京都大学 / 新制・課程博士 / 博士(工学) / 甲第23868号 / 工博第4955号 / 新制||工||1774(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 清水 芳久, 教授 高野 裕久, 准教授 松田 知成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Height is a complex physical trait that displays strong heritability. Adult height is related to length of the long bones, which is determined by growth at the epiphyseal growth plate. Longitudinal bone growth occurs via the process of endochondral ossification, where bone forms over the differentiating cartilage template at the growth plate. Estrogen plays a major role in regulating longitudinal bone growth and is responsible for inducing the pubertal growth spurt and fusion of the epiphyseal growth plate. However, the mechanism by which estrogen promotes epiphyseal fusion is poorly understood. It has been hypothesised that estrogen functions to regulate growth plate fusion by stimulating chondrocyte apoptosis, angiogenesis and bone cell invasion in the growth plate. Another theory has suggested that estrogen exposure exhausts the proliferative capacity of growth plate chondrocytes, which accelerates the process of chondrocyte senescence, leading to growth plate fusion. The overall objective of this study was to gain a greater understanding of the molecular mechanisms behind estrogen-mediated growth and height attainment by examining gene regulation in chondrocytes and the role of some of these genes in normal height inheritance. With the heritability of height so well established, the initial hypothesis was that genetic variation in candidate genes associated with longitudinal bone growth would be involved in normal adult height variation. The height-related genes FGFR3, CBFA1, ER and CBFA1 were screened for novel polymorphisms using denaturing HPLC and RFLP analysis. In total, 24 polymorphisms were identified. Two SNPs in ER (rs3757323 C>T and rs1801132 G>C) were strongly associated with adult male height and displayed an 8 cm and 9 cm height difference between homozygous genotypes, respectively. The TC haplotype of these SNPs was associated with a 6 cm decrease in height and remarkably, no homozygous carriers of the TC haplotype were identified in tall subjects. No significant associations with height were found for polymorphisms in the FGFR3, CBFA1 or VDR genes. In the epiphyseal growth plate, chondrocyte proliferation, matrix synthesis and chondrocyte hypertrophy are all major contributors to long bone growth. As estrogen plays such a significant role in both growth and final height attainment, another hypothesis of this study was that estrogen exerted its effects in the growth plate by influencing chondrocyte proliferation and mediating the expression of chondrocyte marker genes. The examination of genes regulated by estrogen in chondrocyte-like cells aimed to identify potential regulators of growth plate fusion, which may further elucidate mechanisms involved in the cessation of linear growth. While estrogen did not dramatically alter the proliferation of the SW1353 cell line, gene expression experiments identified several estrogen regulated genes. Sixteen chondrocyte marker genes were examined in response to estrogen concentrations ranging from 10-12 M to 10-8 M over varying time points. Of the genes analysed, IHH, FGFR3, collagen II and collagen X were not readily detectable and PTHrP, GHR, ER, BMP6, SOX9 and TGF1 mRNAs showed no significant response to estrogen treatments. However, the expression of MMP13, CBFA1, BCL-2 and BAX genes were significantly decreased. Interestingly, the majority of estrogen regulated genes in SW1353 cells are expressed in the hypertrophic zone of the growth plate. Estrogen is also known to regulate systemic GH secretion and local GH action. At the molecular level, estrogen functions to inhibit GH action by negatively regulating GH signalling. GH treated SW1353 cells displayed increases in MMP9 mRNA expression (4.4-fold) and MMP13 mRNA expression (64-fold) in SW1353 cells. Increases were also detected in their respective proteins. Treatment with AG490, an established JAK2 inhibitor, blocked the GH mediated stimulation of both MMP9 and MMP13 mRNA expression. The application of estrogen and GH to SW1353 cells attenuated GH-stimulated MMP13 levels, but did not affect MMP9 levels. Investigation of GH signalling revealed that SW1353 cells have high levels of activated JAK2 and exposure to GH, estrogen, AG490 and other signalling inhibitors did not affect JAK2 phosphorylation. Interestingly, AG490 treatment dramatically decreased ERK2 signalling, although GH did stimulate ERK2 phosphorylation above control levels. AG490 also decreased CBFA1 expression, a transcription factor known to activate MMP9 and MMP13. Finally, GH and estrogen treatment increased expression of SOCS3 mRNA, suggesting that SOCS3 may regulate JAK/STAT signalling in SW1353 cells. The modulation of GH-mediated MMP expression by estrogen in SW1353 cells represents a potentially novel mechanism by which estrogen may regulate longitudinal bone growth. However, further investigation is required in order to elucidate the precise mechanisms behind estrogen and GH regulation of MMP13 expression in SW1353 cells. This study has provided additional evidence that estrogen and the ER gene are major factors in the regulation of growth and the determination of adult height. Newly identified polymorphisms in the ER gene not only contribute to our understanding of the genetic basis of human height, but may also be useful in association studies examining other complex traits. This study also identified several estrogen regulated genes and indicated that estrogen modifies the expression of genes which are primarily expressed in the hypertrophic region of the epiphyseal growth plate. Furthermore, synergistic studies incorporating GH and estrogen have revealed the ability of estrogen to attenuate the effects of GH on MMP13 expression, revealing potential pathways by which estrogen may modulate growth plate fusion, longitudinal bone growth and even arthritis.

