Os estudos de associação de genoma inteiro têm encontrado alguns marcadores associados a doenças notoriamente hereditárias com herança complexa, mas, muitas vezes, estes marcadores somente explicam uma pequena parte da herdabilidade. Este relativo insucesso é atribuído, entre outras causas, à epistasia, ou seja, interação entre diferentes locos genéticos. A busca por epistasia é complexa e exige intensos recursos computacionais. Diversos métodos têm sido propostos para abordar este problema, incluindo métodos estatísticos tradicionais, busca estocástica e métodos heurísticos. Poucos destes métodos são capazes de processar as grandes massas de dados produzidas nos estudos caso-controle de genoma inteiro, e ainda menos métodos buscam conjuntos de três ou mais marcadores. A busca exaustiva de conjuntos de marcadores epistáticos é inviável hoje em dia para estes conjuntos, mas o algoritmo BOOST (WAN et al., 2010) mostrou que ela é relativamente fácil para pares de locos, em especial com o uso de placas gráficas como processadores (GPGPU). Partindo deste recente sucesso, propomos um algoritmo em fases para a busca de trios de locos que interagem, utilizando a busca de pares como passo inicial, uma abordagem ainda não utilizada. Outra ideia fundamental do algoritmo proposto é a extensão da concepção de trio de marcadores para um trio de blocos haplotípicos, onde cada bloco é formado por marcadores próximos entre si. Usando os dados do WTCCC, o Troost (de TRio+bOOST) sugeriu trios potencialmente epistáticos em todas a sete doenças. Quando submetidos à confirmação em amostra independente, os trios não puderam ser confirmados, exceto os trios para diabetes tipo 1 (T1D). Duzentos e oito trios foram confirmados para T1D, com baixos valores-P e genótipos combinados de risco com altas razões de chances. Os SNPs que compõem estes trios estão todos na região MHC, sabidamente associada à doença, exceto por um deles que está no cromossomo cinco e não havia sido previamente relacionado à T1D. / Genome-wide association studies have found some markers associated with diseases with complex inheritance. However, these markers explain only a fraction of the previously estimated heritability of the trait. This relative failure has been credited, among other causes, to epistasis, i.e. the interaction among genotypes at different loci. The search for epistasis is complex and requires intense computational resources. Many methods have been proposed to approach this problem, including traditional statistics, stochastic search, and heuristic methods. Few of them are capable of extracting, from the large amount of data produced in genome-wide case-control studies, useful information about sets of markers associated with the trait in question. Exhaustive search of sets of interacting markers is unfeasible nowadays for sets of three or more markers, but the BOOST algorithm (WAN et al., 2010) showed that the search is relatively easy for pairs of SNPs, in particular with the use of graphic cards for general processing (GPGPU). Starting from this recent success, we propose an algorithm in phases for the search for trios of interacting loci, using the search for pairs as the initial step, an approach not tried yet, to our knowledge. Another important idea of our algorithm is the extension of the concept of trio of markers to a trio of haplotypic blocks, where each block is formed by neighbor markers. Using data from WTCCC, the Troost (from TRio+bOOST) algorithm suggested potentially epistatic trios in all seven diseases. When submitted to a confirmation in an independent sample, the results could not be confirmed, except for type-1 diabetes (T1D). Two hundred eight trios were confirmed for T1D, with low p-values and risk combined genotypes with high odds ratio. The SNPs that form those trios are all in the MHC region, which is known to be strongly associated to T1D, except by one SNP in chromosome five that has not been previously associated with T1D.

