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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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REDOX POTENTIAL (ORP) REGULATION OF NUTRIENT REMOVAL IN WASTEWATER TREATMENT PROCESSES AND THE STRUCTURE - FUNCTION ANALYSIS OF ACTIVATED SLUDGE FLOCLI, BAIKUN 22 May 2002 (has links)
No description available.
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Chromosome and Genome Evolution in Culicinae MosquitoesMasri, Reem Abed 14 July 2021 (has links)
The Culicinae is the most extensive subfamily among the Culicidae family of mosquitoes. Two genera, Culex and Aedes, from this subfamily have world-wide distribution and are responsible for transmitting of several deadly diseases including Zika, West Nile fevers, chikungunya, dengue, and Rift Valley fevers. Developing high-quality genome assembly for mosquitoes, studying their population structure, and evolution can help to facilitate the development of new strategies for vector control. Studies on Aedes albopitcus as well as on species from the Culex pipiens complex, which are widely spread in the United States, provide excellent models on these topics.
Ae. albopictus is one of the most dangerous invasive mosquito species in the world that transmits more than 20 arboviruses. This species has highly repetitive genome that is the largest among mosquito genomes sequenced so far. Thus, sequencing and assembling of such genome is extremally challenging. As a result, the lack of high-quality Ae. albopictus genome assembly has delayed the progress in understanding its biology. To produce a high-quality genome assembly, it was important to anchor genomic scaffolds to the cytogenetic map creating a physical map of the genome assembly. We first developed a new gene-based approach for the physical mapping of repeat-rich mosquito genomes. The approach utilized PCR amplification of the DNA probes based on complementary DNA (cDNA) that does not include repetitive DNA sequences. This method was then used for the development of a physical map for Ae. albopictus based on the in situ hybridization of fifty cDNA fragments or gene exons from twenty-four scaffolds to the mitotic chromosomes from imaginal discs. This study resulted in the construction of a first physical map of the Ae. albopictus genome as well as mapping viral integration and polyphenol oxidase genes. Moreover, comparing our present Ae. albopictus physical map to the current Ae. aegypti assembly indicated the presence of multiple chromosomal inversions between them.
To better understand population structure and chromosome evolution in Culicinae mosquitoes, especially in the Culex pipiens complex, we studied genomic and chromosomal differentiation between two subspecies Cx. pipiens pipiens and Cx. pipiens molestus. For the species responsible for the spread of human diseases, understanding the population dynamics and processes of taxa diversification is important for an effective mosquito control . Two vectors of West Nile virus, Cx. p. pipiens and Cx. p. molestus, exhibit epidemiologically important behavioral and physiological differences, but the whole-genome divergence between them was unexplored. The first goal of this study was to better understand the level of genomic differentiation and population structures of Cx. p. pipiens and Cx. p. molestus from different continents. We sequenced and compared whole genomes of 40 individual mosquitoes from two locations in Eurasia and two in North America. Principal Component, ADMIXTURE, and neighbor joining analyses of the nuclear genomes identified two major intercontinental, monophyletic clusters of Cx. p. pipiens and Cx. p. molestus. The level of genomic differentiation between the subspecies was uniform along chromosomes. The ADMIXTURE analysis determined signatures of admixture in Cx. p. pipens populations, but not in Cx. p. molestus populations. Thus, our study identified that Cx. p. molestus and Cx. p. pipiens represent different evolutionary units with monophyletic origin that have undergone incipient ecological speciation. The second goal was to study differences at the chromosome level between these two organisms. We first measured whole chromosome and chromosome arm length differences between Cx. p. molestus and Cx. p. pipiens as a basic cytogenetic approach. In addition, we used the novel Hi-C approach to detect chromosomal rearrangements between them since Hi-C was successful in detecting a known inversion in Cx. quinquefasciatus. Cx. p. molestus and Cx. p. pipiens embryos were used to perform the Hi-C technique. Analysis of the Hi-C data showed the presence of two different inversions in Cx. p. pipiens and Cx. p. molestus heatmap, which could explain their different physiology and adaptation in nature. Developing modern genomic and cytogenetic tools is important to enhance the quality of genome assemblies, improve gene annotation, and provide a better framework for comparative and population genomics of mosquitoes; also it is the foundation for the development of novel genome-based approaches for vector control. / Doctor of Philosophy / Mosquitoes are medically important insects because they vector a range of diseases that infect humans. The subfamily Culicinae is responsible for transmitting such diseases as Zika, dengue, and West Nile fevers, which have triggered fatal infections and epidemics in multiple parts of the world. Since 2010-2016, studies have reported exceeding levels of insecticide resistance that slows the disease elimination process. Novel transgenic techniques have a tremendous potential for more efficiently minimizing mosquito-borne diseases and transmission. Availability of high-quality genome assemblies for mosquitoes may help to better understand their population structure and to develop effective and safe vector-control approaches that we urgently need.
