Objetivo: Caracterizar as indicações das gestantes que procuram o serviço de Medicina Fetal do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo que realizaram procedimentos invasivos diagnósticos e avaliar os resultados dos cariótipos fetais e destas gestações no período de fevereiro de 2005 a dezembro de 2009. Método: Estudo observacional retrospectivo das gestantes que realizaram biópsia de vilo corial (BVC), amniocentese e cordocentese. Não foram incluídos outros procedimentos diagnósticos ou terapêuticos. O resultado da gestação foi obtido através de consulta de prontuário eletrônico e/ou físico e/ou contato telefônico. Resultados: Foram realizados 113 BVC, 340 amniocenteses e 260 cordocenteses. A principal indicação para a realização dos procedimentos invasivos foi a presença de malformações fetais (69,8%), seguido por translucência nucal aumentada (13,4%) e idade materna avançada (10,2%). A trissomia do cromossomo 18 foi a aneuploidia mais comum (8,1%), seguido pela trissomia do 21 (6,2%), 45,X0 (4,8%) e a trissomia do 13 (3,8%). Ocorreram 4,9% abortamentos, 25,7% natimortos e 13% neomortos. Oito gestantes optaram pela interrupção judicial. 99% das gestantes cujos fetos não apresentavam malformação e que apresentavam cariótipo fetal normal tiveram nativivos. CONCLUSÃO: A principal indicação para a realização dos procedimentos invasivos foi a presença de malformação fetal em 69,8% das gestantes e presença de anormalidades cromossômicas encontradas nos fetos foi de 26,23%. / Objective: The purpose of this study is to characterize the indications of pregnant women who seek the Fetal Medicine Service of Hospital das Clínicas of São Paulo University to perform invasive diagnostic procedures and evaluate the results of fetal karyotypes and their pregnancies from February 2005 to December 2009. Methods: Retrospective observational study of pregnant women who underwent CVS, amniocentesis or cordocentesis. Other diagnostic or therapeutic procedures were not included. The outcomes of pregnancies were obtained through consultation of medical records and/or telephone contact. Results: 113 CVS, 340 amniocentesis and 260 cordocentesis were performed. The main indication for performing invasive procedure was the presence of fetal anomaly (69.8%), followed by increased nuchal translucency (13.4%) and maternal age (10.2%). The trisomy of chromosome 18 was the most common aneuploidy (8.1%), followed by trisomy 21 (6.2%), 45,X0 (4.8%), and trisomy 13 (3.8%). There were 4.9 % of miscarriage, 25.7% of stillbirth and 13% of neonatal deaths. Eight women opted for legal termination of pregnancies. 99% of pregnant women whose fetus had no structural abnormalities and normal karyotype had a live child. CONCLUSION: The main indication for karyotyping was the presence of fetal malformation in 69.8% of pregnancies and chromosomal abnormalities was found in 26.23% of the fetuses.

Orientador: Carmem Silvia Fontanetti Christofoletti / Banca: Maria Aparecida Marin Morales / Banca: Silvia Tamie Matsumoto / Resumo: A degradação dos recursos hídricos, como os ambientes lênticos, dentre eles, os lagos, é uma das maiores preocupações atualmente, visto que esta pode causar danos diretos ou indiretos à saúde e à sobrevivência dos organismos expostos. Um dos fatores que contribui para a alteração da qualidade das águas de ambientes lênticos é o despejo de efluentes, principalmente àqueles de origem doméstica, portadores de substâncias que chegam a ser tóxicas para o meio aquático. Por meio dos testes citogenéticos, utilizando os mais diversificados organismos-teste, é possível biomonitorar a extensão da poluição e avaliar os efeitos dessas substâncias presentes no ambiente natural. Com esse intuito, o presente trabalho tomou por modelo de estudo, um lago urbano artificial (Lago Azul, Rio Claro-SP) e objetivou avaliar o potencial citotóxico, genotóxico e mutagênico das águas desse ambiente, por meio dos testes de aberrações cromossômicas e micronúcleos, em células meristemáticas de Allium cepa (cebola), em dois tratamentos: o contínuo e o período de recuperação, em água ultra pura; e, pelo teste do micronúcleo associado às anormalidades nucleares e do ensaio do cometa, aplicados em eritrócitos de Oreochromis niloticus (tilápia). Coletas de águas sazonais foram realizadas na estação seca (agosto/2006 e agosto/2007) e na estação chuvosa (março/2007 e fevereiro/2008). Análises físico-químicas foram feitas para uma coleta de cada estação. A partir dos dados obtidos, pode-se inferir que as águas desse ambiente lêntico apresentam potencial citotóxico, genotóxico e mutagênico, nas duas estações de coletas, para os dois organismos-teste empregados. As análises de metais revelaram concentrações acima do permitido pela legislação de Ag, Cd2+, Cu e Fe3+, em ambas as estações. Embora os... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The degradation of water resources, as the lentics environments, among them, the lakes, is a major concern now, because it can cause direct or indirect damages to health and to the survival of the exposed organisms. One factor that can contribute to change the water quality of lentics environments is the dumping of effluents, mainly those of domestic origin, carriers of substances that come to be toxic to the aquatic environment. Through the cytogenetic tests, using the most diverse systems-test, it is possible monitoring the extent of pollution and assess the effects of substances on the natural environment. To that end, this work has taken a model of study, an urban artificial lake (Lago Azul, Rio Claro-SP) and aimed to evaluate the cytotoxic, genotoxic and mutagenic potentials of waters that environment, through tests of chromosome aberrations and micronuclei in meristematic cells of Allium cepa (onion) in two treatments: the continued and the period of recovery, in ultra pure water, and by the micronucleus and nuclear abnormalities test and of the comet assay, applied in erythrocytes of Oreochromis niloticus (tilapia). Seasonal collections of waters were held in the dry season (august/2006 and august/2007) and the rainy season (march/2007 and february/2008). Physical and chemical analyses were made for a collection of each season. From the data obtained, it can be infered that the waters of this lentic environment had cytotoxic, genotoxic and mutagenic potentials in two seasons of collections for the two systems-test employed. Analyses of metals detected high concentrations of Ag, Cd2+, Cu, Fe3+, whose values are higher than permitted by law, in both seasons. Although the cytotoxic, genotoxic and mutagenic potentials have been detected in two seasons, the dry season is that presented the highest risk... (Complete abstract click electronic access below) / Mestre

Objetivo: Descrever as manifestações clínicas de indivíduos com hipótese diagnostica da Sindrome de deleção 22q11 (SD22q11) confirmados por testes genéticos, na primeira avaliação e durante o acompanhamento dos mesmos em avaliações subsequentes para uma melhor definição do curso natural da doença. Local: Laboratório de Genética e Citogenética Humana do HRAC-USP Bauru/SP. Casuística e metodologia: O presente trabalho é retrospectivo e analisou os dados de prontuários de 72 indivíduos cadastrados no HRAC-USP, os quais receberam hipótese diagnóstica da SD22q11 e foram confirmadas por teste genético (MLPA ou FISH). A avaliação envolveu a analise dos dados relatados por todos os setores do HRAC-USP. Resultados e discussão: Foramanalisados 72prontuários deindivíduos com a SD22q11. Constatamos que a idade media dos indivíduos quando do cadastro no HRAC-USP foi de seis anos. Também constatamos que houve um longo período de tempo entre os retornos ao hospital e que, nesses retornos, nem todas as especialidades foram contempladas. Esses fatos prejudicaram a analise da historia natural da anomalia em questão. Com relação às características fenotipicas, observamos a presença de sinais clínicos típicos, como por exemplo: face alongada, lábios finos, hipoplasia alar, anormalidades menores na orelha, dígitos longos e fendas palpebrais, fissuras labiopalatinas, cardiopatias congenitas, dificuldade de aprendizagem, atraso de linguagem e distúrbios comportamentais. A fissura oral foi à manifestação otorrinolaringológica mais frequente, presente em 75% dos pacientes, onde as fissuras submucosas foram as mais frequentes (43%). As características cognitivas como, atraso de fala (87%), dificuldades de aprendizagem (95%) e distúrbios comportamentais (81%), tiveram um resultado significativo, descritas em quase todos os indivíduos. As cardiopatias congênitas estavam presentes em 47,2% dos prontuários analisados. De um modo geral, comparando a frequência