Made available in DSpace on 2015-07-22T20:49:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-01. Added 1 bitstream(s) on 2015-08-11T03:26:17Z : No. of bitstreams: 1 Publico-13210a.pdf: 2089186 bytes, checksum: 4bd9af1812c538db03b2120cffdc516f (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:17Z : No. of bitstreams: 2 Publico-13210a.pdf: 2089186 bytes, checksum: 4bd9af1812c538db03b2120cffdc516f (MD5) Publico-13210b.pdf: 2054432 bytes, checksum: c717885187f0d071e0c0fb8d4bbfe281 (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:17Z : No. of bitstreams: 3 Publico-13210a.pdf: 2089186 bytes, checksum: 4bd9af1812c538db03b2120cffdc516f (MD5) Publico-13210b.pdf: 2054432 bytes, checksum: c717885187f0d071e0c0fb8d4bbfe281 (MD5) Publico-13210c.pdf: 540730 bytes, checksum: fba8c62dcb0177a09d2fde3ef4bace9d (MD5) / A lípase hepática (HL) é uma enzima presente nos sinusoides hepáticos, responsável pela lipólise de lipoproteínas. Contém quatro sítios polimórficos: G-250A, T-710C, 763G-A, e C-514T single-nucleotide polymorphism (SNPs). O último polimorfismo é o foco do presente estudo. Os genótipos associados com o polimorfismo C-514T são CC (homozigoto normal - W), CT (heterozigoto - H) e TT (alelo homozigoto menor - M). A atividade da HL é, significativamente, diminuída nos indivíduos dos genótipos TT e CT. Um total de 58 mulheres pós-menopausas foi estudado. As participantes eram histerectomizadas e submetidas à terapia de reposição hormonal, consistindo de 0,625 mg de estrogênio equino conjugado, uma vez ao dia. Os critérios de inclusão foram: menopausa de até há três anos, resultados de exames de sangue, radiografias, citologia cérvico-vaginal e densitometria óssea normais. O DNA foi extraído a partir de células da mucosa bucal e de células de sangue periférico de todas as pacientes, utilizando-se um conjunto comercialmente disponível (GFX ® - Amersham-Pharmacia, EUA). Resultados: foram encontradas reduções estatisticamente significativas nos triglicérides (t = 2,16; n = 58, p = 0,03), mas não nos níveis de colesterol total (t = 0,14; n = 58, p = 0,89) após o tratamento. Este grupo de bons respondedores eram portadores do alelo T; os genótipos CT e TT estavam presentes com frequência significativamente maior do que no grupo de nãorespondedores (p = 0,02 ou p = 0,07, respectivamente). No entanto, nenhuma diferença significativa nos níveis de HDL-C (t = 0,94; n = 58, p = 0,35) ou LDL-C (t =- 0,83; n = 58, p = 0,41) foi encontrada nestas pacientes. Conclusões: as variações no perfil lipídico associadas ao polimorfismo C-514T são significativas e o alelo T é associado à melhor resposta à TRE. / Background: Hepatic lipase (HL), an enzyme present in the hepatic sinusoids, is responsible for the lipolysis of lipoproteins. Human HL contains four polymorphic sites: G-250A, T-710C, A-763G, and C-514T single-nucleotide polymorphism (SNPs). The last polymorphism is the focus of the current study. The genotypes associated with the C-514T polymorphism are CC (normal homozygous – W), CT (heterozygous – H), and TT (minor-allele homozygous – M). HL activity is significantly impaired in individuals of the TT and CT genotypes. A total of 58 postmenopausal women were studied. The subjects were hysterectomized women receiving hormone replacement therapy consisting of 0.625 mg of conjugated equine estrogen once a day. The inclusion criteria were menopause of up to three years and normal blood tests, radiographs, cervical-vaginal cytology, and densitometry. DNA was extracted from the buccal and blood cells of all 58 patients using a commercially available kit (GFX® - Amersham-Pharmacia, USA). Results: Statistically significant reductions in triglycerides (t=2.16; n=58; p=0.03) but not in total cholesterol (t=0.14; n=58; p=0.89) were found after treatment. This group of good responders were carriers of the T allele; the CT and TT genotypes were present significantly more frequently than in the group of non-responders (p=0.02 or p=0.07, respectively). However, no significant difference in HDL-C (t=0.94; n=58; p=0.35) or LDL-C (t=- 0.83; n=58; p=0.41) was found in these patients. Conclusions: The variation in lipid profile associated with the C-514T polymorphism is significant, and the T allele is associated with the best response to ERT. / TEDE