For the development of high-quality genome assemblies, we need to construct a physical genome map, that shows the physical locations of genes or other DNA sequences of interest along the chromosomes. For this reason, we developed a new gene-based approach for the physical mapping of the mosquito genomes. This method was then used for the development of a physical map for Ae. albopictus. This study resulted in the generation of the first physical map of the Ae. albopictus genome.
To understand population structure in Culicinae mosquitoes, we used mosquitoes from the Culex pipiens complex. Species in this complex transmit different arthropod-borne viruses or arboviruses. Notable is the West Nile Virus, which has triggered fatal infections and epidemics in Eastern and Central Europe, North America and is also known in Asia, Australia, Africa, and the Caribbean. We specifically focused on two subspecies in this complex, Cx. pipiens pipiens and Cx. pipiens molestus that are morphologically identical, but are different physiologically and behaviorally. Although they are spread globally in temperate regions, their population structure and taxonomic status remains unclear. The first goal of this study was to better understand the level of genomic differentiation of Cx. p. pipiens and Cx. p. molestus from different continents. We sequenced and compared the whole genomes of 40 individual mosquitoes from two locations in Eurasia and two in North America. Our study identified that Cx. p. molestus and Cx. p. pipiens represent different evolutionary units that are currently undergoing ecological speciation. The second goal was to study differences at the chromosome level between them. Using the Hi-C approach we detected presence of two different inversions in Cx. p. pipiens and Cx. p. molestus, which could potentially explain their different physiology and adaptation.
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Définition du mécanisme de localisation des ARNm cen et ik2 aux centrosomes chez la DrosophileLegendre, Félix 12 1900 (has links)
L’organisation cellulaire repose sur une distribution organisée des macromolécules dans la cellule. Deux ARNm, cen et ik2, montrent une colocalisation parfaite aux centrosomes. Ces deux gènes font partie du même locus sur le chromosome 2L de Drosophila melanogaster et leur région 3’ non traduite (3’UTR) se chevauchent. Dans le mutant Cen, le transport de Ik2 est perturbé, mais dans le mutant Ik2, la localisation de cen n’est aucunement affectée. Ces résultats suggèrent que cen est le régulateur principal de la co-localisation de cen et ik2 aux centrosomes et que cette co-localisation se produit par un mécanisme impliquant la région complémentaire au niveau du 3’UTR des deux transcrits. La localisation de cen au niveau des centrosomes dans les cellules épithéliales de l’embryon est conservée dans différentes espèces de Drosophile : D. melanogater, D. simulans, D. virilis et D. mojavensis. Cependant, la localisation de ik2 n’est pas conservée dans D. virilis et D. mojavensis, deux espèces dont les gènes cen et ik2 sont dissociés dans le génome. Ces résultats suggèrent que la proximité de Cen et Ik2 dans le génome est importante afin d’avoir un événement de co-localisation de ces deux transcrits. J’ai généré différentes lignées de mouches transgéniques dans lesquelles un transgène contenant la séquence GFP fusionnée à différentes partie de Cen (partie codante, 3’UTR, Cod+3’UTR) qui sont sous le contrôle du promoteur UAS et qui sont gal4 inductibles. La région codante de l’ARNm cen était suffisante pour avoir un ciblage précis du transcrit aux centrosomes. / Messenger RNA (mRNA) localization plays a key role in establishing cellular architecture and function. The centrocortin (cen) and IkB Kinase-like 2 (ik2) mRNAs are co-localized to centrosomes in embryonic epithelial cells. Interestingly, both of these genes are organized in a head-to-head configuration in the genome, with their 3’ untranslated regions (3’UTRs) overlapping on opposite DNA strands. Here we show that gene positioning of cen and ik2 is important for the co-localization of these transcripts during Drosophila embryogenesis. The localization of cen is conserved within the Drosophila phylogeny and ik2 cannot localize when it is separated from the cen locus. Also, loss of function mutants of cen show a complete loss of ik2 localization, proposing that cen is the main driver of the co-localization. Structure-function analysis revealed that the coding region of cen is necessary for its centrosomal targeting, suggesting that a cis-regulatory motif that drives its localization is located in the coding region. This study reveals for the first time the importance of gene positioning for RNA localization. We suggest a model where cen mRNA is the main driver of centrosomal localization, which may occur through post-transcriptional interaction/annealing of these mRNAs via their 3’UTRs.
