Made available in DSpace on 2016-06-07T17:12:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-01-27. Added 1 bitstream(s) on 2016-06-07T17:16:31Z : No. of bitstreams: 1 000860274.pdf: 1576617 bytes, checksum: 77bc3adf366698782a1321577ba4ff16 (MD5) / Introdução: A constipação intestinal é uma queixa extremamente comum em pediatria e está cada vez mais em ascensão. Defecações dolorosas, em 95% dos casos, causam postura retentiva, que podem provocar distensão progressiva da ampola retal, podendo levar ao escape fecal involuntário. Como tratamento inicial, é necessário treinamento e programação das evacuações, e dieta balanceada. Quando não há resposta aos tratamentos, é fundamental considerar sua forma orgânica (5%), sendo a Doença de Hirschsprung o diagnóstico de maior incidência. Seu tratamento é sempre cirúrgico. No entanto, no pós-operatório, parte dos pacientes evolui com constipação crônica. O papel da fibra alimentar na prevenção e no tratamento tem sido discutido com frequência devido aos benefícios à saúde. O amido resistente, encontrado em alguns alimentos, como na banana verde tem sido estudado devido aos potenciais benefícios à saúde humana. Por não ser digerido e não absorvidos no intestino delgado, apresenta comportamento semelhante aos das fibras alimentares. Assim, o objetivo deste trabalho foi avaliar a eficiência terapêutica da complementação alimentar de biomassa da banana verde no tratamento da constipação crônica funcional e em pacientes com constipação orgânica associada ao pós-operatório da Doença de Hirschsprung. Pacientes e Métodos: Foram avaliados 34 pacientes, divididos em dois grupos: G1 - Grupo constipado funcional (22 pacientes) e G2 - Grupo de pacientes em pós-operatório de doença de Hirschsprung com constipação (12 pacientes). O acompanhamento ocorreu por 9 semanas consecutivas. O funcionamento intestinal foi avaliado em dois momentos: 1 semana anterior ao início da complementação (M0) e 8 semanas após (M1), através do preenchimento de questionário com as seguintes variáveis: escala de Bristol, dias com evacuação presente na semana, número de escapes fecais na semana, uso e dosagem consumida... / Introduction: Constipation is an extremely common complaint in pediatrics and is increasingly on the rise. Painful bowel movements in 95% of cases, cause retentive posture, which can cause progressive distention of the rectum, which may lead to involuntary soiling. As an initial treatment, training and scheduling of evacuations and balanced diet is necessary. When there is no response to the treatment, it is essential to consider its organic form (5%), and Hirschsprung's disease is the most common diagnosis. Treatment is always surgical. However, postoperatively, some patients evolve with chronic constipation. The role of dietary fiber in the prevention and treatment has been discussed with frequency because of the health benefits. Resistant starch, found in some foods, such as in green bananas has been studied for potential benefits to human health. Because it is not digested and not absorbed in the small intestine, features similar to those of fiber behavior. The objective of this study was to evaluate the therapeutic efficacy of dietary supplementation of biomass of green banana in the treatment of chronic functional constipation and in patients with organic constipation associated with postoperative Hirschsprung's Disease. Patients and Methods: 34 patients divided into two groups were evaluated: G1- Constipated functional group (22 patients) and G2 - Group of patients in the postoperative Hirschsprung's disease with constipation (12 patients). The program took place for 9 consecutive weeks. The bowel function was assessed at two time points: 1 prior to the start of supplementation (M0) and after 8 weeks (M1) week by completing a questionnaire with the following variables: scale Bristol, days in the week with this evacuation, number of fecal leaks in the week, use and laxative dosage consumed. Patients consumed 2 tablespoons green banana biomass increased in the daily diet for 8 consecutive weeks. Results: With respect to the scale of ...

