Sponge are primitive multi-cellular organisms. They are important sources of secondary metabolites. In the previous studies indicated that the sponges harbor stable symbiotic microbial consortia. The mechanisms for maintenance and transmission of microbial consortia to the next generations are still not fully understood. The sponge, Cinachyrella australinesis, was chosen to further investigate relationship of the symbiotic bacteria within to the host. Fluorescent in situ hybridization ¡]FISH¡^was employed with non-specific ¡]EUB338¡^and specific oligonucleotide probes for bacteria. The sponge was cryo-sectioned¡]1£gm¡^and hybridized with fluorescent probes. The distribution and ratios of the bacteria in the sponge agreed with those of previous studies indicating that the symbiotic bacteria of C. australiensis are stable and endosymbiotic in nature.

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2016-08-30T16:56:46Z No. of bitstreams: 1 Máilla Rebouças Viana dissertação pdf.pdf: 1628670 bytes, checksum: 0f14f7eed7546953dae78ed1447bf742 (MD5) / Made available in DSpace on 2016-08-30T16:56:46Z (GMT). No. of bitstreams: 1 Máilla Rebouças Viana dissertação pdf.pdf: 1628670 bytes, checksum: 0f14f7eed7546953dae78ed1447bf742 (MD5) / INTRODUÇÃO: A Leucemia Mielóide Crônica (LMC) é uma doença mieloproliferativa crônica caracterizada pela presença do cromossomo Philadelphia (Ph). Este cromossomo, observado por técnicas citogenéticas convencionais, é resultante da translocação t(9;22)(q34;q11) que justapõe o rearranjo BCR-ABL, observado por técnicas moleculares. A detecção desse cromossomo ou do rearranjo é fundamental, pois não somente contribui para o diagnóstico, mas também interfere no acompanhamento e no sucesso da terapia utilizada. O rearranjo BCR-ABL possui atividade tirosina quinase elevada, o que levou ao desenvolvimento de novos fármacos inibidores desta ação, conhecidos como inibidores de tirosino quinase (TKI). OBJETIVO: O objetivo deste estudo foi demonstrar a importância da utilização da técnica de citogenética molecular (Fluorescent in situ Hybridization - FISH) aliada a citogenética clássica no monitoramento de pacientes com LMC em terapia com TKI, no Estado da Bahia. METODOLOGIA: Foram analisadas amostras de medula óssea de 20 pacientes com LMC, em tratamento com TKI, encaminhados ao Serviço de Genética Médica do Hospital Universitário Prof. Edgard Santos, utilizando as técnicas de citogenética clássica e FISH. RESULTADOS: Resultados obtidos demonstraram que o cromossomo Ph foi observado em 15%, com a citogenética clássica, e o rearranjo BCR-ABL em 90%, com a citogenética molecular. Outro ponto importante foi que em alguns casos o resultado só foi possível com a técnica molecular, pois a técnica convencional não foi suficiente pela impossibilidade na análise das metáfases, devido a fatores como falta de crescimento celular. CONCLUSÃO: A técnica de FISH demonstrou maior sensibilidade nos casos que a citogenética clássica não detectou o cromossomo Ph, entretanto as duas técnicas foram fundamentais para os resultados obtidos por apresentarem vantagens e desvantagens em relação à outra e, portanto, devem ser usadas de maneira complementar no monitoramento de pacientes com LMC em tratamento com TKI.

abstract: Spatial resolved detection and quantification of ribonucleic acid (RNA) molecules in single cell is crucial for the understanding of inherent biological issues, like mechanism of gene regulation or the development and maintenance of cell fate. Conventional methods for single cell RNA profiling, like single-cell RNA sequencing (scRNA-seq) or single-molecule fluorescent in situ hybridization (smFISH), suffer either from the loss of spatial information or the low detection throughput. In order to advance single-cell analysis, new approaches need to be developed with the ability to perform high-throughput detection while preserving spatial information of the subcellular location of target RNA molecules. Novel approaches for highly multiplexed single cell in situ transcriptomic analysis were developed by our group to enable single-cell comprehensive RNA profiling in their native spatial contexts. Reiterative FISH was demonstrated to be able to detect >100 RNA species in single cell in situ, while more sophisticated approaches, consecutive FISH (C-FISH) and switchable fluorescent oligonucleotide based FISH (SFO-FISH), have the potential for whole transcriptome profiling at the single molecule sensitivity. The introduction of a cleavable fluorescent tyramide even enables sensitive RNA profiling in intact tissues with high throughput. These approaches will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019