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Epigenetic regulation of skin development and postnatal homeostasis : the role of chromatin architectural protein Ctcf in the control of keratinocyte differentiation and epidermal barrier formationMalashchuk, Ogor January 2016 (has links)
Epigenetic regulatory mechanisms play important roles in the control of lineage-specific differentiation during development. However, mechanisms that regulate higher-order chromatin remodelling and transcription of keratinocyte-specific genes that are clustered in the genome into three distinct loci (Keratin type I/II loci and Epidermal Differentiation Complex (EDC)) during differentiation of the epidermis are poorly understood. By using 3D-Fluorescent In Situ Hybridization (FISH), we determined that in the epidermal keratinocytes, the KtyII and EDC loci are located closely to each other in the nuclear compartment enriched by the nuclear speckles. However, in KtyII locus knockout mice, EDC locus moved away from the KtyII locus flanking regions and nuclear speckles towards the nuclear periphery, which is associated with marked changes in gene expression described previously. Chromatin architectural protein Ctcf has previously been implicated in the control of long-range enhancer-promoter contacts and inter-chromosomal interactions. Ctcf is broadly expressed in the skin including epidermal keratinocytes and hair follicles. Conditional Keratin 14-driven Ctcf ablation in mice results in the increase of the epidermal thickness, proliferation, alterations of the epidermal barrier and the development of epidermal pro-inflammatory response. Epidermal barrier defects in Krt14CreER/Ctcf fl/fl mice are associated with marked changes in gene expression in the EDC and KtyII loci, which become topologically segregated in the nucleus upon Ctcf ablation. Therefore, these data suggest that Ctcf serves as critical determinant regulating higher-order chromatin organization in lineage-specific gene loci in epidermal keratinocytes, which is required for the proper control of gene expression, maintenance of the epidermal barrier and its function.
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Etude de la prévalence des aneuploïdies dans les produits d'avortements spontanés : intéret des techniques FISH et MLFA pour la détection des remaniements chromosomiques. / Study of the prevalence of aneuploidies in spontaneous abortion products : FISH and MLFA techniques for the detection of chromosome changes.Haoud, Khadidja 22 January 2014 (has links)
L’avortement spontané (AS) désigne la perte du produit de conception avant sa viabilité, c'est-à-dire avant la 22e semaine d’aménorrhée, ou un poids fœtal inférieur à 500 g. La cause génétique est à l’origine de plus des deux tiers des AS, les aneuploïdies autosomiques, représentant à elles seules jusqu’à 70% des pertes fœtales du 1er trimestre. Le caryotype présente une très bonne sensibilité en ce qui concerne le dépistage des trisomies autosomiques (13, 18 et 21) et des aneuploïdies affectant les chromosomes sexuels, mais il montre d’importantes limites, d’une part en raison des échecs de culture cellulaire et d’autre part en raison de l’existence de remaniements non détectables au caryotype standard. Actuellement plusieurs techniques moléculaires de dépistage rapides des aneuploïdies liées aux échecs de grossesses ont été vérifiées : 1°) la fluorescence in situ par hybridation (FISH) 2°) l’amplification multiplex de sondes nucléiques dépendant des ligatures (MLPA). Ces deux méthodes présentent l’avantage d’être réalisables, sans culture préalable, sur noyaux en interphase ou sur ADN extrait et de permettre la détection d’anomalies cryptiques. Notre étude repose sur l’étude cytogénétique des produits d’AS pour mettre en évidence les anomalies chromosomiques les plus fréquentes à l’origine de ces pertes fœtales et d’en mieux appréhender les mécanismes de survenue. Elle a été réalisée sur 220 patientes âgées de 19 à 45 ans, et était fondée sur l’analyse directe par FISH sur noyaux interphasiques (AneuVysionTM) de prélèvements