Dissertations / Theses on the topic 'Chromosome. eng'

Orientador: Carmem Silvia Fontanetti Christofoletti
Banca: Tatiana da Silva Souza
Banca: Ana Cristina Mielli
Resumo: O lodo de esgoto pode conter substâncias tóxicas, estar contaminado com metais pesados e até mesmo por compostos químicos persistentes, sendo assim, a disposição inadequada desse resíduo pode fazer tais poluentes retornarem ao ambiente e, eventualmente, entrar na cadeia alimentar, caso sejam absorvidos pelas plantas. O conhecimento dos agentes químicos presentes no lodo permite avaliar o risco de contaminação alimentar e ambiental decorrente da utilização de lodos como fertilizantes agrícolas, também chamados de biossólidos. Logo, o presente estudo teve por objetivo avaliar o potencial genotóxico, citotóxico e mutagênico do lodo gerado por uma Estação de Tratamento de Esgoto (ETE), por meio dos sistemas-teste Allium cepa e Tradescantia pallida. As coletas foram realizadas em abril de 2009 e maio de 2010 e os organismos foram expostos ao lodo bruto e seu solubilizado. Quando comparados os resultados obtidos nos bioensaios com as análises físico-químicas, não é possível estabelecer relação de causa e efeito com nenhum composto em específico, uma vez que todos os parâmetros avaliados se encontram dentro dos limites estabelecidos pela Resolução CONAMA 375; no entanto, em ambos os organismos, o lodo apresentou-se genotóxico e mutagênico, alertando sobre a necessidade de cuidado na disponibilização deste tipo de resíduo em solos. Dessa forma é possível concluir que os ensaios de toxicidade genética são capazes de identificar os efeitos diretos do lodo de esgoto e poderiam ser contemplados pela legislação como ferramenta complementar à bateria de testes já estabelecida devido à simplicidade dos testes e custo relativamente reduzido. O trabalho traz ainda uma revisão de literatura sobre a utilização da espécie Tradescantia, suas bases e aplicações das técnicas do pêlo estaminal (Trad-SHM) e teste o do micronúcleo (Trad- MCN), apontando suas... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Sewage sludge can contain toxic substances, may be contaminated with heavy metals and persistent chemicals, so the improper disposal of this waste can return such pollutants to the environment and possibly return to the food chain if absorbed by plants. The knowledge of chemical agents present in the sludge can assess the risk of food contamination and environmental arising from the use of sludge as agricultural fertilizer, also called biosolids. Therefore, this study aimed to evaluate the potential genotoxic, cytotoxic and mutagenic of sludge generated from a Wastewater Treatment Plant (WTP) by the Allium cepa and Tradescantia pallida tests-systems. Samples were collected in April 2009 and May 2010 and the organisms were exposed to raw sludge and its solublilizated samples. Comparing the results obtained in bioassays with the physico-chemical properties, its cannot establish cause and effect relationship with any compound in particular, since all parameters are within limits set by CONAMA Resolution 375, however, in both organisms, the sludge showed genotoxic and mutagenic, and warns about caution in providing this type of waste in soils. Thus, we conclude that the genetic toxicity tests are able to identify the direct effects of sewage sludge and could be provided by the law as a complementary tool to the battery of tests previously established, due to the simplicity of the tests and relatively low cost. The work also contains a review on the use of Tradescantia species, their bases and applications of the techniques of stamen hair (Trad-SHM) and the micronucleus test (Trad-MCN), pointing out its advantages as a tool for monitoring of environmental health
Mestre

Orientador: Ana Silvia Alves Meira Tavares Moura
Banca: Luiz Lehmann Coutinho
Banca: Mônica Corrêa Ledur
Banca: Millor Fernandes do Rosário
Banca: José Roberto Sartori
Resumo: A partir de resultados de um estudo anterior, no qual foram mapeados QTLs para características de peso vivo, peso do coração e pulmões no GGA1, foi definida uma região no intervalo entre os marcadores ADL0234 e LEI0071, abrangendo 82,3 cM. Foram avaliadas três famílias de meios-irmãos paternos que compreendiam sete famílias de irmãos completos, num total de 652 F2 para as características: peso vivo aos 35 e 41 dias de idade, pesos do coração e pulmões e rendimentos de coração e pulmões. Os genótipos de seis marcadores microssatélites foram adicionados aos dez utilizados anteriormente. O mapa de ligação obtido da região compreendeu 110,8 cM com espaçamento médio entre os marcadores de 7,4 cM. Na análise de F2, em um único intervalo (LEI0146-LEI0174), compreendendo 28,8 cM, foram mapeados QTLs para todas as características estudadas, com exceção dos rendimentos de coração e pulmões. Neste intervalo estão localizados o gene IGF1 e o centrômero do cromossomo. A adição de seis marcadores confirmou os QTLs mapeados anteriormente, porém alguns em diferentes posições. A análise de meios-irmãos paternos indicou que os principais QTLs estavam segregando em apenas uma das famílias (7716), na qual cinco QTLs foram mapeados. Na análise de meios-irmãos maternos, duas famílias segregaram QTLs tanto na análise Individual como na Conjunta (7810 e 7971). As diferentes análises permitiram selecionar dois casais F1, que devem ser o alvo dos próximos estudos. Este estudo restringiu a busca por genes candidatos responsáveis pelas características de interesse a uma região de 28,8 cM (9,82 Mb) no GGA1.
Abstract: Based on the results from a previous study, in which QTL for body weight, heart and lungs weights and heart and lungs percentages were mapped to GGA1, a region was defined between markers ADL0234 and LEI0071, spanning 82.3 cM. Three paternal half-sib families, comprising seven full-sib families, totaling 652 F2 were evaluated for body weight at 35 and 41 days of age, heart and lungs weights and heart and lungs yields. Genotypes of six microsatellite markers were added to those of ten previously used. The linkage map of this region spanned 110.8 cM, with average spacing of 7.4 cM between markers. In a single interval (LEI0146-LEI0174), comprising 28.8 cM, QTLs for all traits, except for heart and lungs yields were mapped in the F2 analysis. In this same interval the IGF1 gene, and the chromosome centromere, are located. The use of six additional markers confirmed the same QTLs mapped previously, but some of them, in different positions. The paternal half-sib analysis indicated that the main QTLs were segregating in one of the families only (7716), in which five QTLs were mapped. In the maternal half-sib analysis, two families segregated QTLs both, in the across and within families analyses (7810 and 7971). These analyses allowed the selection of two F1 couples to be the target for future studies. This study restricted the search for candidate genes responsible for the traits of interest to a region of 28.8 cM (9.82 Mb) in GGA1.
Doutor

Orientador: José Maurício Barbanti Duarte
Banca: Fausto Foresti
Banca: Cláudio de Oliveira
Banca: Orlando Moreira Filho
Banca: Vera Fernanda Martins Hossepian de Lima
Resumo: Estudos com veado-mateiro (Mazama americana) mostram que há muitas controvérsias quanto ao número de subespécies ou até quanto ao desdobramento destas em espécies. Em estudo citotaxonômico foram encontradas variações cromossômicas intra e interpopulacionais em populações de M. americana geograficamente distantes, com número diplóide de 48 a 53 e número fundamental de 46 a 57. Com base nisto, o presente estudo visou compreender como ocorreu a reorganização cromossômica dentro das variantes encontradas durante a evolução do grupo. Para isto, estrutura e organização dos cromossomos de M. americana foram analisadas para identificar os rearranjos que originaram a variação intraespecífica através das técnicas de bandamento cromossômico (bandas G, C, Ag-NOR), hibridação in situ (FISH) com sondas teloméricas e pintura cromossômica com o uso de sondas cromossômicas da espécie Mazama gouazoubira. Foram identificados seis citótipos distribrídos em 12 cariótipos diferentes: Rondônia (2n=42 ou 43 e NF=46; 2n=42 e NF=49), Juína (2n= 43, 44 ou 45 e NF=48; 2n=44 e NF=46), Jarí (2n=49; NF=56, Carajás (2n=50 e NF=54), Santarém (2n=51 e NF=56) e Paraná (2n=51,52 ou 53 e NF=56). O cariótipo básico do citótipo Paraná foi utilizado como base comparativa para os demais. Os rearranjos que originaram essas diferenças foram fusões cêntricas, em tandem e inversões pericêntricas. A análise de complexo sinaptonêmico confirmou a existência de um sistema sexual múltiplo do tipo XX/XY1Y2 através da detecção de uma trivalente sexual. Sítios teloméricos intersticiais evidenciam que a ocorrência de eventos de fusões em tandem foi essencial para a evolução cariotípica desta espécie e a homologia de sondas cromossomo-específicas de M. gouazoubira corroboram que o caminho da reorganização cromossômica entre estas espécies foi principalmente através de fusões.
Abstract: Studies with the red brocket deer (Mazama americana) shown that there are a lot of controversies about the number of subspecies or about the unfolding of these in new species. Citotaxonomic studies found intra and interpopulational chromosomal variations, with diploid number varing from 48 to 53 and fundamental number from 46 to 57. Based on these studies, the aim of the present study was understood how the chromosomal reorganization occurred between this variants during the evolution process. For that, we analyzed the chromosomal structure and organization of M. americana, identifying the rearrangements responsible for the intraspecific variation through chromosome banding (G and C-banding, Ag-NOR), in situ hybridization of telomeric probes and chromosome painting using probes of M. gouazoubira species. It were found six different variants: Rondônia (2n=42 or 43 and FN=46; 2n=42 and FN=49), Juína (2n= 43, 44 or 45 and FN=48; 2n=44 and FN=46), Jarí (2n=49 and FN=56), Carajás (2n=50 and FN=54), Santarém (2n=51 and FN=56) and Paraná (2n=51,52 or 53 and FN=56). The basic karyotype of Paraná variant was choosing for comparative analysis. The rearrangements responsible for these chromosomal differences were centric and tandem fusions and pericentric inversions. The synaptonemal analysis sustained the existence of a multiple sexual system (XX/XY1Y2) with detection of a sexual trivalent. Intersticial telomeric sites shown the occurrence of tandem fusions was essential for the karyotype evolution of this species and the homology with the probes of M. gouazoubira corroborated that the way of chromosomal reorganization between these species was mainly through chromosome fusions.
Doutor

Orientador: José Roberto Moro
Banca: Herberte Pereira da Silva
Banca: Luciana Rossini Pinto
Resumo: O Brasil tem uma flora nativa exuberante, muito explorada e pouco estudada. A economia florestal brasileira tem importante papel na qualidade de vida do país sendo necessárias informações que possibilitem uma exploração mais consciente e sustentável das espécies nativas. Uma das formas de se obter esclarecimentos relevantes a respeito das espécies arbóreas do Brasil é por meio de estudos citogenéticos. Esses estudos cromossômicos podem fornecer informações importantes na taxonomia, evolução, genética, melhoramento de plantas e na preservação dos sistemas florestais. Por meio de técnicas de citogenética convencional estabeleceu-se o número cromossômico diplóide de: Balfourodendron riedelianum, 2n = 58 cromossomos, com tamanho médio dos cromossomos de 1,877μm ± 0,44, Cedrela fissilis, 2n = 56 cromossomos, com tamanho médio dos cromossomos de 1,01μm ± 0,26; Hymenaea courbaril var. stilbocarpa, 2n = 24 cromossomos, com tamanho médio dos cromossomos de 3,52μm ± 0,68 ; Myroxylon peruiferum, 2n=26 cromossomos, com tamanho médio dos cromossomos de 1,25μm ± 0,30; Pterogyne nitens, 2n=20 cromossomos, com tamanho médio dos cromossomos de 1,13μm ± 0,27; Tabebuia aurea, 2n = 40 cromossomos, com tamanho médio dos cromossomos de 1,05μm ± 0,23; T. ochracea , 2n=80 cromossomos, com tamanho médio dos cromossomos de 1,02μm ± 0,22 e C. odorata com variação cromossômica de 2n = 42 a 2n = 104 cromossomos. Os resultados obtidos neste trabalho poderão fornecer suporte para futuras pesquisas de manipulação dos cromossomos, comparação em estudos taxonômicos, estudos evolutivos, produção de progênies híbridas para fins comerciais e melhoramento genético de espécies madeireiras
Abstract: Brazil has a lush native flora, much exploited and little studied. The Brazilian forestry economy has an important role in the life quality of the country being necessary information to enable a more conscious and sustainable exploitation of native species. One way to obtain relevant details about the tree species in Brazil is through cytogenetic studies. These chromosome studies may provide important information on taxonomy, evolution, genetics and plant breeding as also as on the conservation of forest systems. Using conventional cytogenetics techniques the diploid chromosome number was established: Balfourodendron riedelianum, 2n = 58 chromosomes, with the average size of chromosomes 1, 877μm ± 0.44, Cedrela fissilis, 2n = 56 chromosomes, with the average size of chromosomes 1, 01μm ± 0.26; Hymenaea courbaril var. stilbocarpa, 2n = 24 chromosomes, with an average size of the chromosomes of 3.52 μm ± 0.68; Myroxylon peruiferum, 2n = 26 chromosomes, with the average size of chromosomes 1, 25μm ± 0.30; Pterogyne nitens, 2n = 20 chromosomes with average size of chromosomes 1, 13μm ± 0.27, Tabebuia aurea, 2n = 40 chromosomes, with an average size of the chromosomes of 1.05 μm ± 0.23; T. ochracea, 2n = 80 chromosomes, with an average size of the chromosomes of 1.02 ± 0.22 μm and Cedrela odorata with chromosome variation of 2n = 42 to 2n = 104 chromosomes. The results of this study may provide support for future research in chromosome manipulation, comparative taxonomy, evolutionary studies, commercial hybrid seed production and breeding timber species
Mestre

Orientador: Carmem Silvia Fontanetti Christofoletti
Banca: Maria Aparecida Marin Morales
Banca: Silvia Tamie Matsumoto
Resumo: 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... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: 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)
Mestre

Orientador: Maria Aparecida Marin Morales
Banca: Carmem Silvia Fontanetti Christofoletti
Banca: Mateus Mondin
Resumo: Os agrotóxicos constituem uma importante estratégia da agricultura, para a obtenção de uma produtividade economicamente viável, pois são substâncias utilizadas no combate de organismos indesejáveis. Os inseticidas são agentes que têm ação de combater insetos, tanto na fase adulta como larval. O fipronil é um composto do grupo fenil-pirazol, toxicologicamente classificado como altamente tóxico, amplamente utilizado em campos agrícolas do estado de São Paulo, bem como nas residências, para combater insetos pragas. A ação deste inseticida se dá pela sua ligação ao canal de cloro, promovendo o bloqueio da ativação da condução dos estímulos nervosos, pelo ácido gama-aminobutírico (GABA), uma substância que controla o fluxo de íons cloro, através da membrana da célula nervosa. Em pequenas concentrações, apresenta uma eficiente ação nos organismos alvos. Esta característica também pode causar problemas ao meio ambiente, muitas vezes, longe até dos lugares onde foi aplicado. O fipronil, em temperaturas moderadas, é estável no ambiente por cerca de um ano. Estudos mostram que esse inseticida pode ser degradado em diversos metabólitos, que são ainda mais tóxicos que ele. Quanto a sua persistência no ambiente, o fipronil apresenta variações decorrentes da sua formulação, mas os seus metabólicos podem ser mais persistentes que o próprio inseticida. Resíduos de fipronil podem ser bioacumulado no tecido adiposo, indicando uma potencialidade de transferência via cadeia trófica. No presente trabalho, foram avaliadas as potencilidades tóxicas, citotóxicas, genotóxicas e mutagênicas do inseticida fipronil, por meio de bioensaios com Allium cepa, cujas sementes foram expostas à germinação em fipronil.
Abstract: The pesticides are an important strategy for agriculture, to obtain productivity economically viable, because they are substances used to eliminate pest organisms. Insecticides are agents which have action to combat insects, in both the adult and larval stage. The fipronil is a group composed of phenyl-pyrazole, toxicologically classified as highly toxic, and it is widely used in agricultural fields in Sao Paulo State, as well as in homes, to combat insect pests. This insecticide acts through its connection to the channel chlorine, promoting blockade in the activation of the conduct of nervous stimuli, the gamma-aminobutyric acid (GABA), a substance that controls the flow of chloride ions through the cell membrane of nerve. In small concentrations, presents an efficient action in the organism targets. This feature can also cause problems to the environment, often far to the places where it is applied. In moderate temperatures, the fipronil is stable in the environment for about a year. Studies show that this insecticide can be degraded in various metabolites, which are even more toxic than the fipronil. Relative to its persistence in the environment, the fipronil presents variations resulting from its formula, but their metabolites may be more persistent than the insecticide. Residues of fipronil can be bioacumulated in adipose tissue, indicating a potential of transference by the food chain. In the present research, it had been evaluated the toxic, cytotoxic, genotoxic and mutagenic potentials of the fipronil insecticide through bioassays with Allium cepa, whose seeds were exposed to germination in this insecticide. The results indicate that the insecticide presented no toxic and cytotoxic effects for the A. cepa species, when we compared the data resulting from the tests performed with the insecticide and with the negative control.
Mestre

