Gene activity is regulated at two separate layers. Through structural and chemical properties of DNA – the primary layer of encoding – local signatures may enable, or disable, the binding of proteins or complexes of them with regulatory potential to the DNA. At a higher level – the structural layer of encoding – gene activity is regulated through the properties of higher order DNA structure, chromatin, and chromosome organization. Cells with abnormal chromosome compaction or organization, e.g. cancer cells, may thus have perturbed regulatory activities resulting in abnormal gene activity. Hence, there is a great need to decode the transcriptional regulation encoded in both layers to further our understanding of the factors that control activity and life of a cell and, ultimately, an organism. Modern genome-wide studies with those aims rely on data-intense experiments requiring sophisticated computational and statistical methods for data handling and analyses. This thesis describes recent advances of analyzing experimental data from quantitative biological studies to decipher the structural layer of encoding in human cells. Adopting an integrative approach when possible, combining multiple sources of data, allowed us to study the influences of chromatin (Papers I and II) and chromosomal aberrations (Paper IV) on transcription. Combining chromatin data with chromosomal aberration data allowed us to identify putative driver oncogenes and tumor-suppressor genes in cancer (Paper IV). Bayesian approaches enabling the incorporation of background information in the models and the adaptability of such models to data have been very useful. Their usages yielded accurate and narrow detection of chromosomal breakpoints in cancer (Papers III and IV) and reliable positioning of nucleosomes and their dynamics during transcriptional regulation at functionally relevant regulatory elements (Paper II). Using massively parallel sequencing data, we explored the chromatin landscapes of human cells (Papers I and II) and concluded that there is a preferential and evolutionary conserved positioning at internal exons nearly unaffected by the transcriptional level. We also observed a strong association between certain histone modifications and the inclusion or exclusion of an exon in the mature gene transcript, suggesting a functional role in splicing.

Despite major advances in our understanding of the biology of childhood acute myeloid leukemia (AML) and the development of new cytotoxic drugs, the prognosis of long-term survival is still only 60-65 %. In the present research, we studied the pharmacokinetics of drugs used in the induction therapy of childhood AML and performed in vitro drug sensitivity testing of leukemic cells from children with AML. The aims of the studies were to correlate the results of the analysis to biological and clinical parameters and to identify subgroups of AML with specific drug sensitivity profiles in order to better understand why treatment fails in some patients and how therapy may be improved. Blood samples were analysed to study the pharmacokinetics of doxorubicin (n=41), etoposide (n=45) and 6-thioguanine (n=50). Doxorubicin plasma concentration and total body clearance were correlated to the effect of induction therapy, and doxorubicin plasma concentration was an independent factor for complete remission, both in univariate and multivariate analysis including sex, age, and white blood cell count at diagnosis. For etoposide and 6-thioguanine no correlation was found between pharmacokinetics and clinical effect. Children with Down syndrome (DS) tended to reach higher blood concentrations of etoposide and thioguanine nucleotides, indicating that dose reduction may be reasonable to reach the same drug exposure as in children without DS. Leukemic cells from 201 children with newly diagnosed AML, 15 of whom had DS, were successfully analysed for in vitro drug sensitivity by the fluorometric microculture cytotoxicity assay (FMCA). We found that samples from children with DS were highly sensitive to most drugs used in AML treatment. In non-DS children, the t(9;11) samples were significantly more sensitive to cytarabine (p=0.03) and doxorubicin (p=0.035) than other samples. The findings might explain the very favorable outcome reported in children with DS and t(9;11)-positive AML. A specific drug resistance profile was found for several other genetic subgroups as well. A detailed study of MLL-rearranged leukemia showed that cellular drug sensitivity is correlated both to partner genes and cell lineage, findings that support the strategy of contemporary protocols to include high-dose cytarabine in the treatment of patients with MLL-rearrangement, both in AML and acute lymphoblastic leukemia (ALL). Our results indicate that drug resistance and pharmacokinetic studies may yield important information regarding drug response in different sub-groups of childhood AML, helping us to optimize future chemotherapy in childhood AML.

No description available.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available in electronic form as viewed 2/16/2009.