Découverte il y a plus d’un siècle, Salmonella n’a cessé d’intriguer les chercheurs. Sa capacité à résister à de nombreux antibiotiques est de plus en plus préoccupante. La surveillance de ce pathogène repose sur un typage rapide et discriminant de façon à identifier le plus précocement possible les sources alimentaires contaminées. Les méthodes classiques sont longues, lourdes et non automatisables. Comprendre l’émergence et l’évolution des Salmonella est la clé pour éradiquer ce pathogène resté l’une des premières causes de diarrhées bactériennes d’origine alimentaire dans le monde. Au cours des dernières décennies, des progrès spectaculaires ont été menés dans le monde de la microbiologie avec l’arrivée des séquenceurs de paillasse, passant du traitement d’une dizaine à des centaines de millions de séquences. L’accès facilité aux séquences génomiques et aux outils qui leurs sont dédiés sont devenus une nécessité. Les outils actuellement disponibles ne sont pas assez discriminants pour sous-typer S. enterica sérotype Typhimurium (STM), sérotype prédominant de Salmonella. Nous avons voulu lors de ce travail, montrer l’intérêt du séquençage entier du génome, pour l’étude génomique de Salmonella. (1) Après avoir séquencé plus de 300 génomes de STM, nous avons mis au point un outil de sous-typage in silico de ce sérotype, basé sur le polymorphisme des CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). La surveillance à haut débit des salmonelloses a été validée en routine sur plus de 800 génomes. L’étude de la coévolution entre le chromosome (SNPs) et les régions CRISPR ont permis d’établir une nomenclature définissant les différentes populations de STM. (2) L’analyse génomique de 280 souches historiques de STM a montré que les gènes de bêta-lactamase conférant une résistance à l’ampicilline et portés par des plasmides étaient répandus chez STM à la fin des années 1950, bien avant l’utilisation de cet antibiotique. La présence de la pénicilline G dans le milieu agricole où ces composés ont été utilisés en tant que promoteurs de croissance ont pu conduire à la sélection des premières souches résistantes à l’ampicilline. (3) L’étude phylogénétique d’un génome issu du cadavre d’une femme décédée il y a plus de 800 ans, probablement à cause de la fièvre entérique et de 219 génomes historiques et récents des sérotypes Paratyphi C, Choleraesuis et Typhisuis ont montré que leurs génomes étaient très similaires au cours des 4000 dernières années. Ainsi, la combinaison des approches génotypique et phylogénétique ont accru nos connaissances sur l’évolution de ce pathogène.Mots clés : Séquençage entier du génome, surveillance épidémiologique, CRISPR, SNP, résistance antibiotique, phylogénie, évolution / Over a century has passed since the discovery of Salmonella and yet, this pathogen still intrigues researchers. Its ability to withstand many antibiotics is of increasing concern. The monitoring of this pathogen is based on a rapid and discriminatory typing to identify the sources of contaminated food as early as possible. The conventional methods are long, heavy and non-automatable. Understanding the emergence and evolution of Salmonella is the key to eradicate this pathogen, which has remained one of the leading causes of foodborne bacterial diarrhea in the world. During the last decades, spectacular progress has been made in the world of microbiology with the arrival of workbench sequencers, passing from a dozen to hundreds of millions of sequences processed. Facilitated access to numerous genome sequences and dedicated tools are mandatory. Tools currently available are not sufficiently discriminating for the subtype of S. enterica serotype Typhimurium, a predominant serotype of Salmonella. Throughout this study, we showed the interest of whole genome sequencing, a multidisciplinary tool, for the genomic study of Salmonella. (1) After sequencing over 300 S. enterica serotype Typhimurium genomes, we have developed an in silico subtyping tool for this serotype, based on the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) polymorphism. High-throughput microbiological monitoring of salmonellosis has been routinely validated on over 800 genomes. The study of coevolution between the chromosome (SNPs of the core genome) and the two CRISPR regions made it possible to establish a nomenclature defining the different populations of this serotype. (2) Genomic analysis of 280 historical strains of S. enterica serotype Typhimurium showed that plasmids carrying beta-lactamase genes, which confer resistance to ampicillin, were widespread within this serotype in the late 1950s, years before ampicillin was first used for clinical purposes. The presence of penicillin G in the farming environment where these compounds were used as growth promoters, may have led to the selection of the first ampicillin-resistant strains. (3) The phylogenetic study of a genome from the corpse of a young woman who died over 800 years ago, probably due to enteric fever, and 219 historical and recent genomes of the serotypes Paratyphi C, Choleraesuis and Typhisuis have shown, despite the differences in host specificity, that their genomes were very similar over the past 4000 years. Thus, the combination of genotypic and phylogenetic approaches has increased our knowledge of the evolution of this pathogen.Key words: Whole genome sequencing, epidemiological monitoring, CRISPR, SNP, antibiotic resistance, phylogeny, evolution

O grande grupo heterogêneo de neuropatias periféricas hereditárias estão entre os casos mais comuns de perda sensitiva e fraqueza muscular em crianças e adolescentes. Pelo menos 84 genes estão envolvidos com neuropatias sensitivo-motoras hereditárias (NSMH), sendo suas formas de herança mais comuns as autossômico-dominantes desmielinizante e axonal e as neuropatias ligadas ao cromossomo X, e as mais raras as autossômicorecessivas desmielinizante e axonal e as formas ainda não classificadas. O gene HINT1, possuinte de 3 exons e localizado no cromossomo 5, codifica a proteína Histidine triad nucleotide binding protein 1, uma variante transcricional (mRNA) regulatória que hidroliza substratos. Recentemente mutações em HINT1 foram também relacionadas à neuropatias axonais com neuromiotonia (ARCMT2-NM), e portanto à CMT. O objetivo deste trabalho foi realizar o screening mutacional do gene HINT1 em uma amostra da população brasileira com quadro clínico de CMT recessivo (CMT2-AR), e foram encontradas 1 mutação silenciosa já previamente descrita, 1 polimorfismo exônico e 1 polimorfismo intrônico, também já conhecidos. Concluiu-se que mutações no gene HINT1 não são portanto responsáveis pela CMT-AR nesta amostra da população brasileira. / The large heterogeneous group of inherited peripheral neuropathies are among the most common causes of sensory loss and muscle weakness in children and adolescents. At least 84 genes are involved in inherited sensorymotor neuropathies (NSMH), being the demyelinating and axonal autosomaldominant and the X-linked neuropathies their most common forms of inheritance, and the demyelinating and axonal autosomal-recessive and not yet classified forms the most rare ones. The HINT1 gene, with 3 exons and located on chromosome 5, encodes the protein Histidine triad nucleotide binding protein 1, a regulatory transcriptional variant (mRNA) that hydrolyzes substrates. Recently, mutations in HINT1 were also related to axonal neuropathy with neuromyotonia (ARCMT2-NM), and therefore to CMT. The objective of this study was the mutational screening of the HINT1 gene in a sample of the Brazilian population with clinical recessive CMT (CMT2-AR), and 1 silent mutation previously described, 1 intronic polymorphism and 1 exonic polymorphism, both also known, were founded. It was then concluded that mutations in the HINT1 gene are not responsible for CMT2-AR in this particular sample of the Brazilian population.