Orientador: Regina Maria Barretto Cicarelli
Banca: Greiciane Gaburro Paneto
Banca: Raquel Mantuaneli Scarel Caminaga
Resumo: A identificação humana pela análise de DNA utiliza o perfil genético de um indivíduo baseado no estudo de uma combinação de marcadores que são herdados de seus progenitores. Os marcadores genéticos mais utilizados na rotina forense estão presentes nos cromossomos autossomos, porém, os marcadores presentes nos cromossomos sexuais (X e Y) e no DNA mitocondrial auxiliam as análises de forma eficiente. Assim, os marcadores do cromossomo Y têm sido muito estudados, pois além do campo forense, estes possuem várias aplicações no campo evolucionário, como na compreensão da genética populacional e exploração da história da evolução humana. Duas categorias principais são atualmente utilizadas para examinar o cromossomo Y: loci bialélico (polimorfismos de nucleotídeo único - SNPs - e inserção Alu) e loci multialélico (minissatélites e microssatélites - STRs). SNPs possuem várias vantagens em relação aos STRs, principalmente com amostras degradadas ou em pequena quantidade, devido a sua alta frequência, simplicidade, menor tamanho e baixa taxa de mutação. Assim, visando à ampliação dos dados da população brasileira em relação aos marcadores genéticos, este estudo teve por objetivo identificar os maiores haplogrupos existentes na população do Estado do Espírito Santo e avaliar as contribuições de Africanos, Ameríndios e Europeus na sua formação, uma vez que esse estado recebeu imigrantes de várias origens. Para isso, foram estudados 35 Y-SNPs em 255 amostras de indivíduos do sexo masculino nascidos no Estado do Espírito Santo - Brasil. A genotipagem foi realizada por PCR seguida por minisequenciamento (SNaPshot Multiplex) e detecção por eletroforese capilar no analisador genético ABI3500 (Applied Biosystems by Life Technologies). Dos 38 haplogrupos possíveis de serem classificados... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Human identification by DNA analysis uses the genetic profile of an individual studying a combination of markers inherited from their parents. The genetic markers most widely used in routine forensic are present in the autosomes, however, the markers present in sex chromosomes (X and Y) and mitochondrial DNA helps analysis efficiently. Thus, the Y chromosome markers have been widely studied because beyond the forensic field, they have several applications in the field of evolution, such as in the understanding of population genetics and exploitation of human evolutionary history. Two main categories are currently used to examine the Y chromosome: biallelic loci (single nucleotide polymorphisms - SNPs - and Alu insertion) and multiallelic loci (minisatellites and microsatellites - STRs). SNPs have several advantages over STRs, especially with degraded or in small quantities samples, due to its high frequency, simplicity, small size and low mutation rate. Thus, aiming to increase the data of the brazilian population related to genetic markers, the objective of this study was to identify the major haplogroups existing in the population of Espirito Santo and evaluate the contribution of Africans, Amerindians and Europeans in their formation, since this state has received immigrants from various origins. For this purpose, 35 Y-SNPs in 255 blood samples from male individuals born in Espirito Santo State-Brazil. Genotyping was performed by PCR followed by minisequencing reaction (SNaPshot Multiplex) and detection by capillary electrophoresis on ABI3500 genetic analyzer (Applied Biosystems by Life Technologies). Of the 38 possible haplogroups to be classified with the 35 SNPs studied, only 19 were detected in this sample. The haplogroup diversity was 0.7794±0.0229 and the most frequent haplogroup was... (Complete abstract click electronic access below)
Mestre

Orientador: Ana Sílvia Alves Meira Tavares Moura
Banca: Heraldo César Gonçalves
Banca: Mônica Corrêa Ledur
Resumo: O objetivo deste trabalho foi identificar locos controladores de características quantitativas (QTLs) nos cromossomos 11 e 13 de galinhas (Gallus gallus) para características de desempenho e carcaça. A partir do cruzamento entre uma linhagem de corte e uma de postura, foi gerada a população experimental F2 na Embrapa Suínos e Aves. Foram avaliadas as seguintes informações fenotípicas: peso ao nascer, peso aos 35, 41 e 42 dias, ganho de peso, consumo de ração, eficiência e conversão alimentar dos 35 aos 41 dias e valores de hematócrito. As carcaças foram evisceradas e avaliados: o comprimento do intestino, peso dos pulmões, do fígado, do coração e da moela. Foram obtidos após quatro horas de resfriamento: peso da carcaça, gordura abdominal, peso de partes: peito, coxas, dorso, asas, cabeça e pés. Quatro e cinco marcadores microssatélites dos cromossomos 11 e 13, respectivamente, foram genotipados num total aproximado de 330 animais F2 em quatro famílias de irmãos-completos. Os mapas de ligação para ambos os cromossomos foram construídos e a análise de mapeamento de QTLs baseada no modelo genético de F2 foi realizada. No cromossomo 11 foram mapeados dois QTLs sugestivos: para peso de pés e de moela, ambos posicionados no intervalo entre ADL0123 e ADL0210. No cromossomo 13 foi mapeado um QTL sugestivo para peso de coração posicionado no intervalo entre MCW0110 e MCW0104.
Abstract: The objective of this study was to identify quantitative trait loci (QTLs) for performance and carcass traits in chicken (Gallus gallus) chromosomes 11 and 13. From the crossbreeding of a broiler and a layer line, an F2 experimental population was generated at the Embrapa Suínos e Aves. The following phenotypic data were recorded: body weights at birth, 35, 41 and 42 d; weight gain, feed consumption and feed conversion from 35 to 41 d; weights of carcass, carcass parts, organs and abdominal fat, hematocrit and length of intestine. Four and five microsatellite markers from chromosomes 11 and 13, respectively, were genotyped in approximately 330 F2 chickens from four full-sib families. The linkage maps for both chromosomes were constructed and the QTL mapping analyses were carried out based on an F2 genetic model. Two suggestive QTLs were mapped to chromosome 11: for feet and gizzard weights, both located in the interval between ADL0123 and ADL0210. On chromosome 13 one suggestive QTL for heart weight was detected in the interval between MCW0110 and MCW0104.
Mestre

Orientador: Maria Aparecida Marin-Morales
Banca: Maria Luiza Silveira Mello
Banca: Cláudia Bueno dos Reis Martinez
Banca: Maria Angélica Maciel Martinho Ferreira
Banca: Edson Luis Maístro
Resumo: 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... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: 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)
Doutor

Resumo: O búfalo de rio (Bubalus bubalis) é uma espécie de interesse econômico pertencente à família Bovidae, utilizado para a produção de carne e leite, além de força de trabalho no campo, constituindo-se em um animal de tripla aptidão. Recentemente, a construção de um painel de células somáticas híbridas irradiadas (linhagens celulares) contendo fragmentos do genoma do búfalo de rio, incorporado ao genoma de hamster (BBURH5000), está permitindo, pela primeira vez, o mapeamento de todos os cromossomos bubalinos. A utilização desse painel de células associado a análises estatísticas, está gerando mapas genômicos de alta resolução contendo diferentes tipos de marcadores moleculares. O presente trabalho teve por objetivo utilizar este painel de células para gerar grupos de ligação com os marcadores mapeados no cromossomo 9 bubalino e assim comparar a organização dos grupos de ligação obtidos com aqueles já descritos para o cromossomo 7 bovino. Para tal, foram testados com DNA bubalino, seqüências de "primers" para PCR de 26 marcadores moleculares previamente mapeados no cromossomo 7 bovino, o qual foi descrito na literatura como o homólogo ao cromossomo 9 de búfalo. Do total de marcadores testados, 18 geraram produtos de PCR adequados ao mapeamento utilizando o painel BBURH5000, sendo que destes 18, foi possível a genotipagem final de 10 marcadores. A partir das análises de ligação com os 10 marcadores genotipados, foi possível distribuir os mesmos em três grupos de ligação no BBU9. A análise comparativa entre os grupos de ligação do BBU9 e do BTA7 revelou a presença dos mesmos grupos de ligação nos cromossomos de ambas espécies, evidenciando conservação de sintenia.
Abstract: The river buffalo (Bubalus bubalis), a member of the Bovidae family, is an economically important livestock specie useful to produce milk and meat, as well as source of labor, comprising an animal of triple aptness. The recent construction of a whole-genome 5000-rad radiation hybrid somatic cell panel for the river buffalo genome (BBURH5000), is allowing for the first time the mapping of all chromosomes from the specie. The use of BBURH5000 panel associated with statistical analyses is generating high-resolution RH maps integrating different types of molecular markers. The goal of this study was to genarate linkage groups with the markers mapped on the river buffalo chromosome 9 (BBU9), using the BBURH5000 panel, and then compare these groups with those already described in BTA7. Previous studies have identified bovine chromosome 7 as homologous to BBU9. Cattle derived PCR primers from 26 markers, previously mapped in cattle, were tested with buffalo DNA. A total of 18 of the 26 markers amplified PCR products suitable for RH mapping. From these 18 markers, it was possible the genotyping of 10. The analyses of linkage with the 10 markers genotyped turned possible to distribute them in three linkage groups on the BBU9. The comparative analyses between the linkage groups of BBU9 and BTA7 revealed the presence of the same linkage groups on the chromosome of both species, revealing conservation of synteny between them.
Orientadora: Maria Elisabete Jorge Amaral
Coorientadora: Vera Fernanda Martins H. de Lima
Banca: Humberto Tonhati
Banca: Simone Cristina Méo Niciura
Mestre

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Jun. 26, 2009). "... in partial fulfillment of requirements for the degree of Doctor of Philosophy in the Department of Biology." Discipline: Biology; Department/School: Biology.

Dissertation presented to obtain the Ph.D degree in Molecular Biology
The ends of eukaryotic chromosomes are protected from illegitimate repair by structures called telomeres. These are comprised of specific DNA repeats bound by a specialized protein complex. When telomere function is compromised, chromosome ends fuse, generating chromosomal abnormalities and genomic instability.(...)

Orientador: Doralice Maria Cella
Banca: Kátia Cristina Machado Pellegrino
Banca: Douglas de Araujo
Resumo: Das subfamílias de Chrysomelidae, Cassidinae sensu lato (s.l.), formada por 6.000 espécies e 43 tribos, possui aproximadamente 100 espécies analisadas citogeneticamente e a maioria delas apresentou 2n=18=16+Xyp, o qual é menor que aquele considerado basal para os Polyphaga, 2n=20=18+Xyp. No entanto, alguns grupos de espécies demonstraram manutenção do número diplóide basal e outros, aumento desse número; algumas espécies do último grupo também exibiram variação em relação ao tipo de sistema cromossômico sexual (SCS). Considerando a revisão taxonômica recentemente realizada para as espécies de Cassidinae s.l., a existência de relação filogenética para algumas espécies dessa subfamília, a alta diversidade de espécies desse grupo registrada para a região Neotropical e o baixo número de espécies que tiveram seus cromossomos estudados, o presente trabalho teve como objetivo verificar os mecanismos envolvidos na evolução cariotípica dessa subfamília através do estudo de sete espécies da fauna brasileira e da revisão dos dados citogenéticos. Os espécimes foram coletados em Avaré e Rio Claro, SP, Nonoai, RS, e Ponta Grossa, PR. As preparações cromossômicas obtidas de embrião e testículos de machos adultos foram coradas com solução de Giemsa. As espécies Agroiconota inedita (2n=42=40+Xyp), Charidotella (sensu stricto) immaculata (2n=22=20+Xyp), Charidotella (sensu stricto) sexpunctata (2n=22=20+Xyp), e Stolas chalybaea (2n=24=22+Xyp) revelaram número diplóide maior do que aquele estabelecido como basal para os Polyphaga e cromossomos com dois braços. Os cariótipos de Cteisella confusa, Deloyala cruciata, e Metriona elatior mostraram a fórmula cromossômica 2n=18=16+Xyp, considerada modal para os Cassidinae s.l., e cromossomos com dois braços. As sete espécies apresentaram cromossomos sexuais facilmente identificáveis por serem... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Among the subfamilies of Chrysomelidae, Cassidinae sensu lato (s.l.) , formed by 6 000 species and 43 tribes, possesses approximately 100 species cytogenetically analyzed and most of them presented 2n=18=16+Xyp, which was smaller than 2n=20=18+Xyp considered basal for Polyphaga. However, some groups of species presented maintenance of the basal diploid number and others showed increase of this number; certain species of the latter group also exhibited variation in the sex chromosome system (SCS). Considering the recent taxonomic revision accomplished for the Cassidinae s.l. species, the existence of phylogenetic relationship for some species of this subfamily, the high diversity of species of this group in the Neotropical region, and the low number of Cassidinae s.l. species karyotyped so far, the aim of the present work was to verify the main mechanisms involved in the karyotype evolution of this subfamily through the study of seven species of the Brazilian fauna and the overview of the cytogenetic data. The individuals were collected in Avare and Rio Claro, SP, Nonoai, RS, and Ponta Grossa, PRo The chromosomal preparations obtained from embryo and testes of adult males were stained with Giemsa solution. The species Agroiconota inedita (2n=42=40+Xyp), Charidotella (sensu stricto) immaculata (2n=22=20+Xyp), Charidotella (sensu stricto) sexpunctata (2n=22=20+Xyp), and Stolas chalybaea (2n=24=22+Xyp) revealed diploid number higher than that established as basal for Polyphaga and biarmed chromosomes. The karyotype of Gteisella confusa, Deloyala cruciata, and Metriona elatior showed the chromosomal formulae 2n=18=16+Xyp considered modal for Cassidinae s.l. and biarmed chromosome. The seven species exhibited easily identified sex chromosomes due to their smallest size in the complement. The analysis of meiotic cells of all the species showed pachytenes with a positively heteropycnotic block... (Complete abstract click electronic access below)
Mestre

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Feb. 17, 2009). "... in partial fulfillment of the requirement for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics." Discipline: Biochemistry and Biophysics; Department/School: Medicine.