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2016-10-04T12:02:05Z No. of bitstreams: 1 DissVBR.pdf: 1898665 bytes, checksum: 77cd0f5baccb8a694beb9e7f230bab15 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-10-04T17:30:10Z (GMT) No. of bitstreams: 1 DissVBR.pdf: 1898665 bytes, checksum: 77cd0f5baccb8a694beb9e7f230bab15 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-10-04T17:30:20Z (GMT) No. of bitstreams: 1 DissVBR.pdf: 1898665 bytes, checksum: 77cd0f5baccb8a694beb9e7f230bab15 (MD5) / Made available in DSpace on 2016-10-04T17:47:42Z (GMT). No. of bitstreams: 1 DissVBR.pdf: 1898665 bytes, checksum: 77cd0f5baccb8a694beb9e7f230bab15 (MD5) Previous issue date: 2014-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / We studied patterns of gene expression in two closely related species of fruit flies of the genus Anastrepha (Diptera: Tephritidea), A. fraterculus and A. obliqua, with the goal of finding candidate genes related to the recent differentiation process between these species. In order to do this, we used the Next-generation sequencing (NGS) with RNA-Seq methodology in head tissues of the two species of flies at different stages of the reproductive life for both sexes. After processing and removal of low quality reads we retained over 140 million paired-end reads. These sequences were assembled into individual transcriptomes for each species and a pooled transcriptome, with 154,787 contigs, representing both species. Based on the results of the assemblies, annotation and mapping we prepared two separate manuscripts, one describing the libraries for each species and a combined analysis and a second that investigate the contigs involved with species differences and their patterns of expression. These data reveal 1991 genes with differential expression in at least one comparison among different reproductive stages. It is noteworthy that we observed twice as many genes with differential expression when contrasting males than with females. Several of these genes were associated to odour, such as Odorant Binding proteins and visgum, suggesting that behavioral changes in food sources, mating choice, or breeding and oviposition sites might be involved with species differences.We also identified two large sets of genes that are differentially expressed between the species, one being underexpressed in A. obliqua and overexpressed in A. fraterculus and the other that had a reverse pattern. We used a differentiation index of SNPs per contig () and pairwise tests of positive selection between the sequences, and analysis of the substitution amino acid type caused by SNPs to identify 7 genes there are candidates to be related to the speciation process between A. fraterculus and A. obliqua. / Investigamos os padrões de expressão gênica em tecidos cefálicos de duas espécies de moscas-das- frutas do gênero Anastrepha (Diptera: Tephritidea), A. fraterculus e A. obliqua, proximamente relacionadas, identificando SNPs com alto grau de diferenciação entre as espécies e realizando análises evolutivas com o objetivo de encontrar genes candidatos relacionados ao recente processo de separação dessas espécies. Para isso, utilizamos as novas tecnologias de sequenciamento em larga escala (NGS) com a metodologia de RNA-Seq em tecidos cefálicos das duas espécies de moscas em diferentes fases da vida reprodutiva dos dois sexos. Após processamento e retirada das sequências com baixa qualidade utilizamos mais de 140 milhões de sequências paired-end para montarmos um transcriptoma conjunto das espécies, com mais de 154 mil contigs, e outros dois separados por espécie. Estes resultados estão apresentados em dois manuscritos distintos, um primeiro que descreve as bibliotecas produzidas para as diferentes espécies e um segundo que investiga padrões de expressão e genes envolvidos na diferenciação das espécies. Estes dados revelaram 1991 genes com expressão diferencial em pelo menos uma comparação de fase de vida reprodutiva entre as espécies, sendo que encontramos duas vezes mais genes com diferença na expressão entre as espécies em machos do que em fêmeas. Diversos destes genes foram associados a genes relacionados ao olfato, como a família gênica das Obps (odorant-binding protein) e o gene visgun, o que pode indicar uma mudança comportamental na preferência por alimento, parceiros ou sítios para cópula e oviposição. Encontramos também dois conjuntos de genes que são diferencialmente expressos entre as espécies, sendo um conjunto super-expresso em A. obliqua e sub-expresso em A. fraterculus e outro conjunto com um padrão revertido. Análises de padrão de seleção nestes genes sugerem sete que apresentam indícios de seleção positiva e ao menos um SNP com altos índices de diferenciação entre as espécies.

