Made available in DSpace on 2016-08-10T10:38:58Z (GMT). No. of bitstreams: 1 ISABELLA LACERDA FERNANDES.pdf: 1467834 bytes, checksum: 985b4aa7bf961993ef7672d3838bc086 (MD5) Previous issue date: 2015-03-31 / Microsatellite markers are short sequences, repetitive, highly polymorphic and hereditary present in the DNA, which follow the Mendelian pattern of segregation. Due to its haplotype heritage has been used to trace the paternal line to be passed from generation to generation without any changes, except in cases of mutation. The stepwise mutation model is more acceptable to mutation in microsatellite markers, assuming that each mutational event the length of a microsatellite changes by one or a few repeating units due to slippage process, which occurs during replication DNA. This study aimed to estimate the rates of change of microsatellite markers of the Y chromosome in a sample of the population and its implications in human identification process. It is a molecular study, which was conducted at Biocroma Laboratory in partnership with LaGene and Replicon in Goiânia-Goiás. Samples of study were selected from 80 cases of investigation of paternity by DNA analysis, undergo mutation analysis in the Y chromosome haplotypes with molecular amplification system PowerPlex® Y23 System - Promega Corporation. Were identified 15 records of germline mutations in the Y chromosome between alleged parents and children related to suspected samples. The results have identified 9 mutations gain and 6 mutations loss of repetitions numbers. The DYS576 marker had the highest number of reported mutations (20%), followed by DYS570, which identified two mutations (13.33%). The markers DYS389 II, DYS391, DYS481, DYS549, DYS438, DYS439, DYS393, DYS458, DYS385 a - b DYS456 showed only 1 (6.66%) mutation record each. In the other markers, DYS389 I, DYS448, DYS19, DYS533, DYS437, DYS635, DYS390, DYS392, DYS643 and Y-GATA-H4 mutations were not identified in the samples analyzed in this study. Thus the identification of mutations increases the tools that are used in genetic analysis link laboratories and can deliver a more reliable result minimizing potential errors in the analyzes. / Os marcadores microssatélites são sequências curtas, repetitivas, altamente polimórficas e hereditárias presentes no DNA, que seguem o padrão mendeliano de segregação. Devido a sua herança haplotípica tem sido utilizado para rastrear a linhagem paterna por ser passado de geração em geração sem nenhuma alteração, exceto em casos de mutação. O modelo step-wise mutation é o mais aceito para mutação nos marcadores microssatélites, admitindo-se que, a cada evento mutacional o comprimento de um microssatélite altera por uma ou poucas unidades de repetição devido ao processo de slippage, que ocorre durante a replicação do DNA. Este trabalho teve como objetivo estimar as taxas de mutações dos marcadores microssatélites do cromossomo Y em uma amostra da população brasileira e suas implicações no processo de identificação humana. Trata-se de um estudo molecular, que foi conduzido no Laboratório Biocroma em parceria com o LaGene e Replicon em Goiânia-Goiás. As amostras de estudo foram selecionadas de 80 casos de investigação de paternidade pela análise do DNA, submetidos a análise de mutações nos haplótipos do cromossomo Y com o sistema de amplificação molecular PowerPlex® Y23 System Promega Corporation. Foram identificados 15 registros de mutações germinativas no cromossomo Y entre supostos pais e supostos filhos referentes às amostras analisadas. Os resultados obtidos permitiram identificar 9 mutações de ganho e 6 mutações de perda de números de repetições. O marcador DYS576 apresentou o maior número de mutações registrados (20%), seguido pelo DYS570, que permitiu identificar 2 mutações (13,33%). Os marcadores DYS389 II, DYS391, DYS481, DYS549, DYS438, DYS439, DYS393, DYS458, DYS385 a-b e DYS456 apresentaram apenas 1 (6,66%) registro de mutação cada. Nos demais marcadores, DYS389 I, DYS448, DYS19, DYS533, DYS437, DYS635, DYS390, DYS392, DYS643 e Y-GATA-H4 não foram identificadas mutações nas amostras analisadas neste estudo. Desta forma a identificação das mutações aumenta as ferramentas que são utilizadas nos laboratórios de análise de vínculo genético e que podem garantir um resultado mais confiável minimizando possíveis erros nas análises.

