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

No description available.

B lymphocytes produce a wide array of antibodies to recognize a countless number of antigens. This highly diverse repertoire is produced during B cell development in the bone marrow from the immunoglobulin heavy chain (Igh) and light chain (Igk and Igl) loci. The mouse Igh is a large (~3Mb) multigene locus that contains 195 variable (V), 10 diversity (D) and 4 joining (J) genes that undergo developmentally regulated V(D)J recombination to produce the variable region of the antibody. Gene expression depends on spatial organisation of chromatin. To ensure that all V genes have a chance to recombine, they are brought into physical proximity to the D-J region by locus contraction and DNA looping. Not all V genes recombine with equal frequencies and we aim to investigate how dynamic changes in 3D structure of the Igh locus facilitate V(D)J recombination. Chromosome conformation capture techniques have revolutionised studies of genome conformation. I have applied a novel form of enriched Hi-C to study both intra-locus (cis) and genome-wide (trans) interactions of the immunoglobulin loci in pro-B and pre-B cells. This method provides a higher resolution than Hi-C and is less biased than 4C and 5C. I have mapped all cis interactions within the Igh locus to produce a comprehensive view of the structure of the locus prior to recombination. This approach has shown that the 3’ superanchor (3’CBEs) and the Intergenic Control Region 1 (IGCR1) containing CTCF sites are the two most interacting regions in the locus making long-range contacts with all V genes. A second major conformational feature is that the distal V genes form a large tightly looped domain forming the centre of mass of the locus to which the 3’CBEs and IGCR1 loop. Thanks to a collaboration on polymer modelling, 5000 single conformations were simulated based on the ensemble Hi-C data. This showed that every structure is different, supporting a model of dynamic and flexible organisation of the locus rather than hierarchical subdomains therein. Moreover, there is only a slight trend for V genes interacting more often with the D-J region to have higher recombination scores, supporting an ‘equal opportunity for all’ model in which participation of V genes in V(D)J recombination is not constrained by linear genomic distance from the DJ region. Nevertheless, CTCF binding level does contribute to V gene recombination frequency. I have also discovered that Igh and Igk loci participate in a highly specialised network of genome-wide (trans) interactions involving genes encoding B cell-specific factors essential for activation and maintenance of B cell identity, including Pax5, Foxo1, Ebf1, and Runx1. I have validated these by 3D DNA FISH and found that at the pro-B cell stage the Igh is involved in many trans interactions, whereas Igk does not make any contacts. In contrast, Igk gains numerous trans interactions at the pre-B cell stage, many of which overlap with the interactions Igh participates in at both developmental stages. Together, these findings reveal a complex developmentally regulated orchestration of genome conformation changes that underpins B cell development.

Made available in DSpace on 2014-06-11T19:22:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-02Bitstream added on 2014-06-13T19:49:27Z : No. of bitstreams: 1 maziviero_gt_me_rcla.pdf: 2136084 bytes, checksum: f88fe503bcf92cfda47c9034c3c0d9c5 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / 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... / 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

Made available in DSpace on 2014-06-11T19:23:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-04-20Bitstream added on 2014-06-13T20:29:39Z : No. of bitstreams: 1 figueiredo_rf_me_arafcf.pdf: 495174 bytes, checksum: 7ab0836eb29809e44df113c6faa89bdc (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / 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... / 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)

