Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2018-10-23T14:00:16Z No. of bitstreams: 1 TESE Mariana Peil VERSÃO CORRIGIDA.pdf: 2685533 bytes, checksum: 792fb6d377fb546168a4c6e1f05c79c9 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-11-13T15:54:17Z (GMT) No. of bitstreams: 1 TESE Mariana Peil VERSÃO CORRIGIDA.pdf: 2685533 bytes, checksum: 792fb6d377fb546168a4c6e1f05c79c9 (MD5) / Made available in DSpace on 2018-11-13T15:54:17Z (GMT). No. of bitstreams: 1 TESE Mariana Peil VERSÃO CORRIGIDA.pdf: 2685533 bytes, checksum: 792fb6d377fb546168a4c6e1f05c79c9 (MD5) Previous issue date: 2018-08-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O aumento da produção no Brasil, assim como no mundo, é uma estratégia para segurança alimentar. No entanto, uma estagnação nos aumentos de produtividade devido, entre outros fatores, a redução da variabilidade genética através da seleção ao passar dos anos. Triticum timopheevii Zhuk. (2n = 28, composição genômica AtAtGG) é um trigo tetraploide e possui alelos desejáveis, principalmente para resistência a doenças, e ele pode ser usado para ampliar variabilidade genética no trigo. Este estudo propôs realizar a introgressão de segmentos de DNA de T. timopheevii. em uma cultivar elite de trigo brasileira. Linhas de T. aestivum/T. timopheevii na geração RC2 foram polinizadas com TBIO Sinuelo (TBIO) e um arranjo de genotipagem Affymetrix Axiom Array juntamente com hibridização genômica in situ (GISH) foram utilizados para detectar e caracterizar as introgressões. No total, 548 cruzamentos foram realizados, e 4.579 sementes foram produzidas possibilitando a geração de um mapa de ligação do T. timopheevii. Este mapa foi usado para identificar 316 introgressões putativas nos híbridos trigo/T. Timopheevii. Foram encontradas introgressões dos grupos de ligação. A análise comparativa confirmou as translocações 4AtL/5AtL herdadas do T. urartu e já reportadas no T. timopheevii e as translocações específicas da espécie 6AtS/1GS 3AtL/4AtL presentes no T. timopheevii. Os dados obtidos aqui mostram que T. timopheevii tem um grande potencial para ser usado em programas de melhoramento porque suas linhas de introgressão apresentam altas taxas de germinação alta fertilidade e boa produção de sementes. / Increasing wheat yield in Brazil, as well as in the world, is a strategy for food security. However, a stagnation has been observed, due to, among other things, the reduction of genetic variability through selection over the decades. Triticum timopheevii Zhuk. (2n = 28, genome composition AtAtGG) is a tetraploid wheat which has desirable alleles, mainly for disease resistance, and it can be used to increase genetic variability in wheat. This study proposed performing the introgression of DNA segments from T. timopheevii lines into a Brazilian elite cultivar. Wheat/T. timopheevii lines in BC2 generation were pollinated with TBIO Sinuelo (TBIO) and Affymetrix Axiom Array along with genomic in situ hybridisation (GISH) were used to detect and characterize introgressions. In total, 548 crosses were performed, and 4,579 seeds were produced enabling the generation of a linkage map of T. timopheevii. This was used to identify 316 T. aestivum/T. timopheevii putative introgressions. Introgressions of all 14 groups of T. timopheevii were found. Comparative analysis showed that T. timopheevii has the 4AtL/5AtL translocations inherited from T. urartu and the species-specific 6AtS/1GS, 3AtL/4AtL already reported in T. timopheevii. From the data obtained T. timopheevii has good potential to be used in introgression programs because its introgression lines present high germination rates, fertility level and good seed production.

