Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-10-01T17:51:19Z No. of bitstreams: 1 475352 - Texto Completo.pdf: 2662157 bytes, checksum: f45c41ca24a0fc661c056b1146ee35a6 (MD5) / Made available in DSpace on 2015-10-01T17:51:20Z (GMT). No. of bitstreams: 1 475352 - Texto Completo.pdf: 2662157 bytes, checksum: f45c41ca24a0fc661c056b1146ee35a6 (MD5) Previous issue date: 2015-08-07 / DNA obtained from body fluids recovered from crime scenes can be used to identify the donor of the biological material but it cannot reveal the tissue source or the possible age of the donor. DNA methylation is an epigenetic modification involved in transcriptional regulation. It is known that methylation is important in cell differentiation and genomic loci are differentially methylated between tissues. Because of this, different methylation patterns between tissues and cells can provide the basis of an assay for body fluid identification. We also know that the ability to determine the age of the sample donor based on DNA would be a powerful tool for forensic investigation. Human aging is associated with epigenetic modifications such as DNA methylation. Several studies have investigated biomarkers for aging which can be used to track donor age, presenting practical implications in forensic analysis. Two genes previously found to be DNA methylation age-associated, NPTX2 and GRIA2, were tested for prediction of age in saliva and blood samples. Both age markers were hypermethylated with the increase of age. The epigenetic predicted age was calculated for both markers, with an average difference of 6.9 years between estimated and observed ages for GRIA2 marker, and an average difference of 9.2 years for NPTX2 marker. Other studies investigated biomarkers for identification of bodyfluids. The most common body fluids found at crime scenes are blood, semen and saliva. A set of epigenetic markers, cg-06379435, ZC3H12D and BCAS4, which produce unique and specific patterns of DNA methylation, can be used to identify these body fluid types. However, to ensure the efficiency of these epigenetic markers, developmental validation studies need to be performed to determine the conditions and limitations of this new tool for forensic analysis. When testing the markers for body fluid identification using different organisms, we did obtain positive results for certain primate samples, however all other tested species were negative. The lowest concentration consistently detected varied from 0.1 to 10ng, depending on the locus. The method also proved to be effective when inhibitors were present in the samples or when samples were degraded by heat. In the case of mixtures, the overall methylation values varied in a consistent and predictable manner when multiple cell types were present in the same sample. Overall, the search of age markers candidates can be an important key in tracking suspects/victims in forensic investigations, and the ability to identify the biological sample using DNA would be a powerful tool. Epigenetic markers will provide the forensic community with new and improved methods to interpret the crime scene. / O DNA obtido a partir de fluidos corporais encontrados em cenas de crime pode ser usado para identificar o doador da amostra, mas n?o revela a fonte de tecido ou a poss?vel idade do doador. A metila??o do DNA ? uma modifica??o epigen?tica envolvida na regula??o da transcri??o. Sabe-se que a metila??o ? importante na diferencia??o celular e loci gen?micos s?o diferencialmente metilados entre os tecidos. Devido a esse fato, diferentes padr?es de metila??o entre tecidos e c?lulas podem proporcionar a base de um ensaio para a identifica??o de fluido corporal. Sabe-se tamb?m que a capacidade de determinar a idade do doador de uma amostra com base no DNA seria uma ferramenta poderosa para a investiga??o forense. O envelhecimento humano ? associado com modifica??es epigen?ticas, como a metila??o do DNA. V?rios estudos investigaram biomarcadores para o envelhecimento, que podem ser usados para estimar a idade do dador, o que apresenta implica??es pr?ticas na an?lise forense. Dois genes, NPTX2 e GRIA2, previamente associados com idade e metila??o do DNA, foram testados para predi??o de idade em amostras de saliva e sangue. Ambos os marcadores mostraram-se hipermetilados com o aumento da idade. A predi??o de idade epigen?tica foi