Orientador: Silvia Regina Rogatto
Banca: Edigard Graner
Banca: Claudia Aparecida Rainho
Resumo: Os tumores de glândulas salivares (TGS) são lesões raras que apresentam heterogeneidade morfológica e sobreposição histológica entre os vários subtipos de tumores. Neste estudo foi utilizada a técnica de hibridação genômica comparativa de alta resolução (HR-CGH) em 64 amostras de TGS, incluindo: 27 adenomas pleomórficos (AP); 11 tumores de Warthin (TW); seis carcinomas ex-adenomas pleomórficos (CXAP); seis carcinomas adenóides císticos (CAC); quatro carcinomas mucoepidermóides (CME); três carcinomas ductais salivares (CDS); três adenocarcinomas (ACAR); dois oncocitomas (ON) e dois carcinomas epiteliaismioepiteliais (CEM). Destas, 47 amostras eram provenientes de tecidos fixados em formalina e em blocos de parafina (FFEP) e 17 de tecidos a fresco. Após a microdissecção, o DNA genômico foi amplificado e marcado por técnica baseada na PCR (SCOMP) e por nick translation, respectivamente. Os cromossomos foram cariotipados usando a imagem DAPI invertido e a intensidade do sinal de hibridação foi determinada ao longo de cada cromossomo. Uma biblioteca com amostras de DNA normais foi construída para selecionar os limites superiores e inferiores para perdas e ganhos cromossômicos. Em todos os casos, as perdas genômicas foram observadas mais freqüentemente do que os ganhos. Na análise comparativa das regiões mínimas comuns detectadas pela HR-CGH envolvendo todos os tipos tumorais, foram observadas alterações genômicas comuns aos diversos tipos, como também exclusivas para os diferentes tipos histológicos. As regiões genômicas consistentemente alteradas nos dois subgrupos tumorais com maior número amostral (adenomas pleomórficos e tumores de Warthin) foram investigadas em bancos de dados para a seleção de genes candidatos que pudessem estar relacionados com a etiologia destes tumores. Os genes NEDD9 (6p24-p25), PPARG (3p25) e c-MYC (8q24.21) foram... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Salivary gland tumors (SGT) are uncommon lesions showing morphologic heterogeneity and histologic overlap among various tumoral subtypes. In the present study, high resolutioncomparative genomic hybridization (HR-CGH) method was performed in 64 SGT samples, including 27 pleomorphic adenomas (PA), 11 Warthin's tumors (WT), six carcinoma ex pleomorphic adenoma (CXPA), six cystic adenocarcinomas (CAC), four mucoepidermoid carcinomas (MEC), three salivary ductal carcinomas (SDC), three adenocarcinomas (ACAR), two oncocitomas (ON), and two epithelium-myoepithelium carcinomas (EMC). Among these samples, 47 were formalin-fixed and paraffin embedded tissues and 17 were fresh tissues. After laser capture microdissection (LCM), the DNA samples were amplified and labeled by PCR-based methods (SCOMP) and nick translation reaction, respectively. Chromosomes were karyotyped based on their inverted DAPI image and the relative hybridization signal intensity was determined along each chromosome. A library with differentially labeled normal samples was built to select the superior and inferior limits for chromosomal gains and losses. Genomic losses were more frequently observed than gains. The comparison among all tumor samples showed common chromosomal imbalances and also exclusive genomic alterations related to the different histological tumor types. Non-random genomic regions involved in pleomorphic adenomas and Warthin's tumors were investigated in databases in order to select candidate genes that could be related to the etiology of these tumors. The NEDD9 (6p24-p25), PPARG (3p25) and c-MYC (8q24.21) genes were selected and the gene transcript levels were evaluated by quantitative real time PCR (qRT-PCR) in PA samples. In accordance to the HR-CGH results, it was observed high transcript levels of NEDD9 in 17 out of 37 PA; low levels of PPARG was detected in 11 out of 36 samples; and high levels... (Complete abstract click electronic access below)
Mestre

Orientador: Maria Tercília Vilela de Azeredo Oliveira
Banca: Alba Regina de Abreu Lima Catelani
Banca: Lilian Castiglioni
Resumo: Os triatomíneos são vetores do protozoário Typanosoma cruzi, agente etiológico da moléstia de Chagas. Esses insetos são hematófagos e pertencem à ordem Heteroptera e à família Reduviidae. Disseminada por grandes extensões do Brasil e de outros países latinoamericanos, a doença de Chagas representa um grave e importante problema de saúde pública, caracterizando limitações e dificuldades aos tratamentos. Isso ocorre devido à precariedade apresentada pela vida dos contingentes humanos mais expostos à infecção. Atualmente, a parasitose tem grande participação entre as doenças cardíacas na América do Sul. Segundo a Organização Mundial de Saúde, estima-se que, até a década de 90, por volta de 20 milhões de indivíduos estavam infectados pelo Trypanosoma cruzi nas áreas endêmicas. Dados atuais revelam que o número de pessoas infectadas foi reduzido para 9,8 milhões graças à intensa erradicação desses insetos. No entanto, a vigilância deve continuar, pois é fundamental para se evitar novos casos. Citogeneticamente, o interesse sobre os triatomíneos está em seus cromossomos holocêntricos e no processo incomum da meiose, cuja segregação dos sexuais é pós-reducional. O número básico de cromossomos nos triatomíneos é de 2n=22. No presente trabalho foi analisado a espermatogênese de duas espécies do gênero Triatoma (Triatoma maculata e Triatoma pseudomaculata), com ênfase aos seguintes aspectos: fases da espermatogênese; estrutura cromatínica e dos cromossomos meióticos e acompanhamento do ciclo nucleolar. Essas espécies estão distribuídas principalmente nos estados do Nordeste brasileiro e são consideradas potencialmente vetores do T. cruzi. As espécies analisadas foram cedidas pelo insetário do Serviço Especial de Saúde de Araraquara (SESA), pertencente ao Departamento de Epidemiologia da Faculdade de Saúde Pública da USP. Os...(Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The triatomines are vectors of the protozoan Typanosoma cruzi, etiological agent of Chagas' disease. These insects are hematophages that belong to the order Heteroptera and to the family Reduviidae. Disseminated through large portions of Brazil and of other Latin American countries, Chagas' disease represents a grave and important public health problem, characterizing limitations and difficulties in treatments. This occurs due to the precariousness presented by life contingent to humans most exposed to the infection. Currently, parasitosis presents high participation among cardiac diseases in South America. According to the World Health Organization, it is estimated that, until the 1990s, approximately 20 million individuals were infected by Trypanosoma cruzi in endemic areas. Current data reveal that the number of persons infected was reduced to 9.8 million by virtue of intense eradiaction of these insects. Nevertheless, vigilance must continue, since it is fundamental to avoiding new cases. Cytogenetically, the interest in triatomines is in its holocentric chromosomes and in its uncommon meiosis process, whose sexual segregation is post-reductional. The basic number of chromosomes in triatomines is 2n=22. The present work analyzed the spermatogenesis of two species of the genus Triatoma (Triatoma maculata and Triatoma pseudomaculata), with emphasis on the following aspects: spermatogenesis phases; structure of chromatin and of meiotic chromosomes and accompaniment of the nucleolar cycle. These species are distributed principally in the states of northeastern Brazil and are considered potential vectors for T. cruzi. The species analyzed were supplied by the insectary of the Special Health Service of Araraquara (SESA), belonging to the Department of Epidemiology in the School of Public Health at USP. The organs studied were...(Complete abstract click electronic access below)
Mestre

Orientador: Maria Tercília Vilela de Azeredo Oliveira
Banca: Hermione Elly Melara de Campos Bicudo
Banca: Patricia Pasquali Parise Maltempi
Resumo: Os heterópteros apresentam a meiose cística nos túbulos seminíferos. Esses possuem o cisto espermatogonial envolto pelas células císticas, as quais desenvolvem a função de nutrição das células em divisão celular. Quanto às características citogenéticas, esses insetos apresentam cromossomos holocinéticos, baixa variabilidade cariotípica e meiose invertida dos cromossomos sexuais. No presente trabalho foram caracterizadas as células císticas quanto a sua localização, ultraestrutura e citogenética e, também, foram analisados os aspectos citogenéticos de quatro espécies do gênero Triatoma. Foram utilizadas as técnicas de microscopia eletrônica de transmissão, citogenética convencional (orceína e AgNOR), bandamento C CMA3/DAPI e a técnica de hibridização in situ fluorescente (FISH), com sonda de DNAr 45S de Drosophila melanogaster. Os resultados indicaram que a célula cística envolve um cisto espermatogonial e apresenta um grande núcleo com invaginações citoplasmáticas. Em todas as espécies foram observados vários graus de ploidia da célula cística. Triatoma infestans e T. infestans melanosoma apresentaram vários blocos heterocromáticos com a periferia CMA3 + e o interior DAPI+. Associada às bordas dos blocos heterocromáticos foram observados os segmentos de DNAr 45S, além da presença de vários nucléolos em cada núcleo. Triatoma matogrossensis, T. rubrovaria e T. brasiliensis apresentaram apenas um bloco heterocromático com as mesmas características, com exceção de T. brasiliensis, que apresentou em algumas células vários blocos CMA3 + dispersos. Nessas espécies foi observado apenas um nucléolo com similaridade na localização dos sítios de DNAr. Quanto aos aspectos citogenéticos, todas as espécies apresentaram 2n = 20A + XY, com decréscimo do tamanho relativo dos cromossomos. Em T. infestans melanosoma os cromossomos foram... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Heteroptera, or "true bugs", exhibit meiosis in their seminiferous tubules. They posses the spermatogonial cysts that are enclosed by cyst cells, which develop the nutritional function of the cells during cell division. In terms of cytogenetic characteristics, these insects possess holokinetic chromosomes, low karyotype variability, and inverted meiosis in the sex chromosomes. In this study, cyst cells from four species of the genus Triatoma were characterized by their location, superstructure, and cytogenetic makeup. Electronic transmission microscopy techniques were used, as well as conventional cytogenetic techniques (Orcein and AgNOR), C-banding with CMA3 and DAPI banding, and Fluorescence in situ Hybridization (FISH) with a 45S DNA probe of Drosophila melanogaster. The results indicated that the the spermatogonial cyst is enclosed by the cyst cell, and that the cyst cell possesses a large nucleus with cytopasmic invaginations. In all species studied, varying degrees of ploidy were observed in the cyst cells. Triatoma infestans and T. infestans melanosoma presented with various heterochromatic blocks, with CMA3 + at the periphery and DAPI+ at the interior. Segments of rDNA 45S were found along the edges of the heterochromatic blocks, along with the presence of various nucleoli in each nucleus. Triatoma matogrossensis, T. rubrovaria and T. brasiliensis presented with only one heterochromatic block with the same characteristics (with the exception of T. brasiliensis, which presented with various dispersed CMA3 + blocks). In these species, only one nucleolus that was similar to the localization of the rDNA sites was found. All species presented with 2n = 20A + XY, with a decrease in size relative to the chromosomes. In the case of T. infestans melanosoma, the chromosomes were split into groups based on their relative sizes. The heterochromatin of this species presented... (Complete abstract click electronic access below)
Mestre

Orientador: Maria Aparecida Marin Morales
Banca: Silvia Tamie Matsumoto
Banca: Mário sérgio Mantovani
Resumo: Os agrotóxicos, substâncias químicas muito utilizadas para combater pragas na agricultura e nas residências, além de contribuírem para o aumento da produtividade e, conseqüentemente, o crescimento economia, também são responsáveis pela contaminação ambiental, quando utilizados indiscriminadamente, e por muitos casos de intoxicações e doenças genéticas nos seres humanos. Dependendo de sua composição química, degradabilidade e persistência no ambiente, os agrotóxicos podem comprometer a cadeia alimentar e afetar o ecossistema como um todo. Dentre os agrotóxicos bastante utilizados em todo o mundo, está o organofosforado malation. Pelo seu amplo uso, torna-se preocupante os possíveis danos que este inseticida possa promover no meio ambiente e nos organismos a ele expostos, pois alguns trabalhos já mostram esta sua ação detrimental. Desta forma, este trabalho investigou o potencial de indução de danos no DNA, para diferentes concentrações de malation, por meio das técnicas de aberrações cromossômicas e teste do micronúcleo em A. cepa e micronúcleo e ensaio do cometa em células HTC (hepatoma tissue culture). Nos testes com células meristemáticas e F1 de A. cepa, expostas por 24 e 48 horas ao malation, foram verificadas freqüências significativamente elevadas de AC e MN. Após 24 horas de exposição, não houve aumento na quantidade de AC, mas sim de MN. A análise das células F1 de A. cepa, juntamente com o teste de AC, forneceu dados importantes sobre a fixação dos danos genéticos induzidos pelo malation. Nenhum resultado relevante de citotoxicidade foi verificado em A. cepa. Após passarem pelo teste de recuperação, somente as células expostas às menores concentrações testadas apresentaram diminuição nos índices de AC e MN.
Abstract: Pesticides, chemicals widely used to combat pest in agriculture and in homes, besides contribute to the increase productivity and the economy, are also responsible for environmental pollution and for many intoxications cases and genetic diseases in human beings. Depending on their chemical composition, degradability and degree of persistence in the environment, the pesticides can endanger the food chain and affect the entire ecosystem. Among the pesticides most used in the world is the organophosphate malathion. Due to its widespread use it has been worrying the possible damages that can promote this insecticide in the environment and in organisms exposed to it. This study investigated the induction potential of DNA damage with several malathion concentrations using chromosomic aberration (CA) techniques, micronucleus test (MN) in A. cepa and micronucleus and comet assay in hepatoma tissue culture (HTC). In A. cepa tests, meristematic and F1 cells exposed for 24 and 48 hours to malathion showed significative frequencies of CA and MN. After 24 hours of exposure, there was no increase in the CA amounts, but was observed an increase to frequency of MN. The F1 cells analysis, together with the CA tests, provided important data on the genetic damages fixation induced by malathion. No relevant results of citotoxicity were verified in A. cepa. After the recovery test, the cells exposed to the smaller tested concentrations of malathion showed reduction of AC and MN indices. In the HTC cells test, was verified by the comet assay, genotoxic effects for all the tested concentrations, after exposure for 24 hours to the insecticide, but by the micronucleus tests, no significative results were found. These data suggest that the DNA lesions were repaired and not elapsed in mutation. With the concomitant realization of different tests, the clastogenic action of malathion could be verified.
Mestre