Made available in DSpace on 2014-06-12T18:01:36Z (GMT). No. of bitstreams: 2 arquivo3081_1.pdf: 3854771 bytes, checksum: 12945eba1d50bff0e15c2bf021c535ae (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A diabetes é uma doença crônico-degenerativa que envolve alterações no metabolismo de carboidratos, lipídios e proteínas. Caracteriza-se por deficiência de secreção e/ou de ação da insulina com consequente hiperglicemia. É preocupante saber que dados da Organização Mundial de Saúde (OMS) prevêem um aumento no número de diabéticos de 171 milhões no ano de 2000 para 336 milhões em 2030, o que se assemelha a uma epidemia. A diabetes mellitus tipo 2 (DM2) é a principal responsável pelo incremento da doença no mundo atual. Este estudo teve como objetivo analisar a ocorrência das principais mutações do gene TCF7L2 (fator de transcrição 2 semelhante ao 7) relacionadas a DM2. Usando a técnica de PCR em tempo real com sondas TaqMan, foram genotipados quatro polimorfismos de um único nucleotídeo (Single Nucleotide Polymorphisms - SNPs) do gene TCF7L2: rs7903146 (C/T), rs7901695 (T/C), rs12255372 (G/T) e rs11196205 (G/C). A partir do resultado da genotipagem dos 340 indivíduos residentes no município de Triunfo - Pernambuco (112 diabéticos e 228 não diabéticos) foram calculadas as frequências alélicas, genotípicas e haplotípicas para o conjunto de SNPs, utilizando o programa UNPHASED. Uma associação significativa foi encontrada entre os SNPs rs7901695 e rs12255372 com DM2 (p = 0,0379 e p = 0,0119, respectivamente) na população estudada. As frequências alélicas corroboraram o que foi observado com os genótipos para o SNP rs7901695. Na análise haplotípica, os pares rs7901695 x rs11196205 e rs11196205 x rs12255372 apresentaram associação com DM2 (p = 0,007 e p = 0,015, respectivamente). Entre os haplótipos triplos, apenas um conjunto (rs7901695 x rs7903146 x rs12255372) não mostrou associação com diabetes (p = 0,2903). Portanto, no presente estudo, os SNPs do TCF7L2 encontraram-se em associação com DM2, sendo que se estes haplótipos forem estendidos, com a adição de outros do mesmo gene, podem-se ter haplótipos que, segregando nas famílias e caracterizando-as, permitam estabelecer um diagnóstico probabilístico preventivo a partir dessas mutações. Uma vez que esses dados sejam replicados, essa associação pode ser utilizada como marcador genético de risco para famílias, cujos ancestrais mais recentes tenham DM2, bem como para monitoração das mesmas. Neste estudo, foi também realizado um levantamento de dados das famílias do distrito de Canaã em Triunfo, a fim de estabelecer a prevalência de DM2 nessa população. Numa amostragem de 198 indivíduos, a prevalência de DM2 correspondeu a 13,6% da população adulta deste distrito, demonstrando que também no sertão nordestino esse é um quadro preocupante

Magister Scientiae - MSc / Autism is a pervasive developmental disorder (PDD) that's incidence is approximately 1 in 158. It is four times more prevalent in males than females and is believed to be caused by both genetic and environmental factors. Research indicates that several genes are involved in autism and it is believed that these genes act together to produce autism. Many genes implicated in this disorder are involved with brain structure formation and brain functioning. Studies have identified the reelin (RELN) gene as necessary for proper formation of brain, which indicates that RELN abnormalities could contribute to the aetiology of several neurogenetic diseases such as schizophrenia, bipolar and autism. The aims of the study were (i) to genotype two SNPs (exonic rs3622691 and intronic rs736707) in the RELN gene using Taqman® SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to detect candidate genes that are over and under-expressed in the samples taken from a SA Caucasian autistic group and compare those with samples taken from a healthy Caucasian group using cDNA microarray. The Taqman® study indicated significant association for the intronic SNP, rs736707, with a p-value of 0.0009 in the total SA group. More so, the Mixed group displayed the highest significance amongst the ethnic groups, with a p-value of 0.00014. The microarray study yielded 21 genes with 95% significance in the Caucasian sample group. Most genes were hypothetical proteins and formed part of the FAM90A family. The LOC83459 showed the highest level of expression in the autistic samples, while the BTNL8 gene was shown to be highly suppressed in the control samples. / South Africa