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2016-10-10T12:36:43Z No. of bitstreams: 1 TeseGHM.pdf: 1501036 bytes, checksum: c01a6f35eb3f6b3347f3be5bfed6db93 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-10-11T14:25:20Z (GMT) No. of bitstreams: 1 TeseGHM.pdf: 1501036 bytes, checksum: c01a6f35eb3f6b3347f3be5bfed6db93 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-10-11T14:25:28Z (GMT) No. of bitstreams: 1 TeseGHM.pdf: 1501036 bytes, checksum: c01a6f35eb3f6b3347f3be5bfed6db93 (MD5) / Made available in DSpace on 2016-10-11T19:59:44Z (GMT). No. of bitstreams: 1 TeseGHM.pdf: 1501036 bytes, checksum: c01a6f35eb3f6b3347f3be5bfed6db93 (MD5) Previous issue date: 2015-01-16 / Não recebi financiamento / The analysis of DNA for the purpose of obtaining genetic profile has become essential for criminal investigation and judicial procedure. Since the origins of the techniques of human identification, nothing has been discovered with greater discriminatory power, between two randomly chosen individuals. Genetic proof has assumed great importance in the courts, however, for the operators of law this whole theme is cloaked in great mystery. The objective of this study was to obtain, and to offer to the operators of Law, a general view on the utilization of genetic profiles, and their consequences for the system, and Brazilian society. However, this powerful tool of human identification, DNA and its interconnections, has been thrust forward by very recent advances, in such volume as to alter in a significant way procedures and results, being obliged to follow the paths beaten by scientists, for a simplified form to put into order the information which has directed the forensic genetics to it's current point in time. A review of human identification was made until the discovery of DNA, its structure, evolution and development. The procedures and the various techniques were analyzed, which were being perfected for the obtaining results from the collecting of biological traces at the crime scene to the laboratory analysis, culminating with automatization by PCR and the elaboration of a genetic profile. DNA in the Forensic world, it's birth and consecration were examined. The presence of the instruments available for working the markets, bestowing greater means of discrimination, the STR tests, the future of the SNPs, the sequence of the second generation, and the obtaining of phenotypic characteristics, were duly dealt with. It was analysed, since its origin and implantation up to the current time, the working of the more significant Genetic Profile Data Banks, its technical and legal concerns, as well as the situation in Brazil, with its advances and obstacles, to become part of the forensic DNA community. Experiments in the testing of carbonized human bones were carried out, for identification in criminal procedure, with full details of the materials utilized, methodology, laboratory equipment and software for analyzing the results, ending up with a conclusive result. Following on the exploratory part was carried out through visits to the most expressive world DNA centres, located in the USA, France, the United Kingdom and Brazil, aimed at by means of interviews to discuss this topic, from technical to ethical aspects, and of the data banks to the operations of the system. Finally, in the discussion, the points observed in the centres of excellence, the results of the interviews, and the possible advantages of each system, in a totally fractioned global posture, were addressed. / A análise do DNA com a finalidade de obtenção de perfil genético tornou-se imprescindível para a investigação criminal e a persecução judicial. Desde os primórdios das técnicas de identificação humana, nada havia sido descoberto com maior poder discriminatório entre dois indivíduos escolhidos ao acaso. A prova genética assumiu grande importância nos tribunais, no entanto, para os operadores do direito, toda essa temática se reveste de grande mistério. O objetivo deste estudo foi obter, e poder oferecer aos operadores do Direito, uma visão geral sobre a utilização de perfis genéticos, e suas consequências para o sistema e a sociedade brasileira. No entanto, essa poderosa ferramenta de identificação humana, o DNA e suas interligações, vêm sendo impulsionada por recentíssimos avanços, de tal monta a alterar de forma significativa procedimentos e resultados, obrigando percorrer os caminhos trilhados pelos cientistas, para de forma simplificada ordenar as informações que nortearam a genética forense até nossos dias. Fez-se uma revisita à identificação humana, até a descoberta do DNA, sua estrutura, evolução e desdobramento. Foram