Tracking seed dispersal using traditional, direct measurement approaches is difficult and generally underestimates dispersal distances. Variation in chloroplast haplotypes (cpDNA) offers a way to trace past seed dispersal and to make inferences about factors contributing to present patterns of dispersal. Although cpDNA generally has low levels of intraspecific variation, this can be overcome by assaying the whole chloroplast genome. Whole-genome sequencing is more expensive, but resources can be conserved by pooling samples. Unfortunately, haplotype associations among SNPs are lost in pooled samples and treating SNP frequencies as independent estimates of variation provides biased estimates of genetic distance. I have developed an application, CallHap, that uses a least-squares algorithm to evaluate the fit between observed and predicted SNP frequencies from pooled samples based on network topology, thus enabling pooling for chloroplast sequencing for large-scale studies of chloroplast genomic variation. This method was tested using artificially-constructed test networks and pools, and pooled samples of Lasthenia californica (California goldfields) from Whetstone Prairie, in Southern Oregon, USA. In test networks, CallHap reliably recovered network topologies and haplotype frequencies. Overall, the CallHap pipeline allows for the efficient use of resources for estimation of genetic distance for studies using non-recombining, whole-genome haplotypes, such as intra-specific variation in chloroplast, mitochondrial, bacterial, or viral DNA.

<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>

Genome-wide Association (GWA) studies have become a widely used method for analyzing genetic data. It is useful in detecting associations that may exist between particular alleles and diseases of interest. This thesis investigates the dataset provided from problem 1 of the Genetic Analysis Workshop 16 (GAW 16). The dataset consists of GWA data from the North American Rheumatoid Arthritis Consortium (NARAC). The thesis attempts to determine a set of single nucleotide polymorphisms (SNP) that are associated significantly with rheumatoid arthritis. Moreover, this thesis also attempts to address the question of whether the one-sided alternative hypothesis that the minor allele is positively associated with the disease or the two-sided alternative hypothesis that the genotypes at a locus are associated with the disease is appropriate, or put another way, the question of whether examining both alternative hypotheses yield more information.

<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>

Brassica carinata is being actively pursued as a new industrial oil crop platform for the Canadian Prairies. A genetic assessment of B. carinata was performed to elucidate its evolutionary origins and create a genetic map to assist in locating genes and traits of interest that would help in marker-assisted breeding. First, genetic analysis using simple sequence repeat (SSR) markers, previously tested on B. juncea and B. napus, was performed, to examine the genetic diversity of 37 B. carinata lines. SSR analysis revealed world accessions were more diverse than lines conditioned to grow in the prairies. Diversity analysis revealed that the parental lines of a double haploid (DH) population, 179 and 345, obtained from the John Innes Centre (JIC), were among the more genetically diverse lines, supporting the use of this population for linkage mapping. Genetic markers created from 3’ targeted SNP discovery between 179 and 345, were tested on the DH population resulting in the generation of a B. carinata genetic linkage map essentially with no prior sequence data knowledge. This genetic map contained 341 SNP and 86 SSR loci identifying eight linkage groups belonging to the B genome, nine belonging to the C genome and two unidentified groups spanning 2041 cM. Comparative mapping of polymorphic markers identified in the amphidiploid B. carinata indicated the orientation of B and C genomes coincide with that of other Brassica species, and the two genomes have remained essentially unaltered, with no major chromosomal rearrangements since the formation of B. carinata. A lesser number of polymorphic markers were detected in the C genome, which suggested the B genome is more genetically diverse in B. carinata. Limited field trials of the 179 x 345 DH population were performed during the 2011 and 2012 growing seasons. Preliminary quantitative trait loci (QTLs) for agronomic traits including flowering time (FT), plant height (PH), and seed quality were identified.