Made available in DSpace on 2016-08-10T10:39:20Z (GMT). No. of bitstreams: 1 Ricardo Goulart Rodovalho.pdf: 490214 bytes, checksum: 86f31d10876ac161981506bf61d01362 (MD5) Previous issue date: 2008-03-27 / Male infertility is under a difficult condition of treatment, because it is not a single entity, but reflecting a variety of different pathological conditions, preventing a unique strategy of treatment. Structural changes in Y chromosome have been responsible for male infertility. We examined 26 family members of 13 patients with male infertility and had deletions in the AZF region. In the family 01, a father and a brother did not present a microdeletion. However, one son present a microdeletion in AZFa (sY84) and spermogram azoospermic but the another son present a microdeletion in AZFa (sY84) and AZFb (sY127) and a normal spermogram. The father of the family 02, a severe oligozoospermic man, presented a microdeletion in AZFa (sY84) and his son, conceived by ICSI process, also presented the same microdeletion. In the other families, only the men with changed spermogram had presented the microdeletion. Probably, in family 01, the father and the brother without microdeletions can to present microdeletions of previous or posterior regions to that one analyzed. The treatment with ICSI can lead to the vertical transmission of microdeletions in AZF region and also it can cause in the expansion of the mutation de novo . This result reinforces the need for an investigation of Y chromosome microdeletion in individual candidates for assisted reproduction, as well as a tracking genetic counseling. / A infertilidade masculina é considerada uma condição de difícil tratamento, o que ocorre pelo fato dela não ser uma entidade única, mas refletir uma variedade de diferentes condições patológicas, dificultando uma estratégia única de tratamento. Alterações estruturais no cromossomo Y têm sido o principal responsável pela infertilidade masculina. Nós investigamos 26 familiares de 13 pacientes portadores de infertilidade masculina que apresentaram deleções na região AZF. Na família 01, o pai e um irmão não apresentaram microdeleção. Entretanto um filho apresentou microdeleção em AZFa (sY84) e espermograma azoospérmico, mas o outro filho apresentou microdeleção em AZFa (sY84) e AZFb (sY127) e um espermograma normal. O pai da família 02, oligozoospérmico severo, apresentou microdeleção na região AZFa (sY84) e seu filho, gerado através da ICSI, também apresentou a mesma microdeleção. Nas outras famílias, apenas os homens com espermograma alterado apresentaram a microdeleção. Provavelmente, na família 01, o pai e o irmão sem microdeleção podem apresentar microdeleções em regiões anteriores ou posteriores àquela analisada. O tratamento com ICSI pode levar à transmissão vertical de microdeleções da região AZF e também pode ocasionar na expansão da mutação de novo . Este resultado reforça a necessidade de uma investigação de microdeleção do cromossomo Y em indivíduos candidatos a reprodução assistida, assim como um acompanhamento e aconselhamento genético.