Orientador: Fabíola Vitti Môro
Banca: Emerson Ricardo Pansarin
Banca: Ricardo Machado da Silva
Resumo: Este trabalho teve como objetivo caracterizar morfologica e citogeneticamente as especies de plantas toxicas: Crotalaria lanceolata E. Mey., Ricinus communis L., Cassia occidentalis L. , Canavalia ensiformis D.C. e Amaranthus spinosus L.. Para a morfologia utilizou-se sementes e plantulas que foram esquematizadas com auxilio de estereomicroscopio equipado com camara clara.Para a citogenetica utilizou-se pontas de raizes, hidroxiquinoleina e coloracao Giemsa. Crotalaria lanceolata E. Mey. apresenta sementes com variados tons de castanhos. A germinacao e epigea e fanerocotiledonar. O embriao e cotiledonar e o endosperma mucilaginoso. Apresenta numero cromossomico 2n = 16 cromossomos, com comprimento medio geral de 3,340 Êm } 0,689. Ricinus communis L. possui sementes com testa mesclada em tons castanhos, com caruncula visivel localizada na parte inferior da semente, germinacao epigea e fanerocotiledonar.O embriao e cotiledonar e o endosperma oleaginoso. O numero cromossomico 2n = 10 cromossomos, com comprimento cromossomico medio de 1,123 Êm } 0,327. Cassia occidentalis apresenta sementes com tons marrom-escuro, embriao cotiledonar e endosperma mucilaginoso. A germinacao e epigea e fanerocotiledonar. Possui 2n = 26 cromossomos com comprimento cromossomico medio e de 1,672 0,400. Canavalia ensiformis D.C apresenta sementes com uma coloracao branca e lignificada, embrião cotiledonar e endosperma mucilaginoso. A germinacao e do tipo epigea e fanerocotiledonar. Apresenta numero cromossomico 2n = 22 cromossomos com comprimento medio de 1,388 Êm 0,249...... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: This work had as objective to characterize morphologic and citogenetics some species of toxic plants: Crotalaria lanceolata E. Mey., Ricinus communis L., Cassia occidentalis L., Canavalia ensiformis D. C. and Amaranthus spinosus L.. For the morphological studies were used seeds and seedlings that were schematized with of stereomicroscoppe equipped with camera lucida. The cytogenetic already used points of rootses, Hidroxiquinoleina and Giemsa coloration. Crotalaria lanceolata E. Mey. present seeds with varied tones of chestnut . Its germination is phanerocotyledonar and epigeous. The seeds are kidney shaped and the embryo is cotyledonary with a mucilaginous endospermic. They present chromosome number 2n = 16 chromosomes, with general medium length of 3,340mm l 0,689. Ricinus communis L. presents seeds with forehead several mixed many tones chestnut, wich visible caruncula located in the inferior part of the seed, germination is phanerocotyledonar and epigeous. The seeds are kidney shaped and the embryo is cotyledonary and an oleaginous endospermic. Its chromosome number is 2n = 10 chromosomes, with length medium chromossomic of 1,123mm l 0,327. Cassia occidentalis presents seed with tones brown-darkness whose interior presents an embryo is cotyledonary and a mucilaginous endospermic. Its germination is phanerocotyledonar and epigeous. The evaluation cytogenetic shows us 2n = 26 chromosomes with length medium chromossomic are of 1,672mm l 0,400. Canavalia ensiformis D.C presents seeds with a white coloration and lignification. In its interior it is located an embryo cotyledonary... (Complete abstract, click electronic access below)
Mestre

Orientador: Cesar Martins
Banca: Luis Antonio Carlos Bertollo
Banca: Eliana Feldberg
Banca: Ligia Souza Lima Silveira da Mota
Banca: André Luis Laforga Vanzella
Resumo: A família Cichlidae tem despertado um grande interesse científico devido à rápida e extensa radiação adaptativa sofrida em alguns de seus grupos e por conter espécies com grande potencial para a aqüicultura, como a tilápia do Nilo, Oreochromis niloticus. O mapeamento físico cromossômico mostra-se promissor como ferramenta para os estudos comparativos e evolutivos entre diferentes espécies. Diante disto, o presente trabalho teve como objetivo realizar mapeamento cromossômico comparativo em ciclídeos utilizando seqüências repetitivas de DNA como sondas. Elementos transponíveis, DNA satélite e seqüências repetidas inseridas em BACs foram utilizados como sondas, através da técnica de FISH, em espécies de ciclídeos africanos e sul-americanos. Os retrotransposons Rex localizaram-se principalmente nas regiões centroméricas de espécies africanas e sul-americanas, com exceção de O. niloticus que demonstrou um padrão de localização disperso destes elementos. O acúmulo de Rex nos centrômeros destas espécies é coincidente com as regiões heterocromáticas, que representam um refúgio para seqüências repetitivas, devido à baixa taxa de recombinação. O DNA satélite SATA hibridou nos centrômeros de todas as espécies analisadas. Esta conservação centromérica mostra um papel importante destas seqüências na organização estrutural e funcional destas regiões nas diferentes espécies. Além disto, em O. karongae, foram observados sinais intersticiais em três pares cromossômicos, corroborando a hipótese de fusões cromossômicas que levaram à redução do número diplóide nesta espécie. O elemento transponível ROn-1 localizou-se intersticialmente no braço longo do par maior de O. niloticus e em posição próxima ao telômero também no braço longo do par meta-submetacêntrico (m/sm) maior dos haplocromíneos e hemicromíneos... (resumo completo, clicar acesso eletrônico abaixo)
Abstract: The Cichlidae family is one of the most species-rich families of fishes. This family has attracted the attention of the evolutionary biologists due the rapid radiation occurred in some species. Moreover, some cichlid species are important for the world aquaculture, such as Nile tilapia, Oreochromis niloticus. The chromosome mapping is useful for comparative and evolutionary studies among different species. To further understand the mechanisms of chromosome evolution in cichlids, repeated sequences were used for the comparative chromosome mapping in cichlid species. Probes containing the transposable elements (TEs) Rex1, Rex3, Rex6 and ROn-1, the SATA satellite DNA, and a BAC-clone enriched of several types of repeated DNAs were used through FISH in the chromosomes of African and South-American cichlids. The TEs Rex were mainly distributed in the centromeric region of all chromosomes in all cichlids, with the exception of O. niloticus, that presented TEs distributed overall in the chromosome arms. The localization of TEs Rex are in coincidence with heterochromatic regions, which can represent an perfect environment for the accumulation of repeated sequences. The satellite DNA was mapped in the centromeres of all cichlid species. The maintenance and centromeric distribution of the SATA satellite DNA in African cichlids suggest that this sequence can play an important role in the organization and function of the centromere in these species. Moreover, in O. karongae, the SATA have shown interstitial signals in three chromosome pairs, corroborating that chromosome fusions were involved in the reduction of diploid number in this species. The transposable element ROn-1 was localized in just one cluster in the largest chromosome of African cichlids, but in different positions, suggesting that different chromosomal rearrangements could have occurred in the origin of the largest chromosomes... (Complete abstract click electronic access below)
Doutor

Resumo: Os triatomíneos são insetos hematófagos de grande importância para a parasitologia humana, pois são transmissores do Trypanosoma cruzi, protozoário causador da doença de Chagas. Além de sua importância médico-sanitária, os triatomíneos destacam-se pela sua citogenética, pois possuem cromossomos holocêntricos e um modelo de meiose incomum, com meiose invertida para os cromossomos sexuais. Recentes pesquisas com marcadores moleculares em triatomíneos tentam compreender a ancestralidade do grupo. Uma das formas de se compreender a evolução entre espécies é a partir da análise de seqüências do DNA ribossômico (DNAr). Por pertencer a famílias multigênicas, cópias individuais do DNAr não acumulam mutações independentemente, resultando em pequena variação intra-específica e relevante diferenciação interespecífica. No presente trabalho foi realizado um estudo comparativo entre as espécies Triatoma maculata e Triatoma pseudomaculata, com base no uso das técnicas citogenéticas convencionais de orceína lacto-acética, impregnação por íons prata, bandamento-C, reação de Feulgen; da técnica de citogenética molecular de bandamento- C CMA/DAPI; e também por meio da análise da região ITS-1 do DNAr, com base no sequenciamento, com o objetivo de avaliar o grau de homologia entre as espécies estudadas. Os cariogramas das duas espécies indicaram dez pares de autossomos (um deles de tamanho maior) e um par de cromossomos sexuais (2n = 22). No ciclo meiótico foi possível observar a fragmentação da região nucleolar no final do estágio difuso. Corpúsculos nucleolares foram observados em alguns dos núcleos em metáfases meióticas de T. pseudomaculata, evidenciando a persistência nucleolar. A técnica de bandamento-C revelou que o cromossomo Y é heterocromático em ambas as espécies. O sequencimento da região ITS-1, indicou que as espécies apresenta... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The triatomines are hematophagous insects of great concern in public health because they are vectors of Trypanosoma cruzi, a protozoan that causes Chagas disease. Triatomines are also of great genetic interest, because that they present holocentric chromosomes and an unusual form of meiosis with post-reductional segregation of sex chromosomes. Recent studies based on molecular markers try to understand the evolutionary history of triatomines. To understand the evolution of a given species, ribosomal DNA (rDNA) analyses are frequently used, which can help to infer evolutionary relationships among species. Individual copies of rDNA do not accumulate mutations independently because they belong to multigene families, resulting in slight intraspecific and important interspecific variation. In this study, a comparative analysis was performed between the species Triatoma maculata and Triatoma pseudomaculata, based on the cytogenetic techniques of lacto-acetic orcein, silver ion impregnation, Cbanding, Feulgen reaction; and CMA/DAPI C-banding. We also compared the species by sequencing the ITS-1 rDNA internal transcribed region in order to evaluate the degree of homology among the studied species. The cariograms of the two species revealed ten autosomes and one pair of sexual chromosomes (2n= 22). In the meiotic cycle, nucleolar fragmentation during the final stages of meiotic prophase I was found. Nucleolar corpuscles were found in some meiotic metaphases of T. pseudomaculata, which is evidence of nucleolar persistence. The C-banding technique revealed that the Y chromosome is heterochromatic in both species. The ITS-1 rDNA sequences showed that the species presented a discharge proximity to each other, and had a high degree of homology (98.5%). The knowledge obtained in this study contributes to the understanding of the interrelation and distribution of those species, and offers... (Complete abstract click electronic access below)
Orientador: Maria Tercília Vilela de Azeredo Oliveira
Coorientador: Lilian Castiglioni
Banca: João Aristeu da Rosa
Banca: Carlos Roberto Ceron
Mestre

Orientador: Hermione Elly Melara de Campos Bicudo
Banca: Otávio Ricci Júnior
Banca: Cláudia Regina Bonini Domingos
Resumo: A Hidroxiuréia (HU) é utilizada como medicamento em várias doenças humanas, incluindo anemia falciforme e câncer. Basicamente, na primeira, atua aumentando a porcentagem de hemoglobina fetal, o que diminui a gravidade do quadro clínico por inibir a polimerização da hemoglobina S e, no segundo, atua impedindo a síntese de DNA na fase S, desta forma bloqueando a divisão celular. Há preocupação com o tratamento longo com essa substância por causar efeitos colaterais, um dos quais se relaciona a problemas na fertilidade masculina. A Drosophila, devido a várias características, como ser o organismo com maior homologia com o homem quanto às doenças genéticas, por utilizar esses genes homólogos nos mesmos processos, mas de forma simplificada e por expressar os genes humanos em construções transgênicas, é hoje muito utilizada em estudos de doenças humanas e de respostas a fármacos, buscando esclarecer mecanismos de ação e conseqüências do uso. Neste trabalho, foram estudados, sob o efeito da HU em duas concentrações (0,1 e 0,25mg/ml de meio de cultura), no modelo biológico mencionado, as características taxa de oviposição, produtividade (número de descendentes), tempo de desenvolvimento, mortalidade, longevidade, tempo de pré-cópula , duração da cópula, presença de alterações morfológicas externas, morfologia do aparelho reprodutor masculino, modificações do peso das moscas, morfologia dos cromossomos politênicos e padrão de expressão das enzimas esterasicas. A produtividade foi analisada em seis gerações consecutivas, visando obter maior compreensão dos efeitos da HU. Cada característica analisada envolveu uma metodologia diferente, descrita no corpo do trabalho. A análise estatística foi baseada na aplicação da ANOVA, e nas comparações de Kruskal-Wallis e Mann-Whitney. Em algumas características o efeito do tratamento mostrou-se... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Hydroxyurea (HU) is used as a medicine in several human diseases, including sickle cell disease (SCD) and cancer. Basically, in the first, HU acts by increasing the percentage of fetal hemoglobin, thus decreasing the severity of the disease symptoms due to inhibition of the hemoglobin S polymerization. In the second mentioned disease, it acts by preventing DNA synthesis in S phase, thereby blocking cell division. There is some concern about side effects caused by the long-term treatment with HU. One of them relates to problems in male fertility. Drosophila, because of its characteristics, such as being the organism with the highest homologies with man as to genetic diseases, since it uses homologous genes in these processes in a simplified form, and also by expressing human genes in transgenic constructs, is now being widely used in studies of human diseases and responses to drugs, seeking for clarifying action mechanisms and consequences of their use. In this work, we studied, in Drosophila biological characteristics under the effect of HU in two concentrations (0.1 and 0.25 mg / ml of culture medium), including oviposition rate, productivity (number of offspring), time development, mortality, longevity, pre-mating and mating duration, presence of morphological changes in external morphology and in the male reproductive system, fly weight changes, polytene chromosome morphology and patterns of esterase enzymes. Productivity was analyzed in six consecutive generations. Each feature analyzed involved a different methodology, described in the body of the work. Statistical analysis was based on the application of ANOVA, and comparisons using the Kruskal-Wallis and Mann-Whitney tests. In some characteristics, the effect of treatment was dosisdependent. In the light of numbers, productivity was higher in control experiments (C) of six generations, and among the treated ones with... (Complete abstract click electronic access below)
Mestre

To prevent re-replication of DNA in a single cell cycle, the licensing of replication origins by Mcm2-7 is prevented during S and G2 phases. Metazoans achieve this by cell cycle regulated proteolysis of the essential licensing factor Cdt1 and formation of an inhibitory heterohexameric complex of Cdt1 with a small protein called geminin. The consequences of either stabilising Cdt1 or ablating geminin in synchronised human U2OS cells are investigated in this PhD Thesis to elucidate the possible contribution of re-replication in gene amplifications or rearrangements commonly seen in human tumours. I show that following geminin loss, cells complete an apparently normal S-phase, but a proportion arrests at the G2/M boundary. When Cdt1 starts to accumulate in these cells, DNA re-replicates, suggesting that the key role of geminin is to prevent re-licensing in G2. Inhibition of cell cycle checkpoints in cells lacking geminin promotes progression through mitosis without detectable levels of re-replication. Checkpoint kinases thereby amplify re-replication into an all-or-nothing response by delaying geminin depleted cells in G2 phase. Comparative Genomic Hybridisation (CGH) array and Solexa Deep DNA sequencing revealed that re-replication after geminin depletion does not appear at preferential genomic regions within the human genome. This is consistent with a recent observation that G2 cells have lost their replication timing information and reduplicate their genome stochastically. In contrast, when Cdt1 is stabilised by the neddylation inhibitor MLN4924, re-replication starts directly from within S-phase raising the question whether alternative mechanisms of may cause distinct genomic consequences.