calculada para ambos os marcadores, com uma diferen?a m?dia de 6.9 anos entre as idades estimadas e observadas para o marcador GRIA2, e uma diferen?a m?dia de 9.2 anos para o marcador NPTX2. Outros estudos investigaram biomarcadores para an?lise de fluidos biol?gicos. Os fluidos corporais mais comuns encontrados em cenas de crime s?o de sangue, s?men e saliva. Um conjunto de marcadores epigen?ticos, cg-06379435, ZC3H12D e BCAS4, que produz padr?es ?nicos e espec?ficos de metila??o do DNA, pode ser usado para identificar estes tipos de fluidos corporais. No entanto, para assegurar a efic?cia desses marcadores epigen?ticos, estudos de valida??o precisam ser realizados para determinar as condi??es e as limita??es desta nova ferramenta para an?lise forense. Em rela??o ? especificidade ao testar os marcadores usando diferentes organismos, resultados positivos foram obtidos para algumas amostras de primatas, entretando os resultados para todas as outras esp?cies testadas foram negativos. Em rela??o ? sensibilidade a concentra??o mais baixa detectada variou de 0,1 a 10 ng, dependendo do locus. Esse ensaio tamb?m se mostrou eficaz quando inibidores estavam presentes nas amostras ou quando as amostras foram degradadas pelo calor. No caso de misturas, os n?veis de metila??o variaram de maneira consistente e previs?vel na presen?a de m?ltiplos tipos celulares em uma s? amostra. No geral, a pesquisa de marcadores de predi??o de idade pode ser uma chave importante na busca de suspeitos/v?timas em investiga??es forenses, assim como a capacidade para identificar uma amostra biol?gica a partir do DNA seria uma ferramenta poderosa. Marcadores epigen?ticos v?o proporcionar ? comunidade forense m?todos novos e aprimorados para interpretar a cena do crime.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2018-03-21T13:09:14Z No. of bitstreams: 1 ClovisCoutinhoDaMottaNeto_TESE.pdf: 24405489 bytes, checksum: f4b763bedf2b803da39f795bc3d80ff3 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2018-03-23T15:58:48Z (GMT) No. of bitstreams: 1 ClovisCoutinhoDaMottaNeto_TESE.pdf: 24405489 bytes, checksum: f4b763bedf2b803da39f795bc3d80ff3 (MD5) / Made available in DSpace on 2018-03-23T15:58:48Z (GMT). No. of bitstreams: 1 ClovisCoutinhoDaMottaNeto_TESE.pdf: 24405489 bytes, checksum: f4b763bedf2b803da39f795bc3d80ff3 (MD5) Previous issue date: 2017-09-29 / O Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A S?rie Percomorpha ? maior divis?o entre os vertebrados, constituindo o maior e mais derivado clado de peixes tele?steos. Dentre suas Ordens, Perciformes e Labriformes constituem modelos adequados ? investiga??o do conservadorismo e diversifica??o cromoss?mica. Em Perciformes o conservadorismo cromoss?mico ? representado por cari?tipos basais com 2n=48 acroc?ntricos, extensivamente compartilhados por uma parcela consider?vel de esp?cies. As causas e extens?o do conservadorismo cariot?pico em v?rias fam?lias desta Ordem n?o s?o inteiramente claras. Diante da diversidade de esp?cies em Labriformes, aspectos mais detalhados de sua evolu??o cariot?pica ou mesmo status taxon?mico de algumas esp?cies merecem particular aten??o. Com vistas a contribuir com novas informa??es sobre essas quest?es foram implementadas an?lises cromoss?micas convencionais (colora??o convencional com Giemsa, bandamento C e Ag-RONs, fluocrocromos base-espec?ficos) e citomoleculares (hibridiza??o in situ com sondas DNAr 18S, DNAr 5S). Oito esp?cies dos g?neros Anisotremus e Haemulon da fam?lia Haemulidae (Perciformes) foram analisadas, incluindo amostras de diferentes ?reas do Atl?ntico, como modelo de evolu??o conservativa. Em 2 esp?cies do g?nero Bodianus da fam?lia Labridae (Labriformes), foram analisados tamb?m aspectos da evolu??o de sequ?ncias repetitivas particulares (DNAr 18S, DNAr 5S, Alu e Tol2) nos cromossomos por hibridiza??o com 5 metilcitosina (5mC). Adicionalmente, foram realizadas compara??es filogen?ticas, utilizando sequ?ncias de DNA mitocondrial (COI e 16S) e nuclear (Rodopsina) para verificar o status taxon?mico da esp?cie Bodianus insularis, end?mica das ilhas Meso-Atl?nticas. Todas as esp?cies de Haemulidae apresentaram 2n=48a, s?tios Ag-RONs simples e heterocromatina centrom?rica reduzida, al?m de consider?vel compartilhamento de s?tios de DNAr 5S e 18S, confirmando a ocorr?ncia de estase cariot?pica na fam?lia. Os padr?es cariot?picos das