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Orientador: Agnes Cristina Fett Conte / Banca: Cleide Largman Borovik / Banca: Cláudia Regina Bonini Domingos / Resumo: Síndromes Mielodisplásicas (SMD) compreendem um conjunto heterogêneo de doenças hematopoéticas caracterizadas por hematopoese ineficaz, que geralmente apresentam citopenias no sangue periférico, medula óssea hipercelular, diferenciação celular displásica e propensão ao desenvolvimento de leucemia mielóide aguda. São classificadas em oito tipos e a incidência anual é estimada entre dois e 12 casos por 100.000 pessoas da população em geral e em até 50 casos por 100.000 indivíduos com idades superiores a 60 anos. A análise cromossômica das células da medula óssea dos doentes ao diagnóstico detecta alterações diretamente relacionadas com o prognóstico em aproximadamente 50% dos casos. Alguns genes também foram relacionados à etiologia e prognóstico das mielodisplasias. O gene FOXO3, um supressor de tumor, embora não estudado anteriormente em SMD, é um dos genes que mais se expressam no tecido hematopoético normal. Alterações neste gene poderiam resultar em hematopoese anormal, pois já foram relacionadas a outros tipos de câncer, com mutações descritas no éxon 1. O objetivo deste trabalho foi estudar células da medula óssea de doentes com SMD de qualquer tipo, ao diagnóstico, para investigar a presença de alterações cromossômicas e de mutações no éxon 1 do FOXO3. A análise citogenética foi realizada em metáfases submetidas ao bandamento GTG, obtidas de culturas de curta duração de células da medula, sem estimulação mitogênica. Para a análise molecular foi extraído o DNA, realizada a amplificação gênica pela Reação em Cadeia da Polimerase e realizado o sequenciamento direto do éxon 1. Entre os 25 casos analisados, três (12%) apresentaram alterações cromossômicas clonais isoladas: deleção intersticial do braço longo do cromossomo 5; monossomia do cromossomo 21 e monossomia do cromossomo 22. Todas puderam ser relacionadas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Myelodysplastic syndrome (MDS) constitute a heterogeneous group of hematopoietic diseases characterized by ineffective hematopoiesis usually with peripheral blood cytopenia, hypercellular bone marrow, dysplastic differentiation and a tendency to evolve to acute myeloid leukemia. They are classified in eight categories by the World Health Organization. The annual incidence is estimated at between two and 12 cases per 100,000 individuals in the general population and up to 50 cases per 100,000 of over 60-year olds. A chromosomal analysis of bone marrow cells at diagnosis identifies changes directly related to prognosis in approximately 50% of cases. Additionally, some genes are also associated to the etiology and prognosis of myelodysplasia. Although not previously studied in respect to MDS, a tumor suppressor, FOXO3, is one of the most commonly expressed genes in normal hematopoietic tissue. Changes in this gene could therefore result in abnormal hematopoiesis, as mutations described in exon 1 have already been associated with other types of cancer. The aim of this study was to investigate chromosomal alterations and mutations in exon 1 of FOXO3 in bone marrow cells from patients diagnosed with any type of MDS. Cytogenetic analysis was performed on metaphases submitted to GTG banding, obtained from short-term cultures of bone marrow cells without mitogenic stimulation. To evaluate mutations in the FOXO3 gene, DNA was extracted from the bone marrow, gene amplification was achieved by polymerase chain reaction and direct sequencing was performed. Of the 25 cases analyzed, three (12%) showed clonal chromosomal abnormalities in isolation characterized as the interstitial deletion of the long arm of chromosome 5, monosomy 21 and monosomy 22. All were correlated to the diagnosis and/or prognosis of patients. No mutations were detected in exon 1, but the 159C>T polymorphism was detected... (Complete abstract click electronic access below) / Mestre

Introdução: Durante as últimas décadas, a utilização de métodos moleculares como Hibridizacão in situ por fluorescência (FISH) e Hibridização Genômica Comparativa (array-CGH) mudou dramaticamente a perspectiva em relação à detecção de rearranjos genômicos submicroscópicos. O número de doenças identificadas como causada por microdeleções/microduplicações cromossômicas aumentou rapidamente, trazendo um papel crucial para a citogenética no diagnóstico destas condições. Objetivo: O objetivo deste estudo foi identificar e caracterizar microdeleções cromossômicas associadas a síndromes de malformações em um laboratório de citogenética de referência de um hospital público do sul do Brasil. Métodos: Estudo retrospectivo e prospectivo, em uma série consecutiva de amostras. O estudo foi baseado em registros hospitalares e laboratoriais de amostras de uma coorte de pacientes com suspeita clínica de microdeleção cromossômica. Foram selecionadas amostras de indivíduos em que o diagnóstico clínico proposto incluia uma suspeita de rearranjo nos cromossomos 4p16.3, 5p15.2, 5q35, 7q11.23, 8q24.12, 15q11-q12, 16p13.3, 17p13.3, 17p11. 