Submitted by Verena Bastos (verena@uefs.br) on 2015-10-08T12:18:43Z No. of bitstreams: 1 Jeanderson_Disserta??o_vers?o final.pdf: 369468 bytes, checksum: 851a8492d73fc0106bfe7784166fdf98 (MD5) / Made available in DSpace on 2015-10-08T12:18:43Z (GMT). No. of bitstreams: 1 Jeanderson_Disserta??o_vers?o final.pdf: 369468 bytes, checksum: 851a8492d73fc0106bfe7784166fdf98 (MD5) Previous issue date: 2013-03-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Exposure to genotoxic agents induces changes in the DNA molecule to commit that genes involved with the repair mechanisms and the control of cell proliferation and differentiation pathways or genes associated with apoptosis can lead to cancer development. Among the many chemicals that have been identified as mutagenic action, include the Androgenic Steroids (AAS), hormones widely used in the search for improved physical performance and increased muscle mass. Objective: In this context, the aim of the development of this study was to evaluate the potential of androgenic anabolic steroid nandrolone decanoate, testosterone propionate and testosterone cypionate to induce chromosome damage, apoptosis and necrosis, using the micronucleus test in exfoliated cells from the oral mucosa of users of AAS with a view to its application as a tool in cancer prevention. Method: The study sample consisted of 55 volunteers, male, divided into two (02) groups, matched for age: 25 subjects (G1) users of nandrolone decanoate, testosterone propionate and testosterone cypionate (alone or simultaneously ) and 30 subjects in the control group (G2). The collection methodology and cytological analysis followed the protocol of Tolbert et al. (1992) and Thomas et al. (2009), which includes, in addition to micronuclei, the computation of degenerative nuclear changes indicative of apoptosis (cariorr?xis, condensed chromatin and pyknosis) and necrosis (karyolysis, cariorr?xis, condensed chromatin and pyknosis). Statistical analysis of the endpoints analyzed (micronucleus, cariorr?xis, condensed chromatin, karyolysis, pyknosis and broken eggs) was performed using the conditional test to compare proportions in situations of rare events. Results: Statistical analysis revealed no significant difference in the occurrence of micronuclei, karyolysis and broken eggs between groups. The occurrence of apoptosis was significantly greater in cells from control individuals. Conclusion: The results show inhibition of apoptosis induced by EAA, suggesting that the described association between the use of these substances and the carcinogenic process can be permeated by this mechanism. / A exposi??o a agentes genot?xicos induz altera??es na mol?cula do DNA que ao comprometerem genes envolvidos com os mecanismos de reparo e com o controle da prolifera??o e diferencia??o celular ou genes associados ?s vias de apoptose, podem levar ao desenvolvimento de c?ncer. Entre os muitos agentes qu?micos que t?m sido identificados como de a??o mutag?nica, incluem-se os Ester?ides Anabolizantes Androg?nicos (EAA), horm?nios amplamente utilizados na busca da melhoria do desempenho f?sico e aumento da massa muscular. Objetivo: Neste contexto, objetivou-se com o desenvolvimento do presente estudo, avaliar o potencial dos ester?ides anabolizantes androg?nicos decanoato de nandrolona, propionato de testosterona e cipionato de testosterona em induzir danos cromoss?micos, apoptose e necrose, atrav?s do uso do Teste de Micron?cleo em c?lulas esfoliadas da mucosa oral de usu?rios de EAA com vista ? sua aplica??o como ferramenta na preven??o do c?ncer. M?todo: A amostra do estudo foi composta por 55 volunt?rios, do sexo masculino, distribu?dos em dois (02) grupos, pareados por idade: 25 indiv?duos (G1) usu?rios de decanoato de nandrolona, propionato de testosterona e cipionato de testosterona (isoladamente ou simultaneamente) e 30 indiv?duos no grupo controle (G2). A metodologia de coleta e an?lise citol?gica seguiu o protocolo de Tolbert et al. (1992) e Thomas et al. (2009), que inclui, al?m de micron?cleos, o computo de altera??es nucleares degenerativas indicadoras de apoptose (cariorr?xis, cromatina condensada e picnose) e necrose (cari?lise, cariorr?xis, cromatina condensada e picnose). A an?lise estat?stica dos endpoints analisados (micron?cleo, cariorr?xis, cromatina condensada, cari?lise, picnose e broken eggs) foi realizada com o uso do teste condicional para compara??o de propor??es em situa??es de eventos raros. Resultados: A an?lise estat?stica revelou que n?o houve diferen?a significativa na ocorr?ncia de micron?cleo, cari?lise e broken eggs entre os grupos. A ocorr?ncia de apoptose foi, significativamente, maior em c?lulas dos indiv?duos do grupo controle. Conclus?o: Os resultados obtidos mostram inibi??o da apoptose induzida pelo uso de EAA, sugerindo que a associa??o descrita entre uso destas subst?ncias e o processo carcinog?nico possa ser permeada por este mecanismo.

