As técnicas de marcadores moleculares têm sido aplicadas em estudos populacionais das espécies bovinas, verificação de genealogia e teste de paternidade. Dentre os marcadores moleculares, os microssatélites (MS) são amplamente utilizados, porém, alguns problemas técnicos têm motivado o desenvolvimento de alternativas, como os marcadores do tipo polimorfismo de nucleotídeos único (SNP). Assim, surgiu a necessidade de identificar haplótipos SNP que estão em concordância com cada alelo MS e então os genótipos MS poderiam ser convertidos em genótipos SNP e vice-versa, por meio da imputação do genótipo. O objetivo deste trabalho foi aplicar um método para imputar alelos MS a partir de haplótipos SNP, para verificação de paternidade, utilizando animais da raça Nelore e também identificar um menor conjunto de SNP, com qualidade suficiente para otimizar e diminuir o custo da genotipagem. Foram realizadas genotipagens em SNP e MS para 99 trios de animais da raça Nelore provenientes da EMBRAPA Pecuária Sudeste e foi verificada a existência de alelos nulos pelo programa MICRO-CHECKER. Foram selecionados SNP que estivessem próximos de cada marcador MS e o programa BEAGLE foi usado para identificar a fase de ligação dos genótipos. Posteriormente, foi realizada a técnica de imputação dos MS a partir de haplótipos SNP e foi verificada a paternidade pelo programa CERVUS. A precisão da imputação dos alelos MS foi verificada através do cálculo da concordância entre os alelos MS imputados e relatados. O marcador SPS115 foi removido da análise por evidências de alelos nulos, devido ao excesso de homozigotos observados. O marcador mais informativo foi o TGLA122, cujo conteúdo de informação polimórfica (PIC) foi 0,8. Foram encontrados desvios do equilíbrio de HW (P<0,05) para os locos ETH225 e TGLA57. Um maior conjunto de SNP foi necessário para imputação de alelos MS para o marcador BM1824. As taxas de verificação de parentesco foram de 97,1% para os alelos MS genotipados e 96,3% para os MS imputados. Somente 4% dos 99 filhos não tiveram a paternidade atribuída, quando a simulação foi feita apenas para o pai conhecido e 1% quando pai e mãe eram conhecidos. Esta técnica obteve precisão maior que 96% para a imputação de dados MS e permitiu imputar dados genotípicos multialélicos a partir de bi-alélicos. Os resultados terão um impacto imediato para os pesquisadores e associações de criadores que visam a transição do MS para SNP baseada em verificação de parentesco. / Molecular markers techniques have been applied in bovine population studies, genealogy verification and paternity test. Among the molecular markers, microsatellites (MS) are widely used, however, some technical problems have motivated alternatives development, as markers type single nucleotide polymorphism (SNP). Thus, the need to identify SNP haplotypes which are in agreement with each MS allele and then MS genotypes could be converted into SNP genotypes and vice versa, through genotype imputation. The objective of this study was to apply a method to impute MS alleles from SNP haplotypes to verify paternity, using Nellore and also identify a smaller set of SNP, with enough quality to optimize and reduce genotype cost. SNP genotyping was performed at and for 99 MS trios Nellore from EMBRAPA Cattle Southeast and was checked for null alleles by MICROCHECKER. SNP were selected that were near each MS marker and the program BEAGLE was used to identify genotypes phase. Subsequently, were applied the MS imputation technique from SNP haplotype and paternity was verified by CERVUS. The accuracy of MS alleles imputation was verified by calculating the correlation between MS alleles imputed and reported. The SPS115 marker was removed from the analysis for null alleles evidence due to homozygote excess observed. The most informative marker was TGLA122 with 0.8 PIC. Deviations from equilibrium HW (P<0.05) were found for the loci ETH225 and TGLA57. A larger set of SNP was necessary to impute MS alleles for the marker BM1824. The verification rates of paternity were 97.1% for genotyped MS alleles and 96.3% for MS imputed. Using imputed MS alleles and when only the sire was considered only 4% of the 99 offspring were not assigned paternity and 1% when both parents were known. The technique achieved greater than 96% accuracy for MS imputation data. This research allow to impute multi-allelic genotypes from bi-allelic data. Our results will have an immediate impact for researchers and livestock associations aiming the transition from MS- to SNP-based parentage verification.