de villosités choriales et sur l’analyse de l’ADN extrait de tissus fœtaux par MLPA afin de révéler d’éventuelles aneuploïdies et micro-remaniements. L’âge gestationnel au moment des prélèvements était compris entre 7 et 38 semaines d’aménorrhée. Sur un total de 151 échantillons analysés par AneuVysionTM, 10 anomalies chromosomiques ont été observées: 3 trisomies 21, 1 trisomie 18, 1 trisomie 13, 1 mosaïque 46,XX/47,XX+21, 3 triploïdies et 1 monosomie X (Turner). Par ailleurs, sur les 69 autres échantillons analysés par MLPA, 6 étaient ininterprétables. Les anomalies trouvées par cette technique étaient: 2 monosomies X. Pour les échantillons restants, la MLPA a été négative. Nous avons en parallèle réalisé une étude rétrospective fondée sur l’analyse comparative d’un échantillon recruté à Sidi Bel Abbès, de femmes ayant subi un AS et admises à la maternité del’hôpital Hassani Abdelkader de Sidi Bel Abbès et d’un échantillon recruté à Clermont-Ferrand de femmes ayant subi un AS et pour lesquelles un prélèvement pour établir le caryotype du produit de fausse-couche avait été adressé dans le service de cytogénétique du CHU Estaing de Clermont-Ferrand. Cette étude a couvert une période de six années, allant de janvier 2005 à décembre 2010. Les techniques de FISH et de MLPA représentent des outils simples, rapides et sensibles pour la détection des remaniements chromosomiques. Elles représentent une alternative très intéressante à la culture cellulaire, et permettent le diagnostic de désordres génomiques indécelables par les techniques conventionnelles. / Spontaneous abortion (SA) is the loss of the product of fertilization before its viability, that is, before22 weeks of gestation or fetal weight less than 500 g. Genetic causes account for more than two thirds of SA, autosomal aneuploidies alone accounting for up to 70% fetal loss. Chromosomal cytogenetic techniques show significant limitations on the one hand because of the failures of cell culture, and secondly because of the existence of undetectable alterations to the standard karyotype. It was therefore planned to use molecular techniques :- Fluorescent in situ hybridization (FISH)- Multiplex ligation-dependent probe amplification (MLPA). Both techniques have the advantage of being achievable without prior culture of cores interphase or DNA extracted and to enable detection of cryptic abnormalities. The project is based on cytogenetic study of AS products to highlight the most frequent chromosomal abnormalities causing fetal losses, and to better understand their occurrence. Our study was performed on 220 patients from 19 to 45 years, and was based on the direct analysis by FISH on interphase nuclei (AneuVysionTM) of chorionic villus sampling and analysis of DNA extracted fetal tissue by MLPA to reveal any aneuploidy and rearrangements. The gestational age of the samples ranged from the 7th to the 38th week of gestation. In a total of 151 samples analyzed by AneuVysionTM, 10 chromosomal abnormalities were observed: three trisomies 21, one trisomy 18, one trisomy 13, one mosaic 46,XX/47,XX+21, 3 triploidies and one monosomy X (Turner). In addition, among the other 69 samples analyzed by MLPA, 6 were uninterpretable. The abnormalities found by this technique were 2 monosomies X. For the remaining samples, the MLPA was negative. We conducted a retrospective parallel study based on the analysis of a sample recruited in Sidi Bel Abbes, women who have had an AS and were admitted to the maternity hospital Abdelkader Hassani, Sidi Bel Abbes ; and a sample recruited in Clermont-Ferrand : women who underwent AS for which a levy to establish the karyotype product miscarriage had been addressed in the Department of Cytogenetics of CHU Estaing, Clermont-Ferrand. This study covered a period of six years, from January 2005 to December 2010. The techniques of FISH and MLPA are simple, rapid and sensitive tools for the detection of chromosomal rearrangements. They represent a very interesting alternative to cell culture and allow diagnosis for genomic disorders undetectable by conventional techniques.