O teste de frequência de micronúcleos (FMN) in vitro é uma das metodologias de escolha no desenvolvimento de testes de segurança toxicológica. Para o seu desenvolvimento no trabalho foram realizadas modificações da técnica convencional, relativas ao substrato de cultivo das células e à sua coloração. As culturas celulares foram desenvolvidas diretamente nas lâminas e a coloração foi realizada com laranja de acridina (AO) ao invés da coloração segundo Giemsa. Foram utilizados controles positivos com potenciais clastogênico (mitomicina C, benzo[a]pireno) e aneugênico (colchicina), recomendados pela OCDE (Organização para Cooperação e Desenvolvimento Econômico). Como moléculas-teste, foram utilizados compostos cuja associação a isótopos radioativos compõem radiofármacos produzidos pelo IPEN. DOTATATO e Ubiquicidina foram testados em diferentes concentrações proporcionais às concentrações máximas utilizadas em pacientes adultos. Para tanto, foram realizadas diluições correspondentes às concentrações 0,1X, 1X e 10X e culturas de células CHO-KI foram expostas a estas concentrações para ensaios de citotoxicidade e FMN. Nenhuma das concentrações induziu citotoxicidade significativa. Para análise de FMN, foram contabilizadas todas as células mononucleadas e multinucleadas até atingir a contagem de 1000 células binucleadas, com ou sem micronúcleos. Desta maneira foi possível analisar a frequência de micronúcleos e o índice de proliferação (CBPI). As concentrações dos fármacos em teste (0,1X, 1X e 10X) não induziram agressão às células. Nenhuma das concentrações revelou citotoxicidade ou genotoxicidade, ou ainda qualquer alteração no ciclo celular em comparação aos controles, comprovando sua segurança conforme os parâmetros exigidos pelas normas internacionais. Os resultados mostraram também uma boa concordância entre a comparação das leituras realizadas por analistas independentes, com pequenas discrepâncias discutíveis, e boa correlação com resultados obtidos com a coloração convencional. Desta maneira as modificações realizadas na técnica de FMN mostraram potencial para cumprir todos os quesitos como teste pré-clínico. / The in vitro micronucleus frequency test (FMN) is one method of choice in the development of toxicological safety tests. For its development, this work carried out modifications of the conventional technique regarding the cultivation substrate of the cells and staining for microscopy evaluation. The cell cultures were grown directly on slides, and staining was performed with acridine orange (AO) instead of the classical Giemsa staining. Positive controls were used for potential clastogenic (mitomycin C, benzo [a] pyrene) and aneugenic (colchicine) effects, recommended by the OECD (Organization for Economic Cooperation and Development). As test molecules, compounds were used whose association with radioactive isotopes make up radiopharmaceuticals produced by IPEN. DOTATATE and Ubiquicidine were tested at different concentrations proportional to the maximum concentrations used in adult patients. Therefore, corresponding to the concentrations dilutions were performed 0.1X, 1X and 10X cultures and CHO-KI cells were exposed to these concentrations for cytotoxicity assays and FMN. None of the concentrations induced significant cytotoxicity. For FMN analysis, it was recorded every mononuclear cells and multinucleated up to 1000 counts binucleated cells with or without micronuclei. In this way it was possible to analyze the frequency of micronuclei and the proliferation index (CBPI). The concentrations of the test drug (0.1X, 1X and 10X) did not induce aggression to cells. None of the concentrations showed cytotoxicity and genotoxicity, or any changes in cell cycle compared to controls, demonstrating their safety according to the parameters required by international standards. The results also showed good agreement between the comparison of readings by independent analysts, with minor discrepancies debatable, and good correlation comparing to classical staining technique. Thus the changes made in FMN technique showed potential to fulfill all requirements as preclinical test.

