by Wai-Chi Kong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 83-92). / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations Frequently Used --- p.v / Chapter Chapter1 --- General Introduction --- p.1 / Chapter Chapter2 --- Long term culture of whole-mount retinal explant --- p.16 / Chapter Chapter3 --- Responses of retinal ganglion cells after peripheral nerve transplantation in vivo and in vitro --- p.46 / Chapter Chapter4 --- Effect of optic nerve or peripheral nerve explants on cultured whole-mount retinal explants --- p.62 / Chapter Chapter5 --- General Discussion --- p.78 / References --- p.83 / Tables --- p.93

Introdução: As particularidades da anatomia e a localização topográfica da orelha a tornam uma estrutura única no corpo humano, existindo diversas situações clínicas em que a sua reconstrução (total ou parcial) pode ser necessária. Devido à dificuldade técnica, as complicações pós-operatórias são freqüentes. Objetivos: Realizar análise epidemiológica dos pacientes submetidos à reconstrução de orelha devido a causas congênitas (microtia) e adquiridas (trauma, queimaduras e outras), com avaliação comparativa dos resultados, para definir qual grupo teria menores índices de complicações. Método: Realizada análise retrospectiva de 279 casos de reconstrução de orelha realizados de 1994 a 2004 na Disciplina de Cirurgia Plástica da Faculdade de Medicina da Universidade de São Paulo. Os pacientes foram separados em portadores de deformidades congênitas ou adquiridas e analisados comparativamente. Resultados: O sexo masculino foi mais prevalente tanto entre os pacientes portadores de deformidades congênitas (61,3%) quanto entre aqueles com deformidades adquiridas (68,75%). A média de idade no início dos procedimentos cirúrgicos foi de 14,3 anos nos pacientes com deformidades congênitas e 29,5 nas deformidades adquiridas. Trauma foi a principal causa de deformidade adquirida (55% dos casos adquiridos), seguido pelas queimaduras (29% dos casos adquiridos) e a única deformidade congênita observada no estudo foi a microtia. Em média, os pacientes dos grupos necessitaram de 4,2 cirurgias, mas aqueles com seqüelas de queimaduras foram submetidos a um número significativamente maior de procedimentos (5,9 - p < 0,01). As principais complicações foram a exposição de cartilagem (15,1% do total de casos), sem diferença entre os grupos, e a brida retroauricular (16,5% do total de casos), sendo esta última mais freqüente nos casos de microtia e seqüelas de queimaduras. Conclusões: Os casos de perda traumática mostraram menor índice de complicações quando comparados àqueles submetidos a reconstrução por microtia ou após queimadura. / Introduction: The distinctive anatomic features and topography render the ear unique in the human body. Total or partial reconstruction of the ear may be required in many clinical conditions, but because technical difficulties are common, the rate of postoperative complications increases. Objectives: To analyze the epidemiologic data of patients who underwent surgery for reconstruction of the ear due to congenital conditions (microtia) or acquired deformities (trauma, burns and others), and to compare the results in order to define which group had the lowest rate of complications. Methods: A retrospective analysis was conducted with 279 cases of ear reconstruction performed between 1994 and 2004 by the Discipline of Plastic Surgery of the University of São Paulo Medical School. The patients were initially separated in two groups, according to their condition (congenital or acquired), to compare their data. Results: Male was the prevailing gender in both groups of ear deformities: congenital (61.3%) and acquired conditions (68.7%). The patients with congenital deformities had a mean age of 14.3 years at the beginning of the treatment, whereas the patients with acquired deformities were 29.5 years old, in average. The major causes of acquired deformities were trauma (55% of the cases in this group) and burns (29%). The only cause of congenital deformity observed was microtia. Patients required an average of 4.2 surgical procedures. However, those with sequelae of burn injuries were submitted to a significantly higher number of procedures (5.9 - p < 0.01). Cartilage exposure (15.1% of the total) and postauricular bridles (16.5%) were the major complications observed in this study. The latter was more common among those cases with microtia and sequelae of burns. Conclusions: Patients with traumatic injuries had a better outcome after surgery than those with microtia or burn injuries, because of a lower rate of complications.