This work has explored the capability of a mixed film composed of oligonucleotides and oligomers to improve the selectivity for the detection of fully complementary oligonucleotide targets in comparison to partially complementary targets which have one and three base-pair mismatched sites. The intention was to introduce a “matrix isolation” effect on oligonucleotide probe molecules by surrounding the probes with oligomers, thereby reducing oligonucleotide-to-oligonucleotide and/or oligonucleotide-to-surface interactions. This resulted in a more homogeneous environment for probes, thereby minimizing the dispersity of energetics associated with formation of double-stranded hybrids. The mixed film was constructed by immobilizing pre-synthesized oligonucleotides onto a mixed aminosilane layer and then growing the oligomer portion by surface-initiated atom transfer radical polymerization (ATRP) of 2-hydroxy methacrylate (PHEMA). The performance of the mixed film was compared to films composed of only oligonucleotides in a series of hybridization and melt curve experiments. Surface characterization techniques were used to confirm the growth of the oligomer portion as well as the presence of both oligonucleotides and oligomer components. Polyatomic bismuth cluster ions as sources for time-of-flight secondary ion mass spectrometry experiments could detect both components of the mixed film at a high sensitivity even though the oligomer portion was at least 200-fold in excess. At the various ionic strengths investigated, the mixed films were found to increase the selectivity for fully complementary targets over mismatched targets by increasing the sharpness of melt curves and melting temperature differences (delta Tm) by 2- to 3-fold, and by reducing non-specific adsorption. This resulted in improved resolution between the melt curves of fully and partially complementary targets. A fluorescence lifetime investigation of the Cy3 emission demonstrated that Cy3-labeled oligonucleotide probes experienced a more rigid microenvironment in the mixed films. These experiments demonstrated that a mixed film composed of oligonucleotides and PHEMA can be prepared on silica-based substrates, and that they can improve the selectivity for SNP discrimination compared to conventional oligonucleotide films.

Interleukin-1 alpha (IL-1a) is a powerful inflammatory cytokine that modulates both innate and adaptive immunity. As such, IL-1a is implicated in the development of multiple inflammatory and autoimmune diseases including atherosclerosis, arthritis and cancer. Therefore, understanding the mechanisms that regulate IL-1a activity is extremely important. For many years, pro-IL-1a was considered to be a fully active alarmin. However, we have previously shown that the removal of the pro-domain by calpain, a protease that is activated upon necrosis, significantly increases IL-1a bioactivity. The work presented in this thesis demonstrates that multiple proteases from diverse biological systems cleave and activate IL-1a. We therefore suggest that IL-1a is an important signalling hub that integrates diverse proteolytic danger signals to alert the immune system. In particular we have identified the inflammatory caspase, caspase-5, as a novel and potent activator of IL-1a. We show that caspase-5 directly cleaves pro-IL-1a during the activation of the non-canonical inflammasome by cytosolic LPS, which mimics intracellular bacterial infection. We also demonstrate that caspase-5-cleaved IL-1a mediates the senescence-associated secretory phenotype (SASP), which drives the deleterious effects of senescent cells in multiple age-related diseases. Therefore, therapeutically targeting caspase-5 may be of interest for pathologies mediated by the non-canonical inflammasome and/or senescent cells. Finally we find that rs17561, a common IL1A polymorphism, reduces active IL-1a release. We find that blood from minor allele homozygotes releases significantly less IL-1a than major allele homozygotes upon LPS stimulation. Therefore, genotyping patients under consideration for anti-IL-1a therapy could predict who would be likely to respond well to the treatment. In conclusion, the work presented in this thesis enhances our understanding of how IL-1a activity is regulated. The identification of both the caspase-5-mediated pathway of IL-1a activation and the defect conferred by the rs17561 SNP could have important clinical implications for the treatment of multiple inflammatory diseases.