Doses de radiação de baixa energia podem induzir quebras de dupla fita no DNA assim como também produzir perfis alterados de expressão de genes relacionados a estas lesões. Os danos não reparados ou mal reparados levam a uma maior suscetibilidade à transformação oncogênica já que os efeitos biológicos mais importantes causados pela radiação ionizante são mutação e carcinogênese. As lesões no DNA provocadas pela radiação podem também ocasionar o surgimento micronúcleos e as células podem ser induzidas à apoptose. O objetivo deste trabalho é estudar in vitro a presença de micronúcleos e a apoptose ocasionados por Raios-X de baixa energia. Pretende-se analisar estes efeitos biológicos em relação à energia equivalente à utilizada em exames mamográficos usando duas linhagens estabelecias de células de mama: a MCF-7 (tumoral) e a HB-2 (não-tumoral). As células, em crescimento exponencial, foram irradiadas no equipamento de arranjo experimental de mamografia do LCR/UERJ. A dose de 5Gy na energia de 30 kV foi aplicada com taxa de 0,1 Gy/seg utilizando filtro de 0,03 mm de molibdênio. As irradiações foram realizadas duas vezes, após as irradiações, as células foram incubadas por 4, 24 ou 48 horas e posteriormente coradas com o corante Hoechst33258 para análise em microscopia de fluorescência. Para cada análise, 1000 células foram categorizadas pela morfologia do núcleo. Os resultados mostraram que a HB-2, utilizada neste estudo como célula mamária normal, apresentou maior sensibilidade aos efeitos da radiação, com 37 % das células em apoptose após 4 hs de incubação, enquanto a MCF-7 apresentou 6,5 %. Nas análises após 24 hs foi possível confirmar a radioresistência da MCF-7 tendo sido observadas 11% de células em apoptose no grupo irradiado. Houve um aumento crescente de micronúcleos radioinduzidos nas duas linhagens de acordo com os tempos de incubação. Na análise de 4 hs a HB-2 apresentou 3%, em 24 hs, 8,5% e 48 hs, 11,5 %. Diante destes resultados, foi possível concluir que a energia do feixe de raios-X utilizada na mamografia pode ser capaz de ocasionar aumento de ocorrência de apoptose e geração de micronúcleos nas duas linhagens estudadas / Radiation doses of low-energy can induce double-stranded breaks in DNA as well resulting in altered profiles of gene expression related to these injuries. The damage is not repaired or poorly repaired leading to increased susceptibility to oncogenic transformation since most important biological effects caused by ionizing radiation are mutation and carcinogenesis. The DNA lesions caused by radiation can also provoke the presence of micronuclei and as result cells can be induced to apoptosis. The aim of this work is to perform a preliminary study "in vitro" to identify the presence of micronuclei caused by low energy. X-rays. It is intended analyze these biological effects caused by x-rays with energy equivalent to the ones used in mammography in two very well established breast: cell lines the MCF-7 (tumor) and HB-2 (non-tumor). Cells in exponential growth were irradiated using an experimental arrangement with a mammography tube of the LCR / UERJ. The dose of 5Gy was used for 30 kVp x-rays using 0.03 mm filter molybdenum with a fixed dose rate of 0.1 Gy / sec. The irradiations were repeated twice and. the cells were incubated for 4, 24 or 48 hours and stained with dye Hoechst 33258 for fluorescence microscopy analysis. For each analysis 1000 cells were categorized by the morphology of the nucleus. The results revealed that the HB-2, used in this study as normal mammary cell have showed high sensitivity to the effects of radiation, with 37% of cells undergoing apoptosis after incubation of 4hs, while MCF-7 showed 6.5%. In the 24 hour-analysis it was confirmed the radiation resistance of MCF-7 were it was observed 11% of cells in apoptosis in the irradiated group. There was an increasing number of radiation-induced micronuclei in both cell lines, according to the incubation times. After 4 hours, HB-2 presented 3% of micronuclei, in 24 hours, the experiment reveled 8.5%, and after 48 hours it increased to 11.5%. As result of this study we may conclude that the energy used in mammography is capable to cause an increase in incidence of apoptosis and in the production of micronuclei in both cell lines studied

Doses de radiação de baixa energia podem induzir quebras de dupla fita no DNA assim como também produzir perfis alterados de expressão de genes relacionados a estas lesões. Os danos não reparados ou mal reparados levam a uma maior suscetibilidade à transformação oncogênica já que os efeitos biológicos mais importantes causados pela radiação ionizante são mutação e carcinogênese. As lesões no DNA provocadas pela radiação podem também ocasionar o surgimento micronúcleos e as células podem ser induzidas à apoptose. O objetivo deste trabalho é estudar in vitro a presença de micronúcleos e a apoptose ocasionados por Raios-X de baixa energia. Pretende-se analisar estes efeitos biológicos em relação à energia equivalente à utilizada em exames mamográficos usando duas linhagens estabelecias de células de mama: a MCF-7 (tumoral) e a HB-2 (não-tumoral). As células, em crescimento exponencial, foram irradiadas no equipamento de arranjo experimental de mamografia do LCR/UERJ. A dose de 5Gy na energia de 30 kV foi aplicada com taxa de 0,1 Gy/seg utilizando filtro de 0,03 mm de molibdênio. As