Common bean is a major dietary component in several countries, but its productivity is negatively affected by abiotic stresses. Dissecting candidate genes involved in abiotic stress tolerance is a paramount step toward the improvement of common bean performance under such constraints. Thereby, this thesis presents a systematic analysis of the DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) gene subfamily, which encompasses genes that regulate several processes during stress responses, but with limited information for common bean. First, a series of in silico analyses with sequences retrieved from the P. vulgaris genome on Phytozome supported the categorization of 54 putative PvDREB genes distributed within six phylogenetic subgroups (A-1 to A-6), along the 11 chromosomes. Second, we cloned four novel PvDREB genes and determined their inducibility-factors, including the dehydration-, salinity- and cold-inducible genes PvDREB1F and PvDREB5A, and the dehydration- and cold-inducible genes PvDREB2A and PvDREB6B. Afterwards, nucleotide polymorphisms were searched through Sanger sequencing along those genes, revealing a high number of single nucleotide polymorphisms within PvDREB6B by the comparison of Mesoamerican and Andean genotypes. The nomenclature of PvDREB6B is discussed in details. Furthermore, we used the BARCBean6K_3 SNP platform to identify and genotype the closest SNP to each one of the 54 PvDREB genes. We selected PvDREB6B for a broader study encompassing a collection of wild common bean accessions of Mesoamerican origin. The population structure of the wild beans was accessed using sequence polymorphisms of PvDREB6B. The genetic clusters were partially associated with variation in latitude, altitude, precipitation and temperature throughout the areas such beans are distributed. With an emphasis on drought stress, an adapted tube-screening method in greenhouse conditions enabled the phenotyping of several drought-related traits in the wild collection. Interestingly, our data revealed a correlation between root depth, plant height and biomass and the environmental data of the location of the accessions. Correlation was also observed between the population structure determined through PvDREB6B and the environmental data. An association study combining data from the SNP array and DREB polymorphisms enabled the detection of SNP associated with drought-related traits through a compressed mixed linear model (CMLM) analysis. This thesis highlighted important features of DREB genes in common bean, revealing candidates for further strategies aimed at improvement of abiotic stress tolerance, with emphasis on drought tolerance / O feijoeiro é um componente essencial na dieta em diversos países, no entanto, sua produção é afetada negativamente por estresses abióticos. O estudo de genes candidatos envolvidos na adaptação aos estresses é uma etapa fundamental para o melhoramento da performance do feijoeiro sob tais estresses. Desse modo, esta tese apresenta uma análise sistemática da subfamília de genes DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB), que reúne genes envolvidos em diversos processos em resposta a estresses, mas pouco estudados no feijoeiro. Primeiramente, uma série de análises in silico com sequências de feijoeiro obtidas da plataforma Phytozome possibilitaram a categorização de 54 genes PvDREB putativos, distribuídos em seis subgrupos (A-1 até A-6) nos 11 cromossomos da espécie. Posteriormente, quatro novos genes PvDREB foram clonados e seus padrões de inducibilidade foram determinados. PvDREB1F e PvDREB5A foram induzidos por desidratação, baixa temperatura e salinidade, enquanto PvDREB2A e PvDREB6B foram predominantemente induzidos por desidratação e baixa temperatura. Polimorfismos de nucleotídeos foram buscados através de sequenciamento por método derivado de Sanger, revelando elevado número de SNP no gene PvDREB6B. A nomenclatura desse gene foi discutida detalhadamente ao longo da tese. A plataforma de marcadores SNP BARCBean6K_3 foi acessada para identificar o SNP mais próximo de cada um dos 54 PvDREB. O gene PvDREB6B foi selecionado para um estudo mais amplo, envolvendo uma coleção de acessos selvagens de origem Mesoamericana. A estrutura populacional destes genótipos foi analisada a partir de polimorfismos na sequência de PvDREB6B. Os grupos genéticos apresentaram associação parcial com variação da latitude, altitude, precipitação e temperatura das áreas em que os acessos naturalmente ocorrem. Com ênfase no estudo do déficit hídrico, uma plataforma de fenotipagem destes acessos em casa de vegetação, utilizando um sistema de tubos plásticos, foi elaborada para a análise de diversos parâmetros relacionados