Objetivo: Em anisométropes, comparar os valores médios individuais dos componentes oculares de ambos os olhos (poder da córnea, profundidade da câmara anterior, poder equivalente do cristalino e comprimento axial), correlacionar as diferenças dos componentes oculares com as diferenças de refração de ambos os olhos; verificar a contribuição total e a seqüência geral de influência das variáveis na diferença refrativa; e identificar o menor número de fatores que contenham o mesmo grau de informações expressas no conjunto de variáveis que influenciam na diferença refrativa. Métodos: Realizou-se um estudo transversal analítico em população de 77 anisométropes de duas ou mais dioptrias, atendida no ambulatório de Oftalmologia do Hospital Universitário da Faculdade de Medicina Nilton Lins, Manaus, Amazonas. Os anisométropes foram submetidos à refração estática objetiva e subjetiva, ceratometria e biometria ultra-sônica A-scan. A análise dos dados foi feita por meio dos seguintes modelos estatísticos: análise univariada, multivariada, de regressão múltipla e fatorial. Resultados: Não houve diferenças significativas na comparação dos valores médios individuais dos componentes oculares entre os olhos. Houve correlação negativa média entre a diferença refrativa e a diferença de comprimento axial (r=-0,64) (P<0,01) e correlação negativa fraca entre a diferença refrativa e a diferença de poder do cristalino (r=-0,34) (p<0,01). As variáveis analisadas responderam, no seu conjunto, por 78% da variação total para a diferença refrativa. A seqüência geral de influência das variáveis na diferença refrativa foi a seguinte: comprimento axial, poder do cristalino, poder da córnea e profundidade da câmara anterior. Foram identificados três fatores para a diferença refrativa: a) fator 1 (refração, comprimento axial); b) fator 2 (profundidade da câmera anterior, poder da córnea) e c) fator 3 (poder do cristalino). Conclusões: O estudo conduzido em 77 indivíduos com anisometropias variando de 2,00 a mais de 19,00 dioptrias, realizado para avaliar a influência dos componentes oculares, mostrou que o comprimento axial foi o principal fator causador das anisometropias, seguido pelo cristalino que contribuiu menos, depois pela córnea e profundidade da câmara anterior, com contribuições ainda menores. A investigação sugere falência no mecanismo adaptativo normal em anisometropia, o que poderia produzir não só descontrole do alongamento do comprimento axial (fator 1), mas também falência no controle do aplanamento da córnea e do aprofundamento da câmara anterior (fator 2) e no achatamento do cristalino (fator 3). / Purpose: To compare the individual means of ocular components of both eyes (corneal power, anterior chamber depth, crystalline lens power and axial length) in patients with anisometropia; to correlate the differences of the ocular components with refractive differences in both eyes; to verify total contribution and the sequence of influence that variables have in refractive differences, and to identify the smallest number of factors that contain the same level of information expressed in the set of variables that influence refractive difference. Methods: An analytical transversal study was carried out in 77 patients with anisometropia of two or more dioptres seen at the Ophthalmologic Clinic, University Hospital, Medical School Nilton Lins, Manaus, Amazon state. All participants were submitted to ophthalmologic exam which included objective and subjective cycloplegic refractometry, keratometry and ultrasonic biometry. Data analysis comprised the following statistical models: univariate, multivariate, multiple and factorial regression analyses. Results: There were no significant differences in the comparison of the individual means of the ocular components. There was negative correlation between refractive difference and difference of axial length (r=- 0.64; p<0.01) and weak negative correlation between refractive difference and crystalline lens power difference (r=-0.34; p< 0.01). The analyzed variables amounted to 78% of the total variation of refractive difference. The general sequence of variables influencing refractive difference was: axial length, crystalline lens power, cornea power, and anterior chamber depth. There were three factors identified for refractive differences: a) factor 1 (refraction, axial length); b) factor 2 (anterior chamber depth, cornea power), and c) factor 3 (crystalline lens power). Conclusions: Seventy-seven cases of anisometropia ranging from 2,00 to over 19,00 dioptres, examined for the individual components of refraction, showed that axial length was the major causative factor; crystalline lens have contributed less, followed by cornea and anterior chamber length. This study has suggested deficit of the normal adaptive mechanism in anisometropia that could produce not only axial elongation (factor 1), but also failure to control flattening of the cornea, deepening of the anterior chamber length (factor 2) and flattening of crystalline lens (factor 3).