irradiações foram realizadas duas vezes, após as irradiações, as células foram incubadas por 4, 24 ou 48 horas e posteriormente coradas com o corante Hoechst33258 para análise em microscopia de fluorescência. Para cada análise, 1000 células foram categorizadas pela morfologia do núcleo. Os resultados mostraram que a HB-2, utilizada neste estudo como célula mamária normal, apresentou maior sensibilidade aos efeitos da radiação, com 37 % das células em apoptose após 4 hs de incubação, enquanto a MCF-7 apresentou 6,5 %. Nas análises após 24 hs foi possível confirmar a radioresistência da MCF-7 tendo sido observadas 11% de células em apoptose no grupo irradiado. Houve um aumento crescente de micronúcleos radioinduzidos nas duas linhagens de acordo com os tempos de incubação. Na análise de 4 hs a HB-2 apresentou 3%, em 24 hs, 8,5% e 48 hs, 11,5 %. Diante destes resultados, foi possível concluir que a energia do feixe de raios-X utilizada na mamografia pode ser capaz de ocasionar aumento de ocorrência de apoptose e geração de micronúcleos nas duas linhagens estudadas / Radiation doses of low-energy can induce double-stranded breaks in DNA as well resulting in altered profiles of gene expression related to these injuries. The damage is not repaired or poorly repaired leading to increased susceptibility to oncogenic transformation since most important biological effects caused by ionizing radiation are mutation and carcinogenesis. The DNA lesions caused by radiation can also provoke the presence of micronuclei and as result cells can be induced to apoptosis. The aim of this work is to perform a preliminary study "in vitro" to identify the presence of micronuclei caused by low energy. X-rays. It is intended analyze these biological effects caused by x-rays with energy equivalent to the ones used in mammography in two very well established breast: cell lines the MCF-7 (tumor) and HB-2 (non-tumor). Cells in exponential growth were irradiated using an experimental arrangement with a mammography tube of the LCR / UERJ. The dose of 5Gy was used for 30 kVp x-rays using 0.03 mm filter molybdenum with a fixed dose rate of 0.1 Gy / sec. The irradiations were repeated twice and. the cells were incubated for 4, 24 or 48 hours and stained with dye Hoechst 33258 for fluorescence microscopy analysis. For each analysis 1000 cells were categorized by the morphology of the nucleus. The results revealed that the HB-2, used in this study as normal mammary cell have showed high sensitivity to the effects of radiation, with 37% of cells undergoing apoptosis after incubation of 4hs, while MCF-7 showed 6.5%. In the 24 hour-analysis it was confirmed the radiation resistance of MCF-7 were it was observed 11% of cells in apoptosis in the irradiated group. There was an increasing number of radiation-induced micronuclei in both cell lines, according to the incubation times. After 4 hours, HB-2 presented 3% of micronuclei, in 24 hours, the experiment reveled 8.5%, and after 48 hours it increased to 11.5%. As result of this study we may conclude that the energy used in mammography is capable to cause an increase in incidence of apoptosis and in the production of micronuclei in both cell lines studied

Made available in DSpace on 2014-06-11T19:22:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-04Bitstream added on 2014-06-13T20:49:39Z : No. of bitstreams: 1 christofoletti_ca_me_rcla.pdf: 3763933 bytes, checksum: d9284f200972936e72e67bb3de745be4 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A degradação dos recursos hídricos, como os ambientes lênticos, dentre eles, os lagos, é uma das maiores preocupações atualmente, visto que esta pode causar danos diretos ou indiretos à saúde e à sobrevivência dos organismos expostos. Um dos fatores que contribui para a alteração da qualidade das águas de ambientes lênticos é o despejo de efluentes, principalmente àqueles de origem doméstica, portadores de substâncias que chegam a ser tóxicas para o meio aquático. Por meio dos testes citogenéticos, utilizando os mais diversificados organismos-teste, é possível biomonitorar a extensão da poluição e avaliar os efeitos dessas substâncias presentes no ambiente natural. Com esse intuito, o presente trabalho tomou por modelo de estudo, um lago urbano artificial (Lago Azul, Rio Claro-SP) e objetivou avaliar o potencial citotóxico, genotóxico e mutagênico das águas desse ambiente, por meio dos testes de aberrações cromossômicas e micronúcleos, em células meristemáticas de Allium cepa (cebola), em dois tratamentos: o contínuo e o período de recuperação, em água ultra pura; e, pelo teste do micronúcleo associado às anormalidades nucleares e do ensaio do cometa, aplicados em eritrócitos de Oreochromis niloticus (tilápia). Coletas de águas sazonais foram realizadas na estação seca (agosto/2006 e agosto/2007) e na estação chuvosa (março/2007 e fevereiro/2008). Análises físico-químicas foram feitas para uma coleta de cada estação. A partir dos dados obtidos, pode-se inferir que as águas desse ambiente lêntico apresentam potencial citotóxico, genotóxico e mutagênico, nas duas estações de coletas, para os dois organismos-teste empregados. As análises de metais revelaram concentrações acima do permitido pela legislação de Ag, Cd2+, Cu e Fe3+, em ambas as estações. Embora os... / The degradation of water resources, as the lentics environments, among them, the lakes, is