region, obtaining data from 2005 to 2015. Of the 45 newborns (NB) with esophageal atresia participants in the study, more than half died, the maternal age with the highest prevalence was 19 to 35 years, with mothers residing in the interior of Goiás, being mostly primipara, with single-fetus gestation with the resolution of gestation by cesarean section. The newborns presented gestational age <37 weeks and weight <2500g, being mostly males. The neonatal complications associated to the ventilatory system were those that had a higher occurrence among newborns with a diagnosis of esophageal atresia. Of the variables related to esophageal atresia, the most frequent type was esophageal atresia with distal tracheoesophageal fistula and the major association with another malformation was with the malformations of the cardiovascular system. A significant death rate was found, mainly in the post-neonatal period. Of the newborns who died most were premature and underweight at birth. We obtained a statistically significant association with death in hospitalized infants <7 days in the NICU, probably due to the severity of the associated diseases. We also found a statistically significant association with the length of hospital stay, with infants who have more malformations along with esophageal atresia, remaining an extended stay. / A atresia esofágica é uma malformação congênita caracterizada pela ausência de continuidade do esôfago, que pode ser associada ou não à comunicação com a traquéia. Os recém-nascidos que apresentam atresia esofágica têm uma boa taxa de sobrevivência em países desenvolvidos, porém ainda há uma importante taxa de mortalidade nos países em desenvolvimento. Objetivou-se analisar as características neonatais e maternas relacionadas à ocorrência de óbito em recém-nascidos com atresia esofágica. Trata-se de um estudo descritivo, transversal com abordagem quantitativa. Os dados utilizados são compostos pelos cadernos de admissão das unidades e pelos prontuários dos recém-nascidos com diagnóstico de atresia de esôfago de um hospital público de grande porte da rede do Sistema Único de Saúde (SUS) da região centro-oeste, obtendo dados de 2005 a 2015. Dos 45 recém-nascidos (RN) com atresia esofágica participantes do estudo, mais da metade foram a óbito, a idade materna com maior prevalência foi 19 a 35 anos, com mães residindo no interior de Goiás, sendo de maioria prímipara, com gestação de feto único com a resolução da gestação por parto cesáreo. Os RN apresentaram idade gestacional < 37 semanas e peso < 2500g, sendo de maioria do sexo masculino. As intercorrências neonatais associadas ao sistema ventilatório foram as que tiveram maior ocorrência entre os recém-nascidos com diagnóstico de atresia esofágica. Das variáveis relacionadas à atresia de esôfago, o tipo mais frequente foi atresia esofágica com fístula traqueoesofágica distal e a maior associação com outra malformação foi com as malformações do aparelho cardiovascular. Foi encontrada uma taxa relevante de óbito, principalmente no período pós-neonatal. Dos recém-nascidos que foram a óbito a maioria eram prematuros e baixo peso ao nascer. Obtivemos uma associação estatisticamente significativa com óbito em recém-nascidos internados com tempo <7 dias na UTIN, provavelmente pela gravidade das doenças associadas. Também encontramos uma associação estatisticamente significativa com o tempo de internação hospitalar, com os RN que possuem mais malformações juntamente com a atresia esofágica permanecendo um tempo prolongado de internação.