Chan, Ka Ki Alex. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 245-263). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese abstract --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vii / Chapter Chapter One --- General introduction --- p.1 / Chapter 1.1 --- Structure and function of the enteric nervous system --- p.1 / Chapter 1.2 --- Neural crest cells (NCC) --- p.5 / Chapter 1.2.1 --- Vagal neural crest cells --- p.7 / Chapter 1.2.2 --- Sacral neural crest cells --- p.10 / Chapter 1.3 --- Prespecialization of the neural crest cells to form ENS --- p.15 / Chapter 1.4 --- Signaling pathways involved in ENS development --- p.19 / Chapter 1.4.1 --- Endothelin signaling pathway --- p.20 / Chapter 1.4.2 --- Ret signaling pathway: GDNF/Ret/GFRa1 --- p.22 / Chapter 1.4.3 --- Ret signaling pathway: NRTN/Ret/GFRa2 --- p.26 / Chapter 1.4.4 --- Phox2b --- p.28 / Chapter 1.4.5 --- Sox10 --- p.29 / Chapter 1.5 --- Hirschsprung's Disease (HSCR) --- p.31 / Chapter 1.6 --- Objective of studies --- p.32 / Figures and legends --- p.35 / Chapter Chapter Two --- The early migratory pathways of mouse sacral neural crest cells --- p.39 / Chapter 2.1 --- Introduction --- p.39 / Chapter 2.2 --- Materials and Methods --- p.46 / Chapter 2.2.1 --- Animals --- p.46 / Chapter 2.2.2 --- Isolation of the mouse embryos at E95 --- p.46 / Chapter 2.2.3 --- Preparation ofWGA-Au --- p.47 / Chapter 2.2.4 --- Preparation of Dil --- p.48 / Chapter 2.2.5 --- Microinjection ofWGA-Au or Dil --- p.48 / Chapter 2.2.6 --- Preparation of rat serum --- p.49 / Chapter 2.2.7 --- Preparation of culture medium --- p.50 / Chapter 2.2.8 --- in vitro whole embryo culture system --- p.50 / Chapter 2.2.9 --- Examination of embryo after culture --- p.51 / Chapter 2.2.10 --- Histological preparation of WGA-Au labelled embryos --- p.51 / Chapter 2.2.11 --- Silver enhancement staining on sections of WGA-Au labelled embryo --- p.52 / Chapter 2.2.12 --- Histological preparation of Dil labelled embryos --- p.53 / Chapter 2.2.13 --- Reconstruction of the mouse embryos --- p.53 / Chapter 2.2.14 --- Cell counting on labelled sacral NCC between the anterior and posterior halves of the somite --- p.54 / Chapter 2.2.15 --- Cell counting on migrating labelled sacral NCC for each somite at different developmental stages --- p.55 / Chapter 2.3 --- Results --- p.57 / Chapter 2.3.1 --- Development of E9.5 mouse embryo in vitro and in vivo --- p.57 / Chapter 2.3.2 --- Labelling of sacral neural crest cells by means of different cell markers --- p.58 / Chapter 2.3.3 --- Migration of sacral neural crest cells at different developmental stages --- p.59 / Chapter 2.3.3.1 --- Distribution of sacral NCC at the 26th somite stage --- p.60 / Chapter 2.3.3.2 --- Distribution of sacral NCC at the 28th somite stage --- p.61 / Chapter 2.3.3.3 --- Distribution of sacral NCC at the 30th somite stage --- p.61 / Chapter 2.3.3.4 --- Distribution of sacral NCC at the 32nd somite stage --- p.63 / Chapter 2.3.3.5 --- Distribution of sacral NCC at the 34th somite stage --- p.64 / Chapter 2.3.4 --- Defined migration pathways of the sacral neural crest cells --- p.65 / Chapter 2.3.5 --- Quantification of migrating sacral NCC at different somite axial levels at different developmental stages --- p.66 / Chapter 2.4 --- Discussion --- p.68 / Chapter 2.4.1 --- E9.5 mouse embryo grew normally in vitro using whole embryo culture --- p.69 / Chapter 2.4.2 --- Migration of sacral neural crest cells at 26th somite stage --- p.70 / Chapter 2.4.3 --- Migration of sacral neural crest cells at 28th somite stage --- p.72 / Chapter 2.4.4 --- Migration or sacral neural crest cells at 30th somite stage --- p.73 / Chapter 2.4.5 --- Migration of sacral neural crest cells at 32nd somite --- p.75 / Chapter 2.4.6 --- Migration of sacral neural crest cells at 34th somite stage --- p.77 / Chapter 2.4.7 --- Majority of sacral neural crest cells migrate along the dorsomedial pathway --- p.80 / Figures and Legends --- p.82 / Tables --- p.136 / Chapter Chapter Three --- The early migratory pathways of Dom mouse sacral neural crest cells --- p.139 / Chapter 3.1 --- Introduction --- p.139 / Chapter 3.2 --- Materials and Methods --- p.145 / Chapter 3.2.1 --- Animals --- p.145 / Chapter 3.2.2 --- In vitro culture of Dom mouse embryos --- p.145 / Chapter 3.2.3 --- Genotyping by polymerase chain reaction (PCR) --- p.146 / Chapter 3.2.4 --- Treatment of the harvested Dom mouse embryos --- p.147 / Chapter 3.2.5 --- Reconstruction of images and cell counting --- p.148 / Chapter 3.2.6 --- Percentage of migrating sacral neural crest cells reduction in Dom mouse embryo --- p.148 / Chapter 3.3 --- Results --- p.150 / Chapter 3.3.1 --- Migration of sacral neural crest cells in Dom mouse embryos at different developmental stages --- p.150 / Chapter 3.3.1.1 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 26th somite stage --- p.150 / Chapter 3.3.1.2 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 28th somite stage --- p.151 / Chapter 3.3.1.3 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 30th somite stage --- p.152 / Chapter 3.3.1.4 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 32nd somite stage --- p.154 / Chapter 3.3.1.5 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 34th somite stage --- p.156 / Chapter 3.3.2 --- Number of migrating sacral NCC of different genotypes of Dom mouse embryos at different developmental stage --- p.158 / Chapter 3.4 --- Discussion --- p.160 / Chapter 3.4.1 --- The use of Dom mouse model to study the etiology of Hirschsprung's disease (HSCR) --- p.161 / Chapter 3.4.2 --- Migration of sacral NCC in Dom mouse embryos --- p.164 / Figures and legends --- p.169 / Tables --- p.230 / Chapter Chapter Four --- General discussion and conclusions --- p.236 / Appendix --- p.241 / References --- p.245

OBJETIVO: O objetivo deste estudo foi predizer o prognóstico perinatal em fetos com derrame pleural isolado por meio da medida do volume pulmonar estimado pela ultrassonografia tridimensional. MÉTODO: Estudo retrospectivo, entre julho de 2005 e julho de 2010, com 19 fetos com derrame pleural isolado (ausência de causas infecciosas, imunes, anomalias cromossômicas ou estruturais associadas) acompanhados no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Os volumes pulmonares foram obtidos pela ultrassonografia tridimensional (Voluson 730 Expert, GE Medical System, Kretzechnick, Áustria) em dois períodos, no momento do diagnóstico (20 26 semanas) e próximo ao parto (duas semanas antecedentes ao parto ou até 36 semanas), e mensurados pela técnica VOCAL (Virtual Organ Computer Aided Analysis) com rotação de 30º. Os volumes obtidos (observados) foram comparados com valores esperados para idade gestacional, e a razão entre o volume total fetal observado/esperado (VPTo/e) foi avaliada de acordo com a mortalidade perinatal e morbidade neonatal (necessidade de ventilação mecânica por mais que 48 horas). RESULTADOS: Dezenove fetos com derrame pleural isolado foram analisados no período do estudo. Doze (63,2%) crianças sobreviveram. Dos sobreviventes, sete (58,3%) apresentaram morbidade respiratória. O VPTo/e no primeiro exame ultrassonográfico não se associou significativamente com mortalidade (VPTo/e: 0,42±0,19 nos sobreviventes contra 0,30±0,08 nos não sobreviventes, p=0,11). No segundo exame, por outro lado, VPTo/e foi significativamente menor nos casos que faleceram (0,24±0,08) em relação aos sobrevivente (0,58±0,21; p<0,01) e nos que necessitaram de ventilação mecânica prolongada (0,35±0,08) comparados aos que não necessitaram (0,68±0,10; p<0.01). CONCLUSÃO: O volume pulmonar fetal medido pela ultrassonografia tridimensional pode ser utilizado para predizer o prognóstico de fetos com derrame pleural isolado. / OBJECTIVE The aim of the present study was to predict the perinatal outcome in isolated pleural effusion using fetal lung volumes assessed by three-dimensional ultrasonography. METHODS: A retrospective study conducted between July 2005 and July 2010, in which 19 fetuses with isolated pleural effusion (absence of infection, immunological causes, chromosomal anomalies and associated structural anomalies) at Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Fetal lung volumes were assessed by three-dimensional ultrasonography (Voluson 730 Expert, GE Medical System, Kretzechnick, Áustria) in two periods: at diagnosis (20-26 weeks) and nears the delivery (2 weeks before delivery or at 36 weeks), by VOCAL technique (Virtual Organ Computer Aided Analysis) with rotation of 30o. The observed volumes were compared to expected values for determine