a major concern now, because it can cause direct or indirect damages to health and to the survival of the exposed organisms. One factor that can contribute to change the water quality of lentics environments is the dumping of effluents, mainly those of domestic origin, carriers of substances that come to be toxic to the aquatic environment. Through the cytogenetic tests, using the most diverse systems-test, it is possible monitoring the extent of pollution and assess the effects of substances on the natural environment. To that end, this work has taken a model of study, an urban artificial lake (Lago Azul, Rio Claro-SP) and aimed to evaluate the cytotoxic, genotoxic and mutagenic potentials of waters that environment, through tests of chromosome aberrations and micronuclei in meristematic cells of Allium cepa (onion) in two treatments: the continued and the period of recovery, in ultra pure water, and by the micronucleus and nuclear abnormalities test and of the comet assay, applied in erythrocytes of Oreochromis niloticus (tilapia). Seasonal collections of waters were held in the dry season (august/2006 and august/2007) and the rainy season (march/2007 and february/2008). Physical and chemical analyses were made for a collection of each season. From the data obtained, it can be infered that the waters of this lentic environment had cytotoxic, genotoxic and mutagenic potentials in two seasons of collections for the two systems-test employed. Analyses of metals detected high concentrations of Ag, Cd2+, Cu, Fe3+, whose values are higher than permitted by law, in both seasons. Although the cytotoxic, genotoxic and mutagenic potentials have been detected in two seasons, the dry season is that presented the highest risk... (Complete abstract click electronic access below)

The objective of this study was to evaluate the effect of thorium (Th) on the metabolism of silver catfish (Rhamdia quelen) through biochemical parameters from the muscle tissue (glycogen, glucose, lactate, protein and ammonia), lipidic peroxidation levels (TBARS), catalase (CAT) and glutathione-S-transferase (GST) in the hepatic and muscular tissues and cytogenetic parameters through the evaluation of nuclear abnormalities in blood cells. Silver catfish juveniles (8.78 ± 0.10cm; 6.41 ± 0.17g) were exposed to different waterborne concentrations of ²³²Th (in μg.L-1): 33.6±8.7; 106.5±37.1; 191.6±19.0 and 758.4±150.4 for 15 days. The levels of muscle glycogen were significantly reduced in fish exposed to 106.5 μg.L-¹ Th, while glucose and protein increased in those exposed to 758.4 μg.L-¹ Th. Lactate levels were higher in fish maintained at 191.6 μg. L-¹ Th and ammonia was higher in those exposed to 33.6, 106.5 and 191.6 μg.L-¹ Th. The lipidic peroxidation levels were diminished in the liver of silver catfish exposed to all tested concentrations of Th. In the muscle lipidic peroxidation was higher in juveniles maintained at 106.5 μg.L-¹ Th and lower in those exposed to 191.6 and 758.4 μg.L-¹ Th. The CAT activity was higher in the hepatic tissue (but not muscle) of fish exposed to all tested concentrations of Th. The GST activity in the liver was lower in fish exposed to 33.6 and 106.5 μg.L-¹ Th, and in the muscular tissue of those maintained at 758.4 μg.L-1 Th. Silver catfish exposed to 106.5 μg.L-¹ presented a significant induction of micronuclei, but no alterations in other erythrocyte abnormalities were observed. These results suggest that exposure to waterborne Th induces changes in the metabolic state, increase of lipidic peroxidation in the liver, some alterations of CAT and GST, and DNA damage. / O objetivo deste estudo foi avaliar o efeito de tório (Th) sobre parâmetros metabólicos (glicogênio, glicose, lactato, proteína e amônia), em tecido muscular de jundiás (Rhandia quelen), níveis de lipoperoxidação (TBARS), catalase (CAT) e glutationa-S-transferase(GST) nos tecidos hepático e muscular e parâmetros citogenéticos através da avaliação de anormalidades nucleares em células sanguíneas. Juvenis de jundiás (8,78±0,10cm; 6,41± 0,17g) foram expostos a diferentes concentrações de ²³²Th 33,6±8,7; 106,5±37,1; 191,6±19,0 e 758,4±150,4 em μg. L-¹ (três repetições por tratamento) por 15 dias. Os níveis de glicogênio muscular diminuíram significativamente a 106,5 μg. L-¹ Th. Glicose e proteína aumentaram na 758,4 μg. L-¹ Th. Os níveis de lactato apresentaram-se elevados em 106,5 μg. L-¹ Th. A amônia aumentou a 33,6; 106,5 e 191,6 μg.L-¹ Th. Os níveis de peroxidação lipídica diminuíram no fígado dos jundiás expostos a todas as concentrações de Th testadas. No músculo esquelético aumentaram a 106,5 μg. L-¹Th e diminuíram a 191,6 e 758,4 μg.L-¹ Th. A atividade da CAT no tecido hepático apresentou aumento em todas as concentrações testadas de Th. Nenhuma alteração foi observada no tecido muscular. A GST diminui no fígado a 33,6 e 106,5 μg.L-¹Th. No tecido muscular diminui a 758,4 μg.L-1 Th. Jundiás expostos a 106,5 μg. L-¹ apresentaram maior indução de micronúcleos. Não foi observadas alterações para outras anormalidades nucleares eritrocíticas. Os resultados obtidos sugerem mudanças nos intermediários metabólitos devido ao estresse provocado pelo Th, aumento da lipoperoxidação no fígado, sendo observado níveis variados no músculo esquelético, alterações das enzimas CAT e GST além de danos no DNA dos peixes expostos ao Th, comparando aos grupos controles.