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Isolamento e caracterização de sequências de PISTILLATA em bromeliaceae e estudo de expressão em tecidos florais de Tillandsia aeranthos (Loisel.) LB Sm.Gaeta, Marcos Letaif January 2016 (has links)
Bromeliaceae é uma família importante entre as monocotiledôneas devido ao seu elevado número de espécies distribuídas no Neotrópico, e por uma riqueza de caracteres adaptativos a diferentes habitats. Flores de bromélias possuem uma grande variação morfológica, frequentemente negligenciada em estudos de morfoanatomia e de filogenia. Para uma melhor compreensão dos mecanismos de desenvolvimento que levam a essas variações, foram analisados aspectos moleculares e evolutivos do fator de transcrição MADS-box PISTILLATA (PI), a partir de sequências de transcritos isolados de inflorescências de bromélias nativas brasileiras. Sequências PI de Bromeliaceae foram comparadas com outras monocotiledôneas, com verificação de expressão em inflorescências de Tillandsia aeranthos (Loisel.) LB Sm. com o uso de hibridação in situ. Sequências de PI mostraram alta conservação, inclusive em um sítio de deleção encontrado para todos os membros analisados da família. Todos os membros da família se agruparam em um único clado em reconstruções filogenéticas. Entretanto, devido a características de taxas de mutações rápidas e divergência antiga do gene, não foi possível obter uma relação precisa entre diferentes famílias ou ordens. Todavia, PI mostrou ter potencial como ferramenta de análise filogenética para espécies proximamente relacionadas. Os transcritos de PI foram localizados principalmente em tecidos meristemáticos de regiões menos desenvolvidas de inflorescências de Tillandsia aeranthos. Flores mais desenvolvidas presentes nas inflorescências mostraram um padrão de acúmulo preferencial de transcritos PI em pétalas e sépalas, assim como esperado para flores com perianto diferenciado em sépalas e pétalas. De qualquer forma, Tillandsia aeranthos se mostrou eficiente para estudos morfoanatômicos com enfoque em desenvolvimento evolutivo. / Bromeliaceae is an important monocotyledon family due to its high number of species in the Neotropic, and a wealth of adaptive characters to different habitats. Bromeliad flowers have large morphological variations, often neglected in morpho-anatomy, floral development and phylogeny studies. For a better understanding of its floral developmental mechanisms that lead to morphological variations, molecular and evolutionary aspects of the transcription factor MADS-box PISTILLATA (PI) encoding gene isolated had been investigated in native bromeliad inflorescences. Bromeliaceae PI sequences were compared with other monocots, and the expression of these transcripts was detected in Tillandsia aeranthos (Loisel.) LB Sm. inflorescences using in situ hybridization. The PI sequences display high conservation, including a specific deletion site found in all family members. Likewise, all Bromeliaceae members grouped into a single clade in phylogeny reconstruction. However, due to rapid mutation rate and ancient divergence in PI, it was not possible to obtain a precise relationship between different monocot families and orders. Nevertheless, PI showed potential as a phylogenetic tool for analysis between closely related species. PI transcripts were located mainly in meristematic tissues from less developed regions of the inflorescences. More developed flowers showed a preferential PI transcripts accumulation in petals and stamens as expected for differentiated perianth, found in Bromeliaceae flowers. Anyway, Tillandsia aeranthos showed good potential skills for morpho-anatomy focused in evolutionary development.