Resumo: O mapeamento cromossômico de seqüências repetitivas de DNA tem se mostrado uma eficiente ferramenta nos estudos comparativos e evolutivos em diversos organismos. Estudos cromossômicos com besouros da subfamília Scarabaeinae têm revelado ampla variabilidade, entretanto a análise da organização cromossômica de DNAs repetitivos neste grupo é escassa e direcionada unicamente ao mapeamento do DNA ribossomal (DNAr) 18S. O presente trabalho teve como objetivo caracterizar cromossomicamente DNAs repetitivos em espécies de Scarabaeinae, utilizando bandeamentos cromossômicos e mapeamento físico cromossômico de seqüências repetitivas, incluindo famílias multigênicas de RNAr 18S, RNAr 5S e histona H3 e a fração de DNA C0t-1. Ampla variabilidade foi observada relacionada ao número/localização dos sítios de DNAr 18S, aparentemente associada a diversificação da heterocromatina. Por outro lado, os genes de RNAr 5S e histona H3, mostraram-se amplamente conservados e co-localizados em um par cromossômico, com aparente intercalação. Análises em representantes de Dichotomius revelaram conservação dos blocos de heterocromatina, entretanto com aparente compartimentalização dos mesmos. O uso da fração DNA C0t-1 confirmou o enriquecimento em DNAs repetitivos da heterocromatina, que se apresentou diversificada entre as espécies, utilizando como referência D. geminatus. Por outro lado, regiões terminais dos cromossomos apresentaram-se amplamente conservadas entre as seis espécies. Além disso, a análise da fração de DNAs repetitivos em D. geminatus indicou origem intraespecífica do cromossomo B desta espécie que possivelmente pode estar sofrendo homogeneização com seqüências encontradas no complemento A. Os resultados indicam distintos padrões de diversificação para o DNA repetitivo nos representantes de Scarabaeinae, sugerindo extensiva reorganizaçãomicrogenômica ao longo
Abstract: The chromosomal mapping of repeated DNAs has been used as an efficient tool in comparative and evolutionary studies in some organism. The chromosomal studies in beetles belonging to the subfamily Scarabaeinae have revealed wide variability, although the analysis of chromosomal organization of repeated DNAs in this group is scarce and directed solely for 18S rDNA mapping. The present study aimed in chromosomal characterization of repeated DNAs in Scarabaeinae species using chromosomal banding and physical chromosome mapping of repeated sequences, including the multigene families for 18S and 5S rRNAs and H3 histone genes and the C0t-1 DNA fraction. Wide variability was observed concerning the number and location of 18S rDNA sites, apparently associated to the heterochromatin diversification. On the other hand, the 5S rRNA and H3 histone genes were widely conserved and co-located in one chromosomal pair, showing apparently interspersion. Analysis in Dichotomius representatives revealed conservation for heterochromatic blocks, although an apparent compartmentalization was observed. The use of C0t-1 DNA fraction confirmed the heterochromatin repeated DNAs enrichment, which is diversified among the species, using as reference D. geminatus. On the other hand, the terminal regions of the chromosomes were highly conserved among the six species. Moreover, the analysis of repeated DNA fraction from D. geminatus indicated intraspecific origin of a B chromosome in this species that possibly could be suffering homogenization with A complement sequences. The results indicate distinct diversification patterns for repeated DNAs in Scarabaeinae representatives, suggesting extensive microgenomic reorganization along the cladogenesis of the group
Orientador: Cesar Martins
Coorientador: Rita de Cássia de Moura
Banca: Marcelo dos Santos Guerra
Banca: Orlando Moreira Filho
Banca: Sanae Kasahara
Banca: Maria Cristina de Almeida
Doutor

Resumo: Devido às crescentes expansões demográficas e industriais observadas nas últimas décadas, o meio ambiente tem recebido uma carga significativamente crescente de efluentes domésticos, industriais e agrícolas, causando impactos severos nos ecossistemas e um potencial comprometimento à saúde humana. Dentre os efluentes domésticos, podemos citar uma gama de poluentes, tais como químicos de diversas categorias, além de contaminações por agentes biológicos diversos. Já os efluentes industriais contêm poluentes orgânicos e/ou inorgânicos, dependendo da atividade industrial. Baseando-se nestes dados, este trabalho teve como objetivo investigar, por meio de ensaios biológicos com dois organismos-teste, a possível presença de contaminantes com potencial citotóxico, genotóxico e mutagênico, que são despejados ao longo do rio Preto, inclusive na Represa Municipal de São José do Rio Preto. O material biológico utilizado neste estudo constituiu-se de sementes de Allium cepa (cebola) e peixes da espécie Oreochromis niloticus (Tilápia). Coletas de águas foram realizadas, sazonalmente, nos meses de agosto de 2006 e 2007 (estação seca) e março de 2007 e 2008 (estação chuvosa), em seis pontos distintos: Ponto 1 (P1), 8 km antes do represamento; Ponto 2 (P2), 1 km antes do represamento; Ponto 3 (P3), local de despejo do esgoto; Ponto 4 (P4), margem oposta do despejo do esgoto; Ponto 5 (P5), saída do represamento; Ponto 6 (P6), 1 km após o represamento. Análises químicas foram realizadas para todas as coletas realizadas. Para a realização do estudo, 100 sementes de Allium cepa foram submetidas à germinação, em placa de Petri, em amostras de águas coletadas nos seis diferentes pontos do rio Preto, em água ultra pura (controle negativo) e em uma substância reconhecidamente aneugênica (Trifluralina - controle positivo), sempre à temperatura ambiente... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Due to increasing population and industrial expansion observed in recent decades, the environment has received a significant increased burden of domestic industrial and agricultural sewerage, which can cause severe impacts on ecosystems, and a potential damage to human health as well. A wide range of harmful pollutants can be found in domestic effluent, such as chemicals from various categories, in addition to contamination by various biological agents. On the other hand, industrial effluents contain organic and / or inorganic pollutants, depending on industrial activity. Based on these data, this study aimed to investigate, by means of biological tests with two test-organism, the possible presence of contaminants with cytotoxic, genotoxic and mutagenic potential, which are dumped along the Preto river, an important river that flows in the region of Sao Jose do Rio Preto/SP. The biological material used in this study consisted of seeds of Allium cepa (onion) and one specie of fish (Tilapia: Oreochromis niloticus). Water samples were taken seasonally in August 2006 and 2007 (dry season) and March 2007, and 2008 (rainy season), in six distinct sites: Site 1 (S1), 8 km before the damming, Site 2 (S2), 1 km before the damming, Site 3 (S3), place of sewerage discharge; Site 4 (S4), opposite margin of sewage discharge, Site 5 (S5), end of the damming; Site 6 (S6) 1 km after damming. Chemical analyses were performed for all collected samples. For the study, 100 seeds of A. cepa were submitted to germination in Petri dishes with samples water from six different sites of the Preto river, Ultra pure water (negative control), and with an aneugenic substance (Trifluralin - positive control). For most of collection points and periods studied, root meristems cells of A. cepa, exposed to water samples collected along the Preto river, showed no significant differences... (Complete abstract click electronic access below)
Orientador: Maria Aparecida Marin-Morales
Coorientador: Maria Tecília Vilela de Azeredo-Oliveira
Banca: Regina Teresa Rosim Monteiro
Banca: Carmem Silvia Fontanetti Christofoletti
Banca: Eduardo Alves de Almeida
Banca: Mary Massumi Itoyama
Doutor

Preservation of genetic integrity during mitotic chromosome segregation requires the attachment of sister chromatids to microtubules emanating from opposite poles of the mitotic spindle. Successful mitosis and cell cycle progression critically depend on the achievement of this attachment state, otherwise called amphitelic attachment or biorientation. Cells therefore operate an error‐correcting system at the kinetochore, which is the main microtubule attachment site on chromosomes. This system selectively removes incorrect attachments while leaving amphitelic attachments unaltered. Error correction of microtubule attachments heavily depends on integration of kinase‐phosphatase signalling. The data presented in this thesis suggest that one mechanism of fine‐tuning this phospho‐regulation at the kinetochore is the regulation of protein phosphatase 2A (PP2A) by the small kinetochore protein Biorientation defective 1 (Bod1).

Mammalian nuclear function depends on the complex interaction of genetic and epi-genetic elements coordinated in space and time. Structure and function overlap to such a degree that they are usually considered as being inextricably linked. In this work I combine an experimental approach with a computational one in order to answer two main questions in the field of mammalian chromosome organization. In the first section of this thesis, I attempted to answer the question, to what extent does chromatin from different chromosome territories share the same space inside the nucleus? This is a relatively open question in the field of chromosome territories. It is well-known and accepted that interphase chromosomes are spatially constrained inside the nucleus and that they occupy their own territory, however, the degree of spatial interaction between neighbouring chromosomes is still under debate. Using labelling methods that directly incorporate halogenated DNA precursors into newly replicated DNA without the need for immuno-detection or in situ hybridization, we show that neighbouring chromosome territories colocalise at very low levels. We also found that the native structure of DNA foci is partially responsible for constraining the interaction of chromosome territories as disruption of the innate architecture of DNA foci by treatment with TSA resulted in increased colocalisation signal between adjacent chromosomes territories. The second major question I attempted to answer concerned the correlation between nuclear function and the banding pattern observed in human mitotic chromosomes. Human mitotic chromosomes display characteristic patterns of light and dark bands when visualized under the light microscope using specific chemical dyes such as Giemsa. Despite the long standing use of the Giemsa banding pattern in human genetics for identifying chromosome abnormalities and mapping genes, little is known about the molecular mechanisms that generate the Giemsa banding pattern or its biological relevance. The recent availability of many genetic and epigenetic features mapped to the human genome permit a high-resolution investigation of the molecular correlates of Giemsa banding. Here I investigate the relationship of more than 50 genomic and epigenomic features with light (R) and dark (G) bands. My results confirm many classical results, such as the low gene density of the most darkly staining G bands and their late replication time, using genome-wide data. Surprisingly, I found that for virtually all features investigated, R bands show intermediate properties between the lightest and darkest G bands, suggesting that many R bands contain G-like sequences within them. To identify R bands that show properties of G bands, I employed an unsupervised learning approach to classify R bands on their genomic and epigenomic properties and show that the smallest R bands show a tendency to have characteristics typical of G bands. I revisit the evidence supporting the boundaries of G and R bands in the current cytogenomic map and conclude that inaccurate placement of weakly supported band boundaries can explain the intermediate pattern of R bands. Finally, I propose an approach based on aggregating data from multiple genomic and epigenomic features to improve the positioning of band boundaries in the human cytogenomic map. My results suggest that contiguous domains showing a high degree of uniformity in the ratio of heterochromatin and euchromatin sub-domains define the Giemsa banding pattern in human chromosomes.

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

The integrity of the genome relies on the maintenance of chromosomes, the structural embodiment of the genetic material. Disruption of chromosome replication can lead to extensive genomic rearrangements, spanning kilobase (Kb) to megabase (Mb) regions. Some chromosome rearrangements are inherently dynamic, beginning as a single unstable rearrangement from which multiple rearrangements emerge. The rare formation and transient behavior of unstable chromosomes renders their study challenging. Here I characterize the genetic ontogeny of unstable chromosomes in a budding yeast model, from initial replication error to unstable chromosome formation to their resolution. I find that the initial error often arises in or near the telomere and frequently forms unstable chromosomes that later resolve to an internal "collection site" in the middle of the chromosome. The initial telomere-proximal unstable chromosome is increased in cells mutant for telomerase, the Tel1 checkpoint kinase and even the Rad9 checkpoint protein, with no known telomere-specific function. Defects in Tel1 and the Rrm3 DNA helicase, or the Tel1-MRX complex and 9-1-1 checkpoint clamp, synergize dramatically to generate unstable chromosomes, further illustrating the consequence of replication error in the telomere. I performed a candidate genetic screen of instability in telomere maintenance and DNA damage response (DDR) proteins to characterize the interplay of pathways regulating senescence and genomic instability. Collectively, my results suggest that unstable chromosomes form in or near damaged telomeres, independently of end degradation (Exo1-independent), by either nonhomologous end joining (partially Lig4-dependent) or by faulty template switch during replication (Lig4- and Rad52-independent). The telomere-proximal unstable chromosomes then rearrange further to the middle of the chromosome. These results implicate telomere replication errors as a common source of widespread genomic changes and make substantial progress to our understanding of the initiation and fate of unstable chromosomes in the eukaryotic genome.

Fetal haemoglobin levels have a major impact on the severity of sickle cell disease (SCD) and Beta-Thalassaemia. Previous studies by our group shows that three loci – the HBS1L-MYB intergenic region (HMIP) on chromosome 6q23, BCL11A on chromosome 2p16 and the β globin cluster on chromosome 11 accounts for up to 50 % of the variation in HbF levels in non-anaemic Europeans. Recent work by our group indicated the presence of regulatory elements in HBS1L-MYB intergenic region (HMIP) evidenced by GATA-1 binding coinciding with strong histone acetylation, RNA polymerase II activity, and erythroid specific DNase I hypersensitive sites. Genetic studies and resequencing of two key individuals from the family that led to discovery of 6q QTL revealed new SNPs in the HBS1L-MYB intergenic region. In silico predictions suggested that a couple of these SNPs altered binding of key transcription factors. We proposed that differential binding of these transcription factors mediated through these SNPs underlie the association with HbF control. Electrophoretic Mobility Shift Assays (EMSAs) and Chromatin immunoprecipitation followed by real time PCR (ChIP-qPCR) and HaploChIP showed that many of the genetically associated variants altered the binding of key transcription factors such as GATA-1, KLF1 and MYB that have crucial roles in globin regulation as well as regulation of erythropoiesis. We have also compared ex-vivo erythroid cell culture kinetics and expression profiles of individuals who are homozygous for the presence or absence of the allelic variant associated with HbF. MYB expression and maturation rate of erythroblasts were noticeably different whereas expression of HBS1L the other flanking gene was not different. We could also demonstrate unequal expression of MYB in individuals heterozygous for the HMIP variants but the direction of the effect should be further investigated. These results further support the regulatory role of HBS1L-MYB intergenic polymorphisms on HbF and erythropoietic kinetics supporting our hypothesis that the effect of the intergenic SNPs is indirect via the flanking MYB gene.

Oral cancer is one of only four major cancers whose predicted mortality rate will significantly increase in the next 15 years. Many patients have potential precursor lesions but the methods for predicting the small number at high risk of cancer do not have sufficient predictive value or are labour intensive. The aim of this project is to investigate tests capable of predicting malignant transformation through detection of chromosomal instability. Image-based DNA image-based cytometry (‘ploidy analysis’) has been used routinely in the diagnostic Oral Pathology Unit but has not been tested on lesions clinically suspected as having high risk and its predictive values are not known for targeted use. Follow up data was compiled for 252 patients with risk lesions from local databases and cancer registries and a follow up study of malignant transformation was performed. Over than half of the dysplastic lesions, which transformed into cancer were aneuploid. Real time qPCR and QuantiGene Plex DNA assays were applied to samples of oral none dysplastic and dysplastic lesions to detect alterations in gene copy number using markers identified in previous studies in the laboratory. Discrepancies between methods were found, with inconsistent results caused by normalised to different housekeeping genes; RnaseP for qPCR, TPM1 for QGPlex and TERT in both techniques. Both these techniques were found to be insufficiently reliable for clinical use. Fluorescence in situ hybridization (FISH) was applied against Cen3/TP63, Cen7/EGFR, Cen8/PTK2, Cen11/CCND1 and 20ptel/MMP9. This proved almost as effective as image based ploidy analysis but was too labour intensive and time consuming for routine use. FISH revealed previously unrecognized zones of variation within dysplastic lesions and variation in clonal structure within them. Our data show that DNA image-based ploidy analysis combined with dysplasia assessment is the most predictive technique for use in a diagnostic setting and should be considered the reference standard.

DNA encodes the genetic instructions for the development and function of organisms and hence maintaining genomic integrity is essential for the propagation of life. However, DNA molecules are under constant threat of metabolic and environmental insults resulting in DNA damages including DNA double strand breaks (DSB), which are considered as a serious threat to cell survival. The majority of these DSB are repaired by Non-homologous end joining (NHEJ). Unrepaired DSB can lead to genomic instability resulting in cell cycle arrest, apoptosis, and mutations. Thus, delineating this DNA repair process is important in understanding the molecular mechanisms of aging and malignant progression. B lymphocytes undergo physiological DNA breaks and NHEJ-mediated DNA repair during their bone marrow differentiation and peripheral class switch recombination (CSR), thus lending them as a good model system in which to delineate the DNA repair mechanisms. To determine the effect of aging on NHEJ, B lymphocytes from old mice were analyzed. The results showed compromised DNA repair in cells from old mice compared to cells from adult mice. These results suggest that NHEJ is compromised during aging and might play critical roles in the aging process and age-associated conditions. To delineate the role of a CT in regulating the immune system, transgenic mice expressing NUP98-HOXD13 (NHD13) were analyzed for B lymphocyte differentiation, peripheral development, CSR, and antibody production. The results showed impaired B cell development and antibody production, which worsened with antigenic stimulation, suggesting the role of NHD13 in immune regulation. These studies explored the possibility of altered NHEJ-mediated DNA repair as a contributing reason for aging process and age-associated conditions. Also, the results from NHD13 study suggested that a primary CT can result in impaired NHEJ and regulate immune cell development and function. Furthermore, the results pointed to the possibility that a primary CT may lead to secondary mutations through altered NHEJ. Thus, these studies shed insight into the molecular mechanisms of altered NHEJ and may help in developing preventive or therapeutic strategies against accumulation of DNA damage, aging process and secondary mutations.
Ph. D.