analisados os procedimentos, e as várias técnicas, que foram sendo aperfeiçoadas, para obtenção de resultados desde a coleta de vestígios biológicos na cena do crime, até a análise laboratorial, culminando com a automatização pela PCR e a elaboração de um perfil genético. Examinou-se o DNA no mundo Forense, seu nascimento e consagração. A presença do instrumental à disposição para trabalhar marcadores, conferindo maior poder de discriminação, o exame dos STRs (Short Tandem Repeats), o futuro com SNPs, o sequenciamento de segunda geração e a obtenção de características fenotípicas foram devidamente abordadas. Verificou-se, desde sua origem e implantação até nossos dias, o funcionamento dos Bancos de Perfis Genéticos mais significativos, sua problemática técnica e legal, assim como a situação do Brasil, com seus avanços e entraves para fazer parte da comunidade forense do DNA. Realizou-se experimentalmente o exame de ossos humanos carbonizados, para identificação em procedimento criminal, com detalhamento dos materiais utilizados, metodologia, equipamentos laboratoriais e software para análise de resultados, finalizando com resultado conclusivo. Na parte exploratória vivencial deste trabalho, foram realizadas visitas aos mais expressivos centros mundiais de DNA, localizados no EUA, França, Reino Unido e Brasil, objetivando por meio de entrevistas discutir a temática, da técnica à ética, e dos bancos de dados à operacionalidade do sistema. Finalmente, na discussão foram abordados os pontos observados nos centros de excelência, o resultado das entrevistas, e as possíveis vantagens de cada sistema, em uma totalmente fracionada postura global.

Morphological markers are useful tools for identifying off-type (atypical plants) in seed production fields. However, environmental factors can cause changes in certain individuals phenotype, turning molecular markers into useful tools for verification of possible genotypic differences between these individuals and the variety studied. The aim of this study was to evaluate the efficiency of SNP markers in identifying off-type based on genotypic and phenotypic (false positive) discrimination in soybean field production. Ten lines developed by Syngenta Breeding Program located in Cascavel-PR, Uberlândia-MG and Lucas do Rio Verde-MT were used. Each line was planted in its adaptation environment in the summer season / 2009-10. Data was collected from 22 morphological features for each line. After properly describing each line, field increases of these ten lines were carried out in Planura-MG, in the winter season / 2010. In the growth stages R6 and R7 the plants phenotypically atypical for each line were collected and evaluated, to which were considered only characteristics controlled by complex genetics. The DNA of the collected plants was analyzed using SNP markers. Twenty SNP assays were used, where each marker represented one soybean chromosome in order to cover the entire genome. Among the plants considered phenotypic off-type using visual morphological traits analyses, only 27.64% were confirmed by molecular tool as genetically distinct from the standard population, with a confidence interval between 19.7% and 35.5%. The use of molecular tools is effective for the discrimination of off-type genotypic and phenotypic (false positive) process due to environmental factors, preventing the write-off of seed lots that may meet the high genetic standards. / Os marcadores morfológicos são ferramentas úteis no processo de identificação de off-type (plantas atípicas) em campos de produção de sementes, mas fatores ambientais podem gerar alteração no fenótipo de determinados indivíduos, sendo os marcadores moleculares ferramentas úteis no processo de validação da real diferença genotípica entre esses indivíduos e a cultivar em estudo. Objetivou-se nesse trabalho avaliar a eficiência do uso de marcadores moleculares SNP s na discriminação de off-type genotípicos e fenotípicos (falso positivos) em campos de produção de sementes de soja. Foram utilizadas dez linhagens desenvolvidas pelos programas de melhoramento genético da Syngenta Seeds Ltda., situados em Cascavel-PR, Uberlândia-MG e Lucas do Rio Verde-MT. Cada linhagem foi semeada em seu ambiente de adaptação na safra de verão do ano 2009/10. Foram então coletados dados de 22 características morfológicas para cada linhagem. Após devidamente descrita cada linhagem, na safra de inverno do ano 2010 foram implantados campos de multiplicação das dez linhagens, na cidade de Planura-MG. Nos estádios fenológicos R6 e R7 foram coletadas as plantas fenotipicamente avaliadas como atípicas para cada linhagem, para qual foram consideradas