Polimorfismos de um nucleotídeo (SNPs) podem alterar, além de códons de leitura da tradução de genes, posições genômicas importantes do processo de regulação gênica, como sítios de iniciação de transcrição, de splicing e, mais especificamente, sítios alvos de microRNAs (miRNAs). Em parti- cular, a identificação de SNPs alterando sítios alvos de miRNAs é um problema em aberto, embora venha ganhando um importante destaque nos últimos anos decorrente dos avanços descobertos sobre a capacidade dos miRNAs como elementos reguladores do genoma, associados inclusive a muitas doenças como o câncer e vários transtornos psiquiátricos. Os recursos computacionais atualmente disponíveis para esta finalidade (alguns bancos de dados e uma ferramenta) estão restritos à análise de SNPs na região 3UTR (UnTranslated Regions) de RNAs mensageiros, onde miRNAs geralmente se ligam para reprimir sua tradução. No entanto, essa é uma simplificação do problema, dado que já se conhece a regulação por miRNAs ativando ou reprimindo a transcrição gênica quando ligados à sua região promotora, aumentando a efetividade da regulação negativa da tradução quando ligados à região codificante do gene ou ainda miRNAs se ligando a RNAs não codificantes. Esses recursos se limitam também à identificação de SNPs na região seed dos sítios de miRNAs, e portanto só identificam criação ou ruptura de sítios. Porém, SNPs localizados fora dessa região não só podem colaborar na criação e ruptura de sítios como também interferir na estabilidade de ligação de miRNAs e, por- tanto, na efetividade da regulação. Além disso, considerando toda a extensão do sítio, não somente a seed , é possível ocorrer mais de um SNP e, sendo assim, a combinação desses SNPs pode ter uma influência ainda maior na ligação com o miRNA. Os recursos atuais também não informam quais alelos dos SNPs, muito menos quais combinações deles, estão causando qual efeito. Por fim, tais recursos estão restritos a Homo sapiens e Mus musculus. Assim, este trabalho apresenta a ferramenta computacional SIMTar (SNPs Interfering in MicroRNA Targets), desenvolvida para identificar SNPs que alteram sítios alvos de miRNAs e que preenche as lacunas mencionadas. Além disso, é descrita uma aplicação de SIMTar na análise de 114 SNPs associados à esquizofrenia, na qual todos foram preditos interferindo em sítios alvos de miRNAs. / Single nucleotide polymorphisms (SNPs) can be involved in alteration of not only open reading frame but also important genomic positions of gene regulation process such as transcription initiation sites, splicing sites and microRNA target sites. In particular, the identification of SNPs interfering on microRNA target sites is still an open problem, despite its increasing prominence in recent years due to the discoveries about the microRNA abilities as regulatory elements in the genome and association with severals diseases such as cancer and psychiatric disorders. The computational resources currently available for this purpose (four databases and one tool) are restricted to the analysis of SNPs in the 3UTR (UnTranslated Regions) of mRNAs, where the microRNAs typically bind in order to repress their translation. However, this is a simplification of the problem, since it is already known the gene transcription activation by microRNAs bound to its promoter region, increasing of the effectiveness of negative regulation of translation when microRNAs are bound to the coding region of the gene or binding of microRNA into non-coding RNAs. These resources are also limited to the identification of SNPs in the seed region of miRNAs, and therefore they can only identify sites creation or disruption. However, SNPs located outside this region can not only create and disrupt target sites but also interfere on the stability of miRNAs binding and therefore on the regulation effectiveness. Moreover, considering the target site length, more than one SNP can occur inside of a site and thus, the combination of these SNPs can have an even greater influence on the microRNA binding. Also, current resources do not display which alleles of SNPs or what combinations of them are causing which effect. Finally, these features are restricted to the Homo sapiens and Mus musculus species. This work presents the computational tool SIMTar (SNPs Interfering on MicroRNA Targets), developed to identify SNPs that alter miRNA target sites and fills the mentioned gaps. Finally, it is described an application of SIMTar on the analysis of 114 SNPs associated with schizophrenia, all of them being predicted interfering with miRNA target sites.