ao estresse por déficit hídrico. Os dados revelaram correlação entre profundidade de raízes, altura das plantas e a biomassa e as variáveis ambientais de cada local. A correlação também foi detectada entre a estrutura populacional estudada por PvDREB6B e os dados ambientais. Finalmente, um estudo de associação genética foi realizado entre os SNP da plataforma e ligados a DREB e os parâmetros fenotípicos, permitindo a identificação de marcadores SNP associados a caracteres específicos, usando um modelo linear misto (CMLM). Esta tese apresentou importantes aspectos sobre os genes DREB em feijoeiro, revelando candidatos para seu uso em estratégias de melhoramento para tolerância a estresses abióticos, com ênfase em déficit hídrico

Os estudos de associação de genoma inteiro têm encontrado alguns marcadores associados a doenças notoriamente hereditárias com herança complexa, mas, muitas vezes, estes marcadores somente explicam uma pequena parte da herdabilidade. Este relativo insucesso é atribuído, entre outras causas, à epistasia, ou seja, interação entre diferentes locos genéticos. A busca por epistasia é complexa e exige intensos recursos computacionais. Diversos métodos têm sido propostos para abordar este problema, incluindo métodos estatísticos tradicionais, busca estocástica e métodos heurísticos. Poucos destes métodos são capazes de processar as grandes massas de dados produzidas nos estudos caso-controle de genoma inteiro, e ainda menos métodos buscam conjuntos de três ou mais marcadores. A busca exaustiva de conjuntos de marcadores epistáticos é inviável hoje em dia para estes conjuntos, mas o algoritmo BOOST (WAN et al., 2010) mostrou que ela é relativamente fácil para pares de locos, em especial com o uso de placas gráficas como processadores (GPGPU). Partindo deste recente sucesso, propomos um algoritmo em fases para a busca de trios de locos que interagem, utilizando a busca de pares como passo inicial, uma abordagem ainda não utilizada. Outra ideia fundamental do algoritmo proposto é a extensão da concepção de trio de marcadores para um trio de blocos haplotípicos, onde cada bloco é formado por marcadores próximos entre si. Usando os dados do WTCCC, o Troost (de TRio+bOOST) sugeriu trios potencialmente epistáticos em todas a sete doenças. Quando submetidos à confirmação em amostra independente, os trios não puderam ser confirmados, exceto os trios para diabetes tipo 1 (T1D). Duzentos e oito trios foram confirmados para T1D, com baixos valores-P e genótipos combinados de risco com altas razões de chances. Os SNPs que compõem estes trios estão todos na região MHC, sabidamente associada à doença, exceto por um deles que está no cromossomo cinco e não havia sido previamente relacionado à T1D. / Genome-wide association studies have found some markers associated with diseases with complex inheritance. However, these markers explain only a fraction of the previously estimated heritability of the trait. This relative failure has been credited, among other causes, to epistasis, i.e. the interaction among genotypes at different loci. The search for epistasis is complex and requires intense computational resources. Many methods have been proposed to approach this problem, including traditional statistics, stochastic search, and heuristic methods. Few of them are capable of extracting, from the large amount of data produced in genome-wide case-control studies, useful information about sets of markers associated with the trait in question. Exhaustive search of sets of interacting markers is unfeasible nowadays for sets of three or more markers, but the BOOST algorithm (WAN et al., 2010) showed that the search is relatively easy for pairs of SNPs, in particular with the use of graphic cards for general processing (GPGPU). Starting from this recent success, we propose an algorithm in phases for the search for trios of interacting loci, using the search for pairs as the initial step, an approach not tried yet, to our knowledge. Another important idea of our algorithm is the extension of the concept of trio of markers to a trio of haplotypic blocks, where each block is formed by neighbor markers. Using data from WTCCC, the Troost (from TRio+bOOST) algorithm suggested potentially epistatic trios in all seven diseases. When submitted to a confirmation in an independent sample, the results could not be confirmed, except for type-1 diabetes (T1D). Two hundred eight trios were confirmed for T1D, with low p-values and risk combined genotypes with high odds ratio. The SNPs that form those trios are all in the MHC region, which is known to be strongly associated to T1D, except by one SNP in chromosome five that has not been previously associated with T1D.