O objetivo deste estudo foi conhecer as experiências de mães de crianças e adolescentes com Doenças de Hirschsprung, de forma a identificar, nos seus cotidianos, situações que necessitam de intervenções de enfermagem, visando melhor qualidade de vida para essas pessoas e suas famílias. Além disso, buscou caracterizar esses pacientes, cadastrados, no período de 1982 a 2007, no Serviço de Gastroenterologia Pediátrica de um Hospital Escola, em um município no interior de São Paulo, segundo variáveis sócio-demográficas e terapêutica. Para atender aos dois primeiros objetivos, procedeuse à análise qualitativa dos dados, utilizando, como instrumento de coleta, entrevistas semi-estruturadas com mães dessas crianças e adolescentes. Os dados para caracterização dos referidos pacientes foram coletados de seus prontuários e apresentados em termos de freqüência. Aqueles gerados a partir das entrevistas foram agrupados ao redor de três temas: conhecendo a doença; experiência do cuidado e redes de apoio. O envolvimento dos pais no cuidado ao filho em situações de doença tem sido um grande desafio para os profissionais de saúde e, assim, para obter sucesso nessa dimensão do cuidar, é necessário haver habilidades nas esferas técnica e interpessoal de ambos os lados pais e profissionais de saúde. No que se refere à caracterização, 67,3% eram do sexo masculino, 43,6% ocupavam a posição de primeiro filho, 76,3% tiveram o diagnóstico no primeiro ano de vida, sendo 20% no primeiro mês e, de um total de 15 óbitos, 11 decorreram de complicações da doença. Os resultados do estudo possibilitaram identificar aspectos que necessitam de intervenção por parte dos membros da equipe de saúde, particularmente da enfermagem, objetivando melhor qualidade de vida para crianças e adolescentes com Doença de Hirschsprung e suas famílias. / This study aimed to know the experiences of mothers of children with Hirschsprung Disease; to identify, in their daily life, situations showing the need of nursing interventions in the search for a better quality of life for these children and their families, and to characterize children and adolescents with Hirschsprung Disease registered at the Pediatric Gastroenterology Service, between 1982 and 2007, according to socio-demographic and therapeutic variables. To accomplish the first two objectives, qualitative data analysis was carried out; semi-structured interviews with 13 mothers of children and adolescents with Hirschsprung Disease, attended at a school-hospital in the interior of the state of São Paulo, were used for data collection. Data for characterization of children and adolescents were collected from their medical records and presented in terms of frequency. Data taken from the interviews were grouped in three main themes: knowing the disease; care experience and support network. Involvement of parents in care to children in situations of disease has been a big challenge for health professionals. To have success in this intervention, technical and interpersonal ability from both parents and health professionals is needed. In the characterization of children and adolescents with Hirschsprung Disease, 67,3% were male; 43,6% were parents first child; 76,3% were diagnosed in the first year of life, 20% in the first month; 11 of the 15 deaths were due to complications of the disease. Results enabled the identification of aspects that need intervention by the health team, specially nursing, aiming a better quality of life for children and adolescents with Hirschsprung Disease and their families.

Tam Wing-yip. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 91-113). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese Abstract --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Hirschsprung's disease --- p.1 / Chapter 1.2 --- Neural crest cells and enteric nervous system --- p.3 / Chapter 1.3 --- Genetics of Hirschsprun´gةs disease --- p.10 / Chapter 1.3.1 --- RET/GDNF/NTN signaling pathway --- p.10 / Chapter 1.3.2 --- EDNRB/EDN3/ECE-1 signaling pathway --- p.13 / Chapter 1.3.3 --- Dominant megacolon and Sox10 --- p.15 / Chapter 1.3.4 --- Other genes involved in intestinal aganglionosis --- p.16 / Chapter 1.4 --- Objectives of the present study --- p.19 / Chapter Chapter 2 --- Enteric Neural Crest Cells Migration in Dominant Megacolon Mouse Embryos --- p.21 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- Animal --- p.26 / Chapter 2.2.2 --- Preparation of rat serum --- p.26 / Chapter 2.2.3 --- Isolation of embryos from pregnant mice --- p.27 / Chapter 2.2.4 --- Preparation of wheat germ agglutinin-gold (WGA-Au) --- p.28 / Chapter 2.2.5 --- Microinjection of WGA-Au conjugate --- p.28 / Chapter 2.2.6 --- Whole embryo culture --- p.29 / Chapter 2.2.7 --- Examination of cultured embryos --- p.30 / Chapter 2.2.8 --- Histological preparation of WGA-Au injected embryos --- p.30 / Chapter 2.2.9 --- Silver enhancement staining and histological examination of the sections --- p.31 / Chapter 2.2.10 --- Genotyping by polymerase chain reaction --- p.32 / Chapter 2.2.11 --- TUNEL assays --- p.33 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- In vivo development of Dominant megacolon mouse embryos of different genotypes --- p.35 / Chapter 2.3.2 --- In vitro development of embryos in control and experimental groups --- p.35 / Chapter 2.3.3 --- Migration of