Local adaptation and phenotypic plasticity are main mechanisms of organisms’ resilience in changing environments. Both are affected by gene flow and are expected to be weak in zooplankton populations inhabiting large continuous water bodies and strongly affected by currents. Lake Baikal, the deepest and one of the coldest lakes on Earth, experienced epilimnion temperature increase during the last 100 years, exposing Baikal’s zooplankton to novel selective pressures. We obtained a partial transcriptome of Epischura baikalensis (Copepoda: Calanoida), the dominant component of Baikal’s zooplankton, and estimated SNP allele frequencies and transcript abundances in samples from regions of Baikal that differ in multiyear average surface temperatures. The strongest signal in both SNP and transcript abundance differentiation is the SW-NE gradient along the 600+ km long axis of the lake, suggesting isolation by distance. SNP differentiation is stronger for nonsynonymous than synonymous SNPs and is paralleled by differential survival during a laboratory exposure to increased temperature, indicating directional selection operating on the temperature gradient. Transcript abundance, generally collinear with the SNP differentiation, shows samples from the warmest, less deep location clustering together with the southernmost samples. Differential expression is more frequent among transcripts orthologous to candidate thermal response genes previously identified in model arthropods, including genes encoding cytoskeleton proteins, heat-shock proteins, proteases, enzymes of central energy metabolism, lipid and antioxidant pathways. We conclude that the pivotal endemic zooplankton species in Lake Baikal exists under temperature-mediated selection and possesses both genetic variation and plasticity to respond to novel temperature-related environmental pressures.

Pyrenophora semeniperda (anamorph Drechslera campulata) is a necrotrophic fungal seed pathogen of a variety of grass genra and species, including Bromus tectorum, an exotic grass that has invaded many natural ecosystems of the U.S. Intermountain West. As a natural seed pathogen of B. tectorum, P. semeniperda has potential as a biocontrol agent due to its effectiveness at killing dormant B. tectorum seeds; however, few genetic resources exist for this fungus. Here, the genome assembly of a P. semeniperda isolate using 454 GS-FLX genomic and paired-end pyrosequencing techniques is presented. The total assembly is 32.5 Mb and contains 11,453 gene models greater than 24 amino acids. The assembly contains a variety of predicted genes that are involved in pathogenic pathways typically found in necrotrophic fungi. In addition, 454 sequence reads were used to identify single nucleotide polymorphisms between two isolates of P. semeniperda. In total, 20 SNP markers were developed for the purposes of recombination assesment of 600 individual P. semeniperda isolates representing 36 populations from throughout the U.S. Intermountain West. Although 17 of the fungal populations were fixed at all SNP loci, linkage disequilibrium was determined in the remaining 18 populations. This research demonstrates the effectiveness of the 454 GS-FLX sequencing technology, for de novo assembly and marker development of filamentous fungal genomes. Many features of the assembly match those of other Pyrenophora genomes including P. tritici-repentis and P. teres f. teres, which lend validity to our assembly. These findings present a significant resource for examining and furthering our understanding of P. semeniperda biology.

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Estimations of heritability and variance explained due to environmental exposures and interaction effects help in understanding complex diseases. Current methods to detect such interactions rely on variance component methods. These methods have been neces- sary due to the m » n problem, where the number of predictors (m) vastly outnumbers the number of observations (n). These methods are all computationally intensive, which is further exacerbated when considering gene-environment interactions, as the number of predictors increases from m to 2m+1 in the case of a single environmental exposure. Novel methods are thus needed to enable fast and unbiased calculations of the variance explained (R2) for gene-environment interactions in very large samples on multiple traits. Taking advantage of the large number of participants in contemporary genetic studies, we herein propose a novel method for continuous trait R2 estimates that are up to 20 times faster than current methods. We have devised a novel method, monsterlm, that enables multiple linear regression on large regions encompassing tens of thousands of variants in hundreds of thousands of participants. We tested monsterlm with simulations using real genotypes from the UK Biobank. During simulations we verified the properties of monsterlm to estimate the variance explained by interaction terms. Our preliminary results showcase potential interactions between blood biochemistry biomarkers such as HbA1c, Triglycerides and ApoB with an environmental factor relating to obesity-related lifestyle factor: Waist-hip Ratio (WHR). We further investigate these results to reveal that more than 50% of the interaction variance calculated can be attributed to ∼5% of the single-nucleotide polymorphisms (SNPs) interacting with the environmental trait. Lastly, we showcase the impact of interactions on improving polygenic risk scores. / Thesis / Master of Science (MSc)

DNA chip technology has drawn tremendous attention since it emerged in the mid 90 s as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges.. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same intensity of excitation light.. The fluorescence contrast is used to quantify the amount of probe-target hybridization. A mathematical model that considers multiple reflections and scattering is developed to explain the mechanism of the fluorescence contrast which depends on the thickness of the PS film. Scattering is the dominant factor that contributes to the contrast. The potential of this assay to detect single nucleotide polymorphism is also tested. / Ph. D.