popula??es de A. virginicus e H. chrysargyreum entre o Caribe e Atl?ntico Sul n?o revelaram varia??es cromoss?micas decorrentes da barreira dos des?gues dos rios Amazonas/Orinoco. Em Bodianus, as an?lises identificaram uma not?vel regi?o descondensada em um par cromoss?mico subteloc?ntrico, denominada regi?o BOD. Entre suas caracter?sticas constitutivas e funcionais particulares, se mostram DAPI-, argentof?lica (Ag+), marcantemente hipometilada e saturada de elementos transpon?veis, sugerindo que a participa??o destes elementos m?veis pode ter contribu?do para sua g?nese e din?mica epigen?tica complexa. Quanto a esp?cie end?mica B. insularis, sua diverg?ncia gen?tica ? muito inferior ? apresentada por esp?cies diferenciadas sugerindo que embora represente um grupo geograficamente isolado, constitua uma sinon?mia de B. pulchellus. A diverg?ncia nos ritmos de diversifica??o cariot?pica entre Haemulidae e Labridae ? aqui discutida ? luz de caracter?sticas cariot?picas intr?nsecas que podem favorecer o tamponamento e a fixa??o de mudan?as cromoss?micas e aspectos biol?gicos das esp?cies que contribuem para as condi??es particulares de evolu??o cariot?pica desses dois grupos de peixes marinhos. / Percomorpha series is the largest division among vertebrates, constituting the largest and most derived clade of teleostean fishes. Among its Orders, Perciformes and Labriformes constitue adequated models for the investigation of conservatism and chromosomal diversification. In Perciformes the chromosomal conservatism is represented by basal karyotypes with 2n = 48 acrocentric, extensively shared by a considerable portion of species. The causes and extent of the karyotypic conservatism in various families of this Order are not entirely clear. In front of the diversity of species in Labriformes, more detailed aspects of its karyotype evolution or even the taxonomic status of some species deserve particular attention. In order to contribute with new information on these issues, it was implemented conventional chromosome analysis (conventional staining with Giemsa, C-banding and Ag- NORs, base-specific fluochromes) and cytomolecular (in situ hybridization with 18S rDNA, 5S rDNA) probes were implemented. Eight species of the genera Anisotremus and Haemulon of the family Haemulidae (Perciformes) were analyzed, including samples from different areas of the Atlantic, as a model of conservative evolution. In 2 species of the Bodianus genus Labridae (Labriformes), it was also analyzed aspects of the evolution of particular repetitive sequences (DNAr 18S, DNAr 5S, Alu and Tol2) in the chromosomes by hybridization with 5 methylcytosine (5mC). Additionally, phylogenetic comparisons were performed using mitochondrial (COI and 16S) and nuclear (Rhodopsin) DNA sequences to verify the taxonomic status of the Bodianus insularis species, endemic to the Meso-Atlantic islands. All Haemulidae species presented 2n = 48a, single Ag-NORs sites and reduced centromeric heterochromatin, besides considerable sharing of rDNA 5S and 18S sites, confirming the occurrence of karyotype stasis in the family. The karyotypic patterns of the populations of A. virginicus and H. chrysargyreum between the Caribbean and the South Atlantic did not reveal chromosomal variations due to the barrier of the discharges of the Amazonas / Orinoco rivers. In Bodianus, the analyzes identified a remarkable decondensed region in a subtelocentric chromosomal pair, denominated BOD region. Among its particular constitutive and functional characteristics, it is DAPI-, Argentof?lica (Ag +), markedly hypomethylated and saturated with transposable elements, suggesting that the participation of these mobile elements may have contributed to its genesis and complex epigenetic dynamics. In relation to the endemic species B. insularis its genetic divergence is much inferior to that presented by differentiated species suggesting that although it represents a geographically isolated group, it constitutes a synonym of B. pulchellus. The divergence in the rates of karyotype diversification between Haemulidae and Labridae is discussed here in the light of intrinsic karyotype characteristics that may favor the buffering and the fixation of chromosomal changes and biological aspects of the species that contributes to particular conditions of karyotype evolution of these two groups of marine fish.