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Aspectos preditivos da progressão da lesão de NIC 1 em biopsias do colo uterino = estudo comparativo entre o metodo da hibridização in situ e a expressão do Ki67, avaliada por metodos morfologicos quantitativos / NIC lesion progression in cervical biopsies : predictive value of in situ hybridization technique versus Ki67 expression, as evaluated by quantitative morphological methodsTriglia, Renata de Marchi 02 March 2010 (has links)
Orientadores: Liliana Aparecida Lucci de Angelo Andrade, Konradin Metze / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T06:41:47Z (GMT). No. of bitstreams: 1
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Previous issue date: 2010 / Resumo: As Neoplasias Intra-epiteliais Cervicais (NIC) são precursoras do carcinoma do colo uterino e, em sua maioria, causadas pelos vírus do Papiloma Humano (HPV) de alto risco oncogênico. A maior parte das NIC1 regride, mas cerca de 15% pode progredir e a integração viral ao genoma da célula tem papel importante neste evento. A hibridização in situ (HIS) detecta o tipo de vírus na lesão e, tem sido atribuído que o sinal difuso desta reação se relaciona à forma epissomal do vírus, enquanto o puntiforme representa o DNA viral integrado ao genoma. O Ki67, marcador de proliferação celular, tem sido usado na graduação das NIC e pode ser um adjuvante na avaliação do comportamento das NIC1. Métodos: 74 biópsias de NIC1 foram estudadas, cujas pacientes apresentaram no acompanhamento biópsias com diagnósticos variados: 19 de cervicite (regressão), 29 com manutenção de NIC1 e 26 com progressão para NIC 2-3. A primeira biópsia foi analisada pela reação de HIS para HPV de alto risco oncogênico (Genpoint®) e pela reação imunoistoquímica para Ki67. Na HIS foram avaliados o número e a distribuição das células nas camadas epiteliais (basal, intermédia ou superficial) e o tipo de sinal (puntiforme ou difuso). A análise do Ki67 foi realizada de 2 formas: a) leitura com avaliação subjetiva da expressão em diferentes níveis do epitélio (camadas basal e para-basal, até a metade e mais da metade da espessura epitelial), b) análise morfológica quantitativa por um programa de computador ("Sociology"), capaz de fazer muitas medidas, como densidade nuclear e relações de vizinhança entre os núcleos. Resultados: a idade da pacientes variou de 16 a 65 anos, sendo a média diferente entre os grupos com e sem progressão (31 e 26, respectivamente, p=0,02). A HIS foi positiva em 22 casos (30%), dos quais 8 progrediram e 14, não. Todos mostraram sinais puntiformes e difusos, entretanto sinal puntiforme na camada basal foi observado em 58,5% dos que progrediram e em apenas 4% dos casos sem progressão (p=0,05). A avaliação subjetiva do Ki67 demonstrou que o
grupo que progrediu expressou positividade em mais da metade do epitélio (p=0.02). A análise morfológica quantitativa do Ki67 evidenciou os seguintes resultados significativos no grupo com regressão: menor densidade nuclear, menor quantidade de vizinhos e maior distância entre os núcleos vizinhos mais próximos (p=0,019; 0,008 e 0,02). As medidas da altura dos núcleos positivos no epitélio e da espessura epitelial não mostraram diferença.