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

Made available in DSpace on 2018-08-01T22:57:27Z (GMT). No. of bitstreams: 1 tese_10051_Dissertação Final Lucas Mesquita Barreto.pdf: 1926946 bytes, checksum: 21145320a1a7d898eb87b86e37b7dd01 (MD5) Previous issue date: 2016-07-18 / Previous cytogenetic studies in Dorstenia mention that the species may have 24 to 72 chromosomes, and suggested a conserved chromosome number 2n = 32 for the Neotropic species. However, some information reported in the literature are dubious or insufficient to assess the potential of cytogenetic data to the better understand of systematics and evolution issues within this genus. Here, eight species of Neotropical Dorstenia had their karyotypes characterized, and the nuclear DNA content measured. Dorstenia bahiensis, D. cayapia, D. grazielae, D. hirta and D. turnerifolia had their karyotypes characterized and the DNA nuclear content measured for the first time. Morphological plant characters and morphometric data were submitted to cluster analysis, followed by a test of group sharpness, and ordination analysis, aiming to support the discussion about the potential of cytogenetic data to infrageneric systematic of Dorstenia. The species showed chromosome number of 2n = 32, varying in chromosomes morphology. The karyotypes least asymmetric were observed in Dorstenia elata, and the more asymmetric were registered in D. bahiensis and D. bonijesu. The 2C value ranged from 3.21 picograms (pg) D. bahiensis to 5.47 pg in D. arifolia. Morphologically similar species, like D. hirta and D. turnerifolia, grouped together based on morphometric data. The sharp groups based on morphometric data correspond to species circumscribed under the sections Dorstenia, Lecania and Emygodia, previously established based on the plant morphology. Our results supports that the chromosome number 2n = 32 is possible conserved in the Neotropical species of Dorstenia, and indicate the potential of cytogenetic data to the systematics of this genus.