Orientador: George Shigueki Yasui / Resumo: A triploidização é uma ferramenta interessante para produzir peixes estéreis. No Mandi amarelo, Pimelodus maculatus isso pode ser aplicada como uma ferramenta de reconstituição de espécies ameaçadas através de transplante de células germinativastronco. No capítulo I, objetivou-se estabelecer um protocolo eficiente para a triploidização da espécie P. maculatus empregando choques de térmicos. As temperaturas testadas foram de 37°C, 38°C e 39°C, 2 minutos pós-fertilização durante 2 minutos. Os embriões intactos serviram como grupo controle diplóide. A ploidia foi confirmada por citometria de fluxo, diâmetro nuclear dos eritrócitos e citogenética. Taxas de fertilização e sobrevivência foram verificadas nos principais estágios de desenvolvimento embrionário (clivagem, blástula, gástrula, segmentação e eclosão), assim como a porcentagem de larvas normais e anormais, e eficiência da triploidização. O choque térmico reduziu significativamente a sobrevivência no estágio de gástrula (P = 0,0178), somito (P = 0,0469) e incrementou o porcentual de larvas anormais (P = 0,0261). A menor sobrevivência foi observada para o tratamento a 39°C. Todos os tratamentos apresentaram altas porcentagens de indivíduos triploides, sendo o maior valor observado para o choque a 38°C (96,7%). Com base nos resultados acima, foi obtido um eficiente protocolo de triploidização em P. maculatus utilizado choque quente (38°C, 2 mpf e duração de 2 minutos). No capitulo II, foram avaliados aspectos relacionados ao... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Triploidization is an interesting tool to produce sterile fish. In the spotted catfish Pimelodus maculatus, this may be used as a tool for reconstitution of endangered species based on stem germ cell transplantation. In Chapter I, the study aimed to establish an efficient protocol for triploidization in the spotted catfish P. maculatus using temperature shock. The temperatures tested were 37°C, 38°C e 39°C, 2 min postfertilization during 2 minutes. Intact embryos served as diploid control. Ploidy status was confirmed by flow cytometry, nuclear diameter of erythrocytes and karyotyping. Fertilization and hatching rates were verified at the main embryo stages (cleavage, blastula, gastrula, somite stage and hatching), the percentage of normal and abnormal larvae and also the efficacy of triploidization. Heat shock decrease the survival at blastula stage (P= 0,0178), somite (P = 0,0469) and increased the percentage of abnormal larvae (P = 0,0261). The lowest survival was observed at 39 °C. All treatments presented high percentages of triploid individuals, and the highest values were observed for heat shock at 38°C (96,7%). Based on results above, it was obtained an efficient protocol for triploidization in P. maculatus using heat shock (38°C, 2 min postfertilization during 2 minutes). In Chapter II, aspects related to the growth and reproductive performance of diploid (2n) and triploid (3n) of P. maculatus were evaluated. The objective was to evaluate growth according with the plo... (Complete abstract click electronic access below) / Mestre

A tirbo Oryzomyini é a mais especiosa dentre os sigmodontíneos e os estudos citogenéticos nesses roedores refletem tal diversidade exibindo uma gama excepcional de variabilidade cromossômica. O número diploide varia de 2n = 16 até 2n = 88, além disso, algumas espécies apresentam polimorfismos de cromossomos autossômicos e sexuais, assim como a presença de supernumerários. De modo a compreender melhor a variabilidade cromossômica do grupo, o presente trabalho tem como objetivo: fazer uma revisão citogenética da tribo; descrever sete novos cariótipos (Euryoryzomys sp. 2N = 58/FN = 92, Neacomys sp. 1 2N = 48/FN = 54, Neacomys sp. 2 2N = 54/FN = 62, Oecomys sp. 1 2N = 54/FN = 84, Oecomys sp. 2 2N = 64/FN = 92, Oecomys sp. 3 2N = 84/FN = 110 e Scolomys sp. 2N = 62/FN = 80); realizar um estudo de pintura cromossômica utilizando sondas cromossomo-específicas