Dados de desenvolvimento ponderal de 3.844 bovinos da raça Nelore, criados em pastagens em duas fazendas do sudoeste do Brasil, dos quais 1.889 tiveram suas carcaças avaliadas por ultra-sonografia e 674 foram confinados por 90 a 120 dias e abatidos por volta dos 24 meses de idade tiveram análises de associação com dezenas de marcadores genéticos realizadas, visando detectar a associação desses marcadores com características economicamente relevantes. Foram analisadas as características de crescimento, peso ao nascer (PNAS), peso a desmama (PDES), peso ao sobreano (PSOB), ganho de peso pós-desmama (GP345), escores visuais de conformação frigorífica (CONF), precocidade de acabamento (PREC), musculosidade (MUSC) e de carcaça área de olho de lombo medida por ultra-sonografia (AOL_US), espessuras de gorduras medida por ultra-som na região lombar (EGS_US) e na picanha (EGP8). Adicionalmente, foram analisadas as características medidas post-mortem, relacionadas a qualidade de carcaça, peso de carcaça quente (PCQ), área de olho de lombo (AOL), espessura de gordura no músculo Longissimus dorsi (EGS) e as características ligadas à qualidade de carne, perdas por exsudação após 7, 14 ou 21 dias de maturação da carne (PEX7, PEX14, PEX21), perdas por cocção e maciez após os mesmos períodos de maturação (PCO7, PCO14 e PCO21, MAC7, MAC14 e MAC21), além de teor de lipídeos totais e colesterol em amostras após 7 dias de maturação. Os genótipos dos polimorfismos de base única (SNP) foram obtidos em laboratórios licenciados por empresa parceira, com uso de placas de micro-arranjos dessa empresa. Foram analisados os efeitos de substituição em análises de marcador único e multi-polimorfismos e os efeitos de aditividade e desvio de dominância de cada marcador genético. Vários dos polimorfismos de DNA analisados apresentaram ou fixação ou freqüências muito altas, maiores que 99%, de um dos alelos impossibilitando as análises de associação. No entanto, muitos outros polimorfismos apresentaram freqüências gênicas adequadas às análises de associação. Cada uma das características avaliadas apresentou, no mínimo, dois marcadores com efeitos significativos (P≤0,05) ou sugestivos (0,05<P≤0,20), o que indica que polimorfismos de DNA podem ser critérios adicionais e auxiliares nos processos seletivos ligados às 24 características de desenvolvimento ponderal, qualidade de carcaça e carne na raça Nelore. Como os efeitos de substituição alélica são responsáveis apenas por parte da determinação de cada característica, em geral uma pequena parte, recomenda-se que as previsões de efeitos de marcadores sejam feitas com análise conjunta dos mesmos. / Data on of 3,844 Nellore cattle, reared under pasture conditions on two different farms in southwestern Brazil, 1,889 of them measured by ultra-sound for carcass traits and 674 bulls finished in a feedlot for 90 to 120 days and slaughtered around 24 month of age were analyzed to verify the association with genetic markers (DNA Single nucleotide polymorphism or SNP) with the objective of detecting association of those markers with traits economically important relevant for Brazilian beef business. Growth traits considered were birth weight (PNAS), weaning weight (PDES), yearling weight, measured at 18 mo (PSOB), weight gain after weaning (GP345), visual scores for carcass conformation (CONF), finishing (PREC) and muscle (MUSC). Carcass traits, measured by ultra-sound were ribeye area (AOL_US), backfat (EGS_US) and fat depth at rump (EGP8). Additionally, carcass traits measured after slaughter were hot carcass weight (PCQ), ribeye area (AOL), fat depth on Longissimus muscle (EGS). Meat quality traits were measured after 7, 14 and 21 days of ageing: weep loss (PEX7, PEX14 and PEX21), shrink loss (PCO7, PCO14 and PCO21) and tenderness (MAC7, MAC14 and MAC21). Total lipids and cholesterol content on samples aged for 7 days, were, also, included on the analysis. The genotypes of DNA markers were carried out in laboratories licensed by a private company using its micro-array panels. Allele substitution effects were estimated in single or multi-polymorphism analysis. Additive and dominance effects were also estimated. Many DNA polymorphisms analyzed showed to be fixed or the frequencies for one of the alleles were too high, more than 99 %. In those cases, analysis could not be performed. However, for many others polymorphisms there was observed variability on allele frequencies what make possible to do the association analysis. All traits analyzed were influenced by, at least, two polymorphisms with statistically significant (P≤0,05) or suggestive (0,05<P≤0,20) effects, thus DNA polymorphisms can be used as additional and auxiliary criteria on selection process of those 24 traits related to animal growth, carcass and meat quality in Nellore cattle. As allele substitution effects explain only a small part of the phenotype, the results of this paper suggest that the effect of those markers should be considered together.