nÃo hà / O elevado consumo de pesticidas no Brasil e no mundo tem levado grupos de pesquisadores a relacionar essa exposiÃÃo a possÃveis danos genÃticos e agravos a saÃde do trabalhador rural. Estudos revelam que o cÃncer à considerado doenÃa genÃtica, vez que resulta do acÃmulo de mutaÃÃes em genes comprometidos com o controle da proliferaÃÃo e da diferenciaÃÃo celular ou de mutaÃÃes em genes envolvidos com os mecanismos de reparo do DNA. O objetivo dessa estudo foi realizar um biomonitoramento genÃtico em indivÃduos expostos ocupacionalmente a pesticidas avaliando a ocorrÃncias de danos cromossÃmicos, atravÃs do teste do MicronÃcleo em cÃlulas esfoliadas da mucosa bucal, teste Cometa e teste de AberraÃÃes CromossÃmicas em linfÃcitos de sangue perifÃrico. Como tambÃm alteraÃÃes hematolÃgicas e hepÃticas. A populaÃÃo estudada incluiu 32 agricultores moradores do povoado Vila Bessa, ConceiÃÃo do JacuÃpe, Bahia, expostos ocupacionalmente a pesticidas e 30 indivÃduos controle, sem historia de exposiÃÃo a pesticidas. O material para anÃlise do teste do micronÃcleo foi coletado por raspagem da mucosa bucal com escova cytobrush, confeccionado um esfregaÃo e posteriormente fixado em soluÃÃo de metanol/Ãcido acÃtico (3:1) e corados pelo mÃtodo FeÃlgen/Fast Green, as lÃminas foram analisadas em teste cego sob microscopia Ãptica em um mÃnimo de 1000 cÃlulas/indivÃduo. Para realizaÃÃo do teste Cometa e de AberraÃÃes, cromossÃmicas e das alteraÃÃes hematolÃgicas e hepÃticas foram coletadas 10 mL de sangue perifÃrico. O teste Cometa foi executado de acordo com a metodologia descrita por por Singh et al. (1988), foram contados 100 cometas por lÃmina e classificados, por anÃlise visual, dentre cinco categorias de danos (0, 1, 2, 3 e 4), e calculado o Ãndice e a FreqÃÃncia de dano. O teste de AberraÃÃes CromossÃmicas foi realizada atravÃs de culturas de linfÃcitos e obtenÃÃo de metÃfases pela interrupÃÃo da citocinese das cÃlulas. Foram analisados o hemograma, e as transaminases TGO, TGP E GGT que foram processados pelo laboratÃrio de anÃlise bioquÃmica de Escola Bahiana de Medicina e SaÃde PÃblica. NÃo houve diferenÃa significativa na ocorrÃncia de micronÃcleos entre os grupos avaliados (p = 0,163), mas alteraÃÃes nucleares indicativos de apoptose e necrose foram encontradas significantemente no grupo exposto a pesticidas (p = 0,001), indicando que uma maior injÃria celular do que simplesmente uma resposta a diferenciaÃÃo e maturaÃÃo do epitÃlio. AberraÃÃes CromossÃmicas numÃricas (3,3%) foi encontrada significantivamente para o grupo exposto (p = 0,001), foram encontrados danos ao DNA avaliados pelo teste Cometa no escore 1 (p< 0.001) no grupo exposto; alÃm do Ãndice de Dano cromossÃmico com mÃdia  SEM 4.032  0.3336 para o grupo controle e mÃdia  SEM 41.05  3.227 para o grupo exposto a pesticida (p<0,0001); e FreqÃÃncia de Dano: mÃdia  SEM grupo controle 4.081  0.3667 e mÃdia  SEM grupo exposto a pesticida 38.44  2.664, com diferenÃas significativas para o grupo exposto (p<0,0001). Os indivÃduos pesquisados estÃo expostos ao glifosato e ao paration-metÃlico, ambos tÃxicos para o organismo humano, e apresentavam-se anÃmicos (p=0,004) e com leucopenia (p < 0,001), porÃm sem alteraÃÃes nas avaliaÃÃes hepÃticas. ConcluÃmos que esses indivÃduos estÃo expostos a agentes potencialmente genotÃxicos, alÃm de apresentarem alteraÃÃes hematolÃgicas, e que a persistÃncia desse contato com os pesticidas poderà levar a desencadeamento dos fenÃmenos envolvidos na iniciaÃÃo e promoÃÃo do cÃncer. / The high consumption of pesticides in Brazil and all over the world have lead researches to relate this exposition to possible genetic and health damages in rural workers. Studies reveal that cancer is considered a genetic disease, once it results of the mutation accrual in genes involved with control of proliferation and cellular differentiation or mutations in genes involved with the DNA repair. The aim of this study was realize a genetic biomonitoring in individuals occupationally exposed to pesticides evaluating the occurrence of chromosomal damages, by the Micronucleus assay in exfoliated cells of buccal mucosa, Comet assay and Chromosome Aberration assay in peripheral blood lymphocytes. Hematologic and hepatic alterations were also evaluated. The studied population included 32 agriculturists living in the village of Vila Bessa, ConceiÃÃo do JacuÃpe, Bahia, occupationally exposed to pesticides, and 30 control individuals, with no history of pesticides exposition. For the micronucleus assay, the material was collected by scaling buccal mucosa with a cytobrush, the smear was made and then fixed in methanol/acetic acid solution (3:1) and colored by FeÃlgen/Fast Green Method, slides were analyzed in blind test by optic microscopy in a minimum of 1000 cells/individual. To realize the assay of Comet and Aberration, Chromosomal and hematologic and hepatic alterations, were collected 10 mL of peripheral blood. Comet test was made according the methodology described by Singh et al. (1988), were counted 100 comet by slides and classified, by visual analyses, into five categories of damages (0, 1, 2, 3 e 4), and then calculated the index and frequency of damage. Chromosome Aberration test was realized with a lymphocytes culture and obtaining of metaphases by interrupting the cells cytokinesis. The hemogram and the transaminases TGO, TGP and GGT were analyzed; those were processed by the biochemical analyses laboratory of Escola Bahiana de Medicina e SaÃde PÃblica. That wasnÂt statistical difference in the occurrence of micronucleus among the evaluated groups (p = 0.163), but nuclear alterations, indicative of apoptosis and necrosis, were significantly found in the pesticide exposed group (p = 0.001), indicating a major cellular injury than a simple answer to epitheliumÂs differentiation and maturation. Numeric Chromosome Aberrations (3.3%) were significantly found in the exposed group (p = 0.001), were found DNA damages evaluated by the Comet assay in score 1 (p<0.001) in the exposed group; as also index of chromosomal damage with media  SEM 4.032  0.3336 to the control group and media  41.05  3.227 to the pesticide exposed group (p<0.0001); and frequency of damage: media  SEM control group 4.081  0.3667 and media  SEM pesticide exposed group 38.44  2.664, with significant differences in the exposed group (p<0.0001). The studied individuals are exposed to glifosate and methyl parathion, both toxic to the human organism, and were anemics (p=0.004) and with leukopenia (p<0.001), however with no alterations in hepatic evaluations. We conclude that these individuals are exposed to potentially genotoxic agents, besides present hematologic alterations, and that the persistence of this contact with the pesticides can trigger to phenomenonâs involved with cancer initiation and promotion.