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Isolamento e caracterização de sequências de PISTILLATA em bromeliaceae e estudo de expressão em tecidos florais de Tillandsia aeranthos (Loisel.) LB Sm.Gaeta, Marcos Letaif January 2016 (has links)
Bromeliaceae é uma família importante entre as monocotiledôneas devido ao seu elevado número de espécies distribuídas no Neotrópico, e por uma riqueza de caracteres adaptativos a diferentes habitats. Flores de bromélias possuem uma grande variação morfológica, frequentemente negligenciada em estudos de morfoanatomia e de filogenia. Para uma melhor compreensão dos mecanismos de desenvolvimento que levam a essas variações, foram analisados aspectos moleculares e evolutivos do fator de transcrição MADS-box PISTILLATA (PI), a partir de sequências de transcritos isolados de inflorescências de bromélias nativas brasileiras. Sequências PI de Bromeliaceae foram comparadas com outras monocotiledôneas, com verificação de expressão em inflorescências de Tillandsia aeranthos (Loisel.) LB Sm. com o uso de hibridação in situ. Sequências de PI mostraram alta conservação, inclusive em um sítio de deleção encontrado para todos os membros analisados da família. Todos os membros da família se agruparam em um único clado em reconstruções filogenéticas. Entretanto, devido a características de taxas de mutações rápidas e divergência antiga do gene, não foi possível obter uma relação precisa entre diferentes famílias ou ordens. Todavia, PI mostrou ter potencial como ferramenta de análise filogenética para espécies proximamente relacionadas. Os transcritos de PI foram localizados principalmente em tecidos meristemáticos de regiões menos desenvolvidas de inflorescências de Tillandsia aeranthos. Flores mais desenvolvidas presentes nas inflorescências mostraram um padrão de acúmulo preferencial de transcritos PI em pétalas e sépalas, assim como esperado para flores com perianto diferenciado em sépalas e pétalas. De qualquer forma, Tillandsia aeranthos se mostrou eficiente para estudos morfoanatômicos com enfoque em desenvolvimento evolutivo. / Bromeliaceae is an important monocotyledon family due to its high number of species in the Neotropic, and a wealth of adaptive characters to different habitats. Bromeliad flowers have large morphological variations, often neglected in morpho-anatomy, floral development and phylogeny studies. For a better understanding of its floral developmental mechanisms that lead to morphological variations, molecular and evolutionary aspects of the transcription factor MADS-box PISTILLATA (PI) encoding gene isolated had been investigated in native bromeliad inflorescences. Bromeliaceae PI sequences were compared with other monocots, and the expression of these transcripts was detected in Tillandsia aeranthos (Loisel.) LB Sm. inflorescences using in situ hybridization. The PI sequences display high conservation, including a specific deletion site found in all family members. Likewise, all Bromeliaceae members grouped into a single clade in phylogeny reconstruction. However, due to rapid mutation rate and ancient divergence in PI, it was not possible to obtain a precise relationship between different monocot families and orders. Nevertheless, PI showed potential as a phylogenetic tool for analysis between closely related species. PI transcripts were located mainly in meristematic tissues from less developed regions of the inflorescences. More developed flowers showed a preferential PI transcripts accumulation in petals and stamens as expected for differentiated perianth, found in Bromeliaceae flowers. Anyway, Tillandsia aeranthos showed good potential skills for morpho-anatomy focused in evolutionary development.
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Linfomas osseos primarios : estudo comparativo com linfomas nodais e linfomas osseos metastaticos quanto a imunoexpressão de proteinas relacionadas a apoptose, regulação do ciclo celular e adesão celular / Primary bone lymphomas : comparison of the immunoexpression of apoptosis related proteins and adhesion molecules with nodal lymphomas and lymphomas metastatic to boneLima, Francisco Pignataro 02 November 2008 (has links)
Orientadores: Eliane Maria Ingrid Amstalden, Jose Vasallo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T14:26:55Z (GMT). No. of bitstreams: 1