dos sinais clínicos encontrados neste trabalho com dados da literatura, constatamos que as frequências encontram-se dentro do esperado. Conclusão: A maioria dos indivíduos cadastrados no HRAC-USP, pertencentes ao grupo de estudo, apresentou idade superior a 06 anos. Portanto, a observação do curso natural da historia da SD22q11 para avaliar características fenotípicas que surgissem ao longo da evolução clinica do indivíduo e que pudessem ajudar no diagnóstico, ficou prejudicada. Mesmo nos casos onde o indivíduo foi cadastrado no HRAC-USP com idade inferior a dois anos, o diagnóstico foi tardio devido a falta de uma ação multidisciplinar e interdisciplinar no hospital. Mesmo não sendo possível avaliar as características fenotípicas surgidas durante a historia natural da doença, constatamos que as manifestações clínicas relatadas nos prontuários cursam com as características da SD22q11 e em frequências que corroboram com as da literatura / To describe clinical manifestations observed in medical records of individuals registered in the hospital with a diagnostic hypothesis of 22q11.2DS confirmed by genetic tests (MLPA OR FISH), since the first assessment in the HRAC-USP and during the follow up of these individuals in subsequent assessments, in order to achieve a better definition to the natural courses of the disease. Local: Laboratory of Human Genetics and Cytogenetics (HRAC-USP Bauru/SP). Methods: This retrospective study analyzed 72 medical records of individuals registered at the HRAC-USP, who were diagnosed with 22q11DS and who had this diagnosis confirmed by a genetic test (MLPA OR FISH). The assessment concerned the analysis of reported data in all sectors of the HRAC-USP. Results and Discussion: 72 medical records of individuals with 22q11DS were analyzed. It was verified that the average age of individuals when registering at the HRAC-USP was six years old. It was also verified that it took a long period of time for these individuals to return to the hospital and, when they did, not all specialties were contemplated. These facts harmed the analysis of the natural history of the anomaly. About the phenotypic characteristics, some typical clinical signs were observed, such as: long face, thin lips, hypoplasia nasal alar, minor abnormalities in the ear, long digits and narrow palpebral fissures, palatal abnormalities, congenital heart defects, learning disabilities, delay speech and behavioral disorders. An oral cleft was the most frequent otorhinolaryngology manifestation, present in 75% of the patients; among which submucous cleft palate were the most frequent (43%). Cognitive features such as, delay speech (87%), learning disabilities (95%) and behavioral disorders (81%) had a significant result, described in almost all individuals. Congenital heart defects were observed at 4% to 48% of individuals with 22q11.2DS, in this study it was observed in 47.2%. In general, comparing the frequency of some clinical signs observed in this study with the literature data, it was verified that the frequencies were within expectations. Conclusion: Most of the individuals registered at the HRAC belonging to the study group were over 6 years old. Therefore, the observation of natural course of the history of 22q11DS to evaluate the phenotypic characteristics that would arise during the clinical evolution of the individual and that could help in the diagnosis was harmed. Even in cases when the individual was registered at the HRAC-USPunder the age of two, the diagnosis was delayed due to lack of a multidisciplinary and interdisciplinary action in the hospital. Even not being possible to measure the phenotypic characteristics that emerged during the natural history of the disease, it was verified that the clinical manifestations reported in the records occur with the 22q11DS characteristics and in frequencies that corroborate with the literature