2,2 e 22q11 que foram analisados por hibridização in situ por fluorescência (FISH). Em 11 amostras com microdeleções, hibridização genômica comparativa (array-CGH) foi realizada. Resultados: Um total de 504 amostras foram avaliadas, sendo as suspeitas mais comuns a deleção 22q11.2 (29,5%), a síndrome de Prader-Willi (21,6%), a síndrome de Williams-Beuren (15%) e da síndrome de Angelman (13 %). Em 120 deles (23,8%) desequilíbrios cromossómicas relacionadas com o diagnóstico clínico foram encontrados. A del7q11.23 foi a alteração mais frequente (8,5%) detectada, seguida por del22q11.2 (5,3%) e del15q11-q12 (4,5%). Conclusões: Nossos achados reforçam à estratégia de que um teste de citogenética molecular sensível associada com uma avaliação clínico qualificado são cruciais para a detecção e caracterização precisa de deleções cromossômicas submicroscópicas. Além disso, nosso estudo enfatiza a necessidade de educação continuada para o desenvolvimento e utilização de novas tecnologias para o diagnóstico citogenético, as quais, em nossa experiência, puderam ser introduzidas com sucesso em um hospital público do sul do Brasil. / Background: During the past decades, the widespread use of FISH and microarray-based technologies dramatically changed our perspective regarding detection of submicroscopic genomic rearrangements. The number of diseases identified as caused by chromosomal microdeletions/microduplications increased quickly, bringing a new and crucial role for cytogenetics in the diagnosis of these conditions. Objective: The purpose of this study was to identify and chraracterize chromosomal microdeletions associated with malformation syndromes in a reference cytogenetics laboratory from a public hospital of Southern Brazil. Methods: Using retrospective and prospective approaches, we evaluated a consecutive series of samples. The study was based in hospital and laboratorial records of samples from a cohort of subjects with clinical suspicion of a chromosomal microdeletion. We selected samples from subjects in whom a clinical diagnosis was proposed, which included deletion of chromosomes 4p16.3, 5p15.2, 5q35, 7q11.23, 8q24.12, 15q11-q12, 16p13.3, 17p13.3, 17p11.2,2 and 22q11 identified by fluorescence in situ hybridization (FISH) analysis. In 11 samples with microdeletions, array-based comparative genomic hybridization (array-CGH) was performed. Results: A total of 504 samples were evaluated, being the most common suspicions the 22q11.2 deletion (29.5%), the Prader-Willi syndrome (21.6%), the Williams-Beuren syndrome (15%) and the Angelman syndrome (13%). In 120 of them (23.8%) chromosomal imbalances related to the clinical diagnosis were found. The deletion 7q11.23 was the most frequently finding (8.5%), followed by del22q11.2 (5.3%) and del15q11-q12 (4.5%). Conclusions: Our findings provide support to the idea that a sensitive molecular cytogenetic test associated with a qualified clinical evaluation are crucial for the detection and precise characterization of submiscroscopic chromosome deletions. Additionally, our study emphasizes the need of continuing 6 education for the improvement of the cytogenetic diagnosis technology, which was successfully introduced in a public hospital of Southern Brazil.

Highly profitable black caviar market and the depletion of wild sturgeon stocks warrant improvements in sturgeon aquaculture. Therefore, chromosomal manipulations, particularly gynogenesis, are focused on for increasing the ratio of females over males in progeny. The present study focused on optimizing chromosomal manipulations in sturgeons, particularly gynogenesis. The reasons of low survival rates were analyzed and the critical steps of gynogenesis induction processes were optimized. In addition, alternative ways of DNA inactivation in sperms were investigated, as well as the influence of native light-dependent DNA repair mechanisms on gynogenesis induction. Methods of interspecific gynogenesis usage for simplifying gynogenetic progeny separation were also proposed. Spectrophotometry analysis was used to investigate the ability of UV light, as the most common DNA inactivating agent, to penetrate into sperm. In addition, investigation of UV-irradiated sperm motility and results of partial gynogenesis induction showed that low transparency of sperms for UV-light can cause significant heterogeneity of UV-irradiation. As a result, a proper dilution of sperm was suggested as a critical step for homogeneous UV-irradiation of samples. Gynogenesis in sterlet was induced with chemical agents that damage sperm DNA, as an alternative to UV irradiation for applied in large-scale production of gynogenotes. All tested substances showed ability to inactivate DNA in spermatozoa, and thus producing gynogenotes. Negative impact of treatments with chemical agents on the sperm motility was observed. Subsequently, these treatments had a low efficiency of gynogenesis induction. The highest percentage of produced gynogenetic larvae 19.8 ? 