The replacement of synthetic polymers with renewable content in emulsion polymerization latexes has been a focus of research over the past several decades. Emulsion polymerization is a more sustainable way to produce polymers for films and resins. Starch is a sustainably sourced material that has proven to be extremely useful as a filler, comonomer, and property modifier for polymer latexes. In this thesis, we attempt to incorporate high levels of starch materials into emulsion latex using waxy and dent sourced (cheaper) vinyl-functionalized regenerated starch nanoparticles (RSNPs). When fed as a batch charge, incorporation (by reaction as opposed to blending) of a grade of waxy RSNPs with 3 wt.% polymerizable sugar-based monomer (PSBM) and medium hydrophobicity (S-3-M) into the polymer matrix was 0-10 wt.% for a 15 wt.% RSNP loaded, 40 wt.% solids latex. Semi-batch feeding of the S-3-M RSNPs resulted in stable latex with the highest loadings of 40 and 50 wt.% (40 wt.% solids) with 0-10 wt.% RSNP incorporation into the synthetic particles, while 40 wt.% loading (20 wt.% incorporation) was achieved with a grade of waxy RSNPs with 6 wt.% PSBM and medium hydrophobicity (S-6-M). Strategies were developed to prepare synthetic latexes with high RSNP loadings and moderate incorporation. Dent sourced RSNPs proved difficult to use in emulsion formulations due in part to the higher percentage of water-soluble linear amylose in the nanoparticles. To reduce the chances of coagulation and minimize the viscosity of the final latex it was important to ensure monomer starved conditions, that the only initiator feed occurred at the seed stage of the reaction (to degrade the soluble starch and prevent later stage coagulation), and that a hydrophobic tie-layer was used to assist in removal of soluble starch from the water phase. Although a successful procedure was devised for creating a dent sourced RSNP loaded latex with a viscosity of 250 cp, it was significantly longer than the procedures used with waxy RSNPs (6 h polymerization + 1 h RSNP dispersion). An additional treatment of the RSNP dispersion prior to the polymerization can lower the final latex viscosity to 100 cp. Higher incorporation of vinyl-functionalized RSNPs may be achievable if the covalently bonded PSBM functional groups are resistant to hydrolysis, the amylose and other small MW starches are completely removed from the water phase, and monomers with more appropriate reactivity and hydrophobicity are employed. To this end, maleic and methacrylic anhydride modified RSNPs were prepared and tested in emulsion polymerizations. The maleic anhydride modified RSNPs were successfully loaded into an emulsion latex at 15 wt.% (40 wt.% solids content) resulting in 20-30 wt.% incorporation when utilizing 2-ethylhexyl acrylate as the tie-layer monomer with 2-ethylhexyl acrylate or butyl acrylate/methyl methacrylate/acrylic acid as the shell layer. This work presents the only comprehensive attempt to incorporate RSNPs into synthetic latexes at high loadings and solids with persulfate initiation without the need to severely reduce the RSNP molecular weight. The learning generated provides a framework for continuing research into increasing the incorporation of RSNPs into polymer particles.

Deficiência intelectual é uma condição heterogênea e complexa, diagnosticada em 1-3% da população mundial. Desequilíbrios cromossômicos e variações no número de cópias (CNVs) são as causas mais frequentes de DI e, até recentemente, a maior parte desse desequilíbrio era averiguado por análises citogenéticas convencionais. Antes da utilização de microarrays cromossômicos (CMA), a causa etiológica da DI ainda permanecia desconhecida em ~60% dos pacientes. A aplicação de CMA tem revolucionado o diagnóstico da DI e de muitas outras doenças congênitas, permitindo explicar a etiologia molecular de parte da DI através da identificação de CNVs patogênicas. Nos países desenvolvidos, CMA é considerado como primeiro teste para avaliar pacientes com múltiplas anomalias congênitas, DI e/ou autismo. Contudo, nos países em desenvolvimento, a detecção de alterações ainda é feita principalmente por métodos citogenéticos convencionais. O objetivo desse estudo foi identificar, através do uso de SNP arrays, o espectro de anomalias cromossômicas presente em uma amostra de 40 pacientes com DI idiopática moderada e grave, apresentando ou não aspectos dismórficos e anomalias congênitas. Em especial, essa coorte de pacientes, em sua maioria (~2/3), não havia sido previamente cariotipada. Embora mundialmente desde 2010 a recomendação seja de realizar arrays antes de cariótipo, a maioria dos pacientes relatados em estudos já havia sido cariotipada antes de array ser oferecido a eles como teste. Foram identificadas alterações raras em 18 pacientes (45%). Em 12 (30%) desses Pacientes, as CNVs eram sabidamente patogênicas; esta taxa diagnóstica está muito acima da taxa de detecção reportada na literatura (~20%) e possíveis causas desta discrepância são discutidas. Outros 6 Pacientes (15%) apresentaram variantes raras de significado incerto (variants of unknown significance - VUS). Um aspecto adicional investigado foram os mecanismos envolvidos na formação de alguns dos rearranjos estruturais; enquanto nosso foco inicial era o uso de arrays para detecção de CNVs, se tornou evidente no decorrer do projeto que o padrão dos SNPs obtido nos arrays revelava, a partir do DNA, informação valiosa sobre a estrutura dos cromossomos e a composição heterogênea de células em uma amostra (mosaicismo). Esses resultados são discutidos em detalhes em duas situações: (1) A descrição de uma deleção terminal 1p36, associada a dissomia uniparental (UPD) em mosaico de segmentos de 1pter de diferentes tamanhos. Sugerimos que essa composição reflita