A combinação do aumento da população mundial e a melhoria nos padrões de vida estão levando a uma maior demanda por alimentos com alto valor nutricional. As variedades de laranjas de polpa vermelha são as únicas laranjas que acumulam licopeno na polpa e no suco. O licopeno é um carotenóide considerado como um composto antioxidante, com ação potencial na prevenção de alguns tipos de câncer. Apesar do valor nutricional agregado as laranjas de polpa vermelha, a caracterização fisiológica e molecular dos carotenóides nessas laranjas permanece pouco estudada. O objetivo deste trabalho foi caracterizar o perfil de transcrição de genes envolvidos na via de biossíntese dos carotenóides e quantificar a concentração dos carotenóides na polpa dos frutos de duas laranjas de polpa vermelha e da laranja Pêra (controle). Para comparação entre variedades, os frutos das laranjas Sanguínea-de-Mombuca (\'SM\') e Bahia Cara-Cara (\'CC\'), que possuem polpa vermelha, e da laranja Pêra (\'P\') foram colhidos aos 270, 300 e 330 dias após o florescimento (DAF), em Cordeirópolis (CCSM). Para identificar a influência climática no acúmulo de carotenóides, frutos da laranja de polpa vermelha \'SM\' foram também colhidos frutos aos 270, 300 e 330 DAF em diversos locais: CCSM, Mogi-Mirim (MM) e São Bento do Sapucaí (SBS). A quantidade dos transcritos dos genes fitoeno sintase (PSY), fitoeno dessaturase (PDS), \'dzeta\'-caroteno dessaturase (ZDS), carotenóide isomerase (CRTISO), licopeno \'épsilon\'-ciclase (LCY\'\'épsilon\'), licopeno \'beta\'-ciclase (LCY\'beta\'), hidroxilase \'beta\'-caroteno (HY\'beta\') e zeaxantina epoxidase (ZEP), que codificam para as enzimas da via de biossíntese dos carotenóides, foram analisados por PCR em tempo real. A quantificação dos principais carotenóides presentes no suco dos frutos foi realizada por meio de cromatografia líquida de alta eficiência. A análise quantitativa de transcritos obtidos a 330 DAF indicaram a existência de maiores níveis de expressão dos genes precursores dos carotenos (PSY, PDS, CRTISO, ZDS, LCY\'beta\') nas variedades de polpa vermelha, principalmente na \'SM\'. A quantidade dos carotenos fitoflueno, licopeno e \'beta\'-caroteno foram maiores nas laranjas vermelhas, em relação ao observado nos frutos de laranja Pêra, correlacionando com os níveis de expressão gênica. Nos frutos da laranja pigmentada \'SM\' coletadas aos 330 DAF pôde-se observar uma possível associação entre o clima do local de cultivo e o conteúdo de transcritos dos genes da via de carotenóides. O cultivo em clima mais quente (MM) resultou em frutos com maiores níveis de transcritos dos genes precursores de carotenos (PSY, PDS, ZDS, CRTISO e LCY\'beta\'), enquanto que o cultivo em região de clima mais frio (SBS) resultou no aumento do nível de transcritos dos genes precursores das xantofilas (HY\'beta\' e ZEP). Como consequência, houve um acúmulo maior dos carotenos fitoflueno, licopeno e \'beta\'-caroteno, nos frutos cultivados em locais quentes, porém, em regiões mais frias, houve maior acúmulo das xantofilas zeaxantina e violaxantina, nos frutos. Com o sequenciamento das regiões de diferentes genes da via de biossíntese de carotenóides, foram identificadas algumas mutações pontuais na sequência de nucleotídeos dos genes PSY, CRTISO e ZDS da variedade pigmentada \'SM\', em comparação com os mesmos genes da laranja Pêra. Algumas dessas mutações resultaram na alteração de aminoácidos na sequência das proteínas e deste modo, supõe-se que estes genes sejam os principais candidatos a serem os responsáveis pela alteração do fenótipo das variedades que produzem frutos com polpa avermelhada, ricos em licopeno / The increasing world population and improvement in living standards are leading to greater demand for foods with high nutritional value. The sweet orange varieties with red-flesh are the only ones that accumulate lycopene in the pulp and juice. Lycopene is a carotenoid compound with antioxidative property which may have a potential action in preventing some cancers. Despite of the high nutritional value in the red-fleshed oranges, the physiological and molecular characterization of carotenoids remains to be elucidated. The present work aimed to characterize the transcriptional profile of genes involved in carotenoids biosynthesis pathway and quantify the carotenoids in red-fleshed oranges and Pêra orange (as a control). For a direct comparison among the varieties, fruits of Sanguínea-de-Mombuca (\'SM\') and Bahia Cara-Cara (\'CC\') red-fleshed sweet oranges, and Pêra orange were harvested at 270, 300 and 330 days after flowering (DAF) in Cordeirópolis station (CCSM). To