Conclusão: o sinal puntiforme da HIS na camada basal, a expressão do Ki67 em mais da metade do epitélio, avaliada subjetivamente, e a maior densidade de núcleos na análise morfológica quantitativa estão mais associados à manutenção e progressão das lesões de NIC1 / Abstract: Cervical Intraepithelial Neoplasias (CIN) are precursory lesions of the cervical carcinoma, and most of them are caused by high-risk Human Papillomavirus (HPV). The majority of CIN1 can regress, but around 15% progress, and viral integration has an important role in this event. Integration can be identified by In situ hybridization (ISH). Literature has considered a punctate signal in ISH as integrated HPV, and the diffuse signal as episomal virus. Ki67, a marker of cellular proliferation, can be useful in graduating CIN and evaluating CIN1 behavior. Methods: 74 CIN1 cases were studied and all patients had another biopsy in the follow-up which revealed: 19 without CIN (regression), 29 with maintenance of CIN1 diagnosis, and 26 that progressed to CIN2-3. ISH with Genpoint® for high risk HPV was evaluated according to the distribution of positive cells at different levels in squamous epithelium: basal, intermediate or superficial layers. The pattern of ISH reaction was classified as diffuse or punctate. Ki67 analysis was performed by 2 different ways: a) interpretation with subjective estimation of the immunostaining in different epithelium levels (basal and parabasal layers, until a half and more than a half of the epithelium thickness); b) quantitative morphological analysis by a software ("Sociology"), capable of performing many measurements, as nuclear density and neighborhood relations among the nuclei. Results: the age varied from 16 to 65 and mean age without progression was 26 and 31 for progression (p=0.02). ISH was positive in 30% (22 cases: 8 with lesion progression and 14 without). All cases showed punctate and diffuse signals, but punctate were observed in 58.5% with progression and only in 4% without (p=0.05). Ki67 subjective analysis revealed that group with progression expressed positive nuclei in more than a half of the epithelium thickness (p=0.02). In the regression group, Ki67 quantitative morphological analysis showed smaller nuclei density, a lower quantity of positive cells and a longer distance between neighbor cells when compared to the others (p=0,019; 0,008 e 0,02). The height of Ki67 positive nuclei and the epithelial thickness were not different among the 3 groups. Conclusion: an ISH punctate signal in the basal layer, Ki67 expression in more than a half of epithelium thickness and the nuclear Ki67 density evaluated quantitatively can be related to CIN1 maintenance and progression / Doutorado / Anatomia Patologica / Doutor em Ciências Médicas
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Estudos cariotípicos em Griffinia Ker Gawl e espécies relacionadas (Amaryllidaceae) / Karyotypic studies in Griffinia Ker Gawl and related species (Amaryllidaceae)Engel, Thaíssa Brogliato Junqueira, 1989- 24 August 2018 (has links)
Orientador: Eliana Regina Forni Martins / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T14:21:30Z (GMT). No. of bitstreams: 1
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Previous issue date: 2014 / Resumo: O gênero Griffinia Ker Gawl pertence à subfamília Amaryllidoideae que, junto às subfamílias Agapanthoideae e Allioideae, compõem a família Amaryllidaceae, com cerca de 73 gêneros e 1605 espécies. As Amaryllidaceae, incluindo as Griffinia, são apreciadas pelas suas flores e cultivadas para a jardinagem e ornamentação. Endêmico do Brasil, esse importante gênero está ameaçado de extinção pela constante degradação de seu ambiente natural, sendo que muitas espécies não foram mais encontradas na natureza e nem em cultivo. A taxonomia de Griffinia é bastante dificultada pela morfologia floral e vegetativa bastante semelhante entre algumas espécies, e pela variação morfológica dentro de uma mesma espécie, ocasionada pelo isolamento entre populações e pela existência de diferentes citótipos. Os objetivos deste trabalho foram obter o cariótipo de diferentes espécies de Griffinia e de espécies proximamente relacionadas, e fornecer à sistemática informações que auxiliem na compreensão das relações filogenéticas e evolutivas das espécies desse gênero. Foram estudadas 10 espécies e duas morfoespécies não identificadas de Griffinia, bem como quatro espécies de gêneros próximos. Pontas de raízes coletadas dessas espécies foram pré-tratadas em colchicina e fixadas em solução Farmer para a produção de lâminas com metáfases mitóticas. Foram determinados número e morfologia cromossômicos e realizados bandamentos com os fluorocromos cromomicina3 (CMA3) e 4',6-diamidino-2-fenilindol (DAPI), e in situ fluorescent hybridization (FISH) com DNA ribossomal (DNAr 5S). Todas as espécies de Griffinia apresentaram 2n=20. Os