Schizophrenia is a poorly understood mental disorder, for which treatment options show variable efficacy and serious side effects. The aetiology of schizophrenia remains unclear, but genetics are known to make a large contribution to risk. It is expected that a third to half of the genetic variation contributing to risk is accounted for by common genetic variants. So far, large-scale genome-wide association studies (GWAS) have implicated 108 common genome-wide significant loci in susceptibility to schizophrenia. While analysis of larger cohorts will likely identify more common variants involved in susceptibility, the functional characterisation of individual risk loci is necessary to identify the risk genes and the functional effects associated with risk variation, potentially exposing novel drug targets. The functional characterisation of a risk locus, on chromosome 10q24, is presented in this thesis. As the best supported risk variants at the locus (rs11191419 and chr10_104957618_I) are noncoding, risk alleles were hypothesised to alter the regulation of one or more genes in the region. Measures of allele-specific expression were used to investigate cisregulatory effects associated with the risk variants on the primary positional candidates BORCS7, AS3MT, CNNM2, and NT5C2 in the human brain. The risk allele of rs11191419 was found to be associated with increased allelic expression of BORCS7 and AS3MT, and with decreased expression of NT5C2. The risk allele of chr10_104957618_I was associated with decreased expression of BORCS7, AS3MT, and NT5C2. A RNA-sequencing pilot study was performed to identify transcripts produced by these genes in brain tissues where cis-regulatory effects associated with risk alleles were observed. This revealed RNA expression of RefSeq transcripts of BORCS7, AS3MT, and NT5C2, as well as of novel transcripts of AS3MT and NT5C2. Using immunohistochemistry, the cytosolic 5'-nucleotidase II, produced by NT5C2, was found to co-localise with neurons, glial cells and neuropil in the adult dorsolateral prefrontal cortex (DLPFC). Knockdown of NT5C2 in neural progenitor cells was found to alter expression of genes involved in the regulation of the cytoskeleton, cellular metabolism and AMPK signalling. These results suggest neurobiological mechanisms through which genetic variation on chromosome 10q24 confers risk to schizophrenia.

Identifying the molecular mechanisms behind reproductive isolation between closely related yeast species provides avaluable understanding of their evolution. Sequence divergence and chromosomal rearrangements are the main post-zygotic barriers behind reproductive isolation within Saccharomyces „sensu strico’ species, where hybrids are readily formed but sterile upon meiosis. Saccharomyces paradoxus and Saccharomyces cariocanus have an almost identical genome in terms of sequence, and therefore provide good model systems to explore the impact of karyotypic rearrangements on reproductive isolation. According to the biological species concept they are considered two different species despite having low sequence divergence. Since the karyotypic analysis revealed that the genomic differences are restricted to four chromosomal translocations, we hypothesized that such rearrangements may be the cause of low spore viability between them. To test this expectation, we engineered two chromosomal translocations in S. paradoxus YPS138, via Cre-loxP mediated recombination event, to render those parts of genome collinear to S.cariocanus UFRJ50816. Our analysis revealed that hybrids between S. cariocanus and engineered S. paradoxus harbouring two translocations showed a significant increase in spore viability (12.7%) compared to control hybrids harbouring five translocations (3.4%) (P=0.0031and P=0.0125, respectively, Two-sample t-test). Consequently, fitness in meiosis was improved four fold by undoing two translocations. Given this result, the prediction for spore viability in complete collinear crossing would be around 50.8 %, which is still far from the value of ca. 100%, which would be expected for strains with very low sequence divergence and belonging to the same species. This indicates that other factors may contribute to meiotic fitness in these hybrids. Further investigation was carried to determine the genome structures by using the PacBio sequencing approach. Our DNA sequencing data revealed other, previously undetected, rearrangements in S. cariocanus strain: one new reciprocal translocation between chromosomes XIII and XIV and 11 inversions distributed in 6 chromosomes. The variations in meiotic viability observed in the engineered hybrids could be because of these 5 chromosomal translocations. Further experiments were also carried out to evaluate the impact of translocations on mitotic fitness and gene expression; we observed a significant drop in the mitotic fitness of engineered translocant strains under different nutritional and temperature stresses. These changes were also accompanied with alteration in genes expression throughout the genome. Our RNA- seq data revealed that many genes were up- or down- regulated because of the translocation. Several genes with altered expression in translocant strains are correlated with morphology changes when they are up- or down- regulated. Therefore, the cell morphology was evaluated under light microscopy and different abnormal cells were detected compared to the wild type. Irregular cell morphology included elongated and clumped cells. Overall, these data confirmed that chromosomal translocations were the cause of reproductive isolation between S. paradoxus and S. cariocanus and play an important role in altering the phenotype and gene expression.

With the full sequence of the human genome now available, anexciting era in biomedical research has started. The sequenceprovides information about all our genes and greatly increasesthe scope to compare genetic activities in different cells, toanalyze genetic variation between individuals and betweendifferent species and, most importantly, to investigatesystematically the whole genome in a gene-by-gene manner, andthus increase our understanding of gene function.

This thesis describes studies in which developments weremade in several areas of functional genomics. Messenger RNAlevels were analyzed by the use of an amplification procedure,in which the 3´-ends of the transcripts were selected inorder to amplify the mRNA population in an unbiased fashion. Bysonicating cDNA originating from expressed mRNA, uniformlysized representatives of the transcripts,“signaturetags”, were obtained. The mRNA levels in the original mRNApopulation correlated well with the levels in the amplifiedmaterial, as verified by microarray analysis and realtimequantitative PCR. The expressed transcripts can be identifiedusing pyrosequencing, by comparing the obtained sequenceinformation from the signature tags to information contained invarious sequence databases. In one of the articles, the use ofpyrosequencing is illustrated by efforts to find genes involvedin the disease progression of atherosclerosis.

More challenging than the study of mRNA levels is to analyzewhen, where and how proteins fulfill their wide-ranging rolesin all the various cellular processes. Proteins are morecomplex biomolecules than mRNA, each having unique properties.Current techniques for studying proteins need much improvement,and are often limited to investigations of a specific portionof the proteome. One approach for studying the whole proteomeis to systematically generate reagents with specific affinityfor the proteins encoded by the genome, one by one. Theaffinity reagents can be used as flags for their targets,providing a flag-specific detection system, so that the targetproteins can be sub-cellularly localized in the majority ofhuman tissues in an array format. One of the articles includedin the thesis presents a pilot project for large-scale affinityreagent production. The aim was to provide a sound basis forwhole proteome studies, but as a pilot study this investigationwas limited to the proteins encoded by human chromosome 21. Allputative genes on the chromosome were subjected to antibodygeneration in a systematic manner. Small, uniform, and easilyproduced representative portions of the full-length proteinswere expressed. These were denoted“Protein EpitopeSignature Tags”and were designed to be unique for theirfull-length counterparts. The antibodies were produced inrabbits and two of the articles in the thesis discuss differentapproaches for affinity purification of the antibodies toachieve the highest possible specificity towards the targets.The resulting“mono-specific”, but still“multi-epitope”, antibodies can be used for a widerange of additional biochemical studies, such as protein arrayand protein pull-out analyses.

Keywords:functional genomics, 3´-end signaturetags, pyrosequencing, amplification, PrEST, chromosome 21,polyclonal antibodies, dual expression, affinitypurification.

Caracterisation d'expressions geniques liees au chromosome x, de 6 sequences genomiques humaines liees au chromosome x; localisation du syndrome coffin-lowry par analyse de linkage et de la dysplasie ectodermique anhidrotique

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES
Schinopsis brasiliensis Engl. and Pseudobombax simplicifolium A. Robins are species widely distributed in the Brazilian semiarid. The pressure on the genetic resources from Caatinga biome, mainly medicinal and for wood, caused by subsistence or commercial use, leads to reduce genetic variability of these species. Based on that, the cytogenetic and the tissue culture can support the characterization and propagation strategies of their populations. The research goals of this study were evaluate growth regulators and culture media effects on the establishment and in vitro micropropagation for the species, and the use of fluorochromes CMA3/DAPI in the cytogenetic characterization of P. simplicifolium. For P. simplicifolium it was evaluated different DKW ingredient concentration, added or not activated charcoal and different concentration of BAP and IBA on the establishment and in vitro multiplication of the species. For S. brasiliensis, it was evaluated the micropropagation media DKW and WPM (with different concentration of BAP and IBA) and the use of activated charcoal and polyvinylpyrrolidone to reduce the oxidation during establishment of in vitro cultivation of the species. In S. brasiliensis, the addition of BAP in the micropropagation media did not promote in vitro multiplication of the species. In P. simplicifolium, the results obtained for variables studied varied depending on the regulators concentrations and among genotypes. Concentrations above 4.92 and 6.64 ?M.L-1 AIB inhibited the multiplication in vitro in both species. The double staining CMA3/DAPI allowed the visualization of 2n = 84 chromosomes and different numbers of CMA+ blocks in the genotypes of P. simplicifolium.
As esp?cies Schinopsis brasiliensis Engl. e Pseudocombax simplicifolium A. Robins encontram-se amplamente distribu?das no semi?rido brasileiro. A press?o sobre os recursos gen?ticos do bioma caatinga, principalmente, medicinal e madeireiro, seja devido ao uso para subsist?ncia ou comercial, tem levado ? perda da variabilidade gen?tica destas esp?cies. Em vista a isto, a cultura de tecidos e a citogen?tica podem auxiliar nas estrat?gias de caracteriza??o e propaga??o de suas popula??es. O objetivo deste trabalho foi avaliar o efeito de reguladores de crescimento e meios de cultura no estabelecimento e multiplica??o in vitro das duas esp?cies, bem como o uso dos fluorocromos CMA3/DAPI na caracteriza??o citogen?tica do imbiru?u. Em P. simplicifolium, avaliou-se diferentes concentra??es dos sais DKW, acrescidos ou n?o com carv?o ativado e diferentes concentra??es de BAP e AIB no estabelecimento e multiplica??o da esp?cie. Em S. brasiliensis, avaliou-se os meios DKW e WPM e o uso do carv?o ativado e da polivinilpirrolidona na redu??o da oxida??o, e o efeito de diferentes concentra??es de BAP e AIB no meio WPM no estabelecimento do cultivo in vitro da esp?cie. Em S. brasiliensis, a adi??o de BAP ao meio de cultura n?o favoreceu a multiplica??o in vitro da esp?cie. Em P. simplicifolium as respostas para as vari?veis estudadas variaram em fun??o das concentra??es dos reguladores e entre os gen?tipos da esp?cie. Concentra??es acima de 4,92 e 6,64 ?M.L-1 de AIB inibiram a multiplica??o in vitro em ambas esp?cies. A dupla colora??o CMA3/DAPI permitiu a visualiza??o de 2n= 84 cromossomos e diferentes n?meros de blocos CMA+ nos gen?tipos estudados de P. simplicifolium.

The fused in sarcoma (FUS) protein has been shown to be a significant disease protein in a subgroup of patients with frontotemporal lobar degeneration (FTLD). Nevertheless, the mechanism underlying FUS associated FTLD is only poorly understood. Recent research has identified a large hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72), reinforcing the association between C9orf72 and FTLD. Moreover, an unusual histopathological change has been observed within the granule cell layer of the cerebellum in chromosome 9p-linked frontotemporal dementia with motor neuron disease. Whether this type of cerebellar pathology is a pathological marker for chromosome 9p-linked families remains unknown. The purpose of this study was to genetically, neuropathologically and biochemically characterize FUS and C9orf72 in FTLD, and also to investigate the association between the cerebellar pathology and chromosome 9p-linked families. The genetic sequencing study searching for potential genetic factors of FUS in FTLD failed to detect any pathogenic mutations or variations. Immunohistochemical study for FUS pathology in FTLD provided strong evidence for FUS being the specific pathological protein in all forms of FTLD-FUS. Immunoblotting for FUS in FTLD detected one novel disease-associated FUS aggregate (~37 kDa) in the urea fraction of atypical FTLD with ubiquitinated inclusions (aFTLD-U) frontal cortical samples, suggesting this unique protein product might be more associated with disease than the full-length protein itself. Immunohistochemical study of C9orf72 in FTLD detected a 'synaptic' staining in CA sectors, as the most prominent histological feature identified. Immunoblotting for C9orf72 protein demonstrated no distinctive bands among different diagnostic groups, in frontal and cerebellar cortical regions. The present study also confirmed the presence of cerebellar p62 neuronal cytoplasmic inclusions (NCI) in a proportion of FTLD-TDP cases. Although most of these cases showed an autosomally dominant pattern of inheritance, not all of them shared a common C9orf72 haplotype, or mutation in C9orf72.Much work is still needed to investigate the underlying pathogenesis of FTLD-FUS. Attention should still be given to identifying possible genetic risk factors in FUS using a large series of FTLD samples and searching for other possible proteins within the FUS immunoreactive neuronal inclusions. Moreover, the target protein within the cerebellar p62 NCI remains unknown, but it is clear that it is not C9orf72 protein.