eventos recorrentes de captura de telômero, embora processo similar nunca tenha sido descrito, e propomos um possível mecanismo responsável por originar esse desequilíbrio complexo. (2) Três dos nossos pacientes apresentam 4 cópias ou uma combinação de 3-4 cópias de segmentos proximais, na maior parte superpostos, de 15q11q13. Possíveis mecanismos de origem desses rearranjos são discutidos / Intellectual disability (ID) is a complex and heterogeneous condition affecting about 1-3% of the general population. Chromosomal imbalances and copy-number variations (CNVs) have been recognized as the most frequent causes of ID and, until recently, most of these imbalances were diagnosed by cytogenetic analysis. Before the application of microarray analysis (CMA), the underlying cause of ID remains unknown in ~60% of patients. The use of CMA has revolutionized the diagnosis of ID and several other congenital disorders, and have made it possible to identify pathogenic CNVs that could explain the molecular etiology of ID. In developed countries, CMA is considered the first-tier technique for the analysis of patients with multiple congenital anomalies, ID, and/or autism spectrum disorders. However, in developing nations, detection of alterations is still performed mainly by conventional cytogenetic techniques. The aim of this study was identifying, using a high-density resolution SNP microarray, chromosomal imbalances in a total of 40 patients presented with moderate-to-severe ID, associated or not with dysmorphic features and congenital anomalies. Particularly, most of the patients in the cohort (~2/3) was not karyotyped previously. Although CMA has been recommended as the first-tier test since 2010 all over the world, the majority of the patients in the reported studies were karyotyped before CMA was offered as a diagnostic test. Rare CNVs were detected in 18 patients (45%). Among those patients, 12 (30%) carried pathogenic CNVs. This yield is much higher than reported in the literature (~20%), and possible causes for this discrepancy are discussed. Six patients (15%) carried variant of unknown significance (VUS). Furthermore, mechanisms involved in structural rearrangements found in some patients were investigated. Even though the main focus of this dissertation was the detection of CNVs using high resolution SNP arrays, throughout the course of this project it was clear that the SNP patterns found could reveal crucial information about the structure of chromosomes and the heterogeneous composition of cells (mosaicism). Those results are discussed in detail in two situations: (1) One description of a terminal 1p36 deletion, associated with mosaic uniparental disomy (UPD) of different sized 1pter segments. We hypothesized that this composition reflects recurrent telomere capture events, although a similar process has never been described so far, and proposed a possible mechanism responsible for originating this complex imbalance. (2) Three of our patients carried four copies or a four-three copies-combination of a proximal, partially overlapping, 15q11q13 segment. Possible mechanisms responsible for this complex rearrangement are discussed

Submitted by RAFAEL ESPIGOLAN (espigolan@yahoo.com.br) on 2017-03-17T22:04:14Z No. of bitstreams: 1 Tese_Rafael_Espigolan.pdf: 1532864 bytes, checksum: c79ad7471b25137c47529f25762a83a2 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-03-22T12:50:50Z (GMT) No. of bitstreams: 1 espigolan_r_dr_jabo.pdf: 1532864 bytes, checksum: c79ad7471b25137c47529f25762a83a2 (MD5) / Made available in DSpace on 2017-03-22T12:50:50Z (GMT). No. of bitstreams: 1 espigolan_r_dr_jabo.pdf: 1532864 bytes, checksum: c79ad7471b25137c47529f25762a83a2 (MD5) Previous issue date: 2017-02-23 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O melhoramento genético animal visa melhorar a produtividade econômica das futuras gerações de espécies domésticas por meio da seleção. A maioria das características de interesse econômico na pecuária é de expressão quantitativa e complexa, isto é, são influenciadas por vários genes e afetadas por fatores ambientais. As análises estatísticas de informações de fenótipo e pedigree permite estimar os valores genéticos dos candidatos à seleção com base no modelo infinitesimal. Uma grande quantidade de dados genômicos está atualmente disponível para a identificação e seleção de indivíduos geneticamente superiores com o potencial de aumentar a acurácia de predição dos valores genéticos e, portanto, a eficiência dos programas de melhoramento genético animal. Vários estudos têm sido conduzidos com o objetivo de identificar metodologias apropriadas para raças e características específicas, o que resultará em estimativas de valores genéticos genômicos (GEBVs) mais acurados. Portanto, o objetivo deste estudo foi verificar a possibilidade de aplicação de modelos semiparamétricos para a seleção genômica e comparar a habilidade de predição com os modelos paramétricos para dados reais (características de carcaça, qualidade da carne, crescimento e reprodutiva) e simulados. As informações fenotípicas e de pedigree utilizadas foram fornecidas por onze fazendas pertencentes a quatro programas de melhoramento genético animal. Para as características de carcaça e qualidade da carne, o banco de dados continha 3.643 registros para área de olho de lombo (REA), 3.619 registros para espessura de gordura (BFT), 3.670 registros para maciez da carne (TEN) e 3.378 observações para peso de carcaça quente (HCW). Um total de 825.364 registros para peso ao sobreano (YW) e 166.398 para idade ao primeiro parto (AFC) foi utilizado para as características de crescimento e reprodutiva. Genótipos de 2.710, 2.656, 2.749, 2.495, 4.455 e 1.760 animais para REA, BFT, TEN, HCW, YW e AFC foram disponibilizados, respectivamente. Após