8.9% was obtained by treatment with aminomethyl-4,5?,8-trimethylpsoralen (AMT) at 50 ?M followed by UV-A (360 nm) irradiation at dose of 900 J/m2. Therefore, this treatment could be used as a substitute for commonly used UV-C irradiation, e.g., in the case of large volumes of sperm. Detailed investigation of photoreactivation in sturgeon sperm revealed a significant level of light-dependent DNA restoration in sperms irradiated with high doses of UV-C light. Induction of gynogenesis with UV-C irradiation followed by exposure to visible light resulted in significant deviations from the typical Hertwig effect. In contrast, the red light with a wavelength of more than 600 nm did not result in decreased DNA damage, instead a moderate increase in damage was observed, i.e., it did not induce photoreactivation. Therefore, the use of infrared light to illuminate work stations during the induction of gynogenesis is suggested. The use of interspecific gynogenesis, particularly gametes of sturgeon species with different ploidy levels, was suggested as a way to simplify the separation of gynogenotes. In addition, application of this method allowed studying the effectiveness of DNA-inactivation and ploidy restoration treatments separately, as well as evaluation of fitness parameters and survival rates in each group of progeny without the physical separation of fish. Finally, the protocol for tetraploidization in sterlet was optimized for the prospective using tetraploid individuals for the induction of gynogenesis and androgenesis with diploid eggs and sperm. In conclusion, the described methods and protocols allowed gynogenesis induction in sturgeons with a survival rate sufficient for aquaculture, taking into consideration their high fertility, although further studies of the consequences of this treatment on fish is required.

A SMD primária hipocelular ocorre com uma frequência de 10-20% dos casos de SMD no adulto, no entanto, é o subtipo mais frequente na infância. O diagnóstico da SMD primária hipocelular é bastante difícil, pois devido à ausência de células na medula óssea esta pode ser confundida com a LMA hipocelular ou AA. O diagnóstico diferencial entre estas entidades hematológicas é de extrema importância devido a maior agressividade da LMA e a possibilidade de evolução da SMD para LMA. Além disso, SMD e AA são indicadas para o TCTH, entretanto, o regime de condicionamento pré-transplante é específico para cada doença. A combinação entre a análise morfológica, realizada através do mielograma e biópsia de medula óssea, e análise citogenética tem desempenhado um papel fundamental no reconhecimento da SMD primária hipocelular. Entretanto, estudos têm sido realizados para tentar melhorar o diagnóstico da doença levando em consideração as características biológicas da SMD como a presença de apoptose. Sendo assim, este estudo teve como objetivo caracterizar o padrão cromossômico da SMD primária hipocelular e correlacionar com aspectos celulares e clínicos. Foram analisados citogeneticamente 86 casos de SMD primária hipocelular, 74 AR, 10 com AREB e 2 com AREB-t. Dentre os pacientes com AR 50% apresentaram cariótipo anormal e todos os pacientes com AREB e AREB-t apresentaram cariótipo anormal. A alteração cromossômica mais frequente foi a del(17p), seguida de alterações envolvendo o cromossomo 7. Nossos resultados sugerem que o padrão cromossômico em SMD primária hipocelular é caracterizado principalmente por perdas parciais e completas de cromossomos (deleções e monossomias). A análise citogenética auxiliou no diagnóstico dos casos com suspeita de SMD primária hipocelular e foi uma importante ferramenta para a escolha do tratamento. O IPSS mostrou ser um bom sistema de escala prognostica para este grupo de pacientes. Alterações envolvendo o cromossomo 17 estiveram associados com o subtipo AR e características displásicas envolvendo o setor granulocítico, no entanto, a del(17p) também pôde ser observada no subtipo AREB. Para análise de apoptose foram utilizadas 42 amostras de pacientes com SMD, 23 com SMD hipocelular, 8 com SMD normocelular e 11 com SMD hipercelular. O índice de apoptose total nos casos de SMD primária hipocelular apresentou uma média de 9,5%, enquanto os pacientes com SMD primária normocelular e hipercelular apresentaram uma média de 12% e 14,1%, respectivamente. Pela análise de linhagens específicas as células já comprometidas com o programa de diferenciação celular parecem ser o principal alvo do programa de apoptose. Apesar dos pacientes com SMD primária hipocelular apresentarem índice de apoptose total mais elevado que os controles eles foram sempre inferiores aos apresentados pela SMD primária normocelular e hipercelular, com exceção dos eritroblastos que foram maiores nos casos de SMD primária hipocelular. O índice de apoptose total foi maior nos estágios iniciais da doença independentemente da celularidade da medula óssea. Os pacientes com del (11q) e del (17p) estiveram associados com a diminuição do índice de apoptose total. Nossos resultados sugerem que a hipocelularidade da medula óssea não é causada pelo processo de apoptose e sim provavelmente por algum defeito no programa de proliferação celular. / Hypocellular primary MDS occurs in a frequency of 10-20% of