números cromossômicos para as espécies analisadas de Eithea, Hippeastrum, Tocantinia e Worsleya foram 2n=18, 44, 22, e 42 respectivamente. Foram observadas espécies de Griffinia com duas, quatro, cinco e seis bandas CMA3+. De um a três sítios de DNAr 5S foram observados em dois a seis pares cromossômicos, em posição terminal, subterminal ou pericentromérica. Para cada espécie, foi observado um padrão único de distribuição de sítios de DNAr 5S, sendo assim possível a delimitação de espécies por meio das técnicas citogenéticas aplicadas. As espécies do grupo reportado na literatura como complexo Liboniana apresentaram semelhanças cariotípicas que podem corroborar a proximidade entre espécies: possuem apenas um par cromossômico com banda CMA3+ e dois pares cromossômicos com sítios de DNAr 5S. Variações cariotípicas foram observadas não apenas entre as espécies, mas também entre populações de uma mesma espécie. Casos de variação intraespecífica são conhecidos para plantas. Contudo, essa variação pode não ser uma variação interpopulacional, mas sim, reflexo da dificuldade taxonômica no gênero. Os resultados obtidos nesse estudo, apontam a citogenética como uma ferramenta útil na delimitação dos gêneros e espécies, e no reconhecimento de grupos dentro de Griffinia. / Abstract: The genus Griffinia Ker Gawl belongs to the subfamily Amaryllidoideae which together with the subfamilies Agapanthoideae and Allioideae, forms the Amaryllidaceae family, with about 73 genera and 1605 species. Amaryllidaceae plants, including Griffinia, are aprecciated because of their flowers and cultivated for gardening and ornamentals. Endemic to Brazil, the genus is endangered by the continuous degradation of their natural environment, and many species have not been found in nature. Taxonomy of Griffinia is very complicated because of its vegetative and floral morphology, which are quite similar between some species. There is also some morphological variation within a single species, caused by the isolation between populations and the existence of different cytotypes. The aim of this study was to obtain the karyotype of different species of Griffinia and closely related species, thus providing for systematic studies some information to assist in the understanding of the evolutionary and phylogenetic relationships of the species of this genus. We studied 10 species and two unidentified morphospecies of Griffinia, as well as four species of closely related genera. Root tips collected from these species were pretreated with colchicine and fixed in Farmer solution for the production of slides with metaphasic cells. We determined the number and chromosomal morphology. We performed banding with chromomicin3 (CMA3) and 4',6-diamidino-2-phenilindole (DAPI) fluorochromes and fluorescent in situ hybridization (FISH) with ribosomal DNA (5S rDNA). All Griffinia species presented 20 chromosomes. Chromosome numbers for the analyzed species of Eithea, Hippeastrum, Tocantinia and Worsleya were 2n= 18, 44, 22 and 42 respectively. Griffinia species presented two, four, five or six CMA3+ bands. One to three 5S rDNA sites were observed in two to six chromosome pairs in the terminal, subterminal or pericentromeric position. For each species, there was a unique pattern of distribution of DNAr 5S sites, so it is possible to delimitate species trough this cytogenetic technique. The species of a group reported in the literature as Liboniana complex showed similar karyotypes that can corroborate the closeness among the species of the group: they have only one chromosome pair with a CMA3+ band and two chromosome pairs with 5S rDNA sites. We observed karyotypic variations not only among species but also between populations of the same species. Cases of intraspecific variation are known for plants. However, this variation may be either an interpopulational variation, or a simple reflection of the difficulty in taxonomy of the genus. The data found at this analysis proved cytogenetic studies to be a quite useful tool in the delimitation of genera and species, and recognition of groups within Griffinia. / Mestrado / Biologia Vegetal / Mestra em Biologia Vegetal
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Estudos citogenéticos e taxonômicos em espécies brasileiras de Swartzia Schreb. (Leguminosae-Papilionoideae) = Cytogenetics and taxonomics studies in Brazilian species of Swartzia Schreb. (Leguminosae-Papilionoideae) / Cytogenetics and taxonomics studies in Brazilian species of Swartzia Schreb. (Leguminosae-Papilionoideae)Pinto, Rafael Barbosa, 1985- 23 August 2018 (has links)
Orientadores: Eliana Regina Forni Martins, Vidal de Freitas Mansano / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T00:22:48Z (GMT). No. of bitstreams: 1