No / Spatial genome organization in the cell nucleus plays a crucial role in the control of genome functions. Our knowledge about spatial genome organization is relying on the advances in gene imaging technologies and the biochemical approaches based on the spatial dependent ligation of the genomic regions. Fluorescent in situ hybridization using specific fluorescent DNA and RNA probes in cells and tissues with the spatially preserved nuclear and genome architecture (3D-FISH) provides a powerful tool for the further advancement of our knowledge about genome structure and functions. Here we describe the 3D-FISH protocols allowing for such an analysis in mammalian tissue in situ including in the skin. These protocols include DNA probe amplification and labeling; tissue fixation; preservation and preparation for hybridization; hybridization of the DNA probes with genomic DNA in the tissue; and post-hybridization tissue sample processing.

No description available.

A história de domesticação da cana-de-açúcar (Saccharum spp.) é atípica. As variedades modernas derivam de um processo que inclui hibridações entre a espécie domesticada S. officinarum e a silvestre S. spontaneum, sucessivos retrocruzamentos, no sentido de recuperar o genoma de S. officinarum e a seleção de progênies superiores. Além disso, as genealogias contemplam cruzamentos entre genótipos e eventualmente espécies, todos com elevado grau de ploidia e número de cromossomos distintos, assim como aneuploidias. Frente ao exposto, este trabalho teve como objetivos estabelecer o número de cromossomos e avaliar o comportamento meiótico da cultivar IACSP93-3046, bem como, identificar as associações cromossômicas em meiose I dos genótipos IACSP93-3046, IACSP95-3018 e de um representante de S. officinarum, Caiana Fita, pela marcação dos centrômeros usando FISH. O número de cromossomos da cultivar IACSP93-3046 foi determinado a partir de preparações do meristema radicular, pré-tratado com 8-hidroxiquinolina (0,03%, 4h), e corado pelo método de Feulgen. As células metafásicas foram analisadas sob microscopia óptica, preferencialmente intactas e com o mínimo de sobreposição de cromossomos. Para a análise do comportamento meiótico utilizou-se a técnica de esmagamento, e as células foram coradas com carmim propiônico. Foram observadas as fases meióticas desde a metáfase I até a telófase II, bem como as tétrades. O pareamento cromossômico em meiose I foi analisado usando a técnica de hibridização in situ fluorescente (FISH). Para tanto, preparações dos genótipos IACSP93-3046, IACSP95-3018 e Caiana Fita foram realizadas pelo gotejamento de uma suspensão de células em diacinese. As sondas foram obtidas por PCR a partir da amplificação da região centromérica de cana-de-açúcar, marcadas com digoxigenina-11-dUTP, por nick translation, e detectadas com anti-digoxigenina-rodamina. As lâminas foram montadas em DAPI-Vectashield e analisadas sob microscopia de fluorescência. O número diplóide 2n = 112 foi observado para a cultivar IACSP93-3046, sendo caracterizado pela primeira vez neste estudo. A microsporogênese de IACSP93-3046 apresentou elevado percentual de irregularidades (68%). De modo geral, as anormalidades foram relativas à segregação dos cromossomos, e incluíram migração precoce para os polos em metáfase I e II, cromossomos retardatários em anáfase (I e II) e em telófase (I e II), cromossomos perdidos em prófase II, e micronúcleos nas tétrades. A análise dos sítios de hibridização permitiu comprovar que os cromossomos se associam predominantemente como bivalentes em IACSP93-3046, IACSP95-3018 e Caiana Fita. As irregularidades na segregação dos cromossomos conduzem a micrósporos aneuploides, como constatado em IACSP93-3046. Sugere-se que a assincronia do processo meiótico entre os genomas que compõem a cana-de-açúcar tem papel relevante na geração dessas irregularidades. / The history of the sugarcane domestication (Saccharum spp.) is atypical. Modern varieties are derived from a hybridization process between the domestic species S. officinarum and the wild species S. spontaneum, successive backcrossings to recover the genome of S. officinarum, and the selection of superior progenies. The genealogies include crossings among genotypes, and possibly Saccharum species, all with a high degree of ploidy and different numbers of chromosomes, as well as aneuploidies. The study aimed to establish the number of chromosomes and evaluate the meiotic behavior of cultivar IACSP93-3046, and identify chromosomal associations in meiosis I of genotypes IACSP93-3046, IACSP95-3018 and Caiana Fita (a representative of S. officinarum) by labeling centromeres using fluorescence in situ hybridization (FISH). The number of chromosomes in cultivar IACSP93-3046 was determined from the root meristem preparations, pretreated with 8-hydroxiquinoline and stained by the Feulgen method. Metaphasic cells, preferably intact and with minimum chromosome overlap, were analyzed under an optical microscope. Meiotic behavior was examined from the preparations by using squashing method and stained with propionic carmine. Meiotic phases were observed from metaphase I to telophase II, and tetrad stages. Chromosomal pairing in meiosis I was analyzed by using the FISH technique. The slides of genotypes IACSP93-3046, IACSP95-3018 and Caiana Fita were produced by dropping a suspension of meiocytes in diakinesis. The probes were obtained by PCR, with amplification of the centromere region, and labeled with digoxigenin-11-dUTP, by nick translation, and detected with anti-digoxigenin-rhodamine. The slides were mounted in DAPI-Vectashield and analyzed under a fluorescence microscope. The diploid number 2n = 112 was observed for cultivar IACSP93-3046 and characterized in this study for the first time. Microsporogenesis of IACSP93-3046 presented a high irregularity percentage regarding chromosome segregation, especially precocious migration to poles in metaphase I and II, laggard chromosomes in anaphase and telophase I and II, lost chromosomes in prophase II, and micronuclei in the tetrad stages. The analysis from the hybridization sites proved that the chromosomal pairing occurred predominantly as bivalents in IACSP93-3046, IACSP95-3018 and Caiana Fita. Chromosomal segregation irregularities led to aneuploid microspores, as confirmed in IACSP93-3046, suggesting the asynchrony in the meiotic process between the sugarcane genomes play an important role in producing these irregularities.