o controle de qualidade, restaram dados de, aproximadamente, 450.000 polimorfismos de base única (SNP). Os modelos de análise utilizados foram BLUP genômico (GBLUP), single-step GBLUP (ssGBLUP), Bayesian LASSO (BL) e as abordagens semiparamétricas Reproducing Kernel Hilbert Spaces (RKHS) e Kernel Averaging (KA). Para cada característica foi realizada uma validação cruzada composta por cinco “folds” e replicada aleatoriamente trinta vezes. Os modelos estatísticos foram comparados em termos do erro do quadrado médio (MSE) e acurácia de predição (ACC). Os valores de ACC variaram de 0,39 a 0,40 (REA), 0,38 a 0,41 (BFT), 0,23 a 0,28 (TEN), 0,33 a 0,35 (HCW), 0,36 a 0,51 (YW) e 0,49 a 0,56 (AFC). Para todas as características, os modelos GBLUP e BL apresentaram acurácias de predição similares. Para REA, BFT e HCW, todos os modelos apresentaram ACC similares, entretanto a regressão RKHS obteve o melhor ajuste comparado ao KA. Para características com maior quantidade de registros fenotípicos comparada ao número de animais genotipados (YW e AFC) o modelo ssGBLUP é indicado. Considerando o desempenho geral, para todas as características estudadas, a regressão RKHS é, particularmente, uma alternativa interessante para a aplicação na seleção genômica, especialmente para características de baixa herdabilidade. No estudo de simulação, genótipos, pedigree e fenótipos para quatro características (A, B, C e D) foram simulados utilizando valores de herdabilidade baseados nos obtidos com os dados reais (0,09, 0,12, 0,36 e 0,39 para cada característica, respectivamente). O genoma simulado consistiu de 735.293 marcadores e 1.000 QTLs distribuídos aleatoriamente por 29 pares de autossomos, com comprimento variando de 40 a 146 centimorgans (cM), totalizando 2.333 cM. Assumiu-se que os QTLs explicavam 100% da variação genética. Considerando as frequências do alelo menor maiores ou iguais a 0,01, um total de 430.000 marcadores foram selecionados aleatoriamente. Os fenótipos foram obtidos pela soma dos resíduos (aleatoriamente amostrados de uma distribuição normal com média igual a zero) aos valores genéticos verdadeiros, e todo o processo de simulação foi replicado 10 vezes. A ACC foi calculada por meio da correlação entre o valor genético genômico estimado e o valor genético verdadeiro, simulados da 12a a 15a geração. A média do desequilíbrio de ligação, medido entre os pares de marcadores adjacentes para todas as características simuladas foi de 0,21 para as gerações recentes (12a, 13a e 14a), e 0,22 para a 15a geração. A ACC para as características simuladas A, B, C e D variou de 0,43 a 0,44, 0,47 a 0,48, 0,80 a 0,82 e 0,72 a 0,73, respectivamente. Diferentes metodologias de seleção genômica implementadas neste estudo mostraram valores similares de acurácia de predição, e o método mais adequado é dependente da característica explorada. Em geral, as regressões RKHS obtiveram melhor desempenho em termos de ACC com menor valor de MSE em comparação com os outros modelos. / Animal breeding aims to improve economic productivity of future generations of domestic species through selection. Most of the traits of economic interest in livestock have a complex and quantitative expression i.e. are influenced by a large number of genes and affected by environmental factors. Statistical analysis of phenotypes and pedigree information allows estimating the breeding values of the selection candidates based on infinitesimal model. A large amount of genomic data is now available for the identification and selection of genetically superior individuals with the potential to increase the accuracy of prediction of genetic values and thus, the efficiency of animal breeding programs. Numerous studies have been conducted in order to identify appropriate methodologies to specific breeds and traits, which will result in more accurate genomic estimated breeding values (GEBVs). Therefore, the objective of this study was to verify the possibility of applying semi-parametric models for genomic selection and to compare their ability of prediction with those of parametric models for real (carcass, meat quality, growth and reproductive traits) and simulated data. The phenotypic and pedigree information used were provided by farms belonging to four animal breeding programs which represent eleven farms. For carcass and meat quality traits, the data set contained 3,643 records for rib eye area (REA), 3,619 records for backfat thickness (BFT), 3,670 records for meat tenderness (TEN) and 3,378 observations for hot carcass weight (HCW). A total of 825,364 records for yearling weight (YW) and 166,398 for age at first calving (AFC) were used as growth and reproductive traits of Nelore cattle. Genotypes of 2,710, 2,656, 2,749, 2,495, 4,455 and 1,760 animals were available for REA, BFT, TEN, HCW, YW and AFC, respectively. After quality control, approximately 450,000 single nucleotide polymorphisms (SNP) remained. Methods of analysis were genomic BLUP (GBLUP), single-step GBLUP (ssGBLUP), Bayesian LASSO (BL) and the semi-parametric approaches Reproducing Kernel Hilbert Spaces (RKHS) regression and Kernel Averaging (KA). A five-fold cross-validation with thirty random replicates was carried out and models were compared in terms of their prediction mean squared error (MSE) and accuracy of prediction (ACC). The ACC ranged from 0.39 to 0.40 (REA), 0.38 to 0.41 (BFT), 0.23 to 0.28 (TEN), 0.33 to 0.35 (HCW), 0.36 to 0.51 (YW) and 0.49 to 0.56 (AFC). For all traits, the GBLUP and BL models showed very similar prediction accuracies. For REA, BFT and HCW, models provided similar prediction accuracies, however RKHS regression had the best fit across traits considering multiple-step models and compared to KA. For traits which have a higher number of animals with phenotypes compared