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Cytogenetic analysis of the malignant clone is clinically important in haematological malignancy. Analysis by metaphase cytogenetics is restricted to the small proportion of malignant cells which are actively dividing. This thesis explores the dynamics of malignant clones using the technique of fluorescence in situ hybridization (FISH) to visualize chromosomal abnormalities in interphase (non-dividing) cells. Hyperdiploid (>46 chromosomes) clones have been investigated by interphase FISH in acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) using appropriate chromosome-specific probes. A hyperdiploid clone was detected in interphase cells in 9/65 patients with ALL in whom metaphase cytogenetics had failed or was normal. A single hyperdiploid cell was identified as clonal in one patient with MDS but not in six others with AML, MDS or ALL. The involvement of different cell lineages in the malignant clone was investigated by simultaneous FISH and identification of the cell type by morphology or monoclonal antibodies. In ALL, hyperdiploid clones were restricted to the lymphoid blasts in 9/9 cases, while Philadelphia (Ph) positive clones, (identified by probes to the genes m- BCR or M-BCR and ABL which fuse as a result of the translocation) were found either in lymphoid blasts alone (1/3 cases) or in both lymphoid and myeloid cells (2/3 cases). In AML trisomy 8 (using a chromosome 8-specific probe) and an 11q23 abnormality (which split YAC 13HH4) were both found only in the myeloid blasts, in 3/3 and 2/2 cases respectively. A sensitive method for the detection of hyperdiploid \geq 50 clones in ALL was developed for minimal residual disease detection. Simultaneous probing of three chromosomes enabled detection of one hyperdiploid cell in 10,000. Heterogeneity in the speed with which the clone was eliminated in remission was seen in 16 patients and early relapse was detected in one patient.

Made available in DSpace on 2014-06-11T19:32:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-31Bitstream added on 2014-06-13T21:03:44Z : No. of bitstreams: 1 poletto_ab_dr_botib.pdf: 1705638 bytes, checksum: 76b096174aaade2107a2ac15d63db4a2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este trabalho teve por objetivo analisar citogeneticamente espécies da família Cichlidae de diferentes origens. Foram feitas análises de uma espécie asiática, de 22 africanas e 30 neotropicais. O número diplóide na família variou de 2n=40 a 2n=60. Os sítios de genes de RNAr 18S variaram de 2 a 9 cromossomos portadores, com sítios terminais para os africanos e em apenas um grande par cromossômico em posição intersticial ou terminal para a maioria dos neotropicais. A hibridação do gene RNAr 5S marcou sítios múltiplos em regiões intersticiais e centroméricas em duas espécies africanas, enquanto que nos ciclídeos neotropicais marcou de um a dois pares, sempre em posição intersticial. Em Haplocromis obliquidens foram encontrados um a dois grandes cromossomos metacêntricos supernumerários e heterocromáticos em 39,6% da população, tanto em fêmea quanto em macho. Este cromossomo B apresentou sítios múltiplos de hibridação de DNAr 18S e sinais leves na região centromérica do DNA satélite centromérico SATA. Sequências repetitivas inseridas em BACs hibridaram de forma difusa por toda a extensão do elemento supernumerário. Esse cromossomo B pode ter se originado a partir de um isocromossomo, ou por acúmulo de DNA repetitivo num pequeno proto-cromossomo B. Em Metriaclima lombardoi um grande cromossomo B metacêntricoe heterocromático foi detectado em 50% das fêmeas e em nenhum macho. Este elemento não apresentou sinais de hibridação de DNAr 18S, porém DNAs repetitivos inseridos em BACs hibridaram nos braços menores do primeiro par do complemento A, fortemente na região centromérica, e de forma difusa ao longo do cromossomo B. Hipotetizamos que este cromossomo B pode ter se originado a partir de um fragmento do primeiro par do complemento A, tendo acumulado DNAs repetitivos como consequência da ausência de recombinação. Sugerimos também que a origem... / This work had the aim of cytogenetic analyses of Cichlidae species from diverse origin. It was analyzed one asian, 22 african and 30 neotropical species. The diploid number ranged between 2n=40 and 2n=60 chromosomes. The 18S rRNA gene sites varied from two to nine bearer chromosomes, in terminal position for the africans ones, and just in one large chromosome pair, in interstitial or terminal position, for the majority of the neotropical ones. The hybridization with 5S rRNA genes labeled multiple sites in two African species, in centromeric and interstitial position, whereas in the Neotropical cichlids it labeled interstitially in one to two pairs of chromosomes. In Haplochromis obliquidens it was found one or two metacentric supernumerary heterochromatic chromosome(s) in 39,6% of the population including males as well as females. This B chromosome showed multiple hybridization sites of 18S rDNA and the SATA centrometromeric satellite DNA slightly labeled in the cetromeric region, while BAC-clone enriched of repeated sequences hybridized scattered along the B chromosome. This B chromosome could have originated from an isochromosome or by accumulation of repetitive DNA in a small proto-B chromosome. In Metriaclima lombardoi a large heterochromatic metacentric B chromosome was detected in 50% of females and none males. This element did not show signals of hybridization of 18S rDNA but BAC-clone enriched of repeated DNAs hybridized in the small arms of first pair of complement, strongly in the centromeric region, and scattered along the B chromosome. We hypothesized that this B chromosome could have originated from a fragment of the A complement, and had acumulated repetitive DNAs as consequence of absence of recombination. We also suggest that the origin of this B from the first chromosome pair could be driving the bearer individuals to present female characteristics independently... (Complete abstract click electronic access below)