características de controle genético complexo. As plantas foram encaminhadas para o Laboratório de Genética Molecular da Syngenta, em Uberlândia-MG, onde suas sementes foram germinadas e o DNA das plântulas analisado com o uso de marcadores moleculares SNP. Foram utilizados 20 SNP assays, sendo que cada marcador representou um cromossomo da soja, visando cobrir todo o genoma. Entre as plantas consideradas off-type fenotípicos por meio da análise visual dos descritores morfológicos, apenas 27,64% foram confirmados pela ferramenta molecular como geneticamente distintos da população padrão, com um intervalo de confiança entre 19,7% e 35,5%. O uso de ferramentas moleculares é útil no processo de distinção de off-type genotípicos e fenotípicos (falso positivos) devido a fatores ambientais, evitando o descarte de lotes de sementes com alto padrão genético. / Mestre em Agronomia

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-09-09T20:17:53Z No. of bitstreams: 2 Dissertação - Daniany Rodrigues Adorno Silva - 2016.pdf: 3317454 bytes, checksum: 2a1e85ca0c09e0b89edfaf2b184ee8e7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-12T14:20:21Z (GMT) No. of bitstreams: 2 Dissertação - Daniany Rodrigues Adorno Silva - 2016.pdf: 3317454 bytes, checksum: 2a1e85ca0c09e0b89edfaf2b184ee8e7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-12T14:20:21Z (GMT). No. of bitstreams: 2 Dissertação - Daniany Rodrigues Adorno Silva - 2016.pdf: 3317454 bytes, checksum: 2a1e85ca0c09e0b89edfaf2b184ee8e7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-08-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The rice (Oryza sativa) is a staple food for the majority of the world's population. One of the main challenges for the breeding programs of this crop is the increase of the yield potential of commercial cultivars. For the development of superior lines and cultivars is necessary to identify and incorporate superior alleles in genetic breeding programs. One of the alterna-tives for the identification of useful genetic variability is the crossing involving genetically unrelated parents, as well as genotyping and phenotyping the segregating populations de-rived from these crossings, and posterior analysis of QTL (Quantitative Trait Locus). This study aimed to identify genes associated with yield of grains in rice through Genotyping by Sequencing (GBS), field experiments and a posterior analysis of QTL involving 232 RIL’s (Recombinant Inbred Lines) derived from the inter-subspecific crossing Epagri 108 (indica) x IRAT 122 (japonica) in two locations (Goianira - State of Goias and Boa Vista - State of Roraima). For the QTL analysis it was mapped 2,382 SNP markers, which identified two QTLs for yield, both located on chromosome 6, exclusively for the experiment of Goianira. The average effects of allele substitutions were 1,365.20 kg.ha-1 and 1,075.49 kg.ha-1, and the proportions of the phenotypic variance explained by the QTLs were 18% and 29%, clas-sified as QTL of large effect. All favorable alleles for yield were derived from genitor IRAT 122. One of the QTL identified to the productivity showed interaction QTL x E, which was expected due to the high significance of interactions G x E detected in the joint analysis. For the experiment of Goianira was also analyzed the trait hundred grain weight, and it were found three QTLs on chromosomes 5, 6 and 12. The average effects of the allelic substitution for the hundred grain weight ranged from 0.12 to 0.14 grams. The proportions of the pheno-typic variance explained by the QTLs ranged from 6 to 8%. Approximately 84% of the QTLs for the hundred grain weight were obtained from the parent IRAT 122. In chromosomal regions identified QTLs for grain yield are contained two genes: the LOC_Os06g16870, a transposon En/Spm, and the LOC_Os06g33320 whose function remains unknown, but whose expression was almost exclusively found in the inflorescences of rice. For the hundred grain weight, the chromosome region of the QTL located on chromosome 5 is located in a linkage block with 82 genes that co-segregate, and whose putative functions include, among others, the adjustment of tillering, pollen formation, grain filling and resistance to abiotic stress. For the QTL located on chromosome 6 it was identified the gene LOC_Os06g16160, which the function still unassigned, but whose expression is located almost exclusively in the root. On chromosome 12, the QTL containing the gene LOC_Os12g41956 expresses a protein of the galactosyl-transferase family, which participates in the synthesis of the RFO’s (Raffinose Family of Oligosaccharides), regulating the levels of reserve oligosaccharides in seeds. / O