investigate the impact of climate variation on carotenoids accumulation, red-fleshed fruits \'SM\' orange were harvested on 270, 300 and 330 DAF in different climate locations: Cordeirópolis station (CCSM), Mogi-Mirim (MM) and São Bento do Sapucaí (SBS). Quantitative transcriptional analysis of the genes phytoene syntase (PSY), phytoene desaturase (PDS), \'dzeta\'-carotene desaturase (ZDS), carotenoid isomerase (CRTISO), lycopene \'épsilon\'-ciclase (LCY\'épsilon\'), lycopene \'beta\'-cyclase (LCY\'beta\'), hydroxylase \'beta\'-carotene (HY\'beta\') and zeaxanthin epoxidase (ZEP) related to the carotenoids biosynthesis pathway were performed by quantitative Real Time PCR. Further, carotenoids quantification in red-fleshed fruits was measured by high performance liquid chromatography. The transcriptional profile at 330 DAF revealed high expression levels for the carotene gene precursors (PSY, PDS, CRTISO, ZDS, LCY\'beta\') in red-fleshed oranges, in particular to \'SM\' orange when compared to Pêra orange. Carotenoids profiling displayed higher phytofluene, lycopene and \'beta\'-carotene concentrations in red-fleshed orange than Pêra orange fruits. Transcript levels of genes related to the carotenoids pathway were responsive to the climate variation in red-fleshed orange harvested at 330 DAF. In warmer climate conditions, increased expression levels were found for the genes related carotene precursors (PSY, PDS, ZDS, CRTISO and LCY\'beta\'), whereas under colder climate conditions, the red-fleshed fruits orange showed higher expression levels for the xanthophylls precursors genes (HY\'beta\' e ZEP). As a consequence, the carotenes phytofluene, lycopene and \'beta\'-carotene accumulated in warmer climate developed fruits, instead of colder climate conditions, where the fruits accumulate increased concentrations of xanthophylls such as zeaxanthin and violaxanthin. Moreover, the sequencing of fragments from different genes related to the carotenoids pathway enabled the identification of point mutations for the genes PSY, CRTISO and ZDS in the red-fleshed variety. These mutations caused amino acid changes in the protein which indicates that these genes might be responsible for the phenotypes red-fleshed fruits and increased lycopene levels shown in the variety \'SM\'

Der "Leukocyte Receptor Complex" (LRC) ist ein DNA-Sequenzabschnitt auf dem Chromosom 19 des Menschen, der eine Länge von über 900.000 Basenpaaren umfaßt. In diesem Chromosomenabschnitt ist eine Vielzahl von Genen lokalisiert, die für die Funktion verschiedener weißer Blutzellen (Leukozyten) von entscheidender Bedeutung sind. Bei den aus diesen Genen synthetisierten Proteinen (Eiweißen) handelt es sich um Strukturen, die auf der Oberfläche dieser Zellen lokalisiert sind und zur Interaktion der Leukozyten mit ihrer Umgebung dienen. Diese auch als Rezeptoren bezeichneten Proteine können mit Oberflächenproteinen auf anderen Körperzellen wechselwirken und daraus resultierende Signale in das Innere der Blutzelle weiterleiten. <br /> In der vorliegenden Doktorarbeit wurde der LRC im Detail untersucht. Hierzu wurde zunächst der gesamte Chromosomenabschnitt aus kleineren, einander überlappenden DNA-Fragmenten rekonstruiert. Aufgrund der in diesen DNA-Fragmenten enthaltenen DNA-Sequenzen war es möglich, den gesamten Chromosomenabschnitt ähnlich einem Puzzle zusammenzusetzen. Die anschließende Analyse des LRC zeigte, daß sich dieser in drei Bereiche, sogenannte Cluster, unterteilen läßt. Diese Cluster sind dadurch gekennzeichnet, daß in ihnen jeweils nur Gene eines Rezeptortyps vorkommen. Hierbei handelt es sich um ‚immunoglobulin-like transcript′ -Gene (ILT) und ‚killer cell Ig-like receptor′-Gene (KIR). Die KIR- und ILT-Cluster werden von weiteren stammesgeschichtlich verwandten Genen unterbrochen und flankiert. Je nach Individuum können im LRC bis zu 31 solcher verwandten Rezeptorgene lokalisiert sein. Auf der Grundlage der Kartierungsdaten und von Daten des humanen Genomprojekts war es zudem möglich, evolutionäre Untersuchungen zur Entwicklung des LRC durchzuführen. Dabei wurde eine Hypothese zur Entstehung des LRC entworfen und zu anderen Spezies in Beziehung gesetzt. <br /> Im zweiten Teil der Arbeit habe ich aufbauend auf der sogenannten HRCA-Methode eine Technik entwickelt, die es erlaubt kleinste Unterschiede zwischen DNA-Sequenzen, sogenannte Einzelbasenpaaraustausche, nachzuweisen. Die entwickelte Methode kann verwendet werden, um sehr ähnliche DNA-Sequenzen, wie z.B. verschiedene KIR-Sequenzen, zu unterscheiden und ihre Menge zu bestimmen. Sie ist außerdem geeignet Mutationen, die mit bestimmten Krankheiten assoziiert