L'œsophage et la trachée proviennent du diverticule endodermique du tube de l'intestin antérieur au cours de l'embryogenèse. Des événements cellulaires et moléculaires bien régulés et organisés entraînent la séparation du tube de l'intestin antérieur en œsophage et trachée. Cette séparation est encore mal connue et la perturbation de ce processus se traduit par une anomalie congénitale sévère telle qu'une l’atrésie de l'œsophage avec ou sans fistule trachéo-œsophagienne (AO/FTO). L'AO/FTO est l'une des malformations congénitales gastro-intestinales les plus courantes affectant 1 naissance sur 3000. Cette malformation nécessite une intervention chirurgicale urgente à la naissance et est fréquemment associée à une morbidité à long terme. Les mécanismes sous-jacents au développement embryonnaire de l'AO/FTO sont mal compris. Les modèles animaux ont été largement utilisés pour comprendre les maladies humaines depuis des décennies et ont considérablement contribué à la compréhension du développement de l'œsophage. Cependant, des différences structurelles et morphologiques clés existent entre l'œsophage humain et animal, ce qui nécessite un modèle plus fiable pour comprendre le développement trachée-œsophagien. Les cellules souches pluripotentes induites par l'homme ont été un outil précieux pour comprendre l'organogenèse en imitant le développement et en déchiffrant les mécanismes qui conduisent à des maladies congénitales et acquises. Cette thèse se concentre donc sur l'utilisation de cellules souches pluripotentes induites (IPS) par des patients pour déchiffrer les mécanismes de signalisation impliqués dans le développement de l'œsophage et les maladies congénitales telles que l’OA/FTO. Il étudie également l'une des maladies œsophagiennes acquises possibles, comme l'œsophage de Barrett. Nous avons orienté la différenciation des IPS saines et dérivées de patients vers différents stades de développement, tels que l'endoderme définitif, l'intestin antérieur, l'épithélium œsophagien et trachéal. De plus, l'épithélium œsophagien a été développé davantage dans un environnement tridimensionnel sans matrice pour générer des organoïdes œsophagiens matures. À chaque étape de la progression du développement, des analyses d'immunofluorescence, de qPCR et de séquençage d'ARN ont été effectuées. Nos résultats suggèrent que l'expression des marqueurs endodermiques CXCR4, SOX17, et GATA4 était similaire dans les cellules différenciées des patients et des cellules saines. Cependant, au stade de l'intestin antérieur, nous avons observé une diminution significative de l'expression des gènes et des protéines du facteur transcriptionnel clé SOX2 dans les cellules dérivées du patient. De plus, en utilisant le séquençage d'ARN à molécule unique, nous avons observé que les gènes critiques GSTM1, et RAB37 impliqués dans la morphogenèse cellulaire et associés à l’OA/FTO étaient dérégulés au stade de l'intestin antérieur dans les cellules dérivées du patient. Nous avons également observé une augmentation significative de l'expression du facteur de transcription NKX2.1 habituellement exprimé uniquement dans les cellules trachéales, dans l'épithélium oesophagien dérivé du patient. NKX2.1 est maintenue dans les organoïdes oesophagiens matures même après 2 mois. Ensuite, nous voulions valider l'utilisation potentielle de nos organoïdes dérivés des IPS pour modéliser les maladies acquises de l'œsophage telles que l'œsophage de Barrett. Nous avons induit une métaplasie ou transformation épithéliale avec surexpression de BMP4 dans des organoïdes de l'œsophage sains et dérivés du patient sur une période d'un mois. Nos résultats préliminaires montrent que les organoïdes de l'œsophage dérivés des patients exprimaient des niveaux d'ARNm plus élevés de MUC5AC, un marqueur épithélial cylindrique par rapport au groupe sain. Cela suggère une plus grande sensibilité de l'organoïde de l'œsophage dérivé du patient aux changements epitheliales métaplasiques. En conclusion, nous avons développé les premiers organoïdes œsophagiens tridimensionnels matures sans matrice différenciés des patients OA/FTO et identifié une signature moléculaire unique dans les cellules dérivées du patient au cours de la différenciation dirigée de l'œsophage. De plus, sur la base des résultats préliminaires, nous avons pu confirmer l'incidence plus élevée de l'œsophage de Barrett chez les patients OA/FTO par rapport au groupe sain. Notre travail met donc en évidence l'importance de l'utilisation des IPS dérivées des patients pour modéliser les maladies œsophagiennes congénitales et acquises afin de fournir de nouvelles informations sur le développement des organes au cours de l'embryogenèse. / The esophagus and trachea originate from the endodermal diverticulum of the anterior foregut tube during embryogenesis. Well-regulated and organized cellular and molecular events result in the compartmentalization of the anterior foregut tube into the esophagus and trachea. This compartmentalization is still poorly understood and disruption in this process results in a severe congenital anomaly such as esophageal atresia with or without tracheoesophageal fistula (EA/TEF). EA/TEF is one of the most common gastrointestinal congenital defects affecting 1 in 3,000 births. This malformation requires urgent surgery at birth and is frequently associated with long-term morbidity. The mechanisms underlying the embryonic development of EA/TEF are poorly understood. Animal models have been widely used to understand human diseases for decades and have significantly contributed to the understanding of esophageal development. However, key structural and morphological differences exist between human and animal esophagus, thus necessitating a more reliable model to understand trachea-esophageal development. Human induced pluripotent stem cells (iPSC) have been a valuable tool to understand organogenesis by mimicking development and deciphering mechanisms that lead to congenital and acquired diseases. This thesis therefore focuses on the use of patient-derived induced pluripotent stem cells to decipher signaling mechanisms involved in esophageal development and congenital diseases such as EA/TEF. It also focuses on one of the possible acquired esophageal diseases, namely, Barrett’s esophagus. We directed the differentiation of healthy and patient-derived iPSCs toward different developmental stages, such as definitive endoderm, anterior foregut, esophageal and tracheal epithelium. Furthermore, the esophageal epithelium was matured further in a matrix free 3-dimensional environment to generate mature esophageal organoids. At each stage of development progression, immunofluorescence, qPCR, and RNA sequencing analysis were performed. Our findings suggest that the expression of endodermal markers CXCR4, SOX17, and GATA4, were similar in both patient and healthy differentiated cells. However, at the anterior foregut stage, we observed a significant decrease in the gene and protein expression of key transcription factor SOX2 in patient-derived cells. Furthermore, using nanopore RNA sequencing, we observed that critical genes GSTM1, and RAB7 involved in cellular morphogenesis and associated with EA/TEF to be dysregulated at the anterior foregut stage in patient-derived cells. We also observed a significant increase in the expression of transcription factor NKX2.1, usually expressed only in tracheal cells, in the patient-derived esophageal epithelium. NKX2.1 expression was maintained in matured esophageal organoids even after 2 months. Next, we wanted to validate the potential use of our PSC-derived organoids to model acquired esophagus diseases such as Barrett’s esophagus (BE). We induced epithelial metaplasia with BMP4 overexpression in healthy and patient-derived esophagus organoids over a 1-month period. Our preliminary results show that patient-derived esophagus organoids expressed higher mRNA levels of MUC5AC, an epithelial columnar marker compared with the healthy group. This suggests a higher susceptibility of patient-derived esophagus organoid to metaplastic changes. In conclusion, we developed the first matrix free mature 3-dimensional esophageal organoids differentiated from EA/TEF patient-derived and identified a unique molecular signature in patient derived cells during directed esophagus differentiation. Furthermore, based on the preliminary results, we could confirm the higher incidence of Barrett’s esophagus in EA/TEF patients compared with the healthy group. Our work therefore highlights the significance of using patient-derived iPSCs to model congenital and acquired esophageal diseases to yield new insights on organ development during embryogenesis. It lays the foundation for a personalized medical approach to other diseases and the ones affecting the whole gastrointestinal system in both children and adults.

Law / LL.M.

Law / LL.M.