arroz (Oryza sativa) é um alimento básico para a maioria da população mundial. Um dos principais desafios para os programas de melhoramento dessa cultura é o aumento do poten-cial produtivo de cultivares comerciais. Para o desenvolvimento de linhagens e cultivares superiores é necessário que sejam incorporados alelos superiores em genitores dos progra-mas de melhoramento. Uma das alternativas para a identificação da variabilidade genética útil é a realização de cruzamentos envolvendo genitores pouco aparentados e a genotipagem e fenotipagem de populações segregantes derivadas desses cruzamentos, com uma posterior análise de QTL (locos de caracteres quantitativos). Esse trabalho teve por objetivo identificar genes associados à produtividade de grãos em arroz por meio da genotipagem por sequenci-amento (GBS), experimentos de campo e uma posterior análise de QTL envolvendo 232 RILs (linhas puras recombinantes) derivadas do cruzamento inter-subespecífico Epagri 108 (indica) x IRAT 122 (japonica) em dois locais (Goianira-GO e Boa Vista-RR). Para a análise de QTL foram mapeados 2.382 marcadores SNPs, os quais identificaram dois QTLs para produtividade, ambos localizados no cromossomo 6, exclusivamente para o experimento de Goianira. Os efeitos médios de substituição alélica foram de 1.365,20 kg.ha-1 e 1.075,49 kg.ha-1, e as proporções das variâncias fenotípicas explicadas pelos QTLs foram de 18% e 29%, classificadas como QTLs de grandes efeitos. Todos os alelos favoráveis para produti-vidade foram provenientes do genitor IRAT 122. Um dos QTLs identificados para a produ-tividade apresentou interação QTL x E, o que já era esperado devido à alta significância das interações G x E detectadas na análise de variância conjunta. Para o experimento de Goianira também foi analisado o peso de 100 grãos, e foram encontrados três QTLs, localizados nos cromossomos 5, 6 e 12. Os efeitos médios de substituição alélica para o peso de 100 grãos variaram de 0,12 a 0,14 gramas. As proporções da variância fenotípica explicadas pelos QTLs variaram de 6 a 8%. Cerca de 84% dos QTLs identificados para o peso de 100 grãos foram provenientes do genitor IRAT 122. Nas regiões cromossômicas dos QTLs identifica-dos para produtividade de grãos estão contidos dois genes: o LOC_Os06g16870, um trans-poson En/Spm, e o LOC_Os06g33320, de função ainda desconhecida, mas cuja expressão foi quase que exclusivamente identificada nas inflorescências de arroz. Para o peso de 100 grãos, a região cromossômica do QTL localizado no cromossomo 5 está contido em um bloco de ligação contendo 82 genes que co-segregam, e cujas funções putativas incluem, dentre outras, a regulação do perfilhamento, formação de pólen, enchimento de grãos e re-sistência a estresse abiótico. Para o QTL identificado no cromossomo 6, nesta região cro-mossômica está presente o gene LOC_Os06g16160, ainda sem atribuição de função, mas cuja expressão está localizada quase que exclusivamente na raiz. Já no cromossomo 12, a região cromossômica do QTL contém o gene LOC_Os12g41956, que expressa uma proteína 15 da família galactosil-transferase, que participa da síntese dos RFOs (oligossacarídeos da fa-mília das rafinoses), regulando os níveis de oligossacarídeos de reserva nas sementes

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2016-09-27T13:58:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_pedro_silveira.pdf: 618469 bytes, checksum: 2d5be3b5e8360e244f71d3c147be68af (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2016-09-28T17:04:11Z (GMT) No. of bitstreams: 2 dissertacao_pedro_silveira.pdf: 618469 bytes, checksum: 2d5be3b5e8360e244f71d3c147be68af (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-28T17:04:11Z (GMT). No. of bitstreams: 2 dissertacao_pedro_silveira.pdf: 618469 bytes, checksum: 2d5be3b5e8360e244f71d3c147be68af (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-02-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Marcadores moleculares, cada vez mais, vem sendo utilizados em programas de melhoramento genético de bovinos leiteiros, incluindo várias características produtivas e sanitárias. Por outro lado, a reduzida atividade da paraoxonase (PON1) na circulação dos animais está associada com maior ocorrência de doenças nestes indivíduos, sendo que há pouca informação quanto a influência do gene da PON1 neste processo. O objetivo deste estudo foi caracterizar a ocorrência de polimorfismos na região promotora do gene da PON1, bem como avaliar a sua relação com a atividade sérica desta proteína no periparto de vacas leiteiras da raça Holandês. Foram utilizadas 28 vacas da raça Holandês, das quais coletou-se sangue para análise da atividade sérica da