The advent of high-throughput sequencing has brought about the creation of an unprecedented amount of research data. Analytical methodology has not been able to keep pace with the plethora of data being produced. Two assays, ImmunoSEQ and the cytokinesisblock micronucleus (CBMN), that both produce count data and have few methods available to analyze them are considered. ImmunoSEQ is a sequencing assay that measures the beta T-cell receptor (TCR) repertoire. The ImmunoSEQ assay was used to describe the TCR repertoires of patients that have undergone hematopoietic stem cell transplantation (HSCT). Several different methods for spectratype analysis were extended to the TCR sequencing setting then applied to these data to demonstrate different ways the data set can be analyzed. The different methods include CDR3 distribution perturbation, Oligoscores, Simpson's diversity, Shannon diversity, Kullback-Liebler divergence, a non-parametric method and a proportion logit transformation method. Herein we also demonstrate adapting compositional data analysis methods to the TCR sequencing setting. The various methods were compared when analyzing a set of 13 subjects who underwent hematopoietic stem cell transplantation. The eight subjects who developed graft versus host disease were compared to the five who did not. There was no little overlap in the results of the different methods showing that researchers must choose the appropriate method for their research question of interest. The CBMN assay measures the rate of micronuclei (MN) formation in a sample of cells and can be paired with gene expression or methylation assays to determine association between MN formation and other genetic markers. Herein we extended the generalized monotone incremental forward stagewise (GMIFS) method to the situation where the response is count data and there are more independent variables than there are samples. Our Poisson GMIFS method was compared to a popular alternative, glmpath, by using simulations and applying both to real data. Simulations showed that both methods perform similarly in accurately choosing truly significant variables. However, glmpath appears to overfit compared to our GMIFS method. Finally, when both methods were applied to two data sets GMIFS appeared to be more stable than glmpath.

Made available in DSpace on 2017-07-24T19:22:10Z (GMT). No. of bitstreams: 1 Ricardo_Kern.pdf: 242426 bytes, checksum: a174b7b70f1d6dd81756070f120c5387 (MD5) Previous issue date: 2006-02-23 / O constante aperfeiçoamento dos testes genéticos, como o Teste de Micronúcleos (MN), os tornaram importantes auxiliares na prevenção do câncer. Com o objetivo de melhor compreender a etiologia do desenvolvimento de neoplasias bucais, o presente trabalho se propôs a estudar a influência do fumo, álcool, trauma mecânico e de substância contidas em colutórios sobre a Freqüência de Micronúcleos (FMN) em células epiteliais bucais de alunos do curso de Odontologia. Para tanto, 40 alunos foram divididos em 4 grupos assim caracterizados G1 – Abstêmios (controle); G2 Alcoolistas; G3 – Usuários de aparelho ortodôntico; G4 – Fumantes alcoolistas, e submetidos ao TMN. Posteriormente estes mesmos alunos receberam de forma aleatória, 4 diferentes tratamentos, por 10 dias, a base de colutórios: T1 (óleos essenciais), T2 (álcool 11%), T3 (álcool, 11% + clorexidina 0,12%), T4 (clorexidina 0,12%), sendo então novamente submetidos ao TMN. Os resultados da FMN demonstraram diferenças significativas entre os 4 grupos G (p = 0,043 Kruskal-Wallis), sendo indicado diferença entre G1 e G3 (Mann-Whitney p ? 0,01) e entre G1 e G4 (Mann-Whitney p ? 0,05), contudo, em relação aos tratamentos com colutórios, todos os tratamentos T não alcançaram resultados significativos (Wilcoxon p > 0,05). Os resultados sugerem que o trauma mecânico causado por aparelho ortodôntico assim como a associação de fumo com bebidas alcoólicas favorecem um aumento na prevalência de MN quando comparados ao controle. A utilização de colutórios bucais, em curto prazo, não foi capaz de causar aumento na freqüência de Micronúcleos