to the number of those with genotypes (YW and AFC), the ssGBLUP is indicated. Judged by overall performance, across all traits, the RKHS regression is particularly appealing for application in genomic selection, especially for low heritability traits. Simulated genotypes, pedigree, and phenotypes for four traits A, B, C and D were obtained using heritabilities based on real data (0.09, 0.12, 0.36 and 0.39 for each trait, respectively). The simulated genome consisted of 735,293 markers and 1,000 QTLs randomly distributed over 29 pairs of autosomes, with length varying from 40 to 146 centimorgans (cM), totaling 2,333 cM. It was assumed that QTLs explained 100% of genetic variance. Considering Minor Allele Frequencies greater or equal to 0.01, a total of 430,000 markers were randomly selected. The phenotypes were generated by adding residuals, randomly drawn from a normal distribution with mean equal to zero, to the true breeding values and all simulation process was replicated 10 times. ACC was quantified using correlations between the predicted genomic breeding value and true breeding values simulated for the generations of 12 to 15. The average linkage disequilibrium, measured between pairs of adjacent markers for all simulated traits was 0.21 for recent generations (12, 13 and 14), and 0.22 for generation 15. The ACC for simulated traits A, B, C and D ranged from 0.43 to 0.44, 0.47 to 0.48, 0.80 to 0.82 and 0.72 to 0.73, respectively. Different genomic selection methodologies implemented in this study showed similar accuracies of prediction, and the optimal method was sometimes trait dependent. In general, RKHS regressions were preferable in terms of ACC and provided smallest MSE estimates compared to other models. / FAPESP: 2014/00779-0 / FAPESP: 2015/13084-3

The publication of a draft equine genome sequence and the release by Illumina of a 50,000 marker single-nucleotide polymorphism (SNP) genotyping chip has provided equine researchers with the opportunity to use new approaches to study the relationships between genotype and phenotype. In particular, it is hoped that the use of high-density markers applied to population samples will enable progress to be made with regard to more complex diseases. The first objective of this thesis is to explore the potential for the equine SNP chip to enable such studies to be performed in the horse. The second objective is to investigate the genetic background of osteochondrosis (OC) in the horse. These objectives have been tackled using 348 Thoroughbreds from the US, divided into cases and controls, and a further 836 UK Thoroughbreds, the majority with no phenotype data. All horses had been genotyped with the Illumina Equine SNP50 BeadChip. Linkage disequilibrium (LD) is the non-random association of alleles at neighbouring loci. The reliance of many genomic methodologies on LD between neutral markers and causal variants makes it an important characteristic of genome structure. In this thesis, the genomic data has been used to study the extent of LD in the Thoroughbred and the results considered in terms of genome coverage. Results suggest that the SNP chip offers good coverage of the genome. Published theoretical relationships between LD and historical effective population size (Ne) were exploited to enable accuracy predictions for genome-wide evaluation (GWE) to be made. A subsequent in-depth exploration of this theory cast some doubt on the reliability of this approach in the estimation of Ne, but the general conclusion that the Thoroughbred population has a small Ne which should enable GWE to be carried out efficiently in this population, remains valid. In the course of these studies, possible errors embedded within the current sequence assembly were identified using empirical approaches. Osteochondrosis is a developmental orthopaedic disease which affects the joints of young horses. Osteochondrosis is considered multifactorial in origin with a variety of environmental factors and heredity having been implicated. In this thesis, a genome-wide association study was carried out to identify quantitative trait loci (QTL) associated with OC. A single SNP was found to be significantly associated with OC. The low heritability of OC combined with the apparent lack of major QTL suggests GWE as an alternative approach to tackle this disease. A GWE analysis was carried out on the same dataset but the resulting genomic breeding values had no predictive ability for OC status. This, combined with the small number of significant QTL, indicates a lack of power which could be addressed in the future by increasing sample size. An alternative to genotyping more horses for the 50K SNP chip would be to use a low-density SNP panel and impute remaining genotypes. The final chapter of this thesis examines the feasibility of this approach in the Thoroughbred. Results suggest that genotyping only a subset of samples at high density and the remainder at lower density could be an effective strategy to enable greater progress to be made in the arena of equine genomics. Finally, this thesis provides an outlook on the future for genomics in the horse.