Une exposition prénatale aux radiations ionisantes est associée au développement de pathologies neurodéveloppementales liées à l’induction de dommages à l’ADN dans les cellules souches et progéniteurs neuraux (CSPN). Ainsi, la stabilité génétique des CSPN est cruciale pour le développement et l’homéostasie du cerveau. Cependant, des altérations génomiques au niveau des CSPN au cours du développement pourraient promouvoir la diversité neuronale. XLF est un composant de la voie de réparation d’ADN par NHEJ (pour Non-Homologous End-Joining). Nous avons montré une augmentation de l’instabilité des CSPN dans le cerveau embryonnaire des souris Xlf-/- qui pourrait perturber la neurogenèse au cours du développement, et ainsi être responsable d’altérations comportementales identifiées chez ces souris à l’âge adulte. A l’aide d’approches cytogénétiques, nous avons comparés la stabilité chromosomique des CSPN et des fibroblastes embryonnaires murins (MEF) exposés à un stress génotoxique aigue (irradiation γ) ou chronique (incorporation de thymidine tritiée dans l’ADN). Nos résultats démontrent que les CSPN maintiennent leur intégrité génétique de façon plus efficace que les MEF. En effet, les CSPN semblent avoir de meilleures capacités de réparation des dommages à l’ADN que les MEF, ce qui leur permet de développer une réponse adaptative à un stress génotoxique chronique. Cette réponse adaptative implique XLF et agit conjointement avec les points de contrôle du cycle cellulaire et l'apoptose pour préserver la stabilité du génome et éliminer les cellules endommagées. L’ensemble de nos résultats apporte la démonstration d’une réponse robuste aux dommages de l'ADN dans les CSPN et souligne l'importance de XLF lors du développement du cerveau
Prenatal exposure to ionizing radiation has been associated with many neurodevelopmental disorders due to the DNA damage induced in neural stem and progenitors cells (NSPC). Thus, genetic stability of NSPC is crucial for brain development and homeostasis. Nevertheless, genomic alterations occurring during development in NSPC may have a potential impact on the physiological neuronal diversity. XLF is a component of the NHEJ (Non-Homologous End-Joining) repair pathway. Here, we show that NSPC from Xlf-/- embryos exhibit increased chromosome instability, leading to premature neurogenesis and consequently neurobehavioral disorders. Using cytogenetic approaches, we compared the chromosome stability of mouse embryonic NSPC and fibroblasts (MEF) exposed to acute (γ-irradiation) or chronic (incorporation of tritiated thymidine into DNA) genotoxic stress. Our results demonstrate the higher capacity of NSPC as compared to MEF to maintain their genomic integrity. We evidenced that NSPC have more efficient DNA repair activity than MEF, allowing them to develop an adaptive response to chronic genotoxic stress. This adaptive response involves XLF and acts together with apoptosis and cell cycle checkpoints to preserve the stability of the genome and to eliminate damaged cells. Altogether, our results provide new insights into the robust DNA damage response in NSPC and highlight the importance of Xlf during brain development

Species belonging to the Saccharomyces ‘sensu stricto’ group present a good model for studying evolution due to their genetic versatility and availability of genomic data. Chromosomal rearrangements play a very important role in evolution of eukaryotes as well as prokaryotes as they can affect phenotypic characteristics and modes of speciation. In nature these rearrangements are most likely caused by highly mobile genetic elements, such as retrotransposons. Karyotypic changes can cause alterations in gene transcription causing genomic instabilities by inactivating or over-expressing particular genes. Only few studies looked at the importance of chromosomal rearrangements and their role on global and local gene expression. The aim of this PhD project is to investigate the impact of chromosomal inversions and translocations on fitness adaptation, gene expression and speciation. I first demonstrated that a single gene inversion (of DAL2) within a co-expressed gene cluster can cause an alteration of the expression of inverted gene as well as the neighbouring genes, ultimately leading to a phenotypic change. I also showed that small and large size pericentric and paracentric inversions do not always alter the gene expression and in general have no effect on growth rate. It was also shown that effect of large inversion on gene expression is not always localized within the inversion but occurs globally. Finally, it is demonstrated that chromosomal translocations can be responsible for the reproductive isolation of Saccharomyces paradoxus and Saccharomyces cariocanus.

Kratki uzastopni ponovci predstavljaju klasu mikrosatelitskih segmenata DNK, rasprostranjenih širom genoma čoveka. Građeni su od uzastopno ponavljajućih sekvenci dužine 2-6 parova nukleotida. Zahvaljujući različitom broju ponavljanja repetitivne jedinice, većina mikrosatelitskih markera pokazuje visok stepen polimorfizma dužine, koji je moguće ispitati primenom tehnike lančane reakcije polimeraze. Pored utvrđivanja spornih srodničkih odnosa, analiza X hromozom mikrosatelitskih markera može se uspešno koristiti i u oblastima kriminalistike, humane identifikacije, populaciono-genetičkim i demografskim istraživanjima i dr. Cilj istraživanja je izrada populacione studije, iz koje će se izračunati broj i frekvencija alela, struktura i frekvencija haplotipova, utvrditi vrednosti relevantnih statističkih parametara, oceniti mogućnost primene analize X-STR markera u slučajevima iz oblasti medicinske kriminalistike, humane identifikacije i veštačenja spornih srodničkih odnosa u populaciji Vojvodine. Istraživanjem je obuhvaćeno 200 odraslih, međusobno nesrodnih osoba. Izolacija DNK materijala iz krvnih mrlja vršena je Chelex metodom, a amplifikacija dobijenih uzoraka DNK metodom PCR, uz korišćenje komercijalnog Mentype® Argus X-12 PCR Amplification Kit – a. Razdvajanje i detekcija dobijenih fragmenata izvršeno je kapilarnom elektroforezom GeneScan i Genotyper programom. Statististička obrada rezultata izvršena je pomoću Arlequin i GENEPOP programa. Za vizuelizaciju interpopulacionih genetičkih odnosa, upotrebljen je program POPTREE2 i koordinatna analiza (Principal Coordinate Analysis - PCoA). Dobijeni rezultati ukazuju da se analiza ispitivanih X-STR markera može uspešno primeniti u slučajevima iz oblasti medicinske kriminalistike, humane identifikacije i veštačenja spornih srodničkih odnosa u populaciji Vojvodine, kao i da mogu poslužiti kao osnova za dalja istraživanja u  populacionogenetičkim, antropološkim, demografskim i drugim oblastima.
Short tandem repeats (STR) represent a class of microsatellites, widely spread throughout the human genome, consisting of tandemly repeated sequences of 2-6 bp. Related to variation in the number of repeat unit displayed, most of microsatellites show a high degree of length polymorphism, investigated by the PCR techniques. The aim of this research is to create a population study, which will be used to calculate allele and haplotype frequencies, determine the value of relevant statistical parameters and assess the possibility of applying X-STR markers analysis in the fields of forensics, human identification and kinship testing. The study included 200 unrelated adults. DNA isolation was performed by Chelex method and DNA amplification by PCR, using commercial Mentype Argus X-12 PCR Amplification Kit. Separation and detection of fragments was obtained by capillary electrophoresis using Gene Scanand Genotyper program. Statistical analysis of the result was performed using Arlequin and GENEPOP program. For visualization of inter population genetic distances POPTREE2 program and coordinate analysis (PCoA) was used. The results show that the analysis of X-STR markers can be successfully applied in the field of forensics, human identification and kinship testing in the population of Vojvodina, as well as to serve as a basis for further research in population genetic, anthropological, demographic and other scientific areas.

UVOD Prenatalna dijagnostika predstavlja skup metoda i postupaka čiji je cilj da potvrde ili isključe postojanje kongenitalnih anomalija ploda. Prenatalni skrining može biti ne invazivni i invazivni. Ne invazivni skrining treba da ima visku senzitivnost i da omogući adekvatnu selekciju trudnica kojima će se predložiti genetsko ispitivanje ploda iz uzoraka dobijenih invazivnim metodama prenatalne dijagnostike. Prenatalni skrining prvog trimestra trudnoće obuhvata ultrazvučni pregled debljine nuhalne translucencije i laboratorijsku analizu dva biohemijska markera od 11 do 14 nedelje trudnoće, Prenatalni skrining drugog trimestra trudnoće koji se zasniva na biohemijskom skriningu i tripl testu iako je jedini koji se primenjuje zbog niske senzitivnosti od 20% do 40%, ne može se smatrati validnim. Integrativni biohemijski test prvog i drugog trimestra imaj veću senzitivnost (od 40 do 60%) ali ni on nije dao očekivane rezultate u adekvatnoj selekciji trudnica za genetsku analizu ploda zbog visoke stope lažno pozitivnih rezultata. Drugi trimestar trudnoće omogućava sonografskim pregledima i biohemijskim analizama dopunski način a u nekim slučajevima i jedini u proceni postojanja rizika Daunovog sindroma ili nekih drugih hromozomskih aberacija ploda Zato je primena integrativnih prenatalnih ne invazivnih metoda prvog i drugog trimestra trudnoće veoma značaja u poboljšanju dijagnostičkih vrednosti prenatalnih skrining testova i ima za cilj da smanji procenat invazivnih procedura zbog mogućih komplikacija i ne potrebnih finansijskih troškova. Daunov sindrom(trizomija 21 para hromozoma) je najčešća hromozomska numerička aberacija praćena mentalnom retardacijom dece (I.Q<70. ) Deca sa Daunovim sindromom su karakterističnog fenotipskog izgleda i sa čestim kongenitalnim anomailjama koje im onemogućavaju normalan život a često su i uzrok njihove prerane smrtnosti. Kongenitalne anomalije su zastupljene kod 2% do 5% živo rođene dece, predstavljaju 25 % perinatalne smrtnosti, četvrtina su uslovljnene hromozomskim aberacijama ili naslednom osnovom, od čega 0, 2%-0, 4% su sa Daunovim sindromom. CILJEVI Ciljevi četrorogodišnjeg istraživanja su bili da se poboljša dijagnostička vrednost postojećih prenatalnih testova, da se potvrdi značaj ultrazvučnog skrininga drugog trimestra trudnoće analizom debljine vratne brazde i dužine butne kosti ploda te da se poboljša njegova senzitivnost korporativnom sonografskom analizom cefaličnog indexa, intraorbitalnog rastojanja i dužine fronto-talamične distance. MATERIJAL I METODE Ukupan broj trudnica obuhvaćen četvorogodišnjim ispitivanjem koje su ultrazvučno pregledane i kojima je savetovano genetsko ispitivanje ploda blio je 4552. Tokom Retrospektivnog dvogodišnjieg ( 2010.2011)bila je 2169 dok je prospektivnom dvogodišnjom analizom (2012, 2013)je bilo obuhvaćeno 2383 trudnica. Ispitivana grupa su bile trudnice kod kojih je genetskom analizom otkriven patološki kariotip ploda, kontrolna grupa je obuhvatila sve ostale trudnice kod je kariotip ploda bio normalan od kojih su 124 trudnice odabrane metodom slučajnog izbora. Retrospektivnom studijom ultrazvučna je pregledana dužina vratne brazde(>6mm i dužina butne kosti<0, 6 od 14 do 22 nedelje trudnoće. Analizirana je cirkulacija fetalne krvi kroz duktus venosus ploda( a talas) i postojanje nosne kosti ploda(+, -). Prospektivnom analizom je ultrazvučnim pregledom ploda dodatno analiziran cefalični index(>85%), i intraorbitalna distanca i duzina fronto-talamične distance(<80%) ploda. Korišćene su metode deskriptivne statističke analize, aritmetička sredina, standardna devijacija, najmanja i najveća vrednost kod parametrijskih obeležja dok su za ne parametrijska postojanje nosne kosti i alfa talasa duktusu venozusu korišćene druge statističke metode, a komparativnim statističkim metodama kod normalnih, patoloških i kariotipova sa Daunovim sindromom ploda. Statistička značajnost je dokazana t testom a definisana nivoom p<0, 05 i p<0, 001 odnosom kod normalnih, patoloških kariotipova i Daunovog sindroma. Multifaktorskom regresivnom logističkom analizom je urađena procena senzitvnosti prenatalnog ultrazvučnog skrininga sa ispitivanim obeležjima drugog trimestra trudnoće REZULTATI I DISKUSIJA Od ukupnog broja ultrazvučno pregledaninh trudnica 4552 kojima je savetovano genetska analiza ploda citogenetskom analizom je otkriveno 66 patoloških kariotipova 1, 49%, sa Dunovim sindromom 31 0, 68%. Deskriptivnom statističkom obradom ultrazvučno ispitivanih obeležja od 14 do 22 nedelje trudnoće, uočeno je odstupanje i potvrđen značaj starije životne dobi trudnica, debljine vratne brazde i dužine frontotalamične distance u odnosu na normalne nalaze katiotipova ploda u predikciji Daunovog sindroma.Vrednosti dužine butne kosti, cefaličnog indeksa i intraorbitalnog rastojanja nisu imala veća odstupanja u poređenju patoškokih i normalnih nalaza kariotipova.Studentovim t testom je i dokazano p<0, 001 za debljinu vratne brazde i dužinu fronto-talamične distance, dok je za stariju životnu dob trudnice potvrđeno a;0, 001. Senzitivnost prenatalnog skrininga drugog trimestra analizom debljine vratne brazde i dužine butne kosti je veća u odnosu na standardno primenjivan biohemijski skrining drugog trimestra tripl testa (senzitivnost 40%-60) sa velikom stopom lažno pozitivnih rezultata.Dokazan je značaj poboljšanja senzitivnosti prenatalnih skrining testova dopunskom analizom tri ultrazvučna parametra, dužine fronto-talamične distance, cefaličnog indeksa i intraorbitalnog rastojanja u predikciji Daunovog sindroma, ali i kod ostalih hromozomskih aberacija ploda u periodu od 14 do 22 nedelje trudnoće primenomi multifaktorske logističke regresivne analize senzitivnost preko 93% sa 7% lažno pozitivnih rezultata. Postojanje korelacije između debljine vratne brazde i dužine fronto-talamične distance ploda poboljšavai senzitivnost prenatalnih ultrazvučnog skrininga. Integrativnim pristupom ultrazvučnog i biohemijskog skrininga drugog trimesra trudnoće, tripl testa očekuje se poboljšati dijagnostičkih vrednosti prenatalnog skrininga senzitivnost ne invazivnog skrininga u predikciji Daunovog sindroma i ostalih hromozomskih aberacija ploda. ZAKLJUČCI 1Potvrđen je značaj starije životne dobi trudnice u povećanju rizika Daunovog sindoma, i ostalih hromozomskih aberacija ploda ( p<0, 001) Potvrđen je značaj zadebljanja vratne brazde ploda >6mm(p<0, 001) i skraćenja butne kosti kod Daunovog sindroma ploda od 14 do 22 nedelje trudnoće u prenatalnom otkrivanju Dunovog sindroma i ostalih hromozomskih aberacija ploda i selekciji trudnica kojima će se predložiti genetsko ispitivanje ploda.Potvrđena je hipoteza da skraćenje fronto-talamične distance poboljšava senzitivnost ultrasonografskog skrininga, jer češće postoji kod Daunovog sindroma ploda ali i ostalih numeričkih hromozomskih aberacija tipa, nego kod normalnih nalaza kariotipa ploda( p<0, 001).Komparativnom analzom ultrazvučnim pregledom fronto-talamična distance debljine vratne brazde i dužine butne kosti ploda od 14 do 22 nedelje trudnoće može se značajno poboljšati vrednost dijagnostičkih prenatalnih testova u predikciji Daunovog sindroma. Postojanje korelacija između fronto-talamične distance i debljine vratne brazde dopunjuje ultrazvučni skrining i povećava njegovu senzitivnost na preko 90%, što je multifaktorskom regresivnom logaritamskom analizom i potvrđeno. Značaj multidisciplinarnog pristupa pogotovo je izražen u predikciji Daunovog sindroma, obzirom na različite specijalnosti koje u njemu učestvuju. Cost – benefit analiza. Visoka senzitivnost ne invazivnog prenatalnog skrininga u predikciji Daunovog sindroma, smanjuje troškove za pojedince i državu jer je njihova cena i do dest puta manja od cene citogenetskih analiza, a i trudnice se ne izlažu riziku mogućih komplikacija prilikom izvođenja invazivnih metoda
INTRODUCTIONS Prenatal diagnostic procedure represent a set of methods and techniques with the aim to afirmate or eliminate the presence of Down’s syndrome and other congenital anomalies Can be non-invasive and invasive methods. Non-invasive methods (laboratory or ultrasonographic) have the aim to make possible the most valid assessment of the risk of presence of an affected fetus in the pregnancy, selected pregnancy for invasive diagnostics procedures and citogenetics analisseskariotipingfoeti. Down’s syndrome, aneuploidy with trisomy 21 chromosomal, is the most common chromosomal numerical aberration associated with mental retardation of children (IQ< 70). Children with Down’s syndrome have characteristic phenotypic appearance with high frequent congenital anomalies that preclude a normal life and are frequently the cause of their earlier death. AIM The aim of the four year long investigation was to confirm the importance of ultrasound screening by the analyses of the basic ultrasound parameters for the second trimester, the thickness of the nuchal fold and the length of the femur of the fetus in the prediction of Down’s syndrome and other chromosomal aberrations of the fetus, as well as to improve other existing ultrasonic screenings of the first and second trimester of pregnancy by ultrasonic examination and analyses of the cephalic index and intraorbital space and the length of the fronto-thalamic distance. MATERIAL AND METODS Retrospective investigation (2010. 2011) and prospective investigation (2012.2013) includes 4655 pregnant women. For all pregnant women the genetic investigation of the fetus was performed. A total of 68 were found with chromosomal aberrations, 38 with Down’s syndrome. The method of haphazard choice in retrospective study and in prospective study ultrasound markers are examined. In retrospective analyses of the nuchal fold (<6mm and the length of femur <0.6, that represent basic ultrasound screening of the second trimester and are analyzed as parametric signs of the second trimester, and are analyzed as parametric markers, and analyses of the circulation of fetal blood through ductus venosus of the fetus. In the retrospective study the length of the nuchal fold (>6mm in length, that represent a basic ultrasound screening of the second trimester, and are analyzed as parametric markers in the prediction of Down’s syndrome and other chromosomal aberrations. RESULTS AND DISCUSION Cytogenetic analyses revealed 66 (1, 49%) pathologic karyotypes and Down syndrome were present in 31 (0, 68%) cases. All pathologic karyotypes were obtained after ultrasound examinations of 4552 pregnant women. Ultrasound markers for period 14th-22nd GW were analyzed with descriptive statistical methods and importance of pregnancy in older women, thickness of nuchal fold and lengths frontal thalamic distance were proofed in case of Down syndrome. Femoral bone lengths, cephalic index and intraorbital distances were similar for both groups, normal and pathologic karyotypes. Student’s t test revealed statistical significance with p<0, 001 values for nuchal fold thickness, frontal thalamic distance and older ages.Three additional ultrasound markers (frontal thalamic distance, cephalic index, intraorbital distance) improve prediction of Down syndrome and other chromosomal aberrations between 14th and 22nd GW as well. Multifactorial logistic regressive analyses revealed 93% sensitivity with 7% false positive results. Corelation between nuchal fold thickness and frontal thalamic distance improve prenatal ultrasound screening sensitivity. Using both ultrasound and biochemical screening (triple test) is way to improve sensitivity of non invasive screening in prediction of Down syndrome and other chromosomal aberrations. CONCLUSIONS Importance of pregnant women ages and higher risk for Down syndrome and other chromosomal aberrations was proofed (p<0, 001).Importance of nuchal fold thickness above 6mm (p<0, 001) and shorter femoral bone marker in period from 14th to 22nd GW in prediction of Down syndrome and other chromosomal aberrations are proofed (p<0, 001). Hypothesses that frontal thalamic distance improve ultrasound screening sensitivity was proofed was proofed (p<0, 001) since it is significantly shorter in Down syndrome and other chromosomal aberrations in comparison with fetuses with normal karyotypes. Comparative analyses of frontothalamic distance, nuchal fold thickness and femoral bone length in period from 14th to 22nd GW can signifi cantly improve prenatal diagnostic testing in Down syndrome prediction. Correlation between frontothalamic distance and nuchal fold thickness improve ultrasound screening sensitivity on 93% that is proofed with multifactorial logistic regressive analyses. Significance of multidisciplinary approach is high in Down syndrome prediction. Cost-benefit: High sensitivity of non invasive prenatal screening in Down syndrome prediction reduces costs for families and government since it costs ten time less than cytogenetic analyses and risk with invasive procedures is avoided.