gestational age, and the observed/expected total fetal lung volume ratio (o/e-TFLV) was evaluated according to perinatal death and neonatal morbidity (need for mechanical ventilation longer than 48 hours). RESULTS: A total of 19 fetuses with isolated pleural effusion were evaluated during the study period. Twelve (63.2%) infants survived. Among the survivors, seven (58.3%) had severe respiratory distress at birth. The o/e-TFLV at the first ultrasound examination was not associated statistically with mortality (o/e-TLFV: 0.42±0.19 in survivors x 0.30±0.08 among those that died, p=0.11). On the second ultrasound examination, on the other hand, the o/e-TFLV was significantly reduced in those cases that died (0.24±0.08) whilst in survivors (0.58±0.21; p<0.01) and in those that needed mechanical ventilation (0.35±0.08) when compared to those that did not need it (0.68±0.10; p<0.01). CONCLUSION: Fetal lung volumes measured by three-dimensional ultrasonography may be useful to predict perinatal outcome in fetuses with primary pleural effusion

Development of a suitable animal model of AIDS is much needed in AIDS research to study infection and pathogenesis as well as to evaluate methods of prevention and treatment of HIV infection. Small animals such as rodents are attractive candidates for AIDS research due to the availability of various inbred and genetically engineered strains, extensive knowledge or their immune system, especially in mice, and the relative ease of breeding and maintaining animal colonies. Transgenic small animal models carrying entire HIV genome or selected genes have been instrumental to understand functions of HIV genes in vivo and their role in HIV pathogenesis. The type of cells in which HIV genes are expressed seems to be an import prerequisite for the study of HIV gene functions in transgenic mice. Mice constitutively expressing the entire HIV-1 genome or HIV-1 nef gene in CD4 + T cells and in the cells of macrophage/dendritic lineage develop an AIDS-like disease very similar to AIDS disease in humans. Similarly, expression of Nef in adult mice, using inducible system, results in the AIDS-like disease. This disease is characterized by thymic atrophy, impaired thymocyte maturation, loss of CD4+ T cells, increased activation and turnover of T cells, which can occur in the absence of lymphypenia, and non-lymphoid organ disease involving the lungs and kidneys. Susceptibility of adult mice to the pathological effects of Nef suggests that the AIDS-like disease in the constitutively expressing Nef Tg mice is not due to developmental defects caused by early expression of Nef. This model highlights the important role of Nef in HIV-1 pathogenesis. The high similarity in the disease in these Tg mice with human AIDS strongly suggest that these mice are a relevant model to study AIDS. This study further evidence that mouse cells can support functions of Nef and these Tg mice represent a unique model to study Nef functions in vivo in the context of the primary immune system. Moreover, the inducible Nef Tg model has given us the ability to control the level and time of expression of Nef which was impossible to do in the previously reported constitutive Nef Tg mouse models. These mice will be useful to study immune reconstitution since Nef expression can be turned off after withdrawal from dox.

Pseudomonas aeruginosa is a common and important pathogen in people with cystic fibrosis (CF). Recently epidemic strains of P. aeruginosa associated with increased morbidity, have been identified. The method of transmission is not clear, but there is evidence of a potential airborne route. The aim of this study was to determine whether different strains of P. aeruginosa isolated from people with CF were able to survive within artificially generated aerosols in an aerobiological chamber. Viable P. aeruginosa could still be detected up to 45min after halting generation of the aerosols. All of the strains of P. aeruginosa expressing a non-mucoid phenotype isolated from people with CF had a reduced ability to survive within aerosols compared to an environmental strain. Expression of a mucoid phenotype by the strains of P. aeruginosa isolated from people with CF promoted survival in the aerosol model compared to strains expressing a non-mucoid phenotype.