de todo o complemento cromossômico de Holochilus sciureus e alguns autossomos de Oligoryzomys moojeni em quinze espécies de Oryzomyini (Cerradomys vivoi 2n = 50, Euryoryzomys sp. 2N = 58, Holochilus sciureus 2n = 56+2Bs, Hylaeamys megacephalus 2n = 54, Neacomys spinosus 2n = 64, Neacomys sp. 1 2n = 48, Neacomys sp.2 2n = 54, Nectomys rattus 2n = 52+1B, Nectomys squamipes 2n = 56+2Bs, Oecomys sp. 1 2n = 54, Oecomys sp. 2 2n = 64, Oligoryzomys moogeni 2n = 70, Pseudoryzomys simplex 2n = 56, Scolomys sp. 2N = 62 e Sooretamys angouya 2n = 58+2Bs); e por fim fazer uma análise filogenética da tribo baseada em dados de pintura cromossômica. Os resultados mostraram uma intensa reorganização genômica envolvendo inversões pericêntricas e/ou reposicionamento centromérico, inversões paracêntricas, rearranjos Robertsonianos, fusões e/ou fissões em tandem e translocações envolvidas na diversidade e evolução cromossômica de Oryzomyini. A utilização de duas abordagens diferentes na análise filogenética mostrou qual é mais confiável e apresenta resultados similares aqueles resultantes das análises morfológicas e moleculares. Além disso, os cromossomos sexuais dessas espécies apresentaram regiões homólogas compartilhadas entre todas as espécies analisadas com amplificação espécie-específica de heterocromatina / Oryzomyini is the most specious tribe of Sigmodontinae subfamily and cytogenetic studies of these rodents reflect such diversity displaying an exceptional range of karyotype variability. Diploid number vary from 2n = 16 to 2n = 88, in addition, some species present autosomal and sex chromosomes polymorphisms, besides the presence of B chromosomes. In order to understand the actual karyotype variability of Oryzomyini we present: a cytogenetic review of the tribe in order to rescue all chromosomal data available for the group; the description of seven new karyotypes (Euryoryzomys sp. 2N = 58/FN = 92, Neacomys sp. 1 2N = 48/FN = 54, Neacomys sp. 2 2N = 54/FN = 62, Oecomys sp. 1 2N = 54/FN = 84, Oecomys sp. 2 2N = 64/FN = 92, Oecomys sp. 3 2N = 84/FN = 110, and Scolomys sp. 2N = 62/FN = 80); a genome-wide comparative study using whole chromosome probes of the entirely chromosome set of Holochilus sciureus and some autosomes of Oligoryzomys moojeni in metaphases of fifteen Oryzomyini species (Cerradomys vivoi 2n = 50, Euryoryzomys sp. 2N = 58, Holochilus sciureus 2n = 56+2Bs, Hylaeamys megacephalus 2n = 54, Neacomys spinosus 2n = 64, Neacomys sp. 1 2n = 48, Neacomys sp.2 2n = 54, Nectomys rattus 2n = 52+1B, Nectomys squamipes 2n = 56+2Bs, Oecomys sp. 1 2n = 54, Oecomys sp. 2 2n = 64, Oligoryzomys moogeni 2n = 70, Pseudoryzomys simplex 2n = 56, Scolomys sp. 2N = 62, and Sooretamys angouya 2n = 58+2Bs) and; a phylogenetic analysis of Oryzomyini using chromosome painting data. The results showed an extensive chromosomal rearrangement, such as pericentric inversions or centromeric shift, paracentric inversions, Robertsonian rearrangements, tandem fusions/fissions, and translocations involved in the karyotype diversity and evolution of Oryzomyini. The use of two different approaches to perform a phylogenetic analysis based on chromosome painting data revealed which one is more trustworthy and present results more similar with molecular and morphological analysis. In addiction, the sex chromosomes of these species present homologous regions between all analysed species with amplification of species-specific heterochromatin

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