A DNA sequence-based phylogenetic tree (Dillon et al., 2004) places the species of the genus Sorghum into two sister lineages, one with x = 5 and the other with x = 10 as a basic chromosome number. It has not been resolved whether or not these lineages are monophyletic or polyphyletic. A repetitive sequence, CEN38, found only in Sorghum and sugarcane, was used to assess evolutionary relationships among Sorghum species. The objectives of this research were to determine the taxonomic distribution of CEN38, its chromosomal position(s), and its organization in DNA. CEN38 was detected by filter hybridization to be present in the DNA of 16 of 21 Sorghum species analyzed, ranging from 15 to ~21,000 copies. It was detected by fluorescence in situ hybridization (FISH) only in chromosomes of species of the section Eu-sorghum, where it had a pericentromeric distribution. The low copy number and/or chromosomal distribution of CEN38 in other Sorghum species apparently does not allow for its detection by FISH. Analysis of restriction enzyme digested DNA with homology to CEN38 and of fragments amplified by PCR using primers selected to amplify S. bicolor CEN38 sequences showed that S. laxiflorum and S. macrospermum have tandemly arranged CEN38 sequences as is found in S. bicolor. This supports the close evolutionary affinity of the species in the x = 10 lineage. In the x = 5 lineage, DNA of 11 of 16 species analyzed hybridized with CEN38 by filter hybridization. In S. versicolor, large DNA fragments (4.36 kb to 23 kb) generated by digestion with restriction enzymes hybridized to CEN38. Since a ladder of smaller fragments was not detected, CEN38 may have been inserted into a transposable element in this species and dispersed throughout the genome. Among species of the x = 5 lineage, PCR using primers for S. bicolor CEN38 amplified only DNA fragments from S. timorense and these formed a ladder based on a ~125 bp repeat. Since hybridization of the CEN38 sequence to DNA of S. timorense was not detected by filter hybridizations, these sequences apparently are not similar to CEN38. Cloning and sequencing of DNA from species of the x = 5 lineage that hybridizes to CEN38 are needed to determine whether or not they are in the CEN38 family. A monophyletic or polyphyletic origin of the x = 5 and x = 10 lineages was not resolved.

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.

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.

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 Pinto_RafaelBarbosa_M.pdf: 4856945 bytes, checksum: 7af922214bf499ea2e8fe2cfb2e9e2f4 (MD5) 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