the adults MDS cases, however it is the most frequent subtype in childhood. Diagnosis of hypocellular primary MDS is very difficult, because the small number of cells in bone marrow and it can be confused with hypocellular AML or AA. The differential diagnosis between these hematologic entities is extremely important because of AML is more aggressive and the possibility of MDS evolve to AML. Besides, MDS and AA are indicated to HSCT, however, conditioning regimens before the transplantation is specific for each disease. The combination between morphologic analysis, carried out through mielogram and bone marrow biopsy, and cytogenetic analysis have been performed a fundamental role on the recognition of hypocellular primary MDS. However, studies have been carried out to try improving the MDS diagnosis considering the biological characteristics as the presence of apoptosis. Thus, the aim of this study was characterize the chromosomal pattern of hypocellular primary MDS and correlate with cellular and clinic aspects. It was analyzed 86 cases of hypocellular primary MDS, 74 RA, 10 RAEB and 2 RAEB-t. Patients with RA presented 50% of abnormal karyotype and all patients with RAEB presented abnormal karyotype as well as RAEB-t patients. The most frequent chromosomal alterations in this study was del(17p) followed by alterations involving chromosome 7. Our results suggest that chromosomal pattern in hypocellular primary MDS is characterized mainly by partial and complete loss of chromosomes (deletion and monosomy). The cytogenetic analysis aided in diagnosis of cases with suspicion of hypocellular primary MDS and it was an important tool for treatment choice. IPSS showed to be a good prognostic scoring system for this group of patients. Alterations involving chromosome 17 were associated with RA subtype and dysplastic characteristics involving granulocytic setor, however, we could see del(17p) in RAEB patients. For apoptosis analysis were used 42 samples of MDS patients, 23 with primary hypocellular MDS, 8 with primary normocelular MDS and 11 with primary hipercelular MDS. The total apoptosis index in cases of hypocellular primary MDS presented a average of 9,5%, whereas patients with primary normocelular MDS and primary hipercelular MDS presented an average of 12% and 14,1%, respectively. For the analysis of specific lineage cells already commited with cellular proliferation program appears to be the main target of apoptosis program. Despite of patients with primary hypocellular MDS presented total apoptosis index more raised than controls they were always lower than primary normocelular MDS and primary hipercelular MDS, except for eritroblasts that were higher in primary hypocellular. The total apoptosis index was higher in initial stage of the disease independently of the bone marrow cellularity. Patients with del(11q) and del(17p) were associated with decreasing of total apoptosis index. Our results suggest that the hypocellularity of bone marrow is not caused by apoptosis process, but probably by probably some defect in cellular proliferation program.

O complexo telomérico, responsável pela integridade genômica, é formado pela interação de DNA com proteínas, que são responsáveis pela proteção desses terminais. O complexo RPA de eucariotos compreende um heterotrímero, que cumpre diversas funções vitais na célula, sendo uma peça fundamental na replicação, reparo e recombinação. A ausência de homólogos de proteínas que protegem o telômero em T. cruzi nos fez investigar se o complexo RPA poderia cumprir essa função. Assim, este trabalho teve como objetivo caracterizar a interação TcRPA-1-telômero. Conseguimos verificar a interação in vitro e in vivo da RPA-1 com o telômero em epimastigotas e tripomastigotas. A ausência de homólogos de proteínas que interagem com o overhang telomérico em tripanosomas, a interação específica RPA-1-telômero, e a sua presença nos telômeros da forma de vida não replicativa, bem como as peculiaridades estruturais da TcRPA-1, levam-nos a propor que essa proteína pode estar envolvida com a proteção dos telomérica neste organismo. / The telomeric complex, responsible for genomic integrity and stability , is formed by the interaction of DNA with proteins that are responsible for maintaining and protecting these terminals. The eukaryotic RPA complex comprises a heterotrimer, which fulfills several vital functions in the cell , being a fundamental player in replication , repair and recombination. The absence of homologous proteins that protect telomeres in Trypanosoma cruzi lead us to hypothesize that RPA complex could fulfill this function. Thus, this study aimed to characterize TcRPA-1-telomere interaction. We could verify in vitro and in vivo interaction of RPA - 1 with telomeres in epimastigote and trypomastigote lifeforms. The absence of homologs of proteins that interact with telomeric overhang in trypanosomes, the specific interaction RPA-1- telomere , its presence in non- replicative lifeform as well as structural peculiarities of TcRPA-1, lead us to propose that this protein may be involved in telomere protection in this organism.