Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / IntroduÃÃo: A anemia falciforme à o resultado de uma mutaÃÃo pontual (GAG&#61614;GTG) no cÃdon do gene da globina &#946;, conduzindo a uma substituiÃÃo de Ãcido glutÃmico por valina na sexta posiÃÃo da cadeia polipeptÃdica. A anemia falciforme apresenta manifestaÃÃes clÃnicas heterogÃneas, que podem ser relacionadas ao tipo de haplÃtipo associado ao gene da HbS e aos nÃveis de HbF. Objetivo: CaracterizaÃÃo clÃnica, hematolÃgica e molecular dos pacientes com anemia falciforme em Fortaleza, CearÃ. Metodologia: Foram analisados 47 pacientes com anemia falciforme, adultos e de ambos os sexos. A determinaÃÃo dos valores hematolÃgicos foi realizada em contador automÃtico de cÃlulas sangÃÃneas; a determinaÃÃo da presenÃa de HbSS foi realizada por eletroforese em pH alcalino em fitas de acetato de celulose e por eletroforese de diferenciaÃÃo em Ãgar-fostato pH 6.2; os nÃveis de HbF foram determinados pela tÃcnica da desnaturaÃÃo alcalina; e a anÃlise dos haplÃtipos da mutaÃÃo ÃS foi realizada por meio da tÃcnica da reaÃÃo em cadeia mediada pela polimerase para polimorfismo dos comprimentos dos fragmentos de restriÃÃo (PCR-RFLP). As anÃlises estatÃsticas foram desenvolvidas no programa GraphPad Prism (versÃo 5.0) e o nÃvel de significÃncia estabelecido foi p < 0,05. Resultados: A distribuiÃÃo dos haplÃtipos do gene da &#946;S-globina, 63% do tipo Bantu, 25% do tipo Benin e 12% do tipo AtÃpico, està em conformidade com a observada para a populaÃÃo brasileira, em que o haplÃtipo Bantu à o mais prevalente, seguido pelo Benin e Senegal. NÃo houve diferenÃa significativa entre o presente estudo e os resultados encontrados no Rio de Janeiro, Porto Alegre, Campinas e RibeirÃo Preto; porÃm uma diferenÃa significativa foi observada quando o estudo foi comparado aos resultados obtidos em Salvador, BelÃm, Amazonas e por outros pesquisadores no CearÃ. A distribuiÃÃo das freqÃÃncias dos haplÃtipos do gene da &#946;S-globina nos diferentes estudos està condizente com a histÃria da formaÃÃo da populaÃÃo brasileira, exceto nos resultados do estudo anterior realizado no CearÃ, que obteve o haplÃtipo Benin com maior prevalÃncia. Conforme os dados histÃricos sobre as origens da populaÃÃo negra trazida ao estado do CearÃ, o haplÃtipo Bantu seria o mais prevalente. Na comparaÃÃo entre os haplÃtipos e as caracterÃsticas hematolÃgicas estudadas, apenas os valores de HbF e Ht apresentaram diferenÃa estatisticamente significativa. Os nÃveis de HbF foram maiores no haplÃtipo Benin, seguido do haplÃtipo Bantu, o que està em conformidade com os dados da literatura. Foi demonstrada maior presenÃa de crises vaso-oclusivas e episÃdios de pneumonia no haplÃtipo Benin/AtÃpico do que no haplÃtipo Bantu/AtÃpico; e maior presenÃa de crises de infecÃÃo urinÃria no haplÃtipo Benin/AtÃpico do que no haplÃtipo Benin/Benin. NÃo houve diferenÃa estatisticamente significativa entre os haplÃtipos Bantu/Bantu e Benin/Benin em relaÃÃo Ãs complicaÃÃes clÃnicas, entretanto foi observado que o haplÃtipo Bantu/Bantu tem uma maior freqÃÃncia em todos os eventos clÃnicos estudados quando comparado ao Benin/Benin. Dentre os resultados foi demonstrada uma tendÃncia de menor nÃmero de pacientes com crises vasos-oclusivas e Ãlceras de perna com o aumento dos nÃveis de HbF. NÃo houve diferenÃa estatisticamente significativa na comparaÃÃo entre os nÃveis de HbF e os valores de HemÃcias, Hemoglobina, HematÃcrito, LeucÃcitos e Plaquetas. ConclusÃes: A determinaÃÃo dos haplÃtipos da anemia falciforme à de grande importÃncia nÃo sà para o acompanhamento e prognÃstico dos pacientes, como tambÃm como ferramenta para estudos antropolÃgicos que contribuam no esclarecimento da origem dos africanos que tanto contribuÃram na formaÃÃo etnolÃgica, econÃmica, cultural e social do Brasil. / Introduction: The sickle cell anemia is the result of a point mutation in the &#946;-globin gene, leading to a substitution of glutamic acid by valine at the sixth position of the polypeptide chain. The sickle cell anemia presents heterogeneous clinical manifestations, which may be related to the type of haplotype associated with the gene for HbS and HbF levels. Objective: Clinical characterization, molecular and haematological patients with sickle cell anemia in Fortaleza, CearÃ. Methods: We analyzed 47 patients with sickle cell anemia, adults of both sexes. The determination of hematological values was performed on blood cells automated meter; the determination of the presence of HbSS was performed by alkaline hemoglobin electrophoresis on cellulose acetate tapes and by differentiation electrophoresis on agar-phosphate pH 6.2; the levels of HbF were determined by alkali denaturation technique; and the analysis of the haplotypes of the ÃS mutation was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Statistical analysis was developed in the program GraphPad Prism (version 5.0) and the level of significance was set p <0.05. Results: The distribution of the haplotypes of &#946;S-globin gene - 63% of the Bantu type, 25% of the Benin type and 12% atypical â was in conformity with that observed for the entire Brazilian population, in which the Bantu haplotype is most prevalent, followed by the Benin and Senegal. There was no significant difference between the results found in this study and those found for the cities of Rio de Janeiro, Porto Alegre, Campinas and RibeirÃo Preto; but there was a significant difference with the results obtained for the cities of Salvador and BelÃm and the state of Amazonas, and by other researchers in CearÃ. The distribution of haplotype frequencies of the &#946;S-globin gene in the different studies is in line with the history of the formation of the Brazilian population, except for the results of a previous study carried out in CearÃ, in which the Benin haplotype was found to be most prevalent. According to the historical information on the origins of the slave population brought to CearÃ, the Bantu haplotype should be the most prevalent. In the comparison between the haplotypes and the haematological characteristics studied, only the values of HbF and Ht showed statistically significant difference. The levels of HbF were higher in the Benin haplotype, followed by the Bantu haplotype, which is in accordance with the literature. Was demonstrated greater presence of painful episodes and episodes of pneumonia in Benin haplotype/Atypical haplotype than in Bantu/Atypical and increased presence of urinary infection crises in Benin haplotype/Atypical haplotype than in Benin/Benin. There was no statistically significant difference between the haplotypes Bantu/Bantu and Benin/Benin for clinical complications, however it was observed that the haplotype Bantu/Bantu has a higher frequency in all studied clinical events when compared to Benin/Benin. Among the results was shown a trend of fewer patients with painful episodes and of leg ulcers with increased levels of HbF. There was no statistically significant difference in the comparison between the levels of HbF and the values of red blood cells, hemoglobin, hematocrit, leukocytes and platelets. Conclusions: The determination of haplotypes of sickle cell anemia is of great importance not only for monitoring and prognosis of patients, but also as a tool for anthropological studies which help in clarifying the origin of Africans who have contributed so much in training ethnological, economic, cultural and social Brazil.

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 pharmacogenetically relevant SLC22A1-3 and SLC47A1 genes in the indigenous African population of South Africa. Secondly, to determine whether allele and genotype frequencies for these SNP were different from that reported for other African, Caucasian, and Asian populations. Thirdly, to infer haplotypes from the genetic information which can potentially be used in future to design and interpret results of pharmacogenetics association studies involving these genes and their substrate drugs. Finally, to determine whether the Xhosa population harbour novel SNPs in the SLC22A2 gene, that encodes the kidney-specific hOCT2. SNaPshot™ multiplex single base minisequencing systems were developed and optimized for each of SLC22A1, SLC22A2, SLC22A3, and SLC47A1 covering the previously identified 78 SNPs. These systems were then used to genotype the alleles of 148 healthy Xhosa subjects residing in Cape Town, South Africa. In addition, the proximal promoter region and all 11 exons and flanking regions of the SLC22A2 gene of 96 of the participants were screened for novel SNPs by direct sequencing. The Xhosa subjects investigated lacked heterozygosity and were monomorphic for 91% of the SNPs screened. None of the SLC22A3 and SLC47A1 SNPs investigated was observed in this study. Sequencing of the SLC22A2 gene revealed 28 SNPs, including seven novel polymorphic sites, in the 96 Xhosa subjects that were screened. The minor allele frequencies of the seven previously identified variant SNPs observed in this study were different compared to that observed for American and European Caucasian, and Asian populations. Moreover, the allele frequencies for these SNPs differed amongst African populations themselves. Eight and seven haplotypes were inferred for SLC22A1 and SLC22A2, respectively, for the Xhosa population from the information gathered with SNaPshot™ genotyping. This study highlights the fact that African populations do not have the same allele frequencies for SNPs in pharmacogenetically relevant genes. Furthermore, the Xhosa and other African populations do not share all reduced function variants of the SLC22A1-3 and SLC47A1 genes with Caucasian and Asian populations. Moreover, this study has demonstrated that the Xhosa population harbours novel and rare genetic polymorphisms in the key pharmacogene SLC22A2. This study lays the foundation for the design and interpretation of future pharmacogenetic association studies between the variant alleles of the SLC22A1-3 and SLC47A1 genes in the Xhosa population and drug disposition and efficacy.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / Tomopterna delalandii occurs throughout the west coast, Western Cape and south coast of South Africa. This range stretches across three distinct biogeographical assemblages. Based on historical records and the fact that Tomopterna is a genus of cryptic frogs, it is possible that there are unknown genetic variations within the species. To investigate whether population structure is present within T. delalandii a mitochondrial gene and nuclear gene, 16S and Tyrosinase, was sequenced from across the range. Haplotype networks and cladograms were constructed to look at the relationship between the genetic samples. This revealed definite population structuring between samples from the western edge of the range and samples from the eastern edge of the range. It also revealed that samples taken from the northern edge of the range, while matching general T. delalandii tadpole morphology, are genetically different. Further study needs to be made into the identity of this new form. This study shows that by using both mitochondrial and nuclear DNA patterns can be discovered about the genetic structure of a species as well as revealing a new one.