vagal neural crest cells in Dom embryos --- p.36 / Chapter 2.3.4 --- Apoptotic cells detection at the vagal region by TUNEL assay --- p.37 / Chapter 2.3.5 --- Migration of sacral neural crest cells in Dom embryos --- p.37 / Chapter 2.3.6 --- Apoptotic cells detection at the sacral region by TUNEL assay --- p.38 / Figures and Tables / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- In vitro culture system supporting the normal development of mouse embryos --- p.40 / Chapter 2.4.2 --- WGA-Au as a cell marker for tracing the NCCs migration --- p.41 / Chapter 2.4.3 --- Vagal neural crest cells migration in Dom mouse embryos --- p.42 / Chapter 2.4.4 --- Apoptotic cell death does not contribute to the total aganglionosis in Dom homozygous embryos --- p.43 / Chapter 2.4.5 --- Sacral neural crest cells migration in Dom mouse embryos --- p.45 / Chapter 2.4.6 --- NCCs migration in zebrafish colourless mutant --- p.47 / Chapter 2.4.7 --- Limitation of the method used in this study --- p.49 / Chapter 2.4.8 --- Conclusions --- p.49 / Appendices / Chapter Chapter 3 --- Migration of Enteric Neural Crest-derived Cells in the Developing Gut of Dominant Megacolon Mouse Embryos --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and Methods --- p.55 / Chapter 3.2.1 --- Isolation of the gut from Dom mouse embryos --- p.55 / Chapter 3.2.2 --- Whole mount immunohistochemistry --- p.55 / Chapter 3.3 --- Results --- p.57 / Chapter 3.3.1 --- PGP9.5 immunoreactivity in the 12.5 d.p.c. Dom embryos --- p.57 / Chapter 3.3.2 --- TH immunoreactivity in the 12.5 d.p.c. Dom embryos --- p.58 / Chapter 3.3.3 --- PGP9.5 immunoreactivity in the 14.5 d.p.c. Dom embryos --- p.59 / Figures and Tables / Chapter 3.4 --- Discussion --- p.61 / Chapter 3.4.1 --- The use of PGP9.5 and TH antibodies as markers for studying the migration of enteric neural crest-derived cells --- p.61 / Chapter 3.4.2 --- Incomplete migration of neural crest-derived cells within the gut of Dom heterozygous embryos --- p.62 / Chapter 3.4.3 --- Failure of sacral NCCs to invade the hindgut of Dom heterozygous embryos --- p.63 / Chapter 3.4.4 --- PGP9.5 and TH positive signals in the gut of Dom homozygous embryos --- p.64 / Chapter 3.4.5 --- Early differentiation of neural crest-derived cells into neurons due to haploinsufficiency of Sox10 --- p.65 / Chapter 3.4.6 --- Conclusions --- p.66 / Chapter Chapter 4 --- Localization of Interstitial Cells of Cajal in the Gut of Dominant Megacolon Mice --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2. --- Materials and Methods --- p.72 / Chapter 4.2.1 --- Isolation of the gut from mouse embryos and adult mice --- p.72 / Chapter 4.2.2 --- Cryosection and immunohistochemistry --- p.73 / Chapter 4.2.3 --- Whole-mount immunohistochemistry --- p.73 / Chapter 4.2.4 --- Total RNA extraction --- p.74 / Chapter 4.2.5 --- Reverse transcription for the first strand cDNA synthesis --- p.75 / Chapter 4.2.4 --- Reverse transcription-Polymerase chain reaction (RT-PCR) --- p.76 / Chapter 4.3 --- Results --- p.77 / Chapter 4.3.1 --- PGP9.5 and c-kit immunoreactivity in the Dom wild type colon --- p.77 / Chapter 4.3.2 --- c-kit immunoreactivity in the Dom heterozygous adult colon --- p.78 / Chapter 4.3.3 --- c-kit and SCF expression during gut development --- p.78 / Figures and Tables / Chapter 4.4 --- Discussion --- p.80 / Chapter 4.4.1 --- The importance in studying the development of ICCs in aganglionic gut --- p.80 / Chapter 4.4.2 --- ICCs development in Dominant megacolon mice --- p.81 / Chapter 4.4.3 --- The relationship between enteric neurons and ICCs development --- p.83 / Chapter 4.4.4 --- Advantages of using confocal microscopy and whole- mount preparations to study the ICCs development --- p.85 / Chapter 4.4.5 --- Conclusions --- p.86 / Chapter Chapter 5 --- General Discussion and Conclusions --- p.87 / References --- p.91

O objetivo deste estudo foi conhecer as experiências de mães de crianças e adolescentes com Doenças de Hirschsprung, de forma a identificar, nos seus cotidianos, situações que necessitam de intervenções de enfermagem, visando melhor qualidade de vida para essas pessoas e suas famílias. Além disso, buscou caracterizar esses pacientes, cadastrados, no período de 1982 a 2007, no Serviço de Gastroenterologia Pediátrica de um Hospital Escola, em um município no interior de São Paulo, segundo variáveis sócio-demográficas e terapêutica. Para atender aos dois primeiros objetivos, procedeuse à análise qualitativa dos dados, utilizando, como instrumento de coleta, entrevistas semi-estruturadas com mães dessas crianças e adolescentes. Os dados para caracterização dos referidos pacientes foram coletados de seus prontuários e apresentados em termos de freqüência. Aqueles gerados a partir das entrevistas foram agrupados ao redor de três temas: conhecendo a doença; experiência do cuidado e redes de apoio. O envolvimento dos pais no cuidado ao filho em situações de doença tem sido um grande desafio para os profissionais de saúde e, assim, para obter sucesso nessa dimensão do cuidar, é necessário haver habilidades nas esferas técnica e interpessoal de ambos os lados pais e profissionais de saúde. No que se refere à caracterização, 67,3% eram do sexo masculino, 43,6% ocupavam a posição de primeiro filho, 76,3% tiveram o diagnóstico no primeiro ano de vida, sendo 20% no primeiro mês e, de um