Orientador: Jose Luiz Donato / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T11:59:27Z (GMT). No. of bitstreams: 1 Roversi_FernandaMarconi_M.pdf: 7284789 bytes, checksum: 9104ecb7671bb8b4b2288503432576a0 (MD5) Previous issue date: 2007 / Resumo: A farmacogenômica utiliza informações genéticas para orientar a escolha da terapia farmacológica em uma base individual, pressupondo que se podem prever diferenças na resposta a agentes terapêuticos entre indivíduos, a partir de sua constituição genética. Os estudos atuais de farmacogenômica objetivam otimizar a eficácia de uma droga e diminuir os efeitos colaterais e a toxicidade. Os estudos de polimorfismos genéticos podem ter um impacto nos ajustes individuais da dose de um determinado fármaco. Os polimorfismos de genes que codificam transportadores de drogas, como o gene MDR1; das enzimas metabolizadoras de fármacos, como a isoforma 2D6 do citocromo P450 (CYP2D6), e de receptores para drogas, relacionam-se às alterações funcionais no fenótipo, contribuindo na resposta a uma droga. O dextromethorphan é um agente antitússico que atua no centro da tosse. Este fármaco é metabolizado, principalmente pela CYP2D6, no metabólito ativo Dextrorphan e sua absorção sofre interferência da proteína codificada pelo gene MDR1. O presente trabalho teve como objetivo a avaliação do genótipo de uma população de voluntários sadios através da análise dos polimorfismos dos exons mais freqüentes (12, 21, e 26) do gene MDR1, correlacionando-se esses polimorfismos com a farmacocinética do fármaco dextromethorphan e do seu principal metabólito dextrorphan. Após uma seleção dos participantes do estudo, feita através de anamnese, exames físico, neurológico, psiquiátrico e laboratorial, e histórico médico, foram realizados experimentos de fenotipagem através da análise quantitativa da droga dextromethorphan e do seu metabólito dextrorphan na urina, em dois períodos distintos (imediatamente após e 12 horas após a administração da droga), por meio de cromatografia líquida de alta eficiência (HPLC) acoplada à espectrometria de massa (LC-MS-MS) e de genotipagem CYP2D6, classificando os voluntários em metabolizadores rápidos ou lentos. Os 27 indivíduos, identificados como metabolizadores rápidos, foram incluídos num protocolo de avaliação farmacocinética dos compostos, ao longo de um período de 48 horas após a administração do xarope dextromethorphan, por meio do método HPLC / LC-MS-MS. A análise da genotipagem MDR1 foi realizada em três etapas sucessivas: extração de DNA, amplificação do gene MDR1 e seqüenciamento automático. Uma vez determinado o genótipo de cada voluntário, uma correlação entre esses dados genotípicos, a farmacocinética do dextromethorphan e algumas características (idade, altura, peso e índice de massa corpórea) foi estabelecida por meio de análise estatística, usando-se o teste Wilcoxon Rank Sum. Os valores obtidos para as Áreas Sob a Curva (AUCs) 0-4 h, 0-24 h, 0-48 h (± DP) foram significativamente mais baixos para os indivíduos 3435TT (1,18 ± 0,05, 2,11 ± 0,83, 2,12 ± 0,83 ngh/ml) em relação aos indivíduos 3435CC (4,49 ± 3,91, 11,07 ± 9,98, 11,33 ± 10,53 ngh/ml) e 3435CT (3,91 ± 3,22, 8,07 ± 7,41, 8,20 ± 7,57 ngh/ml). Os valores referentes a Concentração Máxima (Cmáx) (± DP) também foram significativamente mais baixos nos indivíduos 3435TT (0,45 ± 0,05 ng/ml) em relação aos indivíduos 3435CC (1,74 ± 1,52 ng/ml) e 3435CT (1,52 ± 1,22 ng/ml). Nenhuma diferença significativa foi observada entre os grupos analisados e os fatores idade, peso, altura e IMC. A distribuição genotípica para o exon 26 do gene MDR1 foi 41 % CC, 44 % CT e 15 % TT. Para os exons 21 e 12 desse mesmo gene, essas distribuições genotípicas foram, respectivamente, 56 % GG, 40 % GT, e 4 % TT e 12 % CC, 38 % CT, e 50 % TT. Desse modo, observou-se uma correlação entre o polimorfismo C3435T ¿ exon 26 do gene MDR1 ¿ e alguns parâmetros farmacocinéticos: a Cmáx e as AUCs do fármaco dextromethorphan, após uma única administração oral, apresentaram valores mais baixos nos indivíduos 3435TT. A existência de uma correlação entre a genotipagem e a farmacocinética demonstra que a implementação de técnicas de biologia molecular pode auxiliar alguns testes clínicos, cujo alvo de interesse é a investigação da absorção e da metabolização de drogas / Abstract: Pharmacogenomics is used to study the effects of genetic basis for interindividual differences in drug response. This genetic information can predict and optimize the drug efficacy and/or safety and can minimize the adverse drug reactions and toxicity. Dextromethorphan shares part of the adverse event profile of opioids and is widely used as a probe drug for CYP2D6 phenotyping and for the assessment of its activity. Dextromethorphan is an antitussigen agent that acts in the center of the cough. This drug is mainly metabolized by the CYP2D6 in the active metabolite dextrorphan and its absorption suffers interference from the protein codified for MDR1 gene. In this present study, we investigated the relationship between the MDR1 genotype and the dextromethorphan (DM) pharmacokinetics in 27 healthy Brazilian subjects and determinated the MDR1 genotype frequency in this population. The MDR1 genotype was determined by PCR-DNA sequencing. After single oral administration of 30 mg DM, plasma concentrations of DM and DX were measured and its pharmacokinetic characteristics were compared according to MDR1 genotype. The area under the plasma concentration-time curve 0-4h, 0-24h and 0-48h was significantly lower in subjects with 3435TT (1.18 ± 0.05, 2.11 ± 0.83, 2.12 ± 0.83 ngh/ml) than in those with 3435CC (4.49 ± 3.91, 11.07 ± 9.98, 11.33 ± 10.53 ngh/ml) and 3435CT (3.91 ± 3.22, 8.07 ± 7.41, 8.20 ± 7.57 ngh/ml). The maximum plasma concentrations were lower in subjects with 3435TT (0.45 ± 0.05 ng/ml) than in those with 3435CC (1.74 ± 1.52 ng/ml) and 3435CT (1.52 ± 1.22 ng/ml). There wasn¿t effect of gender or age on the distribution. The distribution of the MDR1 genotype at exon 26, 21 and 12 was, respectively, 41 % CC, 44 % CT, and 15 % TT, 56 % GG, 40 % GT, and 4 % TT and 12 % CC, 38 % CT, and 50 % TT. In conclusion, DM pharmacokinetics was affected by the genetic polymorphisms of the MDR1 gene in healthy Brazilian subjects. These findings may provide a plausible explanation for interindividual variation in the disposition of DM, although our data couldn¿t explain the exact mechanisms by which the polymorphic MDR1 gene paradoxically reduces the plasma levels of DM. Further evaluation is thus warranted. / Mestrado / Mestre em Farmacologia