Atualmente muitas são as técnicas utilizadas na área de citogenética para que os desequilíbrios genéticos potencialmente hereditários sejam identificados ou evitados. Algumas das técnicas são utilizadas apenas para o diagnóstico de enfermidades em humanos, mas seria importante que fossem aplicadas também aos animais domésticos, com a finalidade de se associar uma causa genética ao aparecimento de algumas doenças, como por exemplo, os mastocitomas. Os mastocitomas são formações cutâneas neoplásicas originadas de mastócitos e se apresentam com frequência em cães. Algumas raças apresentam maior susceptibilidade ao aparecimento de mastocitoma, e o Boxer tem a maior incidência nesta espécie. Este trabalho objetivou avaliar comparativamente o cariótipo de cães da raça Boxer e buscar uma possível correlação com o desenvolvimento de mastocitoma. Foram utilizadas técnicas citogenéticas de bandamentos C, G e Ron além de análises em coloração convencional (Giemsa). Para tais análises utilizaram-se células cultivadas de linfócitos periféricos e de mastocitomas de Boxer. O material foi analisado e fotografado em microscópio de imersão da marca Zeiss®, em objetiva de 100x, com filtro verde, equipado com software de análise citogenética Ikaros®. Foram consideradas alterações numéricas e estruturais. Foram analisadas 828 metáfases mitóticas provenientes de linfócitos periféricos e células de mastocitomas. A análise das metáfases provenientes de cultura de células dos linfócitos periféricos não apresentou alterações estruturais e a análise numérica mostrou somente uma metáfase com 2n=77, dentre 476 metáfases analisadas. Na análise das metáfases de células tumorais, entre todas as colorações, observou-se que 70,9% apresentou 2n=78, 17,4% mostraram 2n=77; 9,8% 2n=76. Cinco metáfases (1,3%) apresentaram 2n=79 e duas células apresentaram 2n=75. Todas essas alterações numéricas estão relacionadas com alterações estruturais do tipo fusão cêntrica. Os resultados obtidos mostram que devido ao crescimento descontrolado, as células tumorais promovem rearranjos na tentativa de controlar a divisão celular. Ainda, o aprimoramento das técnicas diagnósticas em citogenética promove o conhecimento da biologia tumoral do mastocitoma canino, e consequentemente, possibilita o desenvolvimento de novas estratégias terapêuticas. / Nowadays there are many techniques applyed in cytogenetics in order to identify or avoid potentially hereditary genetic imbalance. Some of the techniques are used only to diagnose human diseases, but it would be important to apply them also to domestic animals, with the purpose of associating a genetic cause with the appearance of some diseases like, for example, mastocytomas. The mastocytomas are neoplasics cutaneous formation originated from mastocitos and frequently appears in dogs. Some breeds show higher susceptibity to the appearance of mastocytoma, and the boxer breed has shown the highest incidence. The objective of this study is to comparatively evaluate the boxer breed chariotype and search for a possible correlation with the mastocytoma development. There has been utilized cytogenetics bandamentos C,G e Ron as well as conventional (Giemsa) coloring analysis. For those analysis there has been utilized cultivated cells from peripheral lynphocites and boxer´s mastocytomas. The material as been analyzed and photographed in a Zeiss immersion microscope, using a 100x objective with green filter, equipped with Ikaros cytogenetic analysis software. It has been considered numerical and structural alterations. There has been analyzed 828 mithotics metaphases from peripheral lynphocites and mastocytomas cells. The analysis of metaphases from the culture of peripheral lynphocites cells did not show structural alterations and the numerical analysis has shown only one metaphase 2n=77, among 476 analyzed metaphases. In the tumoral metaphase cells analysis, among all colorations, it has been observed that 70,9% presented 2n=78, 17,4% shown 2n=77 and 9,8% 2n=76. Five metaphases (1,3%) presented 2n=79 and two cell presented 2n=75. All these numerical alterations are related with structural alterations from the centric fusion type. The obtained results shows that due to the uncontrolled growth, the tumoral cells promote rearrangements in an attempt to control a cellular division. Yet, the improvement of diagnosis technique in cytogenetics promotes the knowledge of canine mastocytoma tumoral biology, and consequently allows the new therapeutic strategy development.