total de 15 óbitos, 11 decorreram de complicações da doença. Os resultados do estudo possibilitaram identificar aspectos que necessitam de intervenção por parte dos membros da equipe de saúde, particularmente da enfermagem, objetivando melhor qualidade de vida para crianças e adolescentes com Doença de Hirschsprung e suas famílias. / This study aimed to know the experiences of mothers of children with Hirschsprung Disease; to identify, in their daily life, situations showing the need of nursing interventions in the search for a better quality of life for these children and their families, and to characterize children and adolescents with Hirschsprung Disease registered at the Pediatric Gastroenterology Service, between 1982 and 2007, according to socio-demographic and therapeutic variables. To accomplish the first two objectives, qualitative data analysis was carried out; semi-structured interviews with 13 mothers of children and adolescents with Hirschsprung Disease, attended at a school-hospital in the interior of the state of São Paulo, were used for data collection. Data for characterization of children and adolescents were collected from their medical records and presented in terms of frequency. Data taken from the interviews were grouped in three main themes: knowing the disease; care experience and support network. Involvement of parents in care to children in situations of disease has been a big challenge for health professionals. To have success in this intervention, technical and interpersonal ability from both parents and health professionals is needed. In the characterization of children and adolescents with Hirschsprung Disease, 67,3% were male; 43,6% were parents first child; 76,3% were diagnosed in the first year of life, 20% in the first month; 11 of the 15 deaths were due to complications of the disease. Results enabled the identification of aspects that need intervention by the health team, specially nursing, aiming a better quality of life for children and adolescents with Hirschsprung Disease and their families.

As Neoplasias endócrinas múltiplas (NEMs) são síndromes herdadas de modo dominante e causadas por mutações germinativas em genes específicos. Caracterizam-se pela presença de tumores em um conjunto de glândulas endócrinas, conjunto este típico de cada tipo-específico de NEM. Dentre os diferentes tipos de NEMs, há a neoplasia endócrina múltipla tipo 2 (NEM2) que envolve os fenótipos, carcinoma medular de tireóide (CMT), hiperparatiroidismo primário (HPT), feocromocitoma (FEO) e megacólon congênito (doença de Hirschsprung, HSCR). Apesar da prevalência da NEM2 na população em geral ser baixa (~ 1:30.000), o número de casos afetados por família pode ser expressivo, uma vez que sua penetrância é praticamente completa (~100%). A doença de HSCR ou aganglionose intestinal congênita quando ocorre está geralmente associada à mutações RET nos códons 609, 618 e 620; apresenta ampla variação fenotípica, padrão de herança complexa e baixa penetrancia. Poucos casos de HSCR podem apresentar mutações em outros genes. Mutações no gene RET são responsáveis por aproximadamente metade (~50%) dos casos familiares de HSCR e alguns casos esporádicos (~10-20%), sugerindo fortemente que a HSCR seja doença poligenica. Tem-se também sugerido que polimorfismos genéticos no RET podem influenciar o fenotipo da NEM2/HSCR. No presente estudo, analisamos o gene RET no sentido de investigar se o desenvolvimento de megacólon em pacientes com a mutação germinativa RET p.C620R estaria associado à presença de ou a) a uma segunda mutação germinativa ou b) a um SNP, ou c) a um haplótipo informativo, que possivelmente poderia estar potencialmente interagindo genicamente com a mutação RET principal e eventualmente modulando o fenótipo HSCR / The multiple endocrine neoplasias (MENs) are inherited multi-tumoral conditions caused by germline mutations in specific genes. Specifically, the multiple endocrine neoplasia type 2 (NEM2) is a hereditary endocrine disorder transmitted dominantly and involving three main tumors, medullary thyroid carcinoma (CMT), primary hyperparathyroidism (HPT) and pheochromocytoma (PHEO). Despite the low prevalence of MEN2 in general population, the number of affected individuals per family can be significant as the penetrance of MEN2 is almost complete (~100%). In addition to CMT, PHEO and HPT, other conditions as congenital megacólon (Hirschsprung disease, HSCR or congenital intestinal aganglionosis) may occur in MEN2 (HSCR/MEN2). HSCR/MEN2 usually is due to RET mutations in codons 609, 618 and 620. HSCR has a wide phenotypic variation; is a complex multigenic disease; and has a low penetrance. Mutations in the RET gene are responsible for approximately 50% of the familial HSCR cases and ~10% of the sporadic HSCR cases, supporting that HSCR is a polygenic disease and this is confirmed by a few HSCR cases associated with mutations in the EDNRB and EDN3 genes. In the present study, we focused in the analysis of the RET gene in order to investigate whether the development of congenital megacólon in patients with RET mutation p.C620R is associated with the presence of, a) a second RET germline mutation, b) a SNP, or with a haplotype that co-segregate with the disease