Submitted by Cristiane Chim (cristiane.chim@ucpel.edu.br) on 2017-12-15T11:22:16Z No. of bitstreams: 1 RAFAELY FERREIRA SEVERO.pdf: 1126351 bytes, checksum: 6458d3583e0c5a75240f2e91f00a55c4 (MD5) / Made available in DSpace on 2017-12-15T11:22:16Z (GMT). No. of bitstreams: 1 RAFAELY FERREIRA SEVERO.pdf: 1126351 bytes, checksum: 6458d3583e0c5a75240f2e91f00a55c4 (MD5) Previous issue date: 2017-08-29 / Oral cancer is a multifactorial chronic disease and it is generally accepted that oral cancer development may be preceded by oral potentially malignant lesions (OPML). Studies have reported an increased risk for OPML development, associated with smoking and drinking alcohol, where genetic studies are being carried out in order to uncover the variety of phenotypes related to habits of dependence, which may occur due to genetic polymorphisms. The serotonin or 5-hydroxytryptamine (5-HT) molecule regulates some of the brains functions by inhibiting or stimulating the GABA system; and thus it is able to regulate alcoholism and smoking habits. In this study, we investigated the T102C polymorphism at the 5HT2A receptor in oral dysplasia in OPML and their association with smoking and alcohol habits. This case-control study included patients with OPML histopathologically diagnosed with dysplasia and within this group, patients with and without smoking and alcohol consumption habits. Cell samples from the oral lesions were collected with the patients previously anesthetized using disposable cytological brushes. DNA extraction was performed and the polymorphism was genotyped in real-time PCR allelic discrimination assays. A total of 109 individuals were included in this study (37 with dysplasia and 72 controls). The genotype (p=0.016), allele (p=0.020) and smoking habits (<0.001) distribution differed significantly between dysplasia and control group, where the CT and TT genotype, and the T allele showed a higher frequency in dysplasia (65.6, 18.8 and 84.4%, respectively) than in controls (55.7, 4.9 and 60.7%). In regard to smoking habits the higher frequency was in the dysplasia group. In the multivariate logistic regression analysis associating variables of interest and the presence of dysplasia showed that individuals with smoking habits present 7.58 increase risk to develop dysplasia then non-smokers; and individual carrying the T allele for the T102C polymorphism have a 4.6 increase risk to develop oral dysplasia in OPML. Overall the results in our study indicated a relationship between smoking habits and oral dysplasia in OPML. In addition our study indicated, for the first time, a possible relationship between the T102C polymorphism and oral dysplasia in OPML. Keywords: oral cancer, oral potentially malignant lesions, rs6313, serotonin. / O câncer bucal é uma doença crônica multifatorial e é amplamente aceito que o desenvolvimento de câncer oral pode ser precedido por lesões orais potencialmente malignas (LOPM). Estudos têm relatado um risco aumentado para o desenvolvimento de LOPM, associado com o hábito de fumar e beber álcool, onde estudos genéticos estão sendo realizados a fim de descobrir a variedade de fenótipos relacionados aos hábitos de dependência, que pode ocorrer devido a polimorfismos genéticos. A molécula de serotonina ou 5-hidroxitriptamina (5- HT) regula algumas das funções do cérebro, inibindo ou estimulando o sistema GABA; e assim é capaz de regular os hábitos de alcoolismo e o tabagismo. Neste estudo investigamos o polimorfismo T102C no receptor 5HT2A (rs6313) em displasia oral em LOPM e a sua associação com hábitos de fumo e álcool. Este estudo de caso-controle incluiu pacientes com LOPM histopatologicamente diagnosticados com displasia avaliando hábitos de consumo de tabaco e álcool. As amostras de células das lesões orais foram coletadas com os pacientes previamente anestesiados usando escovas citológicas descartáveis. A extração de DNA foi realizada e o polimorfismo foi genotipado em ensaios de discriminação alélica de PCR em tempo real. Um total de 109 indivíduos foram incluídos neste estudo (37 com displasia e 72 controles). A distribuição do genótipo (p=0,016), alelos (p=0.020) e o hábito de fumar (<0.001) diferiram significativamente entre os grupos de displasia e controle, onde o genótipo CT e TT e o alelo T mostraram maior frequência na displasia (65,6, 18,8 e 84,4%, respectivamente) do que nos controles (55,7, 4,9 e 60,7%). No que se refere aos hábitos tabagistas a maior frequência foi no grupo de displasia. Na análise de regressão logística multivariada associando as variáveis de interesse e a presença de displasia mostrou que indivíduos com hábitos tabagistas apresentam um risco 7,58 vezes maior de desenvolver displasia do que não fumantes; e individuais carregando o alelo T para o polimorfismo T102C tem um risco 4,6 vezes maior de desenvolver displasia oral em LOPM. Em geral os resultados do nosso estudo indicaram uma relação entre hábitos tabagistas e a displasia oral em LOPM. Além disso, nosso estudo indicou, pela primeira vez, uma possível relação entre o polimorfismo T102C e a displasia oral em LOPM.

Fusarium head blight (FHB) is a serious disease of wheat (Triticum aestivum) and other small grains; disease severity is affected by temperature and rainfall. This research comprised three studies: an artificially warmed experiment during 2016-2017, a morphology study and an FHB resistance screening study in 2015-2016, using approximately 250 wheat cultivars and breeding lines from programs in the eastern US. The location was the University of Kentucky Spindletop Research Farm in Lexington, KY. Higher levels of Fusarium damaged kernels and the toxin deoxynivalenol (DON) were observed in the warmed treatment compared to the control, and plant development was accelerated. In the FHB resistance screen, significant (p < 0.05) genotype differences for all traits were observed. A GWAS identified 16 SNPs associated with resistance and susceptibility, ranging from -2.14 to 4.01%. Three DON-associated SNPs reduced toxin levels by 3.2, 2.1, and 1.5 ppm. In the morphology study, negative correlations were observed among morphological and disease traits. Small effect SNPs were identified for all morphological traits, which might be useful in genomic selection; traits like spike length, spikelet number and inclination could be used in phenotyping. Response to warming indicates that existing resistance sources may be less effective in a warming climate.