Lam, Man Ting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 128-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lee Siu-wah. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves [106]-116). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Figures --- p.ix / List of Tables --- p.x / Abbreviations --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.2 --- Etiology of Hepatocellular Carcinoma --- p.5 / Chapter 1.2.1 --- Viral Infection --- p.5 / Chapter 1.2.1.1 --- Hepatitis B Virus --- p.5 / Chapter 1.2.1.2 --- Hepatitis C Virus --- p.7 / Chapter 1.2.2 --- Cirrhosis and Chronic Inflammation --- p.8 / Chapter 1.2.3 --- Aflatoxin --- p.9 / Chapter 1.3 --- Genetic Studies of Hepatocellular Carcinoma --- p.9 / Chapter 1.3.1 --- Conventional Cytogenetics --- p.9 / Chapter 1.3.2 --- Molecular Cytogenetics --- p.12 / Chapter 1.3.3 --- Molecular Genetic Studies --- p.12 / Chapter 1.3.3.1 --- Proto-oncogenes --- p.12 / Chapter 1.3.3.2 --- Tumour Suppressor Genes --- p.13 / Chapter 1.3.3.3 --- Cell Cycle Genes --- p.14 / Chapter 1.4 --- Background of Study --- p.16 / Chapter 1.5 --- Objectives of Study --- p.17 / Chapter Chapter 2 --- Material and Methods --- p.18 / Chapter 2.1 --- Materials --- p.19 / Chapter 2.2 --- Analysis of Chromosomal Imbalances by Comparative Genomic Hybridization --- p.23 / Chapter 2.2.1 --- Comparative Genomic Hybridization --- p.23 / Chapter 2.2.2 --- Methods / Chapter 2.2.2.1 --- Preparation of Normal Metaphases --- p.30 / Chapter 2.2.2.2 --- Extraction of High Molecular Weight DNA --- p.30 / Chapter 2.2.2.3 --- Labeling of DNA by Nick Translation --- p.31 / Chapter 2.2.2.4 --- Labeling Efficiency --- p.31 / Chapter 2.2.2.5 --- Preparation of Probe --- p.33 / Chapter 2.2.2.6 --- Hybridization --- p.33 / Chapter 2.2.2.7 --- Washing and Detection of Signals --- p.35 / Chapter 2.2.2.8 --- Image Acquisition and Analysis --- p.35 / Chapter 2.2.2.9 --- Control Experiments --- p.36 / Chapter 2.3 --- Positional Mapping of Novel Amplicon by Interphase Cytogenetics --- p.39 / Chapter 2.3.1 --- Fluorescence in situ Hybridization --- p.39 / Chapter 2.3.2 --- Using Yeast Artificial Chromosomes (YAC) as Probe --- p.41 / Chapter 2.3.3 --- Methods --- p.48 / Chapter 2.3.3.1 --- Culture of Yeast Artificial Chromosomes --- p.48 / Chapter 2.3.3.2 --- Extraction of Total YAC DNA --- p.48 / Chapter 2.3.3.3 --- Verification of YAC Clones for Chimerism by FISH --- p.49 / Chapter 2.3.3.4 --- Inter-Alu-PCR --- p.49 / Chapter Chapter 3 --- Assessment of Genetic Changes in HCC by Comparative Genomic Hybridization (CGH) --- p.57 / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and Methods --- p.58 / Chapter 3.2.1 --- Patients and Specimens --- p.58 / Chapter 3.2.2 --- Comparative Genomic Hybridization --- p.60 / Chapter 3.2.3 --- Statistical Analysis --- p.60 / Chapter 3.3 --- Results --- p.61 / Chapter 3.3.1 --- Overall Copy Number Aberrations in 67 HCC and Surrounding Cirrhotic Tissues --- p.61 / Chapter 3.3.2 --- TNM Staging --- p.61 / Chapter 3.3.3 --- Tumour Size --- p.72 / Chapter 3.3.4 --- Serum AFP Elevation --- p.72 / Chapter 3.3.5 --- Chromosomal Aberrations in HCC arising from Cirrhotic and Non- cirrhotic Livers --- p.72 / Chapter 3.4 --- Discussion --- p.73 / Chapter 3.4.1 --- Recurrent Gains --- p.73 / Chapter 3.4.2 --- Recurrent Losses --- p.75 / Chapter 3.4.3 --- Tumour Progression --- p.76 / Chapter 3.5 --- Conclusion --- p.78 / Chapter Chapter 4 --- Positional Mapping of a Novel Amplicon on Chromosome 1q21-q25 by Interphase Cytogenetics --- p.79 / Chapter 4.1 --- Introduction --- p.80 / Chapter 4.2 --- Materials --- p.82 / Chapter 4.3 --- Methods --- p.82 / Chapter 4.3.1 --- Preparation of Paraffin-embedded Tissue Sections --- p.82 / Chapter 4.3.2 --- Verification of YAC Probes for Chimerism --- p.83 / Chapter 4.3.3 --- Hybridization Efficiency of Test and Reference Probes --- p.83 / Chapter 4.3.4 --- Slide Pretreatment and FISH with YAC Probes --- p.88 / Chapter 4.3.5 --- Scoring of FISH Signals --- p.88 / Chapter 4.4 --- Results --- p.89 / Chapter 4.4.1 --- Relative Copy Number Gain --- p.89 / Chapter 4.4.2 --- Intratumour Heterogeneity --- p.90 / Chapter 4.5 --- Discussion --- p.90 / Chapter 4.6 --- Further Studies --- p.104 / Chapter 4.6.1 --- Fine Mapping of Chromosomal Region 1 q21 --- p.104 / Chapter 4.6.2 --- Isolation of Novel Genes in the Amplicon --- p.105 / Chapter 4.6.3 --- Expression Status of the EDC Genes --- p.105 / References --- p.106