In vivo DNA is compacted tightly, via its association with histones and non-histone proteins, into higher-order chromatin structure. In this state, the DNA is refractory to the cellular factors that require access to DNA. The repressive nature of chromatin is alleviated in part by the action enzymes that modify chromatin structure. There are two major groups of chromatin modifying enzymes: those that post-translationally modify histones by the addition of small chemical moieties and those that utilize the energy derived from ATP hydrolysis to physically disrupt chromatin structure. The SWI/SNF enzyme belongs to this latter group. The SWI/SNF complex was identified originally in yeast. Several of its subunits are required for the expression of a subset of inducible genes. The ATPase activity is provided by the SWI2/SNF2 protein. In mammals, there are two biochemically separable SWI/SNF complexes that contain either BRG1 or BRM, both homologs of yeast SWI2/SNF2. The yeast and mammalian SWI/SNF complexes are able to disrupt the Dnase I digestion pattern of in vitro assembled mononucleosomes and arrays, as well as facilitate the accessibility of restriction nucleases and transcription factors. The mechanism by which SWI/SNF functions has yet to be elucidated. SNF5 is a component of the yeast SWI/SNF complex. It is required for sucrose fermentation and mating type switching. The mammalian homolog of Snf5 is SNF5/INI1. SNF5/INI1 was identified simultaneously by two groups as a protein that shares homology with Snf5 and via a yeast two hybrid assay as a protein that interacts with HIV integrase (INtegrase Interactor). INI1 is a component of all mammalian SWI/SNF complexes purified to date. In humans, mutations and/or deletions in INI1 are associated with a variety of cancers, including malignant rhabdoid tumors, choroid plexus carcinomas, medullablastomas, primitive neuralectodermal tumors, and some cases of leukemia. Furthermore, constitutional mutations within INI1in individuals presenting with these tumors support the role of INI1 as a tumor suppressor. In this thesis, we show that Ini1 also functions as a tumor suppressor in mice. Approximately 20% of mice heterozygous for Ini1 present with tumors. Most of these tumors are undifferentiated or poorly differentiated sarcomas with variable rhabdoid features. All tumors examined to date show loss of heterozygosity at the Ini1 locus. We also show that Ini1 is essential for embryonic development. Mice homozygous-null for Ini1die between days 4 and 5.5 post-fertilization due to an inability to adhere to their substratum, form trophectoderm, and expand their inner cell mass. We further characterize the function of Ini1 in tumor suppression by generating mice heterozygous for both Ini1 and either Rb or p53. While heterozygosity at the Ini1 locus appears to have no effect on the rate of tumorigenesis in Rb-heterozygous mice, many of the tumors arising in compound heterozygous mice present with an altered morphology. This finding suggests that Ini1 may contribute to tumor progression due to loss of Rb. In contrast, mice compound heterozygous for Ini1 and p53 show a marked reduction in the rate of tumorigenesis compared to p53-heterozygous mice. Furthermore, the tumor spectrum is altered in these compound heterozygous mice. These findings suggest that Ini1 may function normally to repress p53 activity. Lastly, we show that expression of the Ini1 tumor suppressor itself is regulated tightly. Tissues and cells heterozygous for Ini1 express roughly equivalent levels of Ini1 protein and mRNA as their wild-type counterparts. We further show that this compensation is mediated by an increase in the rate of transcription from the wild-type Ini1 allele. Moreover, when exogenous Ini1 is introduced into Ini1-heterozygous cells, expression from the Ini1 promoter is reduced. These data indicate that a compensatory mechanism exists to ensure that the steady-state levels of Ini1 are constant. In summary, research detailed in this thesis has contributed to our understanding of the regulation of Ini1 as well as the role this protein plays in mammalian development and tumor suppression.