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Previous issue date: 2013 / Resumo: Swarzia é um gênero basal da subfamília Papilionoideae (Leguminosae). Apesar do seu posicionamento ter gerado debate entre muitos autores no passado, estudos sistemáticos recentes confirmam a monofilia de Swartzia, compondo o clado swartzióide juntamente com mais sete gêneros. A diversidade morfológica e a ampla distribuição geográfica na região neotropical tornam o gênero um interessante objeto de estudos taxonômicos e sistemáticos. Embora Swartzia apresente centro de diversidade amazônico, também possui alta riqueza de espécies na região extra-amazônica, apresentando complexos de espécies, com difícil delimitação morfológica de alguns táxons, necessitando de ferramentas adicionais para uma melhor compreensão da evolução no grupo. A citogenética, mediante estudos cromossômicos, fornece informações importantes na elucidação de relações supra e infra genéricas e, através de uma abordagem citotaxonômica, pode contribuir para o esclarecimento de problemas sistemáticos e taxonômicos. O presente trabalho visa ampliar os estudos do gênero, contribuindo com um inédito estudo citogenético e ampliando estudos taxonômicos das Swartzia na região extra-amazônica brasileira. Para o capítulo 1 foram coletadas sementes de 18 espécies distribuídas no território brasileiro para análise cromossômica e no capítulo 2 é apresentado um estudo taxonômico de Swartzia na região extra-amazônica brasileira, com chave de identificação. Swartzia apresentou número cromossômico constante entre as espécies analisadas (2n=2x=26). Entretanto, S. leptopetala demonstrou potencial de autopoliploidização ao apresentar sementes 2n=2x=26 e 2n=4x=52 numa mesma árvore, configurando processos de poliploidização em meristemas isolados. O tamanho dos cromossomos (tamanho relativo dos cromossomos e comprimento total de cromatina - TCL) foi medido para todas as 18 espécies coletadas. No geral, os cromossomos são pequenos, sendo o menor cromossomo encontrado em S. acuminata (0.25?m), enquanto o maior foi encontrado em S. euxylophora (1.41?m). Os números de bandas CMA+/DAPI- (2) e sítios de rDNA 45S (2) e 5S (2) também não apresentaram variação interespecífica. Swartzia euxylophora, cuja inclusão no gênero havia sido anteriormente questionada, apresentou as características citogenéticas semelhantes às demais Swartzia e, somadas à morfologia observada em campo, sustentam o posicionamento do táxon dentro do gênero. Os dados cariotípicos (número e tamanho cromossômicos, e número de bandas CMA/DAPI e de genes ribossomais) não permitem a diferenciação das espécies em nível de sessão. Até o momento são disponibilizadas informações cromossômicas para cerca de 10% das Swarztia, não sendo possível sugerir mecanismos de evolução cariotípica no gênero. Mediante a análise dos dados citogenéticos fornecidos neste trabalho e disponíveis na literatura é possível afirmar que os gêneros do clado swartzióide apresentam números cromossômicos diferentes, sendo este um caráter diagnóstico. No capítulo 2, os estudos taxonômicos para as Swartzia extra-amazônicas resultaram na descrição de cinco novas espécies e na elaboração de uma chave de identificação para táxons da região. Quatro delas, S. alagoensis, S. arenophila, S. revoluta e S. submontana, pertencem à seção Acutifoliae que se destaca por possuir alta diversidade e por ser exclusivamente brasileira. Swartzia thomasii pertence à seção Glabriplantae, anteriormente uma seção exclusivamente amazônica / Abstract: Swartzia is a basal genus of subfamily Papilionoideae (Leguminosae). Although the positioning of the genus has been a controversial issue among some authors in the past, recent systematic studies confirm the monophily of Swartzia as being part of a swartzioid clade with other seven genera. The morphological diversity and the widespread geographical distribution at the Neotropical region, make the genus an interesting object of taxonomic and systematic studies. Although Swartzia present an amazonian diversity center, it also has high species richness at extra-amazonian region, presenting species complex, with hard morphological delimitation of some taxa, requiring additional tools for a better comprehension of evolution within the group. Cytogenetics, by studying chromosomes, provides important informations for elucidating supra- and infrageneric relations and, by a citotaxonomic approach, it can contribute to solve systematic and taxonomic problems. The present study aims to increase the studies of the genus, contributing with an inedit cytogentic study and extending taxonomic studies of Brazilian extra-amazonian Swartzia. For chapter 1, there were collected 18 species distributed through Brazilian territory for chromosomal analyses and in chapter 2 we presenting a taxonomic study of Swartzia in extra-Amazonian region of Brazil with a description key. Swartzia presented a conservated chromosome number among species (2n=2x=26). However, S. leptopetala demonstrated an autopolyploidization