La vigne est soumise à de nombreuses maladies impliquant l'utilisation de produits phytosanitaires en grande quantité dont l'utilisation est néfaste pour l'environnement et la santé des utilisateurs. Un enjeu est donc de développer des méthodes alternatives à la lutte chimique. La protéine codée par le gène NPR1 (Nonexpressor of pathogenesis-related gene 1) joue un rôle clef dans la résistance à large spectre chez les plantes. Des éliciteurs tels que l'acide salicylique ou des agents pathogènes influencent l'activation de NPR1 dans le cytoplasme. La translocation de NPRl dans le noyau et son interaction avec des facteurs de transcription TGA induit l'expression des gênes PR (Pathogenesis-related). Nous avons identifié sept homologues potentiels des gènes NPR1 et TGA chez Vitis vinifera (VvNPR1.1, VvNPR1.2, VvTGA1 à 5). L'étude de la diversité de séquences dans les exons de 15 accessions de Vitaceae indique qu'ils sont soumis à une forte pression de sélection purificatrice. De plus, l'analyse in silico des régions promotrices des VvNPR1 montre la présence, d'éléments cis-régulateurs potentiels, en réponse aux stress biotiques et abiotiques ainsi que des motifs de liaison à des facteurs de transcription. Une étude plus poussée des introns montre quelques éléments transposables et un faible polymorphisme dans six accessions de Vitis vinifera. Ces résultats argumentent en faveur d'une pression de sélection forte agissant sur ces gènes. Ceci nous a mené à formuler des hypothèses fonctionnelles et à réaliser une étude d'interaction avec les facteurs de transcription VvTGA1 et VvTGA4 par la technique du double hybride. Ces derniers n'interagissent pas avec VvNPR 1.1. / Numerous diseases affect grapevine, resulting in the use of phytochemicals in large quantities that are harmful for environment and user's health. In the long term, the aim is to develop alternative methods to chemicals. The protein encoded by NPR1 (Nonexpressor of pathogenesis-related gene 1) plays a pivotal role in conferring broad spectrum pathogen resistance in plants. Activation of NPR 1 in the cytoplasm is influenced by elicitors such as salicylic acid or pathogens associated with the accumulation of reactive oxygen species. Translocation of NPR1 into the nucleus and interaction with TGA transcription factors induce the expression of PR (Pathogenesis-related) genes. Using a candidate gene approach, we have identified seven putative homologs to NPR1 and TGA in the grapevine genome (VvNPR1.1, VvNPR1.2, VvTGA1 to 5). The study of sequence diversity in exons of 15 accessions of the Vitaceae family indicates that these exons are subjected to a strong purifying selection pressure. Moreover, in silico analysis in the promoters of VvNPR1 shows putative cis-regulator elements, in answer to biotic and abiotic stresses as well as link patterns to transcription factors. An intron study shows transposable elements and a low polymorphism in six accessions of Vitis vinifera. These results suggest a strong selection pressure on these genes. Functional hypotheses were formulated, and an interaction study with transcription factors VvTGA1 and VvTGA4 was conducted using a method based on yeast two hybrid, showing that they do not interact with VvNPR1.1.

Die Arbeitsgruppe Zimmer am Institut für Klinische Biochemie und Pathobiochemie der Universität Würzburg detektierte im Gen der Dihydrolipoamid-Dehydrogenase (DLD) eine bisher nicht bekannte Mutation, die für die Entwicklung einer familiären Form der dilatativen Kardiomyopathie (DCM) verantwortlich ist. Die DLD spielt als Teil von mitochondrialen Enzymkomplexen eine wichtige Rolle im Energie- und Aminosäurestoffwechsel der Zelle. Mutationen im DLD-Gen führen dabei meist zu neurologischen Syndromen mit Erscheinungsbildern wie mentaler Entwicklungsverzögerung, Krampfanfällen und spastischen Bewegungsstörungen. Fälle von Herzinsuffizienz und frühkindlicher Hypertropher Kardiomyopathie wurden ebenfalls beschrieben. Von den zahlreichen Gendefekten, die als Auslöser für die dilatative Kardiomyopathie bekannt sind, ist bisher noch keiner im Gen der DLD beschrieben worden. Vielmehr sind DCM-Mutationen in Genen zu finden, die für muskelspezifische Proteine kodieren. Dadurch führen sie oft zu einer Beeinflussung der Kraftübertragung im Sarkomer. Die durch die Arbeitsgruppe Zimmer beschriebene DLD-Mutation wurde in einer portugiesischen Großfamilie entdeckt, in welcher das Auftreten der dilatativen Kardiomyopathie über mehrere Generationen hinweg zu verfolgen ist. Ähnliche Fälle außerhalb dieser Familie sind nicht bekannt. Demnach gibt es keine Daten, die die Häufigkeit DCM-assoziierter Mutationen im Gen der Dihydrolipoamid-Dehydrogenase beschreiben. Die vorliegende Arbeit beschäftigte sich damit weitere Zusammenhänge zwischen genetischen Alterationen im DLD-Gen und dem Auftreten einer DCM aufzudecken. In diesem Rahmen wurden DCM-Patienten, die erwiesenermaßen an einer familiären Form dieser Erkrankung leiden, gezielt auf Veränderungen in den Exons des DLD-Gens untersucht. Insgesamt wurden die Exons von 88 Patienten auf das Vorhandensein heterozygoter Mutationen überprüft. Hierfür wurden PCR-Produkte, die die jeweiligen Exons enthielten, mit Hilfe der Denaturing High-Performance Liquid Chromatography (DHPLC) untersucht. Diese Methode ermöglicht eine hochsensitive und gleichermaßen äußerst spezifische Detektion heterozygoter Mutationen. Auffällige Ergebnisse wurden anschließend mittels Sequenzierung verifiziert. Insgesamt wurden bei elf Patienten fünf unterschiedliche Mutationen nachgewiesen. Es handelte sich um vier bereits bekannte Einzelnukleotid-Polymorphismen und eine bisher nicht beschriebene Mutation. Dabei lagen vier Mutationen in nicht näher bezeichneten Intron-Bereichen, eine Mutation in einer 3‘ Spleißstelle und eine weitere Mutation in der 3‘ UTR (untranslated region) der mRNA. Somit befanden sich also einige Mutationen an für die Regulation der Genfunktion strategisch wichtigen Positionen. Da es sich dabei um bekannte Polymorphismen handelte, wurde mit Hilfe der Daten des HapMap Projekts überprüft, ob es bereits Hinweise für eine klinische Assoziation gab. Die Daten zeigten, dass bisher keiner der hier detektierten SNPs mit klinischen Erscheinungsbildern in Verbindung gebracht