INTRODUÇÃO: Pacientes com deficiência de GH e síndrome de Turner, associados a baixa estatura, são beneficiados com o tratamento com GH. Há controvérsias sobre a atuação deletéria do GH no crescimento craniofacial, porém a maioria dos trabalhos é retrospectiva. Nosso objetivo foi realizar estudo prospectivo para avaliar o crescimento craniofacial de pacientes em tratamento com GH e o possível desenvolvimento de traços acromegálicos. CASUÍSTICA: 30 pacientes com idade cronológica de 4,6 a 23 anos e idade óssea de 1,5 a 13 anos divididos em 3 grupos baseados no diagnóstico e uso de GH: grupo 1- pacientes virgem de tratamento com GH portadores de hipopituitarismo e deficiência isolada (n=6); grupo 2: pacientes já em tratamento com GH: portadores de hipopituitarismo e deficiência isolada (n=16); grupo 3: pacientes com síndrome de Turner em tratamento com GH (n=8). A dose do GH utilizada foi de 0.1 a 0.15 U/kg/dia, via subcutânea, à noite, por 2 a 11 anos. MÉTODOS: medidas antropométricas (altura, pés e mãos), radiografia panorâmica, telerradiografia seguida pela análise cefalométrica de Ricketts e medidas lineares da base do crânio, altura facial, terço inferior da face, mandíbula e maxila, e fotografia facial de frente e perfil anualmente, por no mínimo 3 anos. As medidas lineares citadas foram comparadas com a média da população brasileira e entre si para avaliar o desenvolvimento craniofacial individual. As medidas de mãos e pés foram comparadas com atlas de morfometria e consideradas alteradas quando >P97. Os níveis de IGF1 e IGFBP3 foram mensurados a cada 6 meses para adequação da dose de GH. Os resultados foram analisados estatisticamente tomando-se como significantes valores de p<0,05. RESULTADOS: grupos 1 e 2 (deficiência isolada de GH ou hipopituitarismo): 3 pacientes com perfil desarmonioso obtiveram harmonia, 2 pacientes devido ao crescimento mandibular e um paciente devido ao crescimento maxilar, nenhum paciente desenvolveu desarmonia facial; observamos aumento significante da base posterior do crânio, mandíbula e terço inferior da face (p<0,05). Grupo 3 (síndrome de Turner): 2 pacientes com face desarmoniosa obtiveram harmonia, devido ao crescimento mandibular e nenhuma paciente desenvolveu desarmonia facial. Todos os pacientes, quando comparadas a análise cefalométrica de Ricketts inicial e final, mantiveram o mesmo padrão de crescimento facial. Observamos aumento das mãos em 2 pacientes (1 do sexo masculino com deficiência de GH e outra com síndrome de Turner), enquanto que o aumento dos pés foi observado em 50% das pacientes com síndrome de Turner e em 32% dos pacientes com deficiência de GH. CONCLUSÕES: A comparação das medidas cefalométricas do grupo de pacientes com deficiência de GH, virgem de tratamento, demonstrou maior atuação do GH no crescimento da base posterior do crânio e mandíbula; todos os pacientes mantiveram o mesmo padrão de crescimento craniofacial durante o acompanhamento; não houve correlação estatisticamente significante entre as medidas cefalométricas e a harmonia da face, portanto a associação dos métodos de cefalometria e análise facial por fotografia é necessária para avaliar a atuação do GH no crescimento craniofacial, houve melhora da harmonia facial em 28% dos pacientes retrognatas, devido ao crescimento mandibular, portanto pacientes retrognatas podem ser beneficiados com o tratamento com GH; não observamos desenvolvimento de desproporções faciais e nenhum paciente desenvolveu desarmonia facial no decorrer do tratamento com doses padronizadas de GH. Observamos, no entanto, aumento das extremidades, principalmente dos pés. / INTRODUCTION: Patients with GH deficiency and Turner syndrome, associated to short stature can benefit from GH treatment. There are controversies on the deleterious effect of GH on craniofacial growth; however, most of the studies are retrospective. Our objective was to carry out a prospective study to evaluate the craniofacial growth of patients in treatment with GH and the possible development of acromegalic features. PATIENTS: 30 patients with chronological age of 4.6 to 23 years and bone age of 1.5 to 13 years divided in 3 groups based on the diagnosis and GH use: group 1- patients with hypopituitarism and isolated GH deficiency naïve to GH treatment (n=6); group 2: patients with hypopituitarism and isolated GH deficiency (n=16) and group 3: patients with Turner syndrome, both already on GH treatment (n=8). GH treatment (0.1 to 0.15 U/kg/day, subcutaneously) was carried out at the night for 2 to 11 years. METHODS: Anthropometrical (height, hands and feet) measurements, panoramic x-ray, teleradiography followed by cephalometric analysis according to Ricketts and linear measurements of the skull base, facial height, lower third of the face, lower jaw and maxilla, and frontal and profile analysis of face by photography were made annually, for at least 3 years. The mentioned linear measurements