Centromere identity is integral for proper kinetochore formation and chromosome segregation. In most species chromosomes have a centromere at a defined locus that is propagated across generations. The histone H3 variant CENP-A acts as an epigenetic mark for centromere identity in most species studied. CENP-A is absent from the inactivated centromere on dicentric chromosomes and present at neocentromeres that form on non-centromeric sequences. Thus, the canonical centromere sequence is neither necessary nor sufficient for centromere function. Nevertheless, centromeres are generally associated with particular sequences. Understanding the organisation of centromeric sequence features will provide insight into centromere function and identity. In this study I use the fission yeast Schizosaccharomyces pombe model system to address the relationship between CENP-ACnp1 and centromeric sequence features. These analyses reveal that CENP-ACnp1 nucleosomes are highly positioned within the central domain by large asymmetric AT-rich gaps. The same sequence features underlying CENP-ACnp1 positioning are conserved in the related species S. octosporus, but are not found at neocentromeres, suggesting that they are important but non-essential for centromere function. CENP-ACnp1 over-expression leads to ectopic CENP-ACnp1 incorporation primarily at sites associated with heterochromatin, including the sites where stable neocentromeres form. Ectopic CENP-ACnp1 also occupies additional sites within the central domain that are not occupied in cells with wild-type CENP-ACnp1 levels. In wild-type cells CENP-ACnp1 occupied sites are likely also occupied by H3 nucleosomes or the CENP-T/W/S/X nucleosome-like complex in a mixed population. Several candidate proteins were investigated to determine a protein residing in the large gaps between CENP-ACnp1 nucleosomes could be identified. No proteins could be localised to the AT-rich gaps between CENP-ACnp1 nucleosomes, but the origin recognition complex in a promising candidate. The results presented in this thesis demonstrate that nucleosomes within the fission yeast centromere central domain are highly positioned by sequence features in a conserved manner. This positioning also allows for another complex, possibly the origin recognition complex, to bind to DNA. Nucleosome positioning, DNA replication, and transcription could individually and collectively influence CENP-ACnp1 assembly and centromere function. Further experiments in fission yeast will continue to provide insight into the general properties of centromere function and identity.

Abstract In tumourigenesis, genetic alterations accumulate in the genes responsible for cell growth, proliferation and DNA repair: proto-oncogenes, tumour suppressor and DNA repair genes. Inactivation of tumour suppressor gene function is commonly recognised as a deletion of one of the two alleles; LOH, loss of heterozygosity. In the present study, LOH of several chromosomal regions was studied in both breast and ovarian cancer. LOH for chromosome region 11q was examined in a large breast cancer consortium cohort (N = 988) and in a Finnish ovarian cancer cohort (N = 78), and the clinical significance of these alterations was evaluated. In breast cancer, LOH of the studied markers at 11q23.1, harbouring approximately 2 Mb of DNA, was seen to be associated with shortened cancer-specific survival. Two candidate genes, ATM (the ataxia telangiectasia disorder gene) and DDX10 (a putative RNA helicase gene) map to this chromosomal region. In ovarian cancer, LOH at 11q23.1–q24 was assigned mainly to two chromosomal regions, A and B, which are proximal and distal to 11q23.2–q23.3, respectively. Only the distal B region was seen to be associated with an aggressive disease course. Therefore, it appears that inactivation of the ATM or DDX10 genes is not crucial for determining the outcome of ovarian cancer. The CHK1 gene at 11q24, encoding a protein kinase required for DNA damage checkpoint function, is a putative target gene at the B region. On the basis of the present results, the main TSG in the studied region involved in the progression of breast cancer maps to 11q23.1 and the corresponding gene for ovarian cancer more distally to 11q23.3-q24. In addition, LOH at 3p, 6q, 8p, 11p, 16q and 17p was examined and their role in the genetic evolution of ovarian cancer was evaluated. Of the studied chromosomal regions, LOH at 17p appeared to be an early event and LOH at 16q24.3, 11q23.3/q24 and 11p appear to occur later in the progression of ovarian cancer.