As Neoplasias endócrinas múltiplas (NEMs) são síndromes herdadas de modo dominante e causadas por mutações germinativas em genes específicos. Caracterizam-se pela presença de tumores em um conjunto de glândulas endócrinas, conjunto este típico de cada tipo-específico de NEM. Dentre os diferentes tipos de NEMs, há a neoplasia endócrina múltipla tipo 2 (NEM2) que envolve os fenótipos, carcinoma medular de tireóide (CMT), hiperparatiroidismo primário (HPT), feocromocitoma (FEO) e megacólon congênito (doença de Hirschsprung, HSCR). Apesar da prevalência da NEM2 na população em geral ser baixa (~ 1:30.000), o número de casos afetados por família pode ser expressivo, uma vez que sua penetrância é praticamente completa (~100%). A doença de HSCR ou aganglionose intestinal congênita quando ocorre está geralmente associada à mutações RET nos códons 609, 618 e 620; apresenta ampla variação fenotípica, padrão de herança complexa e baixa penetrancia. Poucos casos de HSCR podem apresentar mutações em outros genes. Mutações no gene RET são responsáveis por aproximadamente metade (~50%) dos casos familiares de HSCR e alguns casos esporádicos (~10-20%), sugerindo fortemente que a HSCR seja doença poligenica. Tem-se também sugerido que polimorfismos genéticos no RET podem influenciar o fenotipo da NEM2/HSCR. No presente estudo, analisamos o gene RET no sentido de investigar se o desenvolvimento de megacólon em pacientes com a mutação germinativa RET p.C620R estaria associado à presença de ou a) a uma segunda mutação germinativa ou b) a um SNP, ou c) a um haplótipo informativo, que possivelmente poderia estar potencialmente interagindo genicamente com a mutação RET principal e eventualmente modulando o fenótipo HSCR / The multiple endocrine neoplasias (MENs) are inherited multi-tumoral conditions caused by germline mutations in specific genes. Specifically, the multiple endocrine neoplasia type 2 (NEM2) is a hereditary endocrine disorder transmitted dominantly and involving three main tumors, medullary thyroid carcinoma (CMT), primary hyperparathyroidism (HPT) and pheochromocytoma (PHEO). Despite the low prevalence of MEN2 in general population, the number of affected individuals per family can be significant as the penetrance of MEN2 is almost complete (~100%). In addition to CMT, PHEO and HPT, other conditions as congenital megacólon (Hirschsprung disease, HSCR or congenital intestinal aganglionosis) may occur in MEN2 (HSCR/MEN2). HSCR/MEN2 usually is due to RET mutations in codons 609, 618 and 620. HSCR has a wide phenotypic variation; is a complex multigenic disease; and has a low penetrance. Mutations in the RET gene are responsible for approximately 50% of the familial HSCR cases and ~10% of the sporadic HSCR cases, supporting that HSCR is a polygenic disease and this is confirmed by a few HSCR cases associated with mutations in the EDNRB and EDN3 genes. In the present study, we focused in the analysis of the RET gene in order to investigate whether the development of congenital megacólon in patients with RET mutation p.C620R is associated with the presence of, a) a second RET germline mutation, b) a SNP, or with a haplotype that co-segregate with the disease

Les cellules de la crête neurale se caractérisent par leur capacité de migration dansl’embryon et la variété des types cellulaires qu’elles sont capables de générer (mélanocytes,système nerveux entérique (SNE) et périphérique). Chez l’homme, plusieurs maladiescongénitales affectant des organes et tissus divers, ont pour origine une anomalie demigration, prolifération, survie ou différenciation de ces cellules. Au laboratoire, nousétudions deux d’entre elles, le syndrome de Waardenburg-Hirschsprung (WS4- anomalie depigmentation, surdité et maladie de Hirschsprung (HSCR : anomalie entérique)) et lesyndrome de Mowat et Wilson (MWS – retard mental sévère, dysmorphie faciale avec ousans HSCR). A l’heure actuelle, quatre gènes ont été impliqués : l’endothéline 3 (EDN3) etson récepteur à sept domaines transmembranaires EDNRB et les deux facteurs de transcriptionZEB2 et SOX10. Au cours de ma thèse, nous avons montré que des délétions de SOX10 sontégalement responsables de 15% des cas de WS2 (défauts de pigmentation et surdité sansHSCR), élargissant le spectre des phénotypes liés à une mutation au sein de ce gène(Bondurand, Dastot-Le Moal, Stanchina et al. Am. J Hum. Genet, 2007).Parallèlement à ces études génétiques, nous avons souhaité mieux définir la fonction et lesinteractions entre les différents gènes impliqués dans le WS4 (SOX10, EDN3 et EDNRB).Pour cela, nous avons croisé les modèles murins invalidés pour ces gènes, et comparé lephénotype des simples et doubles mutants. A travers cette analyse phénotypique, nous avonsdémontré qu’une interaction entre ces molécules est nécessaire au développement normal duSNE et des mélanocytes dérivés de la crête neurale. En effet, par rapport aux simples mutants,les doubles mutants Sox10;Edn3 et Sox10;Ednrb présentent une augmentation de ladépigmentation, et une forte aggravation du phénotype entérique. Le suivi du devenir descellules formant le SNE au cours du développement nous a permis de montrer quel’aggravation du phénotype entérique est due à une diminution du pool de cellulesprogénitrices par apoptose (Stanchina et al. 2006).Dans la continuité des travaux déjà réalisés, nous avons voulu améliorer notrecompréhension du rôle joué par le gène du MWS : ZEB2, et étudier ses interactions avec