were compared with the average Brazilian population and among themselves to evaluate the individual craniofacial development. The hand and foot size measurements were compared with a morphometric atlas and were considered increased when >P97. The levels of IGF1 and IGFBP3 were measured each 6 months for GH dose adequacy. The results were analyzed statistically and p values < 0.05 were considered statistically significant. RESULTS: Group 1 and 2 with isolated GH deficiency or hypopituitarism: 3 patients with disharmonious profile attained harmony, 2 due to the mandibular growth and 1 patient due to maxillary growth; no patient developed facial disharmony; we observed a significant increase of the posterior skull base, inferior jaw and lower third of the face (P<0.05). Group 3 with Turner syndrome: 2 patients with facial disharmony obtained harmony due to the mandibular growth and no patient developed facial disharmony. All of the patients maintained the same pattern of facial growth when the initial and final cephalometric analyses according to Ricketts were compared. Hand size increase was observed in 2 patients (1 with GH deficiency and another with Turner syndrome); foot size increase was observed in 50% of the patients with Turner syndrome and in 32% of the patients with GH deficiency. CONCLUSIONS: The comparison of the cephalometric measurements of the group with GH deficiency naïve to GH treatment, demonstrated a greater GH effect on the growth of the posterior skull base and jaw; all of the patients had kept the same craniofacial growth pattern during the follow-up; there was no statistically significant correlation between the cephalometric measurements and facial harmony; therefore, the association of the methods of cephalometric and facial analysis through photography is mandatory to evaluate the effect of GH on craniofacial growth. There was an improvement in the facial harmony in 28% of the retrognathic patients due to mandibular growth; therefore, patients with mandibular retrognathism can benefit from GH treatment. None of the patients treated with standardized doses of GH developed facial disharmony during treatment. We observed however, an increase of the extremities, mainly of the feet.

A morfogênese das glândulas salivares envolve eventos complexos e coordenados, dependentes da interação epitélio-mesênquima e do microambiente. Fatores de crescimento coordenam vários desses processos biológicos e o fator transformador de crescimento-beta (TGF-) mostra-se relevante. Utilizando imunoistoquímica e imunofluorescência, a distribuição do TGF-1, 2 e 3 foi mapeada e sua expressão comparada com a expressão de marcadores de maturação em glândulas salivares humanas obtidas de fetos que tinham entre 4ª e 24ª semanas de vida intra-uterina. O TGF-1 foi detectado durante a fase pseudoglandular no mesênquima. Nas outras etapas da diferenciação glandular esse fator foi expresso no citoplasma das células acinares até a glândula salivar adulta. O TGF-2 foi detectado desde o estágio de botão inicial da glândula salivar. Sua expressão foi observada nas células ductais e sua presença aumentada ao longo da diferenciação glandular. O TGF-3 foi visto durante a fase pseudoglandular das glândulas salivares, inicialmente fraco nas células ductais e foi o único detectado em células mioepiteliais. A troca de subunidades de TGF- durante a maturação das glândulas salivares sugere mudanças estimuladas durante os complexos estágios de desenvolvimento dessas glândulas. O presente estudo reafirma essa evidência, e mostra que as subunidades do TGF- são fatores importantes durante a diferenciação de glândulas salivares. / Morphogenesis of salivary glands involves complex coordinated events. Synchronization between cell proliferation, polarization and differentiation, which are dependent on epithelialmesenchymal interactions and on the microenvironment, is a requirement. Growth factors mediate many of these orchestrated biological processes and transforming growth factor-beta (TGF- ) appears to be relevant. Using immunohistochemistry and immunofluorescence, we have mapped the distribution of TGF- 1, 2 and 3 and compared it with the expression of maturation markers in human salivary glands obtained from fetuses ranging from weeks 4 to 24 of gestation. TGF- 1 first appeared during pseudoglandular stage in the surrounding mesenchyme and, in the more differentiated stages, was expressed in the cytoplasm of acinar cells throughout the adult gland. The TGF- 2 was detected since the bud initial stage of the salivary gland. Its expression was observed in ductal cells and increased along gland differentiation. The TGF- 3 was detected from the pseudoglandular stage of the salivary gland, being weakly expressed on ductal cells, and it was the only factor detected on myoepithelial cells. The data suggest that TGF- have a role to play in salivary gland development and differentiation.