Lam Wai-Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves [64]-[73]). / Abstracts in English and Chinese. / Abstract (in English) --- p.i-ii / Abstract (in Chinese) --- p.iii -iv / Acknowledgements --- p.v / Table of contents --- p.vi -ix / List of Figures --- p.x / List of Tables --- p.x / Abbreviations --- p.xi -xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatocellular Carcinoma (HCC) --- p.1-2 / Chapter 1.2 --- Major risk factors of HCC / Chapter (1) --- Hepatitis B Virus (HBV) --- p.2-4 / Chapter (2) --- Hepatitis C Virus (HCV) --- p.5-6 / Chapter (3) --- Cirrhosis --- p.6 / Chapter (4) --- Dietary alfatoxin B1 (AFB1) --- p.6 -7 / Chapter (5) --- Alcoholic consumption --- p.7 / Chapter (6) --- Iron overload --- p.8 / Chapter 1.3 --- Genetic aberrations in HCC --- p.8-9 / Chapter (1) --- Chromosomal loss --- p.10-13 / Chapter (2) --- Chromosomal gains --- p.13-15 / Chapter 1.4 --- roposed study --- p.15 / Chapter (1) --- Hypomethylation of heterochromatin in chromosome 1q copy number gain. --- p.16 / Chapter (2) --- ositional mapping on 1q21 - q22 by interphase cytogenetics. --- p.16-17 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Southern Blot Analysis for Satellite DNA Hypomethylation. --- p.18-19 / Chapter 2.1.2 --- ositional Mapping by Interphase Cytogenetics. --- p.19 -24 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Southern Blot Analysis for Satellite DNA Hypomethylation / Chapter (1) --- Extraction of high molecular weight DNA --- p.25 / Chapter (2) --- DNA digestion with methyl-sensitive restriction enzyme --- p.25 -26 / Chapter (3) --- Control for the complete DNA digestion. --- p.26 / Chapter (4) --- Southern Blotting. --- p.26 -27 / Chapter 2.2.2 --- ositional Mapping by Interphase Cytogenetics / Chapter (1) --- Yeast Artificial Chromosome (YAC) --- p.28 -29 / Chapter (i) --- YAC culturing --- p.29 -30 / Chapter (ii) --- YAC DNA extraction --- p.30 -31 / Chapter (iii) --- Inter-Alu-Polymerase Chain Reaction --- p.32 -33 / Chapter (2) --- -1 derived Bacterial Artificial Chromosome (PAC) --- p.34 / Chapter (i) --- AC culturing and DNA extraction --- p.34 -35 / Chapter (3) --- FISHrobe labeling by nick translation. --- p.35 / Chapter (4) --- FISHrobereparation --- p.36 / Chapter (5) --- Dot-blot analysis. --- p.36 -37 / Chapter (6) --- Verification of the YAC andACrobes by metaphase FISH --- p.37 / Chapter (7) --- Hybridization efficiency test --- p.38 / Chapter Chapter 3 --- Southern Blot Analysis for Satellite DNA Hypomethylation / Chapter 3.1 --- Introduction --- p.39 -40 / Chapter 3.2 --- Materials and Methods / Chapter (1) --- atients --- p.41 / Chapter (2) --- Mathyl-sensitive restriction enzyme digestion. --- p.42 / Chapter (3) --- Classical satellite 2 DNArobe labeling and hybridization. --- p.42 -43 / Chapter (4) --- Membrane washing and signal detection. --- p.43 / Chapter (5) --- Signal detection and reference ratio determination. --- p.43 -44 / Chapter (6) --- Comparative Genomic Hybridization (CGH) --- p.44 -45 / Chapter 3.3 --- Results / Chapter (1) --- Heterochromatin hypomethylation and 1q12 breakpoint. --- p.45 / Chapter (2) --- Heterochromatin hypomethylation in adjacent hepatitis Infected liver tissue. --- p.46 / Chapter 3.4 --- Discussion --- p.47-51 / Chapter Chapter4 --- ositional Mapping of 1q21 - q22 by Interphase Cytogenetics / Chapter 4.1 --- Introduction --- p.52-53 / Chapter 4.2 --- Materials and Methods / Chapter (1) --- atients --- p.53 / Chapter (2) --- YAC clones --- p.53 -54 / Chapter (3) --- AC clones --- p.55 / Chapter (4) --- Formalin-fixedaraffin-embedded tissue sections pretreatment. --- p.55 / Chapter (5) --- Hybridization --- p.56 / Chapter (6) --- Signal detection --- p.56 -57 / Chapter 4.3 --- Results / Chapter (1) --- Relative copy number gain on YAC examined. --- p.57 -59 / Chapter (2) --- AC findings --- p.60 / Chapter 4.4 --- Discussion --- p.60 -63 / References

Human pluripotent stem cells are increasingly used for cell-based regenerative therapies worldwide, with the use of embryonic and induced pluripotent stem cells as potential treatments for a range of debilitating and chronic conditions. However, with the level of chromosomal aneuploidies the cells may generate in culture, their safety for therapeutic use could be in question. This study aimed to develop sensitive and high-throughput assays for the detection and quantification of human pluripotent stem cell aneuploidies, to assess any changes in their positioning in nuclei, as well as investigate the possible roles of lamins in the accumulation of aneuploidies. Using Droplet Digital PCR™, we optimised the detection of aneuploid cells in a predominantly diploid background. An assay was established for the sensitive detection