lesgènes du WS4. Dans un premier temps, nous avons analysé l’effet de l’expression constitutiveou l’inhibition de ce facteur sur la survie, prolifération et différenciation des cellulesprogénitrices du SNE à l’aide d’un système de culture de progéniteurs entériques disponibleau laboratoire. Nos résultats suggèrent un effet répresseur de ZEB2 sur la différenciationneuronale. Ce facteur pourrait donc être nécessaire au maintien du pool de cellulesprogénitrices dans un état indifférencié. Nous avons ensuite étudié les interactions entre ZEB2et les gènes du WS4. Nous avons croisé les souris portant une invalidation du gène ZEB2 avecles souris invalidées pour SOX10 ou portant une mutation de EDN3 ou EDNRB, et démontréqu’une interaction entre ZEB2 et SOX10 est nécessaire au développement normal du SNE. Eneffet, par rapport aux simples mutants, les doubles mutants présentent une forte aggravationdu phénotype entérique, due à une diminution de la prolifération des cellules progénitrices et àune augmentation de la différenciation neuronale. L’analyse phénotype des mutantsZeb2;Edn3 et Zeb2;Ednrb suggère également l’existence d’une interaction entre ces troismolécules, mais l’origine du défaut entérique reste inexplorée.Ces études nous ont permis de mieux appréhender les réseaux moléculaires mis en place aucours du développement du SNE, de comprendre l’origine des anomalies entériques observéeschez les patients, améliorant leur prise en charge. / To understand in more details the molecular and cellular bases of hereditary diseases resulting from defects of neural crest (NC) development, we study several neurocristopathies, in particular Waardenburg syndrome (WS – pigmentary abnormalities and hearing loss), and Mowat-Wilson syndrome (MWS, severe mental retardation, facial dysmorphy, with or without HSCR (congenital megacolon)). To date, about ten causative genes have been identified, among which are the seven transmembrane domain receptor EDNRB and its ligand endothelin 3 (EDN3), the two transcription factors SOX10 and ZEB2.We contributed to the research efforts engaged to unravel these disorders. In particular, we identified the first mutations of SOX10 in patients presenting with WS4 (association of WS with HSCR disease) and WS2 (Bondurand, Dastot-Le Moal, Stanchina et al. Am. J Hum. Genet, 2007), and participated to functional studies describing its role during enteric nervous system (ENS) development. More recently, we identified the gene ZEB2 as responsible for MWS. The goal of my thesis was to understand the function of these genes and their interaction during the development of NC and ENS in particular. For this purpose, we combined an in vitro approach (isolation of ENS progenitors) to in vivo experiments (phenotype analysis of simple and double mutant mice). We demonstrated that an interaction between SOX10, EDN3 and EDNRB is necessary for the normal development of the ENS and melanocytes (Stanchina et al. 2006), and then focused our efforts in understanding the function of ZEB2 during the development of the ENS as well as its interactions with WS4 genes. Preliminary results suggest that ZEB2 inhibition accelerates neuronal differentiation in vitro. In the same time, generation of Zeb2;Sox10, Zeb2;Edn3 and Zeb2;Ednrb have been realized. Through phenotype analysis of Sox10;Zeb2 double mutants, we showed that a coordinated and balanced interaction between these two genes is required for normal ENS development. Indeed, double mutants present with more severe ENS defects due to decreased proliferation of enteric progenitors and increased neuronal differentiation from E11.5 onwards. These data revealed that crosstalks between these two transcription factors are crucial for proper ENS development. Analysis of Zeb2;Edn3 and Zeb2;Ednrb double mutant suggest also an interaction between these genes. Future experiments will help us to confirm these results and to determine the cellular and molecular origin of these interactions. These studies will enable us to better apprehend the molecular bases of these diseases, and to understand the origin of the enteric anomalies observed in patients. This knowledge may also help to develop new therapeutic strategies

Next, the influences on the migration of neural crest cell from the microenvironment of the hindgut through which the neural crest cells migrate were studied. An organ culture system was established to recombine different gut segments together at E11.5 for gut culture in order to trace the migration of neural crest cells from the midgut of the +/+ or Dom/+ embryo to the hindgut of the same or different genotypes. At E11.5, the midgut of both +/+ and Dom/+ embryos had already been fully colonized by neural crest cells, thus an explanted midgut segment (donor midgut) could serve as the source of the neural crest cells, while the caudal half of the hindgut (recipient hindgut) acted as the recipient of the neural crest cells from the donor midgut segment because at this stage, the caudal half of the hindgut was completely devoid of neural crest cells. After three days of culture, when a segment of midgut from the +/+ embryo was used as the donor of migratory vagal neural crest-derived cells and combined with an aneural segment of the hindgut (segment without neural crest-derived cells) from Dom/+ or Dom/Dom embryos, neural crest-derived cells