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-17Bitstream added on 2014-06-13T20:01:10Z : No. of bitstreams: 1 ventura_bc_dr_rcla.pdf: 1747414 bytes, checksum: d6fd71edeabed9a63deeb67289ab785e (MD5) / Os azocorantes são substâncias químicas extremamente utilizadas em indústrias têxteis que podem induzir mudanças no material genético de organismos expostos, mesmo que essas alterações no DNA não se expressem de imediato. Foram avaliadas as citotoxicidade, genotoxicidade e mutagenicidade de diferentes concentrações (1, 10, 100 e 1000 mg/L – na ausência dos microorganismos – e 50 e 200 mg/L – na presença dos microorganismos) do azocorante Black Dye Commercial Product (BDCP), antes e após tratamentos de biodegradação por diversos microrganismos (1. “Pool” de bactérias heterotróficas provenientes de uma estação de tratamento biológico de efluentes, 2. Levedura Candida viswanathii, e 3. Fungo Basidiomiceto Phanerochaete chrysosporium), por meio de diferentes técnicas citogenéticas (coloração convencional, bandamento C, bandamento RON, bandamento por fluorocromos base-específicos CMA/DAPI e hibridação in situ fluorescente – FISH) aplicadas sobre o organismo teste Allium cepa. Pela técnica citogenética de coloração convencional, foi possível verificar que o corante, com e sem ação microbiana, induziu apoptose, necrose, células micronucleadas, aberrações cromossômicas e alterações nucleares. As aberrações cromossômicas e alterações nucleares foram visualizadas em todos os estágios do ciclo celular: na intérfase foram observados brotos nucleares e células poliplóides; na prófase, perdas de material genético; na metáfase, aderências cromossômicas, perdas cromossômicas, C-metáfases e poliploidias; na anáfase e telófase, multipolaridades, pontes e perdas cromossômicas. Os brotos nucleares apareceram com maior freqüência nas células submetidas aos testes do azocorante tratado com microorganismos, sendo que esse tipo de alteração deve estar associado à presença dos metabólitos do corante. As freqüências de micronúcleos... / Azo dyes are chemical substances extremely used by textile industries that may induce changes in the genetic material of exposed organisms, even if these changes in the DNA do not express themselves immediately. Cytotoxicity, genotoxicity and mutagenicity evaluations of the different azo dye (BDCP) concentrations were performed (1, 10, 100 e 1000 mg/L – without microorganisms – and 50 and 200 mg/L – with microorganisms), before and after biodegradation tests, using different microorganisms (1. Heterotrofic bacteria ”pool” proceeding from an effluent biological treatment station, 2. Candida viswanathii - Yeast, and 3. Phanerochaete chrysosporium - Basidiomicet Fungi), by means the different cytogenetical assays (conventional staining, C-banding, RON-banding, CMA/DAPI banding and fluorescent in situ fluorescent hybridization), using Allium cepa as test organism. By the conventional staining cytogenetic assay, it was possible to verify that the azo dye induced apoptosis, necrosis, micronuclei, chromosome aberrations and nuclear alterations. The chromosome aberrations and nuclear alterations were visualized in all phases of the cell cycle: in the interphase were observed nuclear buds and polyploidizated cells; in the prophase were observed genetic material losses; in the metaphase were observed chromosome adherences, chromosome losses, C-metaphases and polyploid cells; and in the anaphase and telophase were observed multipolar cells, chromosome bridges and chromosome losses. The frequencies of nuclear buds were the higher when the cells had been submitted to the azodye treated with microorganisms, suggesting that this kind of alteration must be associated to the presence of the azodye metabolites. The micronuclei and chromosome breaks frequencies... (Complete abstract click electronic access below)