of up to 1% of mosaicism and was used for the monitoring of low-level chromosome copy number changes across different cell lines, conditions and passages in the human pluripotent stem cells. In addition, fluorescence in-situ hybridisation was used to map genes ALB and AMELX on chromosomes 4 and X, respectively, in karyotype-stable chromosome X aneuploid lymphoblastoid cell lines. Our results demonstrated significant alternations in the gene loci positioning in the chromosome X aneuploid cell lines. Using the same established method, the positioning of ALB and AMELX was monitored, alongside the genomic instability with ddPCR™, in the different human pluripotent stem cell lines, conditions and passage. We demonstrated a highly plastic nuclear organisation in the pluripotent stem cells with many changes occurring within a single passage. Furthermore, these results were not exclusive to a single cell line or condition, regardless of the presence or absence of feeder cells and of passage number, and the flexibility of the chromatin organisation remained throughout the duration of the study. We demonstrated high levels of genomic instability with recurrent gains and losses in the AMELX copy number in the human embryonic stem cells during the course of our study, however no significant changes in their gene loci positioning from these abnormalities were observed. xvi | P a g e Additionally, we observed reduced levels of lamin B2 in the aneuploid lymphoblastoid cell lines and complete loss in some hPSC samples. Our results support recent findings that suggest a link between lamin B2 loss and the formation of chromosome aneuploidies in cell culture. In conclusion, our data demonstrates several key novel findings. Firstly, we have established a sensitive technique for the detection of up to 1% mosaicism, which to our knowledge is the most sensitive assay currently available. Secondly, we showed significant changes in the gene loci positioning between aneuploid and diploid cell lines. Thirdly, utilising our novel ddPCR™ assay, we demonstrated the karyotypical instability of hPCSs with consistent gains and/or loses of gene copy numbers in a short period of time in culture. When studying the effects of different growth conditions, we showed that the karyotypical instability was not exclusive to a single condition or a combination of conditions, and what is more, the karyotypical abnormalities detected were not observed to change the gene positioning of hPSCs significantly, with the genome organisation remaining plastic. Finally, our results support a potential association of lamin B2 loss and karyotypical instability. We conclude that more sensitive and robust techniques need to be readily used by clinicians for the screening of potential therapeutic hPSCs.

Leung Ho-yin. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 104-118). / Abstracts in English and Chinese. / Acknowlegements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iv / "Table of Contents," --- p.vi / List of Figures --- p.xi / List of Tables --- p.xii / Abbreviation --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 . --- Cancer Incidences in Hong Kong --- p.2 / Chapter 1.2. --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.3. --- "Etiological Risk Factors," --- p.7 / Chapter 1.3.1. --- Liver Cirrhosis / Chapter 1.3.2. --- Chronic Viral Hepatitis / Chapter 1.3.2.1. --- Hepatitis B Virus (HBV) / Chapter 1.3.2.2. --- Hepatitis C Virus (HCV) / Chapter 1.3.3. --- Dietary Aflatoxin B1 exposure / Chapter 1.3.4. --- Heavy Alcohol Consumption / Chapter 1.3.5. --- Hemochromatosis / Chapter 1.4. --- Genetic Aberration in HCC --- p.12 / Chapter 1.4.1. --- Chromosomal Gains / Chapter 1.4.2. --- Chromosome Losses / Chapter 1.5. --- Epigenetic Changes --- p.18 / Chapter 1.6. --- Aims of Thesis --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter 2.1. --- Materials --- p.23 / Chapter 2.1.1. --- Culture of Cell Lines / Chapter 2.1.2. --- Preparation of Normal Human Metaphase / Chapter 2.1.3. --- DNA Extraction from Cell Lines / Chapter 2.1.4. --- DNA Extraction from Tissues / Chapter 2.1.5. --- DNA Extraction from Blood / Chapter 2.1.6. --- Nick Translation / Chapter 2.1.7. --- Dot Blot / Chapter 2.1.8. --- Probe Preparation / Chapter 2.1.9. --- Fluorochrome-conjugated antibodies / Chapter 2.1.10. --- Fluorescence Microscopy and