Uma população experimental F2 foi desenvolvida a partir do cruzamento de sete machos de uma linhagem não endogâmica de frangos de corte (TT), com sete fêmeas de uma linhagem não endogâmica de postura (CC), gerando vinte famílias F1 TC, com aproximadamente 100 progênies F2 cada obtidas em 17 incubações. Foram obtidos dados fenotípicos para vinte e oito características de desempenho e qualidade da carcaça em 2063 aves F2. As aves F1 TC foram genotipadas para 30 marcadores microssatélites posicionados nos cromossomos 3 e 5 para determinar o grau de informação dos marcadores. Os marcadores informativos foram empregados na genotipagem seletiva das aves F2 que apresentaram valores fenotípicos extremos (4,5 % superiores e 4,5 % inferiores), dentro de cada uma das 20 famílias, para a característica peso vivo aos 42 dias de idade ajustado (PV42aj). Os genótipos obtidos foram comparados através do teste de qui-quadrado. Foram encontradas seis regiões no cromossomo 3 e três regiões no cromossomos 5 com marcadores apresentando ligaçãio sugestiva (P < 0,10) com QTL para PV42aj. Foram selecionadas seis famílias mais informativas para a maioria dos marcadores significativos, e 90 progênies F2 de cada família (n=540) foram genotipadas. Os genótipos foram empregados para construção de mapas de ligação específicos para os cromossomos. Os fenótipos ajustados para efeitos fixos, os mapas de ligação e os genótipos foram empregados para mapeamento de QTL por análise de regressão, utilizando o programa QTL Express. Foram encontrados no cromossomo 3 10 QTL siginificativos para peso corporal aos 35, 41 e 42 dias de idade, para ganhos de peso do nascimento aos 35, 41 e 42 dias de idade, para peso de asas e coxas com sobrecoxas, e para peso de gordura abdominal e porcentagem de gordura. Foram observados um QTL no cromossomo 3 com ligação sugestiva para peso de pés, e três QTL no cromossomo 5 para consumo de ração, peso do coração, peso de gordura abdominal e porcentagem de gordura abdominal. Não houve interações significativas do QTL com efeito de família ou sexo. Os QTL significativos para peso de gordura abdominal e porcentagem de gordura abdominal apresentaram forte efeito de imprinting gamético. Os efeitos dos QTL foram na sua maioria aditivos, com o alelo originado da linhagem de corte aumentando o valor para a característica. Características de carcaça apresentaram efeito aditivo negativo, indicando que os alelos provenientes da linhagem de postura diminuíram o valor dessas características. A população experimental foi adequada para o mapeamento de QTL significativos para características de desempenho e carcaça no cromossomo 3, e QTL sugestivos para características de carcaça no cromossomo 3 e características de desempenho e carcaça no cromossomo 5. / An F2 chicken population was established from a cross of a broiler-sire line (TT) and an egg laying line (CC). This population was used for detecting and mapping quantitative trait loci (QTL). Over 2000 F2 TC offspring from 17 hatches were reared to slaughter at 6 wk of age. Twenty-eight performance and carcass traits were measured. The DNA were extracted from blood samples and informativeness of 50 selected microsatellite markers along chromosomes 3 and 5 were obtained in F1s. Data of 2063 individuals from 20 families were used to chosen offspring with high and low phenotypes for BW42, for selective genotyping. Twenty markers were used in the complete genotyping of 566 individuals from 6 families most informative. Interval mapping QTL analyses were carried out by regression method, using QTL Express software. Significant QTL at the genome were mapped on chromosome 3 for body weigh at 35, 41 and 42 days, gain between birth and 35, 41 and 42 days, abdominal fat weight, abdominal fat percentage, weight of wings and weight of thighs. Suggestive QTL were identified for weight of feet on chromosome 3 and for abdominal fat weight, abdominal fat percentage, heart weight and feed consumption on chromosome 5. Significant QTL for abdominal fat weight and abdominal fat percentage showed strong imprinting effect. There was no evidence for interactions of the QTL with sex and family, or for two QTL on the same chromosome for any of the traits. Genetic effects were generally additive, with the broiler alleles increasing performance traits. Additive effects were negative for carcass traits, indicating that the layer line decreased these traits. Experimental population was adequate to mapping significant QTL for performance and carcass traits on chromosome 3, and suggestive QTL for carcass traits on chromosome 3 and for performance and carcass traits on chromosome 5.