 Acorus calamus L. је вишегодишња зељаста биљка влажних подручја, чија су лековита својства одавно позната. Дрогу иђирота чини ризом који се употребљава као чај, прах, сок, гел, уље или крема. Због прекомерне експлоатације и високог степена угрожености ове дивље лековите биљне врсте у Србији је уведена забрана његовог сакупљања из природе. Да би се задовољила повећана потражња од стране индустрија које га користе, гајење иђирота се намеће као једно од најпрагматичнијих решења. За потребе утврђивање начина гајења, прво се приступило истраживању услова успевања и варирање својстава иђирота са пет локалитета природних станишта: Обедска бара, Делиблатска пешчара, Засавица, Раковац и Дубовац. Потом је заснован двогодишњи пољски оглед на ком је испитиван утицај примене основних агротехничких мера, односно густине садње и ђубрења азотом, на својства биљака. Оглед је постављен 2013. године, на подручју засеока Ћумуране у насељу Рипањ. Код биљка са природних станишта и са огледног поља праћена су следећа својства: висина биљака, дужина ризома, број и дужина бочних грана на ризому, број пупољака на ризому и бочним гранама, број нодуса на ризому и бочним гранама, проценат суве материје ризома, маса свежег и сувог ризома, однос масе свежег и сувог ризома. Код гајених биљака праћен је и принос свежег и сувог ризома. Код свих узорака иђирота испитиван је садржај и састав етарског уља из ризома. Идентификоване су, такође, најзаступљеније компоненте и утврђен је садржаја β-азарона. За утврђивање нивоа плоидије популација из Србије рађено је пребројавање хромозома. Испитивањем биљака са природних станишта, констатовано је да на већину њихових морфолошких одлика, локалитет није имао утицаја. Утицај локалитета испољен је само на: број нодуса на ризому (највећи је био у Дубовцу - 58 а најмањи на Обедској бари - 15), проценат суве материје (највећи је био у Раковцу - 50,2%, а најмањи на Делиблатској пешчари - 37,9%) и однос масе свежег и сувог ризома (највећи је на Делиблатској пешчари - 2,64, а најмањи у Раковцу - 1,90). У пољском огледу добијени су следећи резултати: висина биљака је била највећа при најмањој густини усева (35000 биљака/ha) и најмањој дози азота (60 kg/ha) дужина ризома, као и број и дужина њихових бочних грана су највећи при средњој густини садње (48000 биљака/ha) и најмањој дози азота; број пупољака на ризому има највећу вредност при најмањој густини садње и најмањој дози азота. Број пупољака прве бочне гране је био највећи на највећој густини садње (62000 биљака/ha) и при најмањој дози азота, а број нодуса на ризому није зависио од густине садње, као ни од доза ђубрења. Највећа маса свежег и сувог ризома остварена је при средњој густини садње (48000 биљака/ha) и најмањој (62000 биљака/ha) дози азота, док је проценат суве материје био највећи при највећој густини и најмањој дози азота (60 kg/ha). На однос масе свежег и сувог ризома утицај нису имали ни ђубрење ни густинасадње усева, док су приноси свежег и сувог ризома били највећи на највећој густини садње и при најмањој дози азота. Садржај етарског уља иђирота из природе није показао значајна одступања између локалитета. Садржај етарског уља ризома гајеног иђирота опадао је са порастом густине садње, док различите дозе азота нису утицале на ову особину. Максимална концентрација β-азарона у етарском уљу иђирота са природног станишта била је 17,07 % (локалитет Раковац), док је највећи садржај β-азарона код гајеног иђирота био 21,41 %. Повећана концентрација β- азарона се објашњава интензивнијим метаболизмом азота, због повећане количине из ђубрива. обијени резултати потврђују да је концентрација β-азарона у етарском уљу ризома иђирота слична резултатима из других земаља Европе. Бројањем хромозома утврђено је да иђирот припада триплоидном, европском варијетету Аcоrus cаlаmus vаr. cаlаmus.
 Acorus calamus L. je višegodišnja zeljasta biljka vlažnih područja, čija su lekovita svojstva odavno poznata. Drogu iđirota čini rizom koji se upotrebljava kao čaj, prah, sok, gel, ulje ili krema. Zbog prekomerne eksploatacije i visokog stepena ugroženosti ove divlje lekovite biljne vrste u Srbiji je uvedena zabrana njegovog sakupljanja iz prirode. Da bi se zadovoljila povećana potražnja od strane industrija koje ga koriste, gajenje iđirota se nameće kao jedno od najpragmatičnijih rešenja. Za potrebe utvrđivanje načina gajenja, prvo se pristupilo istraživanju uslova uspevanja i variranje svojstava iđirota sa pet lokaliteta prirodnih staništa: Obedska bara, Deliblatska peščara, Zasavica, Rakovac i Dubovac. Potom je zasnovan dvogodišnji poljski ogled na kom je ispitivan uticaj primene osnovnih agrotehničkih mera, odnosno gustine sadnje i đubrenja azotom, na svojstva biljaka. Ogled je postavljen 2013. godine, na području zaseoka Ćumurane u naselju Ripanj. Kod biljka sa prirodnih staništa i sa oglednog polja praćena su sledeća svojstva: visina biljaka, dužina rizoma, broj i dužina bočnih grana na rizomu, broj pupoljaka na rizomu i bočnim granama, broj nodusa na rizomu i bočnim granama, procenat suve materije rizoma, masa svežeg i suvog rizoma, odnos mase svežeg i suvog rizoma. Kod gajenih biljaka praćen je i prinos svežeg i suvog rizoma. Kod svih uzoraka iđirota ispitivan je sadržaj i sastav etarskog ulja iz rizoma. Identifikovane su, takođe, najzastupljenije komponente i utvrđen je sadržaja β-azarona. Za utvrđivanje nivoa ploidije populacija iz Srbije rađeno je prebrojavanje hromozoma. Ispitivanjem biljaka sa prirodnih staništa, konstatovano je da na većinu njihovih morfoloških odlika, lokalitet nije imao uticaja. Uticaj lokaliteta ispoljen je samo na: broj nodusa na rizomu (najveći je bio u Dubovcu - 58 a najmanji na Obedskoj bari - 15), procenat suve materije (najveći je bio u Rakovcu - 50,2%, a najmanji na Deliblatskoj peščari - 37,9%) i odnos mase svežeg i suvog rizoma (najveći je na Deliblatskoj peščari - 2,64, a najmanji u Rakovcu - 1,90). U poljskom ogledu dobijeni su sledeći rezultati: visina biljaka je bila najveća pri najmanjoj gustini useva (35000 biljaka/ha) i najmanjoj dozi azota (60 kg/ha) dužina rizoma, kao i broj i dužina njihovih bočnih grana su najveći pri srednjoj gustini sadnje (48000 biljaka/ha) i najmanjoj dozi azota; broj pupoljaka na rizomu ima najveću vrednost pri najmanjoj gustini sadnje i najmanjoj dozi azota. Broj pupoljaka prve bočne grane je bio najveći na najvećoj gustini sadnje (62000 biljaka/ha) i pri najmanjoj dozi azota, a broj nodusa na rizomu nije zavisio od gustine sadnje, kao ni od doza đubrenja. Najveća masa svežeg i suvog rizoma ostvarena je pri srednjoj gustini sadnje (48000 biljaka/ha) i najmanjoj (62000 biljaka/ha) dozi azota, dok je procenat suve materije bio najveći pri najvećoj gustini i najmanjoj dozi azota (60 kg/ha). Na odnos mase svežeg i suvog rizoma uticaj nisu imali ni đubrenje ni gustinasadnje useva, dok su prinosi svežeg i suvog rizoma bili najveći na najvećoj gustini sadnje i pri najmanjoj dozi azota. Sadržaj etarskog ulja iđirota iz prirode nije pokazao značajna odstupanja između lokaliteta. Sadržaj etarskog ulja rizoma gajenog iđirota opadao je sa porastom gustine sadnje, dok različite doze azota nisu uticale na ovu osobinu. Maksimalna koncentracija β-azarona u etarskom ulju iđirota sa prirodnog staništa bila je 17,07 % (lokalitet Rakovac), dok je najveći sadržaj β-azarona kod gajenog iđirota bio 21,41 %. Povećana koncentracija β- azarona se objašnjava intenzivnijim metabolizmom azota, zbog povećane količine iz đubriva. obijeni rezultati potvrđuju da je koncentracija β-azarona u etarskom ulju rizoma iđirota slična rezultatima iz drugih zemalja Evrope. Brojanjem hromozoma utvrđeno je da iđirot pripada triploidnom, evropskom varijetetu Acorus calamus var. calamus.
  Acorus calamus L. is a perennial herbaceous plant found in wet areas, whose medicinal properties have been long known. The drug of sweet flag is made from the rhizome which is used as tea, powder, juice, gel, oil or cream. Because of overexploitation and the high degree of endangerment of this wild medicinal plant species in Serbia collecting was banned. In order to meet the increased demand for this plant by industries that use it, cultivation of sweet flag is emerging as one of the most pragmatic solutions. For the purpose of determining the method for plant growth, the first approach was studying the growing conditions and the variation of properties of sweet flag from five natural habitats: Obedska pond, Deliblato Sands, Zasavica, Rakovac and Dubovac.After that a two-year field experiment was designed in which the effect of application of basic agrotechnical measures, i.e., different planting density and doses of nitrogen fertilization on the plants, was studied. The experiment was set up in 2013, in the area of the hamlet Ćumurana in the settlement Ripanj. In plants from natural habitats and the experiment, the following properties were measured: the height of the plants, the length of the rhizome, the number and length of lateral branches on the rhizome, the number of buds on the rhizome and lateral branches, the number of leaf scars on the rhizome and lateral branches, the percentage of dry matter of the rhizome, the mass of the fresh and the dry rhizome, the ratio of mass between the fresh and the dry rhizome. In the experiment with cultivated plants, the yield of the fresh and the dry rhizome was also measured. For all samples of sweet flag, the content and composition of the essential oil from the rhizome was measured. Also, the main components were identified and the content of the β-asarone was determined. For the purposes of identifying the ploidy, i.e., which varieties the sweet flag from Serbia belongs to, counting of chromosomes was done. By examining plants from natural habitats, it was noted that the site had no influence on the majority of morphological features of plants. The impact of the sites was manifested only in the number of leaf scars (the largest number was in Dubovac-58,0 and the smallest number was in Obedska pond-15,0), percentage of dry matter (the largest percentage was in Rakovac-50.2% and the smallest percentage was in Deliblato Sands-37,9 %) and the ratio of mass between a fresh and a dry rhizome (the largest ratio was in Deliblato Sands-2,6 and the smallest ratio was in Rakovac-2,2). In the field experiment, the following results were obtained: the height of the plants is the largest at the lowest planting density (35000 plants/ha) and lowest nitrogen dosage (60 kg/ha), the length of the rhizome, as well as number and length of lateral branches of the rhizome are largest at medium planting density (48000 plants/ha) and the lowest dosage of nitrogen (60 kg/ha), the number of buds on the rhizome has the highest value at the lowest planting density (35000 plants/ha) and the lowest dosage of nitrogen (60 kg/ha). The number of buds on the first lateral branch was largest at the largest planting density (62000 plants/ha) and the lowest dosage of nitrog (60 kg/ha) , and the number of leaf scars on the rhizome doesnot depend on the planting dosage or the dosage of fertilizing. The largest mass of the fresh and the dry rhizome is accomplished at medium planting density and the lowest dosage of nitrogen, while the percentage of dry matter was largest at the largest density and the lowest dosage of nitrogen. The fertilization and the crop density had no effect on the ratio of mass between the fresh and the dry rhizome, while the yields of the fresh and the dry rhizome were largest at the largest planting density and the lowest dosage of nitrogen. The content of essential oils of natural sweet flag showed no significant discrepancies between the sites. The content of essential oil of cultivated sweet flag rhizomes declined with the increase of planting density, while different nitrogen doses had no effect on this property. The maximum concentration of β-asarones in the essential oil of natural sweet flag was 17,07 % (Rakovac), while the largest content of β-asarones in cultivated sweet flag was 21,41 %. The larger concentration of β-asarones is explained through increased nitrogen metabolism, because of increased amounts of fertilizer. The results confirm that the concentration of β-asarone in the essential oil of sweet flag rhizomes is similar to the concentration of essential oil of sweet flag rhizomes from Europe. By counting the chromosomes it is established that sweet flag belongs to the triploid, European variety of Аcоrus cаlаmus vаr. cаlаmus.