potential, presenting seeds with 2n=2x=26 and 2n=4x=52 in the same tree, being a polyploidization process in isolated meristems. Chromosome length (relative chromosome length and total chromatin length - TCL) were measured for all 18 species collected. In general, Swartzia chromosomes are small, being the shortest chromosome found in S. acuminata (0.25?m) and the longest in S. euxylophora (1.41?m). The number of CMA+/DAPI- bands (2) and rDNA sites 45S (2) and 5S (2) did not present interspecific variation too. Swartzia euxylophora, which the inclusion in the genus was questioned, presented all cytogentics characteristics similar to all other analyzed Swartzia and together with morphological features observed in the field, it supports the taxon as belonging to the genus. The karyotypic data (number and size of chromosomes, and number of CMA/DAPI and ribosomal genes) do not allow the differentiation of species at section level. Until now, there is chromosomal information for about 10% of Swartzia species available, not being possible suggest karyotypic evolution mechanisms in the genus. By analyzing cytogentic data provided by this study and available in the literature, it is possible to say that genera in swartzioid clade have different chromosome number, being a diagnostic character. In chapter 2, the taxonomic studies of extra-Amazonian Swartzia resulted in a description of five new species and the elaboration of a regional key for the regional taxa. Four of them, S. alagoensis, S. arenophila, S. revoluta and S. submontana, belong to section Acutifoliae, which stands out for having high diversity and for be exclusively Brazilian. Swartzia thomasii belongs to section Glabriplantae, otherwise an exclusively Amazonianian section / Mestrado / Taxonomia Vegetal / Mestre em Biologia Vegetal
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Estudo cromossômico da tribo Dalbergieae sensu Klitgaard & Lavin (2005) com ênfase no clado Dalbergia s. str. (Leguminosae, Papilionoideae) = Chromosome studies of the tribe Dalbergieae sensuKlitgaard & Lavin (2005) with emphasis on Dalbergia sensu strictoclade (Leguminosae, Papilionoideae) / Chromosome studies of the tribe Dalbergieae sensuKlitgaard & Lavin (2005) with emphasis on Dalbergia sensu strictoclade (Leguminosae, Papilionoideae)Polido, Caroline do Amaral, 1983- 23 August 2018 (has links)
Orientadores: Eliana Regina Forni Martins, Ana Paula de Moraes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T11:58:38Z (GMT). No. of bitstreams: 1
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Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital quando for liberada / Abstract: The abstract is available with the full electronic document when available / Doutorado / Biologia Vegetal / Doutora em Biologia Vegetal
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Investigação laboratorial da síndrome velocardiofacial e possíveis fenocópias / Laboratory investigations of the velocardiofacial syndrome and phenocopies possibleSgardioli, Ilária Cristina 19 August 2018 (has links)
Orientador: Vera Lúcia Gil da Silva Lopes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T02:52:06Z (GMT). No. of bitstreams: 1
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Previous issue date: 2011 / Resumo: A Síndrome Velocardiofacial (SVCF), uma das formas do espectro da Síndrome de deleção 22q11.2, possui incidência de 1/4.000 a 1/6.000 nascimentos. Embora a microdeleção em 22q11.2 seja a principal causa da síndrome, cerca de 10 a 20% dos pacientes com características clínicas da SVCF não a apresentam. Em alguns indivíduos com características clinicas da SVCF e sem microdeleção em 22q11.2, foram encontradas outras aberrações cromossômicas e mutações no gene TBX1. Existem evidências em modelos animais que aventam a associação de alterações no gene FGF8 ao fenótipo da SVCF em humanos. No entanto, até o momento, o gene FGF8 ainda não havia sido estudado em pacientes com SVCF sem microdeleção. Os objetivos deste trabalho foram: 1 - investigar as causas genéticas em pacientes com suspeita clínica da SVCF por meio de cariótipo e triagem de microdeleções, utilizando a técnica de MLPA e FISH; 2 - verificar a presença de alterações nas seqüências codificantes dos genes TBX1 e FGF8 em pacientes sem deleção 22q11.2. Foram incluídos 109 indivíduos com suspeita clínica de SVCF avaliados clinicamente por médico geneticista e os métodos utilizados foram: cariótipo com bandas G; MLPA, FISH; seqüenciamento direto das regiões codificantes dos genes TBX1 e FGF8 e SNP-array. Dos 101 casos em que o exame de cariótipo foi realizado, quatro apresentaram aberrações cromossômicas não relacionadas à SVCF, sendo que em duas foi possível a confirmação dos resultados por SNP-array. Realizou-se MLPA de 106 casos, sendo que 29 foram positivos para a deleção. Dos casos negativos, selecionou-se 31 indivíduos após