Made available in DSpace on 2018-08-01T22:57:30Z (GMT). No. of bitstreams: 1 tese_9189_Dissertação Final Paulo Marcos Amaral Silva20160628-103338.pdf: 832244 bytes, checksum: de4cc3d0c60078e1f76f3d4f95f3b534 (MD5) Previous issue date: 2015-08-14 / The dioecy occurs in 6% of the angiosperms, including all the Myrsine species (Primulaceae) that show male and female individuals distinguishable based in sexual organs and morphology of sepals and petals. The sexual system fixed in this genus, was the motivation in research if the existence of sexual chromosomes culminate in dioecy for Myrsine. Employing tissue culture, flow cytometry and cytogenetic tools, the aimed this study was characterize the karyotype of Myrsine coriacea (Sw.) R.Br. ex Roem & Schult., Myrsine umbellata Mart., Myrsine guianensis (Aubl.) Kuntze and Myrsine parvifolia A.DC. From leaves of male and female individuals collected in the field and leaves and roots of unknown sex obtained of in vitro culture, were found mean values of DNA content statistically identical. These data were attributed to the presence of secondary metabolites reported for the genus. However, an intraspecific variation was observed in cytogenetic analysis in the four Myrsine species. Slides exhibited metaphases with 2n = 45 and 2n = 46 chromosomes in different individuals. These results were observed consistently from different times of exposition to anti-mitotic agent and distinct treatments of enzymatic maceration. Thus, the chromosome number variation found, associated with the sexual system well defined, can be concerning to the sexual chromosomes in Myrsine genus. These data suggest a chromosomal system of sex 9 determination with multiple X and/or Y described in some plant species. The system ZW also is possible, as well as X0 or Z0, systems still not reported in vegetal groups. The present work provided the first data about the nuclear DNA content by flow cytometry in Myrsine and supplied cytogenetics evidences that indicate the existence of sexual chromosomes.

In Western Europe and North America, acute myeloid leukaemia (AML) is predominantly a disease of the elderly, with a median age at the time of presentation in excess of 60 years. However, many clinical trials in AML fail to recruit elderly adults due to a combination of strict entry criteria, or physician or patient bias. Thus, clinical outcome data from many trials may not be readily applicable to older patients with the disease. Furthermore, because the clinical outcome for many older patients with AML is frequently poor, elderly patients who receive intensive chemotherapy with curative intent are frequently selected for treatment on clinical criteria rather than on objective prognostic criteria that may define clinical outcome. The karyotype at the time of presentation may be considered one of the most important prognostic factors in adult AML. Therefore, the aim of this thesis were firstly to analyse the clinical outcome data from a cohort of elderly patients managed at a single centre in order to document the cytogenetic features of AML in an elderly population, to define the prognostic importance of presentation karyotype in the elderly, and to identify other prognostic factors. Retrospective analysis clearly demonstrated improved clinical outcome for older patients with AML over time, primarily as a consequence of improved supportive care and the delivery of more intensive chemotherapy. In addition, 'unfavourable' presentation karyotype, increasing age and raised serum LDH were found to correlate with poor clinical outcome Molecular cytogenetic techniques based upon fluorescence in-situ hybridisation technology offer the chance to detect and analyse cytogenetic aberrations at a higher resolution than can be achieved with conventional techniques. The cytogenetic data provided by comparative genomic hybridisation and mulitplex fluorescence in-situ hybridisation when used in the analysis of elderly patients with AML were found to correlate well with results obtained by conventional methods. Importantly, additive cytogenetic data were more likely to be provided if multiplex-fluorescence in-situ hybridisation was used in the analysis of cases with marker chromosomes or in cases with complex karyotype, although the technique was limited by an inability to reliably detect telomeric translocations. In addition, although both techniques can be used to complement conventional G-banding analysis, conventional FISH methods are often required to confirm the results.