PON1. Estas avaliações foram feitas nos dias -21, -7, 0, 3, 6, 9, e 23 (dia 0 = dia do parto). Foi realizada a extração de DNA dos leucócitos circulantes e a partir daí foi feito o PCR dessas amostras, visando a amplificação de um fragmento de DNA contendo 828 pb no promotor da PON1. Após a eletroforese em gel de agarose, as amostras foram purificadas e sequenciadas para identificação dos polimorfismos. Foram encontrados seis SNPs no promotor do gene da PON1, localizados nas posições -22, -105, -221, -392, -611 e -676. Destes, os SNPs -105, -221 e -611 foram correlacionados com a atividade sérica da PON1 no período avaliado (p<0,05), sendo que os genótipos PON1-105(AA), PON1-221(AA) e PON1-611(CC) apresentaram os maiores níveis de PON1 (p<0,05). Além disto, os SNPs -105 e -221 apresentaram diferenças nos níveis de PON1 durante o pré-parto(p<0,05). Em conclusão, foram identificados 6 SNPs na região do promotor do gene da PON1, dos quais três estão associados com a atividade da enzima. / Molecular markers, increasingly are being used in breeding programs of dairy cattle, including several production and health characteristics. On the other hand, the reduced activity of paraoxonase (PON1) in the circulation of animals is associated with increased occurrence of diseases in these individuals, there is little information regarding the influence of the PON1 gene in this process. The aim of this study was to characterize the occurrence of polymorphisms in the promoter region of the PON1 gene and to evaluate its relationship with serum PON1 activity in peripartum dairy Holstein cows. For that blood was collected from 28 Holstein cows for analysis of serum activity of paraoxonase (PON1). The sampling was performed on days -21, -7, 0, 3, 6, 9 and 23 (day 0 = day of calving). DNA extraction was performed in circulating leukocytes and PCR of these samples was performed, targeting the amplification of a DNA fragment containing 828 bp in the PON1 promoter. After agarose gel electrophoresis, the samples were purified and sequenced to identify the polymorphisms. Six SNPs were found in the promoter of the PON1 gene, located at positions -22, -105, -221, -392, -611 and -676. From these, SNPs -105, -221 and -611 were associated with serum PON1 activity during the study period (p < 0.05), whereas the PON1-105(AA), PON1-221(AA) and PON1 -611(CC) genotypes had higher levels of PON1 (p < 0.05). Moreover, SNPs -105 and -221 had differences in the levels of PON1 already during the prepartum period (p < 0.05). In conclusion, 6 SNPs were identified in the promoter of the PON1 gene, three of which are associated with the activity of the enzyme.

The tilapias are a group of African and Middle Eastern cichlid fish that are widely cultured in developed and developing countries. With many different species and sub-species, and extensive use of interspecies hybrids, identification of tilapia species is of importance in aquaculture and in wild populations where introductions occur. This research set out to distinguish between tilapia species and sub-species by retrieving species-specific nuclear DNA markers (SNPs) using two approaches: (i) sequencing of the coding regions of the ADA gene; and (ii) next-generation sequencing, both standard RADseq and double-digest RADseq (ddRADseq). The mitochondrial DNA (mtDNA) marker cytochrome c oxidase subunit I (COI) was used to verify tilapia species status. ADA gene sequence analysis was partially successful, generating SNP markers that distinguished some species pairs. Most species could also be discriminated using the COI sequence. Reference based analysis (RBA: using only markers found in the O. niloticus genome sequence) of standard RADseq data identified 1,613 SNPs in 1,002 shared RAD loci among seven species. De novo based analysis (DBA: based on the entire data set) identified 1,358 SNPs in 825 loci and RBA detected 938 SNPs in 571 shared RAD loci from ddRADseq among 10 species. Phylogenetic trees based on shared SNP markers indicated similar patterns to most prior phylogenies based on other characteristics. The standard RADseq detected 677 species-specific SNP markers from the entire data set (seven species), while the ddRADseq retrieved 38 (among ten species). Furthermore, 37 such SNP markers were identified from ddRADseq data from a subset of four economically important species which are often involved in hybridization in aquaculture, and larger numbers of SNP markers distinguished between species pairs in this group. In summary, these SNPs are a valuable resource in further investigating hybridization and introgression in a range of captive and wild stocks of tilapias.

No description available.