A epigenética aborda o controle da expressão gênica através de diversos fatores que agem sob a cromatina, os melhor estudados são a metilação do DNA e a acetilação em histonas, relacionadas à repressão e ativação gênica, respectivamente. Em mamíferos, existem dois fenômenos epigenéticos interessantes: a inativação do cromossomo X (ICX) em fêmeas, que garante o equilíbrio transcricional gênico entre os sexos, e o imprinting genômico, caracterizado pela expressão monoalélica dependente da origem parental. No presente estudo, propusemos verificar a manutenção do controle epigenético em células humanas normais e transformadas em condições semelhantes de hipometilação do DNA e hiperacetilação em histonas (após uso das drogas 5-aza-2-\'deoxicitidina (5-aza-dC) e ácido valproico, respectivamente), através do monitoramento da expressão alelo-específica pelo uso de polimorfismos de única base presentes em regiões codificadoras. Em células normais houve manutenção da ICX e do imprinting genômico, enquanto que em células transformadas hipometiladas foram observadas indução de XIST, e perda de imprinting dos genes IGF2, H19 e PEG10. Observamos que ambas as drogas podem diminuir a expressão de DNMT1, e 5-aza-dC altera o equilíbrio entre acetilação e desacetilação da histona H4. Ainda, a ordem de adição dos reagentes ocasionou diferenças no nível de acetilação da histona H4 e na expressão gênica de XIST e PEG10. Nossos dados sugerem que: células humanas normais apresentam maior estabilidade do controle epigenético comparadas às células humanas transformadas, genes submetidos à ICX e \"imprintados\" não apresentam diferenças na rigidez do controle de expressão, e a cascata de reação seguida após perturbação de marcas epigenéticas pode ser alterada dependendo da modificação inicial. / Epigenetics refers to mechanisms related to gene activity through conformational modifications in DNA without changes in the nucleotide sequence. Key players in the epigenetic control are DNA methylation and histone acetylation, which are related to gene activation and repression, respectively. Two striking epigenetic phenomena in mammalians are X chromosome inactivation (XCI) and genomic imprinting. XCI triggers the transcriptional silencing of all but one X chromosome in each female cell, while genomic imprinting is a process that leads to mono-allelic gene expression based on parental origin. In the present study, we intended to verify the maintenance of epigenetic control in normal and transformed human cells under the same conditions of epigenetic disturbance. For this purpose, 5-aza-2\'-deoxycytidine (5-aza-dC) and valproic acid (VPA) were used to cause DNA hypomethylation and histone hyperacetylation, respectively. By monitoring allelic-specific expression using single nucleotide polymorphisms present in coding regions, we were able to check the effects of the modifications in the expression pattern of imprinted or subjected to XCI genes. While in female normal cells XCI and genomic imprinting were not affected by VPA or 5-aza-dC treatments, transformed male cells showed XIST activation and loss of imprinting of PEG10, IGF2 and H19 genes in the hypomethylation scenario. In addition, both drugs can decrease the expression of DNMT1, and 5-aza-dC alters the balance between acetylation and deacethylation of histone H4. Furthermore, we could see different degrees of histone H4 acetylation levels and of XIST and PEG10 expression, depending on which of the drugs was added first. Our data suggest that the epigenetic control in normal human cells is more stable when compared to transformed human cells. In addition, both XCI and genomic imprinting are epigenetic features equally hard to disturb. Finally, depending on the initial epigenetic modification (global demethylation or acethylation), it will induce different epigenetic control networks, with consequence to the final status of gene expression.

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