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Previous issue date: 2008 / Resumo: Cerca de 20% a 40% dos linfomas não-Hodgkin (LNH) B são extranodais e caracterizam-se por comportamento biológico distinto, de acordo com o seu sítio de origem e variante histológica. Linfoma ósseo primário (LOP), uma forma extranodal rara, apresenta-se na maior parte dos casos como LNH B de alto grau e evolui com bom prognóstico. Os estudos sobre o LOP são escassos e suas características imunofenotípicas e citogenéticas ainda não estão bem definidas. Neste trabalho, foram analisados três grupos de linfomas difusos de grandes células B (LDGCB): a) ósseos primários, B) nodais e c) metastáticos (com envolvimento ósseo secundário). Foram investigados, nesses grupos, dados clínicos e de sobrevida, aspectos morfológicos, imunofenotípicos e citogenéticos. Na análise imunoistoquímica, foi verificado o estudo das moléculas envolvidas na apoptose e regulação do ciclo celular (Bcl-2, Bax, p53, Bcl-6, p21, pRB) moléculas envolvidas na adesão celular (CD29, CD62L, CD44, CD51) índice de proliferação celular (Ki-67) e expressão das subpopulações de células infiltrantes do sistema imune (CD3, CD4, CD5, CD8, CD57, CD68, Foxp3) com objetivo de melhor caracterizar o LOP. Foram selecionados no total 138 casos de LDGCB, provenientes dos departamentos de Anatomia Patológica FCM-UNICAMP, Hospital AC.-Camargo - Fundação Antônio Prudente, Instituto de Traumatologia e Ortopedia da USP e Centre Hospitalier Universitarie (CHU Purpan). Do total de 138, 76 casos eram linfomas ósseos primários, 46 casos linfomas nodais e 16 casos linfomas com envolvimento ósseo secundário. Nossos resultados demonstraram que o LOP possui características evolutivas que diferem dos demais LDGCB extranodais. Observamos que a maioria dos LDGCB ósseos primários (66%) pertenceram ao imunofenótipo centro germinativo (CG) e não exibiram diferenciação plasmacítica. Os três grupos de LDGCB analisados exibiram forte imunoexpressão das proteínas relacionadas à apoptose e ao ciclo celular enquanto que a imunoexpressão de moléculas de adesão foi fraca ou ausente, na maioria dos casos. O estudo da sobrevida, envolvendo a ausência da imunoexpressão das moléculas de adesão CD29, CD62L e CD51, nos três grupos, revelou-se significativamente associada com melhor sobrevida. O significado biológico deste achado ainda está para ser esclarecido. O CD44 foi o mais fortemente expresso nas células tumorais. As moléculas envolvidas na apoptose Bax e p53 foram relacionadas à pior evolução e o pRB à evolução favorável nos LOP. As proteínas Bcl-2 e Bcl-6 foram expressas nos três grupos de linfomas. Com a análise em 32 casos de LOP das principais translocações em LNH (MYC, BCL2/IGH, BCL6, ALK, IGH/CCND1 e PAX5), conseguimos caracterizar melhor esta entidade. Rearranjos, envolvendo o BCL2 e MYC foram encontrados, entretanto não ocorreram rearranjos do BCL6. Os demais genes não demonstraram translocações. Estes achados indicam que o LOP deve ser considerado uma categoria distinta de LDGCB extranodal, com aspectos genéticos e moleculares mais próximos dos nodais / Abstract: The incidence of extranodal lymphomas is increasing in the last decades. Around 20 to 40% of B-cell non-Hodgkin lymphomas (NHL) arise outside the lymph nodes and may present distinct biological behavior, as compared to the corresponding nodal counterpart of similar histological grade. The heterogeneity of large B-cell lymphomas (LBCL) has been matter of various studies, although morphology alone may be insufficient to separate groups of clinical relevance, immunophenotyping and molecular techniques have demonstrated that different variants of LBCL may present diverse clinical and prognostic features. Primary bone lymphoma (PBL) is rare form of extra nodal NHL, which generally presents a more favorable prognosis than the nodal counterpart. The reason for this is unclear. We selected a large series (n=138) of nodal NHL (N=46), primary (n=76) and metastatic (n=16) bone diffuse large cell lymphomas collected in tissue micro-arrays from several centers in France and Brazil. We investigated to be comparing clinical, morphological and immunophenotypic features. Immunohistochemical detection of proliferation markers, apoptosis related proteins and adhesion molecules was be marked. Our results demonstrated that primary bone LDGCB possesses peculiar clinical characteristics that you differ of the other extranodal LDGCB as the longest evolution and smaller recurrence tax. The three groups of LDGCB exhibited strong immunoexpression of the related proteins the apoptosis and cellular cycle while the immunoexpression of adhesion molecules was weak or absentee in most of the cases. These cases were classified according to the expression of antigens with germinal center (GC) (n=37/62) or non germinal center (non-GC) (n=26/62) stages of B-cell differentiation. The study of the survival involving the absence of the imunoexpressão of the adhesion molecules CD29, CD62L and CD51 in the three groups was revealed significantly associated with better outcome. The biological meaning of this discovery is still to be explained. CD44 was it more strongly expressed in the tumor cells and it demonstrated relationship away