reavaliação clínico-dismorfológica com a hipótese clínica mantida e foi realizado seqüenciamento das regiões codificantes dos genes TBX1 e FGF8. No gene TBX1 foram encontradas variações normais na seqüência e no gene FGF8 não foram detectadas alterações, com exceção dos exons 1 e 2, em que não foi possível análise por problemas técnicos. O exame de cariótipo contribuiu na investigação inicial e permitiu concluir a investigação por outras técnicas. O MLPA se mostrou eficiente para a investigação diagnóstica da deleção 22q11.2, identificando, também, outras alterações; FISH confirmou 82,1% dos resultados obtidos por MLPA. Não foram identificadas alterações patogênicas nas regiões codificantes dos genes TBX1 e em 90% das regiões codificantes do gene FGF8 / Abstract: Velocardiofacial Syndrome (SVCF), one of the forms of the 22q11.2 Microdeletion Syndrome spectrum, has an incidence of 1/4.000 to 1/6.000 births. Although the 22q11.2 microdeletion is the main cause of the syndrome, approximately 10 to 20% of the patients with clinical features of SVCF don't present it. In a few individuals with SVCF clinical features and without 22q11.2 microdeletion other chromosomal aberrations and changes in TBX1 gene were found. There is evidence in animal models that suggests the associated changes in FGF8 gene in humans. However, the FGF8 gene had not been studied in patients with SVCF without microdeletion yet. The purpose of this work was: 1 - To investigate the genetic causes in patients with clinical suspicion of SVCF through the karyotype and microdeletion screening using MLPA and FISH techniques; 2 - To check the presence of changes in coding sequences of the TBX1 gene and FGF8 gene in patients without 22q11.2 deletion. 109 individuals with clinical suspicion of SVCF and clinically evaluated by a clinical geneticist were included, and the following methods were used: karyotype with GTG banding, MLPA, FISH, direct sequencing of coding regions of TBX1 and FGF8 gene and SNP-array. Four out of 102 cases in which the tests of karyotype were performed presented chromosomal aberrations not related to SVCF, two of them confirmed by SNP-array. The MLPA of 106 cases was performed, 29 of them positive to deletion. 31 individuals were selected among negative cases, after clinical reevaluation based on the same clinical hypothesis maintained, and direct sequencing of coding regions of TBX1 and FGF8 gene were performed. Normal variations in sequence were found in TBX1 gene and alterations were not detected in FGF8 gene except for 1 and 2 exons because of technical problems. The karyotype test contributed in the first investigation and permitted us to conclude the investigation by means of other techniques. The MLPA proved to be effective for the diagnostic investigation of 22q11.2 deletion, identifying other alterations as well; FISH confirmed 82,1% results obtained by MLPA. Pathogenic alterations in coding regions of TBX1 gene and in 90% of the coding regions of FGF8 gene were not identified / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas
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An automated multicolour fluorescence in situ hybridization workstation for the identification of clonally related cellsDubrowski, Piotr 05 1900 (has links)
The methods presented in this study are aimed at the identification of subpopulations (clones) of genetically similar cells within tissue samples through measurement of loci-specific Fluorescence in-situ hybridization (FISH) spot signals for
each nucleus and analyzing cell spatial distributions by way of Voronoi tessellation and Delaunay triangulation to robustly define cell neighbourhoods.
The motivation for the system is to examine lung cancer patient for
subpopulations of Non-Small Cell Lung Cancer (NSCLC) cells with biologically meaningful gene copy-number profiles: patterns of genetic alterations statistically
associated with resistance to cis-platinum/vinorelbine doublet chemotherapy treatment.
Current technologies for gene-copy number profiling rely on large amount of cellular
material, which is not always available and suffers from limited sensitivity to only the
most dominant clone in often heterogeneous samples. Thus, through the use of FISH, the
detection of gene copy-numbers is possible in unprocessed tissues, allowing identification of specific tumour clones with biologically relevant patterns of genetic aberrations.
The tissue-wide characterization of multiplexed loci-specific FISH signals,
described herein, is achieved through a fully automated, multicolour fluorescence imaging microscope and object segmentation algorithms to identify cell nuclei and FISH spots within. Related tumour clones are identified through analysis of robustly defined cell neighbourhoods and cell-to-cell connections for regions of cells with homogenous
and highly interconnected FISH spot signal characteristics.