sind, nachzuweisen und könnte somit in der Diagnostik Anwendung finden. / The Leukocyte Receptor Complex (LRC) is a DNA region on human chromosome 19 with a length of approximately 900.000 base pairs. A number of genes, which are located in this chromosomal region, are known to be important for the function of some types of white blood cells (leukocytes). The products of theses genes are proteins, which are located on the surface of these cells and enable them to interact with their environment. These proteins are also called receptors. They can bind to cell surface proteins on other cells and transmit resulting signals into the leukocyte. <br /> During my work I analyzed the chromosomal organization of the LRC in detail. To do so, I reconstructed the whole chromosomal region from smaller overlapping DNA fragments. Due to the DNA sequences contained within these fragments it was possible to put the whole chromosomal region together like a puzzle. The following analyses of the LRC showed that it is mainly composed of three regions, so called clusters. These clusters are characterised by the presence of only one receptor family. These are the immunoglobulin-like transcripts (ILTs) and the killer cell Ig-like receptors (KIR) respectively. In the LRC the KIR- and ILT-Clusters are flanked by additional receptor genes, which are evolutionary related to KIRs and ILTs. The number of receptor genes in the LRC varies between individuals, their can be up to 31 genes on each chromosome. <br /> On the basis of the data obtained in this work as well as data from the human genome project it was also possible to draw conclusions concerning the evolutionary development of the LRC. I developed a hypothesis of the origin of the LRC and discussed this in comparison to other species. <br /> In the second part of my thesis I developed a new technique based on the so-called ’hyper-branched rolling circle amplification′ (HRCA). This technique allows the detection of small differences between two or more DNA molecules, so called ’single nucleotide polymorphisms′ (SNPs). With this newly developed method it is possible to distinguish very similar variants of a gene, e.g. two KIR sequences, and to determine their relative concentration. The method can also be used to detect mutations, which are associated with certain diseases and could therefore be used for diagnostic purposes.

研究指出，在不同人類個體的DNA序列中，只有0.1%的基因組排列是相異的，而其餘的序列則是相同的。這些相異的基因組排列則被稱為單一核苷酸(SNP)。Affymetrix公司發展出一種DNA晶片技術稱之為Affymetrix GeneChip Mapping 100K SNP set，此晶片可用來決定單一核苷酸資料的基因類型(genotype)。Affymetrix公司採用預設「動態模型演算法」(DM)來決定基因型態。本論文的研究目的是探討與示範對於DM方法中預設的S值的四種修正方式。而這四種修正的方法分別是： (1) Standardized L value，(2) Median-polished L value，(3) Median-center L value，和(4) Median-standardized L value。為了比較S值與四種改進方法，本研究藉由SNP的模擬資料來進行比較。資料的模擬是基於利用改寫過的階層式之Bolstad模型（2004），而模擬模型的參數估計是利用華人細胞株及基因資料庫中95位台灣人的100K SNP資料。根據AA模型與AB模型模擬資料的基因型態正確率，Standardized L value是最好的判斷基因型態之方法。在另一方面，因為DM方法並不是設計來決定Null模型的基因型態，因此對於Null模型模擬資料的基因型態判斷會有問題。關於Null模型的基因型態判斷，本論文提供了一些簡短的討論與建議。然而，依然需要進一步的研究探討。 / It is known there is only 0.1% in the DNA sequences that is different among human beings, and the rest of them are the same. These differences in DNA sequences are defined as SNPs (Single Nucleotide Polymorphism). The Affymetrix, Inc. had developed a DNA chip technology called Affymetrix GeneChip Mapping 100K SNP set for SNP data used to determine the genotype call. The default algorithm applied by Affymetrix, Inc. to decide genotype calls is the Dynamic Model-based (DM) algorithm. This study aimed to investigate and demonstrate four different ways to modify the basic component used in DM algorithm, namely, the S value. These four modified methods include: (1) Standardized L value, (2) Median-polished L value, (3) Median-centered L value, and (4) Median-standardized L value. In order to compare the S value with the four modified L values, a simulation study was conducted. A hierarchical version of Bolstad’s model (2004) was adopted to simulate the SNP Data. The parameters for the simulation model were estimated based on 95 Taiwanese 100K SNPs data from Taiwan Han Chinese Cell and Genome bank. The Standardized L value was proven to be the best method based on the accuracy of the genotype calls determined according to the simulated data of AA model and AB model. On the other hand, the genotype call for simulated data under Null model is problematic since the DM approach is not designed to determine the Null model. We have given some brief discussion and remarks of the genotype call for Null model. However, further research is still needed. /

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