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Epidemiology and Biostatistics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

The reliability of genome analysis and proficiency of genetic manipulation in vivo and in vitro are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, orientation with respect to telomeres, and linear alignment with respect to chromosomal features and dimensions. I undertook five studies aimed at integrating sorghum genomics and cytogenetics at several levels. The results help establish an entirely new "cyto-genomics" resource, impacts of which are likely to be broad. In the first study, I developed a FISH-based karyotyping system for Sorghum bicolor Moench. I used integrated structural genomic resources, including linkage maps and large-insert clonal libraries of sorghum genomic DNA to develop a 17-locus probe cocktail for simultaneous fluorescent in situ hybridization (FISH). This probe enabled facile identification of all chromosome pairs in mitotic chromosome spreads. Perhaps just as important, I established time-efficient means to select sorghum BAC clones for multi-probe FISH. Thus, an integrated cyto-genomics system for sorghum can be constructed without need of chromosome flow sorting or microdissection, both of which are difficult and costly. In the second study, hybridization of DNA clones from 37 different genomic regions enabled the assignment of linkage groups and orientation of linkage maps to chromosomes. Comparisons between genetic and physical distances throughout the genome enabled a new nomenclature for linkage group designation in sorghum. The results provide an integrated nomenclature system of Sorghum bicolor chromosomes and linkage groups. In the third study, I created high-resolution maps by FISH to pachytene bivalents for two linkage groups (B and H), and defined relationships between pericentromeric heterochromatin, centromeres, mapped markers and recombination rates. These relationships will help guide the development and use of sorghum genomics. In the fifth study, I used FISH in two ongoing gene-targeted efforts. For the maturity gene ma5 and fertility restoration gene rfl, I estimated physical lengths between currently available flanking molecular markers. This enables estimation of recombination densities in these regions and assessment of the applicability of map-based and -assisted cloning.

To enhance the scientific utility and practical application of the honey bee genome and assign the linkage groups to specific chromosomes, I identified chromosomes and characterized the karyotype of the sequenced strain DH4 of the honey bee. The primary analysis of the karyotype and ideogram construction was based on banding and Fluorescence In Situ Hybridization (FISH) for rDNA detection. FISH confirmed two locations for the NOR on telomeric regions of chromosomes 6 and 12 plus an additional less frequent signal on chromosome 1, all three of which were confirmed with silver staining (AgNO3). 4’6-diamidino-2phenylindole (DAPI), and CBanding methods were used to construct the primary ideograms that served as a basis to further identify the chromosomes and locate important structures. The primary map was compared with Giemsa banding, AgNO3-banding, Trypsin banding, and R-banding. The karyotype of the honey bee was established as two metacentric chromosomes (1 and 10), two submetacentric with ribosomal organizer (6 and 12), four submetacentric heterochromatic chromosomes (16, 15, 4 and 13), four euchromatic subtelocentric chromosomes (2, 8, 11 and 14) and four acrocentric chromosomes (3, 5, 7 and 9). In situ nick-translation banding methods were used to verify the heterochromatin distribution. The cytogenetic maps of the honey bee karyotype represented in the ideograms were subsequently used to place 35 mapped BACs (Solignac et. al. 2004) of Solignac’s BAC library. As the BACs hybridized to multiple sites, the mapping was based on strength and frequency of the signals. Location and position of the BACs was compared with those published in the different version of Map Viewer of the NCBI and BeeBase web sites. 10 BACs were confirmed with the last version of Map Viewer V4, 12 BACs were mapped based on high frequency and agreement with the earlier version of Map Viewer. 14 BACs were mapped as confirmed based on moderate frequency of the signal and agreement with the last version of MVV, most of these BACs hits as a secondary signal.

The reliability of genome analysis and proficiency of genetic manipulation in vivo and in vitro are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, orientation with respect to telomeres, and linear alignment with respect to chromosomal features and dimensions. I undertook five studies aimed at integrating sorghum genomics and cytogenetics at several levels. The results help establish an entirely new "cyto-genomics" resource, impacts of which are likely to be broad. In the first study, I developed a FISH-based karyotyping system for Sorghum bicolor Moench. I used integrated structural genomic resources, including linkage maps and large-insert clonal libraries of sorghum genomic DNA to develop a 17-locus probe cocktail for simultaneous fluorescent in situ hybridization (FISH). This probe enabled facile identification of all chromosome pairs in mitotic chromosome spreads. Perhaps just as important, I established time-efficient means to select sorghum BAC clones for multi-probe FISH. Thus, an integrated cyto-genomics system for sorghum can be constructed without need of chromosome flow sorting or microdissection, both of which are difficult and costly. In the second study, hybridization of DNA clones from 37 different genomic regions enabled the assignment of linkage groups and orientation of linkage maps to chromosomes. Comparisons between genetic and physical distances throughout the genome enabled a new nomenclature for linkage group designation in sorghum. The results provide an integrated nomenclature system of Sorghum bicolor chromosomes and linkage groups. In the third study, I created high-resolution maps by FISH to pachytene bivalents for two linkage groups (B and H), and defined relationships between pericentromeric heterochromatin, centromeres, mapped markers and recombination rates. These relationships will help guide the development and use of sorghum genomics. In the fifth study, I used FISH in two ongoing gene-targeted efforts. For the maturity gene ma5 and fertility restoration gene rfl, I estimated physical lengths between currently available flanking molecular markers. This enables estimation of recombination densities in these regions and assessment of the applicability of map-based and -assisted cloning.