with inferior survival without statistical significance. The molecules involved in the apoptosis Bax and p53 were related the worst evolution and the pRB the favorable evolution in PBL. The proteins Bcl-2 and Bcl-6 were expressed in the three lymphomas groups and associated the worst evolution, however without significance. By fluorescence in situ hybridization, we found a substantial number of cases with a rearrangement of BCL2 (n=9/32) and MYC (n= 3/32) whereas PAX5, BCL6, BCL-1/Cyclin D1 and ALK genes were in germ line configuration. Interestingly, one case, with GC phenotype, showed a dual BCL2 and MYC rearrangement. We observed that the majority of the cases with rearrangements were of GC phenotype. These results, associated with the lack of BCL6 rearrangement, suggest that bone DLBCL represents a specific group within extranodal B-cell lymphomas. Keywords: diffuse large cell lymphoma, adhesion molecules, apoptosis, fluorescent in situ hybridization / Doutorado / Anatomia Patologica / Doutor em Ciências Médicas
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Isolamento e caracterização de sequências de PISTILLATA em bromeliaceae e estudo de expressão em tecidos florais de Tillandsia aeranthos (Loisel.) LB Sm.Gaeta, Marcos Letaif January 2016 (has links)
Bromeliaceae é uma família importante entre as monocotiledôneas devido ao seu elevado número de espécies distribuídas no Neotrópico, e por uma riqueza de caracteres adaptativos a diferentes habitats. Flores de bromélias possuem uma grande variação morfológica, frequentemente negligenciada em estudos de morfoanatomia e de filogenia. Para uma melhor compreensão dos mecanismos de desenvolvimento que levam a essas variações, foram analisados aspectos moleculares e evolutivos do fator de transcrição MADS-box PISTILLATA (PI), a partir de sequências de transcritos isolados de inflorescências de bromélias nativas brasileiras. Sequências PI de Bromeliaceae foram comparadas com outras monocotiledôneas, com verificação de expressão em inflorescências de Tillandsia aeranthos (Loisel.) LB Sm. com o uso de hibridação in situ. Sequências de PI mostraram alta conservação, inclusive em um sítio de deleção encontrado para todos os membros analisados da família. Todos os membros da família se agruparam em um único clado em reconstruções filogenéticas. Entretanto, devido a características de taxas de mutações rápidas e divergência antiga do gene, não foi possível obter uma relação precisa entre diferentes famílias ou ordens. Todavia, PI mostrou ter potencial como ferramenta de análise filogenética para espécies proximamente relacionadas. Os transcritos de PI foram localizados principalmente em tecidos meristemáticos de regiões menos desenvolvidas de inflorescências de Tillandsia aeranthos. Flores mais desenvolvidas presentes nas inflorescências mostraram um padrão de acúmulo preferencial de transcritos PI em pétalas e sépalas, assim como esperado para flores com perianto diferenciado em sépalas e pétalas. De qualquer forma, Tillandsia aeranthos se mostrou eficiente para estudos morfoanatômicos com enfoque em desenvolvimento evolutivo. / Bromeliaceae is an important monocotyledon family due to its high number of species in the Neotropic, and a wealth of adaptive characters to different habitats. Bromeliad flowers have large morphological variations, often neglected in morpho-anatomy, floral development and phylogeny studies. For a better understanding of its floral developmental mechanisms that lead to morphological variations, molecular and evolutionary aspects of the transcription factor MADS-box PISTILLATA (PI) encoding gene isolated had been investigated in native bromeliad inflorescences. Bromeliaceae PI sequences were compared with other monocots, and the expression of these transcripts was detected in Tillandsia aeranthos (Loisel.) LB Sm. inflorescences using in situ hybridization. The PI sequences display high conservation, including a specific deletion site found in all family members. Likewise, all Bromeliaceae members grouped into a single clade in phylogeny reconstruction. However, due to rapid mutation rate and ancient divergence in PI, it was not possible to obtain a precise relationship between different monocot families and orders. Nevertheless, PI showed potential as a phylogenetic tool for analysis between closely related species. PI transcripts were located mainly in meristematic tissues from less developed regions of the inflorescences. More developed flowers showed a preferential PI transcripts accumulation in petals and stamens as expected for differentiated perianth, found in Bromeliaceae flowers. Anyway, Tillandsia aeranthos showed good potential skills for morpho-anatomy focused in evolutionary development.
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