Η ακεραιότητα και η λειτουργία της μιτωτικής συσκευής διαδραματίζει ουσιώδη ρόλο για τον ορθό προσανατολισμό και την ολίσθηση των χρωμοσωμάτων στους πόλους της ατράκτου, οδηγώντας στον ισομερή διαχωρισμό των χρωμοσωμάτων κατά τη μιτωτική ή μειωτική διαίρεση. Τροποποιήσεις του δικτύου των μικροσωληνίσκων (α- και β-τουμπουλίνη) και των κέντρων οργάνωσης αυτών (ΜΤΟC, γ-τουμπουλίνη) είναι δυνατόν να προκαλέσουν βλάβες στη μιτωτική συσκευή, διαταράσσοντας το χρωμοσωματικό αποχωρισμό, με αποτέλεσμα το σχηματισμό ανευπλοειδικών κυττάρων. Η διουρητική φαρμακευτική ένωση υδροχλωροθειαζίδιο (HCTZ) χορηγείται κατά της υπέρτασης και είναι γνωστό ότι προκαλεί μη αποχωρισμό σε διπλοειδή στελέχη του μύκητα Aspergillus nidulans. Πρόσφατες μελέτες της ερευνητικής μας ομάδας έχουν δείξει ότι επάγει αυξημένες συχνότητες μικροπυρήνων και διαταράσσει το χρωμοσωματικό αποχωρισμό σε καλλιέργειες ανθρωπίνων λεμφοκυττάρων in vitro. Στην παρούσα εργασία μελετήθηκε η επίδραση του HCTZ στην οργάνωση του δικτύου των μικροσωληνίσκων κατά τη μεσόφαση και τη μίτωση, με συνδυασμένη εφαρμογή μεθόδων διπλής ανοσοσήμανσης των πρωτεϊνών των μικροσωληνίσκων, του κεντροσώματος και του κινητοχώρου. Εφαρμόστηκε επίσης in situ υβριδοποίηση με φθοροχρώματα (FISH), με α-satellite πανκεντρομερικό ανιχνευτή, για τον εντοπισμό μη ενσωματωμένου (lagging) χρωμοσωματικού υλικού. Η μελέτη πραγματοποιήθηκε σε κυτταρικές σειρές μυός C2C12 και ανθρώπου HFFF2. Παρατηρήθηκε ότι το HCTZ προκαλεί μείωση του ρυθμού διαίρεσης, αποδιοργάνωση του δικτύου των μικροσωληνίσκων και αυξημένη συχνότητα ανώμαλων μεταφάσεων με ποικίλο αριθμό σημάτων γ-τουμπουλίνης. Αυξάνει το ποσοστό των μεταφάσεων και μειώνει το ποσοστό των ανα-τελοφάσεων, προκαλώντας συσσώρευση των κυττάρων στο στάδιο της μετάφασης. Επίσης, επάγει τη χρωμοσωματική καθυστέρηση, καθώς αυξάνει τη συχνότητα των μικροπυρήνων που παρουσιάζουν τόσο σήμα κινητοχώρου όσο και σήμα κεντρομέρους. Η γενετική δράση του στα κύτταρα C2C12 δε φαίνεται να επηρεάζεται σημαντικά από τη χρονική διάρκεια έκθεσης των κυττάρων σ’ αυτό. Από τις δυο κυτταρικές σειρές που χρησιμοποιήθηκαν φαίνεται ότι τα κύτταρα C2C12 είναι περισσότερο ευαίσθητα στην απόκριση στο HCTZ. Τα αποτελέσματα μας, υποδεικνύουν ανευπλοειδογόνο δράση της φαρμακευτικής ένωσης, επιβεβαιώνοντας και ενισχύοντας προηγούμενα ευρήματα της ερευνητικής μας ομάδας. / The integrity and function of mitotic apparatus play essential role for the equitable orientation and the slipping of chromosomes to the spindle poles, indicating normal distribution of chromosomes during mitotic or meiotic division. Modifications of the microtubule network (α- and β- tubulin) and microtubule-organizing centers (MTOC, γ- tubulin) may cause severe damage to the mitotic apparatus, disturbing the segregation of chromosomes and resulting to aneuploid cells. The diuretic drug hydrochlorothiazide (HCTZ) is used for the treatment of hypertension and has been found to induce non-disjunction in diploid strains of Aspergillus nidulans. Recent studies of our team have shown that HCTZ produces increased frequencies of micronuclei and disturbs chromosome segregation in human lymphocytes cultures treated in vitro. In the present study was investigated the effect of HCTZ on the organization of the microtubule network during mesophase and mitosis, with combined application of double immunofluorescence staining assay, for the visualization of microtubules, centrosomes and kinetochore proteins. Fluorescence in situ hybridization (FISH), with α-satellite pancentromeric probe, was also applied, for the localization of not integrated (lagging) nuclear material. The study was realized in C2C12 mouse cells and HFFF2 human cells. Our results revealed that HCTZ causes decrease of the cell division rate, disorganization of the microtubule network and increased frequency of abnormal metaphases with various γ-tubulin signals. HCTZ increases the percentage of metaphases and decreases the percentage of ana-telophases, indicating a metaphase arrest. Also, induces chromosome delay, as was shown from the high frequency of micronuclei that presents kinetochore and centromere signal. The genetic activity of HCTZ in C2C12 cells does not appear to be significantly influenced by the duration of the cell’s exposure time to the drug. It appears that C2C12 mouse cells are more sensitive in their response to the HCTZ. These results indicate aneugenic activity of this drug, confirming and enhancing our previous findings.