Image Analysis / Chapter 2.1.11. --- Primer Labeling / Chapter 2.1.12. --- Polymerase Chain Reaction / Chapter 2.1.13. --- Gel Preparation / Chapter 2.1.14. --- Gel Electrophoresis / Chapter 2.2. --- Sample --- p.28 / Chapter 2.2.1. --- Patients / Chapter 2.2.2. --- Cell Lines / Chapter 2.3. --- Comparative Genomic Hybridization --- p.30 / Chapter 2.3.1. --- Method / Chapter 2.3.1.1. --- Preparation of Normal Human Metaphase / Chapter 2.3.1.2. --- DNA Extraction / Chapter 2.3.1.3. --- Nick Translation / Chapter 2.3.1.4. --- Labeling Efficiency / Chapter 2.3.1.5. --- Probe Preparation / Chapter 2.3.1.6. --- Slide Preparation / Chapter 2.3.1.7. --- Hybridization / Chapter 2.3.1.8. --- Post Hybridization Wash / Chapter 2.3.1.9. --- Image Capturing and Analysis / Chapter 2.3.1.10. --- Control Experiment / Chapter 2.4. --- Microsatellite Analysis --- p.46 / Chapter 2.4.1. --- Method / Chapter 2.4.1.1. --- Fluorescent-Labeled Polymorphic Markers / Chapter 2.4.1.1.1. --- Polymerase Chain Reaction / Chapter 2.4.1.1.2. --- Gel Preparation / Chapter 2.4.1.1.3. --- Gel Electrophoresis / Chapter 2.4.1.1.4. --- Data Analysis / Chapter 2.4.1.2. --- Radioisotope-Labeled Polymorphic Markers / Chapter 2.4.1.2.1. --- Primer Labeling / Chapter 2.4.1.2.2. --- Polymerase Chain Reaction / Chapter 2.4.1.2.3. --- Gel Preparation / Chapter 2.4.1.2.4. --- Gel Electrophoresis / Chapter 2.4.1.2.5. --- Autoradiography and Data Analysis / Chapter 3. --- Chapter 3 Genetic Imbalances on Chromosome 1 --- p.55 / Chapter 3.1. --- Introduction --- p.56 / Chapter 3.2. --- Methods --- p.57 / Chapter 3.2.1. --- Patients and Cell Lines / Chapter 3.2.2. --- CGH / Chapter 3.2.3. --- MSA with Fluorescent-labeled Polymorphic Markers / Chapter 3.2.4. --- Refinement of lp36 loss / Chapter 3.2.5. --- Investigation of Homozygous Deletion in lp36 / Chapter 3.3. --- Results --- p.63 / Chapter 3.3.1. --- CGH / Chapter 3.3.2. --- MSA on Primary HCC Cases / Chapter 3.3.3. --- Refinement of lp36 loss / Chapter 3.3.4. --- Investigation of Homozygous Deletion in lp36 / Chapter 3.3.5. --- CGH vs MSA / Chapter 3.4. --- Discussion --- p.74 / Chapter 4. --- Chapter 4 Genetic Imbalances on Chromosome 4q --- p.78 / Chapter 4.1. --- Introduction --- p.79 / Chapter 4.2. --- Methods --- p.82 / Chapter 4.2.1. --- Patients and Cell Lines / Chapter 4.2.2. --- CGH / Chapter 4.2.3. --- MSA with Radioisotope-labeled Polymorphic Markers / Chapter 4.3. --- Results --- p.86 / Chapter 4.3.1. --- CGH / Chapter 4.3.2. --- MSA / Chapter 4.3.2.1. --- MSA on Primary HCC cases / Chapter 4.3.2.2. --- MSA on In-house developed HCC cell lines / Chapter 4.3.2.3. --- Combined MSA Results / Chapter 4.4. --- Discussion --- p.94 / Chapter 5. --- Chapter 5 Proposed Future Studies --- p.99 / Chapter 5.1. --- "Microarray Analysis," --- p.101 / Chapter 5.2. --- Functional Studies --- p.102 / Chapter 6. --- Bibliography --- p.104

Objetivo: Caracterizar as indicações das gestantes que procuram o serviço de Medicina Fetal do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo que realizaram procedimentos invasivos diagnósticos e avaliar os resultados dos cariótipos fetais e destas gestações no período de fevereiro de 2005 a dezembro de 2009. Método: Estudo observacional retrospectivo das gestantes que realizaram biópsia de vilo corial (BVC), amniocentese e cordocentese. Não foram incluídos outros procedimentos diagnósticos ou terapêuticos. O resultado da gestação foi obtido através de consulta de prontuário eletrônico e/ou físico e/ou contato telefônico. Resultados: Foram realizados 113 BVC, 340 amniocenteses e 260 cordocenteses. A principal indicação para a realização dos procedimentos invasivos foi a presença de malformações fetais (69,8%), seguido por translucência nucal aumentada (13,4%) e idade materna avançada (10,2%). A trissomia do cromossomo 18 foi a aneuploidia mais comum (8,1%), seguido pela trissomia do 21 (6,2%), 45,X0 (4,8%) e a trissomia do 13 (3,8%). Ocorreram 4,9% abortamentos, 25,7% natimortos e 13% neomortos. Oito gestantes optaram pela interrupção judicial. 