from the midgut segment successfully crossed the combination junction and migrated normally along the hindgut segment to reach its caudal end within a normal developmental time frame. However, the migration of neural crest-derived donor cells from the Dom/+ midgut segment was abnormal in the recipient hindgut with a genotype of +/+, Dom/+ or Dom/Dom as evidenced by the retarded rostrocaudal progression of the vagal neural crest-derived cells and the reduced number of migratory cells in the recipient hindgut segment. These results thus indicate that the migration of the vagal neural crest-derived cells is minimally influenced by the migratory environment of the hindgut of the Dom embryo, and that the neural crest cells themselves may be defective in migration leading to the retarded migration in the hindgut of Dom mouse embryos. / The vagal neural crest cells originating from the region of the neural tube adjacent to somites 1 to 7 migrate along defined pathways to the gastrointestinal tract and then colonize the gut to give rise to the majority of neurons and glia of the enteric nervous system. Mutation of Sox10 in the Dominant megacolon (Dom) mouse, which is an animal model of Hirschsprung's disease, leads to aganglionosis (absence of ganglia) in varying lengths of the hindgut. To investigate the underlying cellular mechanism of aganglionosis, the migration of vagal neural crest cells from the neural tube to the gut (pre-enteric migration) in Dom mouse embryos at E8.5 was firstly traced with extrinsic cell markers, such as wheat germ agglutinin gold conjugates (WGA-Au) or fluorescent dye DiI. After the vagal neural crest cells entered the gut at E9.5, their migration was then followed by the examination of the expression of specific markers for undifferentiated neural crest cells with immunohistochemical staining. It was found that, although vagal neural crest cells in embryos of the three genotypes examined migrated along similar pre-enteric pathways at a similar migratory rate, the numbers of neural crest cells in embryos heterozygous (Dom/+) and homozygous (Dom/Dom) for the Sox10 mutation were significantly reduced when compared with the number of neural crest cells in wild-type (+/+) embryos. After vagal neural crest had entered the gut and from E10.5 onwards, no neural crest-derived cells were found in the gut of Dom/Dom embryos, and the migration of neural crest cells along the Dom/+ gut was significantly retarded from E12.5 onwards as compared with the migration in stage-matched +/+ embryos. / To further trace the cause of defective migration of neural crest cells in the Dom embryo, the proliferation and survival of neural crest cells were investigated with BrdU labeling and TUNEL assay. It was found that, although there was no obvious difference in the proliferating ability of vagal neural crest cells in embryos of all the three Dom genotypes studied during the pre-enteric migration and the migration in the gut, more apoptotic neural crest cells were found along the pre-enteric migratory pathway of Dom/Dom embryos than Dom/+ and +/+ embryos. Therefore, the decreased surviving ability, but possibly not the reduced proliferating ability, of neural crest cells during their pre-enteric migration may be partly responsible for aganglionosis in the hindgut of the Dom mouse. / Wang Liang. / "June 2006." / Adviser: W. Y. Chan. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1380. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 287-307). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Made available in DSpace on 2014-06-11T19:33:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-08-22Bitstream added on 2014-06-13T18:45:05Z : No. of bitstreams: 1 lourencao_plta_dr_botfm.pdf: 2438220 bytes, checksum: 2ab7ae217c75319cb0f431af1a533ca6 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A identificação pré-operatória da zona de transição na Doença de Hirschsprung (DH) tornou-se um passo fundamental para o planejamento cirúrgico, especialmente para a técnica de abaixamento endorretal transanal. O presente estudo tem como objetivo investigar prospectivamente o valor da determinação colonoscópica da zona de transição na avaliação pré-operatória dos pacientes com DH. A colonoscopia foi realizada em doze pacientes com diagnóstico de DH previamente confirmado pela manometria anorretal, enema opaco e biópsia de sucção do reto. Endoscopicamente, o primeiro local com ausência de peristaltismo foi identificado como o início da zona agangliônica. Pouco acima deste ponto, a zona de transição foi marcada através de uma tatuagem com tinta nanquim. Durante o abaixamento endorretal transanal, uma biópsia de congelação envolvendo a espessura total da parede foi sempre realizada. Os resultados da determinação colonoscópica da zona de transição foram comparados com os obtidos pelo enema opaco. A colonoscopia permitiu a identificação da zona de transição em todos os 12 casos (100%). O enema opaco revelou a presença da zona de transição em apenas 7 pacientes (58,3%). A análise das amostras de congelação, obtidas pouco acima das áreas endoscopicamente marcadas, revelou a presença de células ganglionares em todos os casos. A análise histopatológica das peças cirúrgicas confirmou o diagnóstico de DH em todos os casos, assim como a localização da zona de transição no mesmo local