Em um cenário em que a qualidade da informação é essencial para a sustentação do processo de gestão do sistema ou do processo de seleção animal, corretas atribuições de paternidade se tornam importante para a manutenção das informações de pedigree e, consequentemente, para a estimativa dos valores genéticos dos animais em programas de seleção. Com o advento do uso de marcadores moleculares do tipo SNP na seleção genômica e em diferentes painéis de teste de parentesco, maiores estudos se fazem cada vez mais necessários. Dessa maneira esse estudo buscou analisar os ganhos em acurácia dos valores genéticos para características produtivas em um cenário de seleção real entre avaliações genéticas que continham ou não animais com atribuições de paternidade por teste de DNA e além disso, verificar os possíveis conflitos de seleção entre elas. Como resultados foram encontrados ganhos em acurácia principalmente em animais mais jovens, mas de maneira geral todos os animais foram beneficiados e apresentaram ganhos acima de 8% os animais diretamente submetidos ao teste. Conflitos de seleção variaram entre 14 a 52% quando considerada somatória das diferenças de seleção nas duas avaliações genéticas, mostrando também, que existiu uma alteração na ordem de classificação dos animais nas características analisadas. Como o fator custo é um ponto importante quando se fala em avaliação genética e tudo que envolve a nova fase dos marcadores moleculares, um painel de paternidade eficiente é aquele que apresenta o melhor desempenho na determinação da paternidade sob um menor custo. Dessa forma, foram comparados alguns grupos de marcadores e o painel de 195 SNP proposto pelo grupo ISAG se mostrou eficiente, assim como outros, na determinação de paternidade em um grupo especifico de animais. Os resultados deste trabalho indicam também que o teste de parentesco por DNA é uma ferramenta que pode aumentar a precisão do arquivo pedigree por aumentar a conexão dos animais, ou seja, por criar maiores ou mais laços genéticos entre os indivíduos. Assim, poder-se-ia melhorar o desempenho da avaliação genética em um programa de melhoramento genético bovino através de pequenos grupos de marcadores moleculares com um custo-benefício atrativo. / In a scenario where information quality is essential to support a process management system or an animal selection process, correct paternity assignments become important for maintaining pedigree information and hence to estimate animals genetic values in breeding programs. With the advent of the use of SNP molecular markers in genomic selection and different kinship test panels, larger studies are increasingly necessary. Thus, this study investigated the gains in accuracy of breeding values for production traits (in a real selection scenario) between the genetic evaluations of animals with or without parenting assignment by DNA testing. It also verified possible selection conflicts between them. The following results showed gains in accuracy, especially in younger animals. However, in general, all animals benefited having gains greater than 8% where animals were directly impacted. Checking conflicts ranged from 14-52% when the sum of the differences of selection in both genetic evaluations was considered. This also showed that there was a change in the ranking of animals in the analyzed characteristics. As cost is also an important factor in genetic evaluations using new molecular markers, an effective parenting panel would be one that had the best performance in the paternity determination under a lower cost. Therefore, we compared some small marker groups and the 195 SNP panel proposed by ISAG group and determined this was efficient in estimating paternity on a specific group of animals. These results also suggest that kinship testing by DNA is a tool that can increase the accuracy of the pedigree file to increase the animals\' connection, or to create larger or more genetic links between individuals. This study shows that testing with small groups of molecular markers improves the performance of genetic evaluation in a bovine genetic selection program with an attractive cost-benefit.