99% das gestantes cujos fetos não apresentavam malformação e que apresentavam cariótipo fetal normal tiveram nativivos. CONCLUSÃO: A principal indicação para a realização dos procedimentos invasivos foi a presença de malformação fetal em 69,8% das gestantes e presença de anormalidades cromossômicas encontradas nos fetos foi de 26,23%. / Objective: The purpose of this study is to characterize the indications of pregnant women who seek the Fetal Medicine Service of Hospital das Clínicas of São Paulo University to perform invasive diagnostic procedures and evaluate the results of fetal karyotypes and their pregnancies from February 2005 to December 2009. Methods: Retrospective observational study of pregnant women who underwent CVS, amniocentesis or cordocentesis. Other diagnostic or therapeutic procedures were not included. The outcomes of pregnancies were obtained through consultation of medical records and/or telephone contact. Results: 113 CVS, 340 amniocentesis and 260 cordocentesis were performed. The main indication for performing invasive procedure was the presence of fetal anomaly (69.8%), followed by increased nuchal translucency (13.4%) and maternal age (10.2%). The trisomy of chromosome 18 was the most common aneuploidy (8.1%), followed by trisomy 21 (6.2%), 45,X0 (4.8%), and trisomy 13 (3.8%). There were 4.9 % of miscarriage, 25.7% of stillbirth and 13% of neonatal deaths. Eight women opted for legal termination of pregnancies. 99% of pregnant women whose fetus had no structural abnormalities and normal karyotype had a live child. CONCLUSION: The main indication for karyotyping was the presence of fetal malformation in 69.8% of pregnancies and chromosomal abnormalities was found in 26.23% of the fetuses.

Cellular immortalization is a major hallmark of cancer and is a multi-step process that requires numerous cell-type specific changes, including inactivation of control mechanisms and stabilization of telomere length. The telomeres at the chromosome ends are essential for genomic stability, and limit the growth potential of most cells. With each cell division, telomeres are shortened. Short telomeres may induce an irreversible growth arrest stage called senescence, or a growth crisis stage characterized by high genomic instability and cell death. Only very rarely do cells escape from crisis and become immortal, a stage that has been associated with the activation of the telomerase enzyme which can elongate and stabilize the telomeres. The processes leading to senescence bypass, growth crisis escape and finally immortalization are only beginning to be elucidated. Most of our knowledge of the immortalization process is based on analyses of human fibroblast and epithelial cell cultures immortalized by genetic modification. In this thesis, spontaneously immortalized human T lymphocytes derived from patients with Nijmegen Breakage Syndrome and a healthy individual were used to identify critical events for senescence bypass and immortalization. Genetic analysis showed a clonal progression and non-random genetic changes including the amplification of chromosomal region 2p13-21 as an early event in the immortalization process. Telomere length gradually shortened at increasing population doublings and growth crisis was associated with critically short telomeres. The clone(s) that escaped growth crisis demonstrated a logarithmic growth curve, very short telomeres and, notably, no increase in telomerase activity or expression of the telomerase catalytic gene, hTERT. Instead, upregulation of telomerase activity and telomere length stabilization were late events in T lymphocyte immortalization. Escape from crisis was associated with downregulation of DNA damage response genes and altered expression of cell cycle regulators and genes controlling the cellular senescence program. These data indicated that a number of layers of regulation are important in the process of immortalization and to provide further mechanistic detail, epigenetic analysis was carried out. Genome wide methylation array analysis identified early and step-wise methylation changes during the immortalization process. Interestingly, applying these findings to tumors of T cell origin revealed commonly methylated CpG sites in transformed cells. Deregulated gene expression of the polycomb complexes may have contributed to the epigenetic changes observed. Taken together, our analysis of spontaneously immortalized T cell cultures identified several steps in the immortalization process including genetic, epigenetic, gene expression and telomere/telomerase regulatory events, contributing further insights to the complexity of cancer cell immortalization.