previamente tatuado endoscopicamente. O exame colonoscópico pré-operatório demonstrou ser uma ferramenta útil para determinar a localização da zona de transição em pacientes com DH / Preoperative identification of the transition zone in Hirschsprung’s disease (HD) has become an essential issue for surgical planning, especially for Transanal Endorectal Pull- Through (TEPT) procedure. The present study aimed to investigate prospectively, the value of endoscopic marking of the transition zone between normal and aganglionic bowel, as a landmark of the location of pull-through procedure for treatment of HD. Colonoscopy was performed on twelve patients with HD diagnosis, previously confirmed by anorectal manometry, contrast enema and rectal suction biopsies. Endoscopically, the first site with absence of motility was identified as the beginning of the aganglionic area. Just above this point, the transition zone was marked with an Indian Ink tattooing. During the TEPT, a full-thickness biopsy for frozen section analysis was performed just above this mark. The results of colonoscopic making were compared with contrast enema. Colonoscopy allowed the identification and tattooing of the junction between normal bowel with peristalsis and aganglionic bowel without peristalsis in all 12 cases (100%). Barium enema revealed the transition zone in 7 patients (58.3%). Frozen samples, obtained just above the marked areas revealed the presence of ganglion cells in all cases and the histopathological analysis of surgical specimens confirmed the diagnosis of HD in all cases and checked the location of the transition zone at the same site previously identified by colonoscopy. Colonoscopic marking of the transition zone may be a useful tool to set the location of pull-through procedure for treatment of HD

Orientador: Maria Aparecida Marchesan Rodrigues / Coorientador: Bonifácio Katsunori Takegawa / Banca: Erika Veruska Paiva Ortolan / Banca: Márcia Guimarães Silva / Banca: Cleverson Teixeira Soares / Banca: Gisele Alboghetti Nai / Resumo: A identificação pré-operatória da zona de transição na Doença de Hirschsprung (DH) tornou-se um passo fundamental para o planejamento cirúrgico, especialmente para a técnica de abaixamento endorretal transanal. O presente estudo tem como objetivo investigar prospectivamente o valor da determinação colonoscópica da zona de transição na avaliação pré-operatória dos pacientes com DH. A colonoscopia foi realizada em doze pacientes com diagnóstico de DH previamente confirmado pela manometria anorretal, enema opaco e biópsia de sucção do reto. Endoscopicamente, o primeiro local com ausência de peristaltismo foi identificado como o início da zona agangliônica. Pouco acima deste ponto, a zona de transição foi marcada através de uma tatuagem com tinta nanquim. Durante o abaixamento endorretal transanal, uma biópsia de congelação envolvendo a espessura total da parede foi sempre realizada. Os resultados da determinação colonoscópica da zona de transição foram comparados com os obtidos pelo enema opaco. A colonoscopia permitiu a identificação da zona de transição em todos os 12 casos (100%). O enema opaco revelou a presença da zona de transição em apenas 7 pacientes (58,3%). A análise das amostras de congelação, obtidas pouco acima das áreas endoscopicamente marcadas, revelou a presença de células ganglionares em todos os casos. A análise histopatológica das peças cirúrgicas confirmou o diagnóstico de DH em todos os casos, assim como a localização da zona de transição no mesmo local previamente tatuado endoscopicamente. O exame colonoscópico pré-operatório demonstrou ser uma ferramenta útil para determinar a localização da zona de transição em pacientes com DH / Abstract: Preoperative identification of the transition zone in Hirschsprung's disease (HD) has become an essential issue for surgical planning, especially for Transanal Endorectal Pull- Through (TEPT) procedure. The present study aimed to investigate prospectively, the value of endoscopic marking of the transition zone between normal and aganglionic bowel, as a landmark of the location of pull-through procedure for treatment of HD. Colonoscopy was performed on twelve patients with HD diagnosis, previously confirmed by anorectal manometry, contrast enema and rectal suction biopsies. Endoscopically, the first site with absence of motility was identified as the beginning of the aganglionic area. Just above this point, the transition zone was marked with an Indian Ink tattooing. During the TEPT, a full-thickness biopsy for frozen section analysis was performed just above this mark. The results of colonoscopic making were compared with contrast enema. Colonoscopy allowed the identification and tattooing of the junction between normal bowel with peristalsis and aganglionic bowel without peristalsis in all 12 cases (100%). Barium enema revealed the transition zone in 7 patients (58.3%). Frozen samples, obtained just above the marked areas revealed the presence of ganglion cells in all cases and the histopathological analysis of surgical specimens confirmed the diagnosis of HD in all cases and checked the location of the transition zone at the same site previously identified by colonoscopy. Colonoscopic marking of the transition zone may be a useful tool to set the location of pull-through procedure for treatment of HD / Doutor