Philosophiae Doctor - PhD / Solute carrier transporters belonging to the major facilitator family of membrane transporter are increasingly being recognized as a possible mechanism to explain inter-individual variation in drug efficacy and response. Genetic factors are estimated to be responsible for approximately 15-30% of inter-individual variation in drug disposition and response. The aims of this study were to determine the minor allele frequencies of 78 previously identified single nucleotide polymorphisms in the pharmacogenomically relevant SLC22A1-3 and SLCO1B1 genes in the Admixed population of South Africa. Thereafter, to determine whether allele and genotype frequencies for these SNP were different from that reported for other African, Caucasian, and Asian populations. The inferred haplotypes from the genetic information possessed the potential to subsequently be used in future to design and interpret results of pharmacogenomic association studies involving these genes and their substrate drugs. Furthermore, to determine whether the Cape Admixed population harbour novel SNPs in the proximal promoter regions of SLC22A1- 3 and SLCO1B1-3 genes, that encodes hOCT1-3 and hOATP1 and hOATP3, respectively. SNaPshot™ multiplex single base mini-sequencing systems were developed and optimized for each of SLC22A1, SLC22A2, SLC22A3, and SLCO1B1 genes covering the previously identified 78 SNPs. These systems were then used to genotype the alleles of 130 healthy Cape Admixed subjects residing in Cape Town, South Africa. In addition, the proximal promoter regions of the SLC22A1-3 and SLCO1B1-3 genes of 96 of the participants were screened for novel SNPs by direct sequencing. The Cape Admixed subjects investigated displayed a lack of variation and were monomorphic for 78% of the SNPs screened. None of the SLC22A3 SNPs investigated was observed in this study. Sequencing of the proximal promoter regions of the SLC22 and SLCO genes did not reveal any novel SNPs in the 96 Cape Admixed subjects that were screened. This study highlights the fact that African populations do not have the same allele frequencies for SNPs in harmacogenomically relevant genes. Furthermore, the Cape Admixed and other African populations do not share all reduced-function variants of the SLC22A1-3 and SLCO1B1-3 genes with Caucasian and Asian populations. In addition, previously identified novel regulatory variants in SLC22A2 did not exhibit a significant effect on the ability of the promoter to drive transcription. However, it must be noted that these results were observed at 95% confidence interval, and that a 99% confidence interval the significance may increase theoretically. Additionally, it should be noted that more intensive studies are required to determine the potential effect these novel variants may well cause. This study lays the foundation for the design and interpretation of future pharmacogenomic association studies between the variant alleles of the SLC22A and SLCO genes in the Cape Admixed population, as well as optimizations for future expression, and more importantly, drug transport assays with respect to drug disposition and efficacy. / National Research Foundation (NRF) and the Medical Research Council (MRC)