This study presents experiments which demonstrate the system’s ability to
accurately quantify FISH spot signals in various tumour tissues and in up to 5 colours
simultaneously or more through multiple rounds of FISH staining. Furthermore, the
system’s FISH-based cell classification performance is evaluated at a sensitivity of 84% and specificity 81% and clonal identification algorithm results are determined to be comparable to clone delineation by a human-observer. Additionally, guidelines and procedures to perform anticipated, routine analysis experiments are established. / Science, Faculty of / Physics and Astronomy, Department of / Graduate
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Generation of a MOR-CreER knock-in mouse line to study cells and neural circuits involved in mu opioid receptor signaling / ミューオピオイド受容体(MOR)のシグナル伝達および神経回路制御機構解析を目的とするMOR-CreERノックインマウスの開発Okunomiya, Taro 23 March 2020 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22366号 / 医博第4607号 / 新制||医||1043(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 岩田 想, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Homoeologous Recombination-Based Chromosome Engineering for Physical Mapping and Introgression in Wheat and Its Relatives Aegilops speltoides and Thinopyrum elongatumZhang, Mingyi January 2020 (has links)
Wheat (genome AABBDD) is one of the essential crops, offering approximate 20% of human calorie consumption worldwide. Allopolyploidization of three diploid ancestors led to hexaploid wheat with narrowed genetic variation. Chromosome engineering is an applicable approach to restore the evolutionarily-omitted genetic diversity by homoeologous chromosomes recombination between wheat and its relatives. Two diploid relatives of wheat, Thinopyrum elongatum (genome EE) and Aegilops speltoides (genome SS), containing favorable genes, are used as gene resources for alien introgression and genome diversification in wheat. An advanced and effective experiment procedure was developed and applied for the production, recovery, detection, and characterization of homoeologous recombinants. Meanwhile, a novel recombinant chromosome recovery strategy was exploited with improved efficiency and accuracy.
In this study, recombinants of wheat chromosomes 3B and 7B with their homoeologous chromosomes in Th. elongatum and Ae. speltoides (i.e. 3B-3E, 7B-7E, and 7B-7S) were produced and detected. Totolly, 81 3B-3E recombinants and four aberrations involving in distinct chromosomal regions were developed in three recombination cycles by fluorescent genomic in situ hybridization (FGISH). The secondary and tertiary recombination breakpoints occurred toward the proximal regions comparing to the primary recombination under this advanced recombination procedure. A novel recovery strategy was used to recover 7B-7E and 7B-7S homoeologous recombinants by chromosome-specific markers and FGISH verification. Marker-based pre-screening and subsequent FGISH verification identified 29 7B-7E and 61 7B- 7S recombinants, seven 7B-7E and four 7B-7S Robertsonian translocations, one 7E and five 7S telocentric chromosomes, and three 7S deletions. All the recombinants and aberrations were genotyped by high-throughput wheat 90K single nucleotide polymorphism (SNP) assay and the recombination breakpoints were physically mapped to wheat chromosome 3B or 7B according to their FGISH patterns, SNP results, and wheat reference genome sequence. Chromosome 3B was physically partitioned into 38 bins with 429 SNPs. Meanwhile, 44 distinct bins were resolved for chromosome 7B with 523 SNPs. A composite bin map was constructed for chromosomes 3B and 7B, respectively, with a comprehensive analysis of FGISH and SNPs results. In summary, this project provides a unique physical framework for further wheat genome studies and diversifies the wheat genome for germplasm development in wheat breeding.
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Nuclear Structure Studied by Fluorescence Hybridization: Visualization of Individual Gene Transcription and RNA Splicing: A ThesisXing, Yigong P. 01 April 1993 (has links)
The overall objective of this study has been to address some of the longstanding questions concerning functional organization of the interphase nucleus. This was achieved by using recently developed high-resolution fluorescence in situ hybridization techniques for a precise localization of specific DNA and RNA sequences in conjunction with immunocytochemistry and biochemical fractionation. This study is based on the philosophy that new insights may be gained by an approach that attempts to interrelate genomic organization, spatial arrangement of RNA metabolism, and nuclear substructure within the mammalian cell nucleus.
The nuclear distribution of an exogenous, viral RNA (Epstein-Barr Virus, EBV) within nuclear matrix preparations was studied by developing an approach which couples in situhybridization with biochemical fractionation procedures. EBV RNA molecules accumulate in highly localized foci or elongated tracks within the nucleus of lymphoma cells. These RNA tracks were retained with spatial and quantitative fidelity in nuclear matrix preparations even after biochemical fractionation which removes 95% of cellular protein, DNA, and phospholipid. This provided direct evidence that the primary transcripts are localized via their binding to, or comprising part of, a non-chromatin nuclear substructure.