werden konnte. Es gab auch keine Hinweise dafür, dass die erfassten SNPs im Patientenkollektiv häufiger vorkamen als in der Normalbevölkerung. Einer der hier beschriebenen Mutationen war nicht in den verwendeten Datenbanken aufgeführt. Daher kann ein pathologischer Einfluss dieser Mutation zwar nicht ausgeschlossen werden, erscheint aber aufgrund ihrer Lage in einem weit vom Exon entfernten Intron-Bereich nicht offensichtlich. Es ließen sich also für keine der detektierten Mutationen pathogene Eigenschaften nachweisen. In Protein-kodierenden Sequenzbereichen konnten keine Mutationen nachgewiesen werden. Abschließend lässt sich also sagen, dass eine Assoziation zwischen Mutationen im DLD-Gen und dem Auftreten einer familiären DCM im Rahmen dieser Arbeit nicht bestätigt werden konnte. Es ist jedoch möglich, dass DCM-assoziierte Mutationen im DLD-Gen nur äußerst selten auftreten oder aber die durch die Arbeitsgruppe Zimmer detektierte Mutation im DLD-Gen die bisher einzige ist, die mit DCM in Verbindung gebracht werden kann. Um diese Frage zu klären müssen Untersuchungen mit größeren Patientenkollektiven angeschlossen werden. / The working group Zimmer at the Institute of Clinical Biochemistry and Pathobiochemistry at the University of Wuerzburg detected an until now unknown mutation in the gene of dihydrolipoamide dehydrogenase (DLD), which causes a familial dilated cardiomyopathy (DCM). The DLD plays an important role in the metabolism of carbohydrates and amino acids. In most cases mutations in the DLD gene lead to neurological symptoms like mental developmental delay, seizures and spastic movement disorders. Cases of heart failure and an early childhood hypertrophic cardiomyopathy are also known. Among the numerous genetic defects, which are known to be responsible for the development of a dilated cardiomyopathy, there is no one known in the gene of DLD. To a greater degree DCM mutations are found in genes, which encode for muscle-specific proteins. In this way they often lead to an affectation of the force transmission in the sarcomer. The mutation, which was described by the working group Zimmer was found in an portuguese extended familiy. In this family the presence of a dilated cardiomyopathy could be followed up for several generations. Similar cases outside of this family are not known. So there is no data about the frequency of DCM associated mutations in the gene of dihydrolipoamide dehydrogenase. The work in hand dealt with revealing further relationships between genetic variances in the DLD gene and the presence of DCM. In this context the exons of the DLD gene of DCM patients with a familial form of this disease were examined. Overall the DLD exons of 88 patients were investigated looking for heterozygous mutations. For this purpose the Denaturing High-Performance Liquid Chromatography was used. This method allows a highly sensitive and also exceedingly specific detection of heterozygous mutations. Afterwards conspicuous results were verified by sequencing. Altogether five different mutations in eleven patients were found. It was about four already known single nucleotide polymorphisms and one until know not known mutation. Four mutations were located in not further described intronic regions. One was found in a 3' splice site and one in a 3' UTR. In this way some mutations were located in regions which are important for the regulation of gene function. Because it was about known SNPs, it was checked if there is evidence for associations with clinical phenotypes. Therefor the data of the HapMap project was used. The data shows that no one of the detected SNPs is known to be associated with clinical phenotypes. In protein coding sequences no mutation was detected. Conclusively it can be said that within this work an association between mutations in the DLD gene and the presence of a familial DCM could not be confirmed. It is possible that mutations in the DLD gene associated with DCM are very rare or unique in this family. To reason this out, investigations with greater patient populations are necessary.

The emergence of MNAzymes (Multi-component nucleic acid enzymes) provides a new approach for detection of target analytes in various applications. In this thesis, three novel MNAzyme-based methodologies were developed to expand the range of the applications of MNAzymes. MNAzymes can be coupled with DNA or RNA ligands called aptamers to generate an apta-MNAzyme system, which can be used for the detection of non-nucleic target analytes such as small molecules and proteins. Direct detection using apta-MNAzyme system is performed in a format, which was isothermal, fluorescent, rapid, and requires no protein enzymes. Apta-MNAzymes can be coupled with a signal amplification cascade to increase the sensitivity of the reaction. Another MNAzyme-based methodology termed truncated MNAzyme arm system was developed to discriminate the presence of a single base mismatch of two closely related sequences. The system employs a partzyme with a truncated sensor arm and a stabiliser oligonucleotide that binds adjacently to the truncated sensor arm to stabilise the active MNAzyme structure. Truncated MNAzyme real-time PCR system is capable of discriminating the presence of a single base mismatch in a target DNA with high specificity and sensitivity (down to approximately 10 gene copies). The generic nature of the system enables simultaneous detection of three SNP targets in a multiplex format. MNAzymes was also investigated with various strategies to discriminate DNA sequences that are either methylated or unmethylated. In this thesis, bisulphite-treated DNA samples present in as low as 0.032 % of methylated DNA in a background of unmethylated DNA were discriminated using MNAzyme real-time methylation specific PCR (MSP) system. Furthermore, the presence of 5-methylcytosines in a target sequence increases the melting temperature of the duplex DNA. This was exploited further to directly discriminate DNA methylation status of target sequences using the truncated MNAzyme arm system without the need for bisulphite modification. Findings in this thesis have broadened the scope of MNAzymes as versatile tools for many possible applications and flexible alternative to the current technologies.