Then the nuclear distribution of RNA from an endogenous gene, fibronectin, was investigated using fluorescence techniques modified for more sensitive detection of endogenous RNAs within nuclear morphology. A series of in situhybridization experiments were performed using different combinations of intron, cDNA, and genomic probes for RNA/RNA or RNA/DNA analysis in intact cells. Fibronectin RNAs were highly localized in the nucleus, forming foci or tracks. Both intron and exon sequences were highly concentrated at the same site within the nucleus, indicating the presence of primary unspliced transcripts. Double-color hybridization using a nontranscribed 5' flanking sequence probe and a genomic DNA probe showed that the gene and RNA track for fibronectin were spatially overlapped, with the gene consistently towards one end of the track. These results provided evidence that the accumulation of RNA molecules occurs directly at or near the site of transcription, and further indicated a structural polarity to the RNA track formation with the gene towards one end. It was further discovered that within a single cell, cDNA probes produced longer tracks than those formed with intron probes, i.e. intron signals were generally confined to a smaller part of the track than the exon signals, indicating that splicing occurs within the RNA track. Additional experiments using poly(A) RNA hybridization or anti-SC-35 antibody staining combined with fibronectin RNA hybridization have shown that the fibronectin tracks were associated with recently discovered transcript domains enriched in poly(A) RNA and splicing factors.
To further determine whether other specific genes and RNAs are functionally organized within the nucleus, the nuclear distribution of several active or inactive genes was analyzed in terms of their spatial relationship to transcript domains. The results indicated that in addition to fibronectin, the genes or their primary transcripts from two other active genes, collagen and actin, were also closely associated with the domains. For both of these, over 90% of the gene/RNA sites were either overlapping or directly contacting the domains. In contrast. for two inactive genes, cardiac myosin heavy chain and neurotensin, it was found that both genes were separated from the domains in the majority of nuclei. Histone genes, which have several unique features, showed a relatively complex result with about half of the gene signals extremely close to the domains. Therefore, three actively expressed genes were demonstrated to be tightly associated with the domains and, moreover, their RNAs showed distinct and characteristic spatial relationships with the domains. In contrast, two inactive genes were not associated with the domains. One potential implication of these finding is that active genes may be preferentially localized in and around these transcript domains.
The nuclear localization of another RNA, XIST, standing for X-inactivation specific transcript, was studied because of its potentially unique biological role. XIST is the only gene which is known to be expressed from the inactive human X chromosome but not from the active X chromosome, and was believed to be important in X inactivation. Using fluorescence in situhybridization, it was found that XIST RNA was highly localized within the nucleus and always completely overlapped the Barr body which is the condensed, inactive X chromosome. The different fine distribution pattern of XIST RNA within the nucleus as compared to other protein coding RNAs suggested a unique function for this RNA, possibly involving a structural role in inactivating the X chromosome.
The final area of my thesis research was to study and acquire expertise in the applications of fluorescence in situ hybridization in gene mapping and cancer genetics. A retinoblastoma (RB)-related putative tumor suppressor gene, p107, was mapped to human chromosome 20 in band q11.2. Localization of p107 to 20q11.2 was of particular interest because of the correlation of breakpoints in this area with specific myeloid disorders such as acute nonlymphocytic leukemia and myelodysplastic syndrome. Other applications of in situ hybridization including the search for unknown genes at a known chromosomal breakpoint, detection of deletions, translocations or other chromosomal rearrangements associated with specific tumors were also explored and reviewed.
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A Synergy of Spatiotemporal Transcriptomic Techniques for Non-Model Organism Studies: Something Old, Something New, Something Borrowed, Something Ocean BlueWatson, Kelly 07 1900 (has links)
In situ hybridization (ISH) has played a crucial role in developing a spatial transcriptomic
understanding of emerging model organisms in the past, but advancing high-throughput
RNA-sequencing (RNA-seq) technology has pushed this method into the shadows, leading
to a loss of data resolution. This shift in research towards the exclusive use of RNA-seq
neglects essential considerations for transcriptomic studies including the spatial and
temporal expression of transcripts, available budget, experimental design needs, and
validation of data. A synergy of spatiotemporal transcriptomic techniques is needed,
using the bulk and unbiased analysis of RNA-seq and the visual validation and
spatiotemporal resolution of ISH. Integration of this synergistic approach can improve our
molecular understanding of non-model organisms and establish the background data
needed for advancing research techniques. A prime example lies within an emerging
model of the marine science and symbiosis fields, where I present a case study on a
threatened coral reef keystone – the cnidarian-dinoflagellate symbiosis. Establishing a
whole-mount ISH protocol for the emerging cnidarian model Aiptasia (sea anemone) will
help future studies reveal the gene regulation underpinning the establishment,
persistence, and breakdown of this complex symbiotic relationship.
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