L'utilisation de lignées de soya possédant différents gènes Rps (« Resistant to Phytophthorasojae ») demeure la meilleure méthode de lutte pour combattre la pourriture phytophthoréenne. La résistance conférée par les gènes Rps repose sur le concept gène-pour-gène où une relation existe entre un gène de résistance (gène Rps) chez la plante et un facteur d'avirulence correspondant (gène Avr) chez l'agent pathogène. Sept gènes Rps ont été introduits avec succès dans les cultivars commerciaux, soit les gènes Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps3a et Rps6. À long terme, l'agent pathogène arrive à contourner la résistance conférée par ces différents gènes Rps par la diversification génétique de ces gènes d'avirulence, entraînant la complexification des pathotypes de P. sojae. Afin d'exploiter efficacement ce type de résistance, il devient donc primordial de pouvoir identifier ces pathotypes pour connaître l'interaction des différentes souches de P. sojae avec les gènes Rps utilisés. Ce projet de doctorat avait donc comme objectif une meilleure compréhension de la diversité haplotypique des populations de P. sojae, concernant l'interaction avec les gènes Rps d'intérêt commercial, afin de permettre aux producteurs de faire un choix éclairé qu and vient le temps de choisir des lignées résistantes à la maladie. Dans un premier volet de recherche, notre hypothèse stipulait que l'analyse des variations nucléotidiques et structurales associées aux sept principaux gènes d'avirulence de P. sojae permettrait d'identifier le spectre des haplotypes associés aux phénotypes des isolats, relativement à leur compatibilité avec les gènes de résistance Rps correspondants. Le séquençage du génome complet de 31 isolats représentant la diversité des pathotypes canadiens a permis d'identifier les différents haplotypes des sept principaux gènes d'avirulence (1a, 1b, 1c, 1d, 1k, 3a et 6) et d'y associer un profil de virulence pour chaque gène Rps correspondant. L'utilisation d'une méthode de phénotypage en bassin hydroponique a permis de constater que le test traditionnel d'inoculation de l'hypocotyle menait à une proportion importante de faux positifs, ce qui a pu nuire à la compréhension de certaines interactions Rps-Avr par le passé. Notre étude a révélé que l'utilisation unique des signatures génomiques permettait de prédire 99,5 % des interactions possibles et que ces dernières pouvaient donc être utilisées pour prédire précisément le profil de virulence des isolats de P.sojae. Ces résultats ont mené à notre deuxième volet de recherche qui visait à démontrer que les marqueurs discriminants identifiés dans le premier volet de l'étude permettraient de s'affranchir du test de phénotypage, par le développement d'un outil moléculaire de type multiplex PCR, capable de prédire le pathotype des isolats de P. sojae. Des amorces spécifiques permettant d'amplifier les allèles associés à l'avirulence envers chaque gène Rps ont été conçues et multiplexées. Le multiplex PCR développé a démontré une efficacité de 97 % lorsque testé sur un panel d'isolats au profil inconnu de virulence. Le troisième volet de recherche visait à mieux comprendre l'interaction de P. sojae avec le gène de résistance Rps8. Notre hypothèse suggérait que l'avirulence de P. sojae envers le produit du gène de résistance Rps8 était déterminée par un effecteur RXLR, codé par un gène d'avirulence correspondant. L'analyse d'une descendance F2 en ségrégation par génotypage par séquençage couplée à l'utilisation des données de séquençage des 31 isolats de P. sojae a permis de cibler Avr3a comme le meilleur gène candidat. En utilisant la méthode d'édition de génome médiée par CRISPR/Cas9, nous avons démontré que le knock-out complet du gène Avr3a chez P. sojae induisait un gain de virulence envers Rps8. Nous avons aussi démontré qu'un allèle spécifique d'Avr3a était reconnu de façon différentielle par Rps3a et Rps8, ce qui représente la première distinction nette entre ces deux gènes de résistance. L'ensemble de ce projet de doctorat a permis de fournir de nouvelles informations sur la complexité des gènes Avr en plus de démontrer que leurs signatures génomiques pouvaient être utilisées pour prédire précisément l'interaction de l'agent pathogène avec les différents gène Rps du soya. Le développement d'un test moléculaire simple et précis offre aux producteurs et sélectionneurs une solution concrète afin d'exploiter efficacement l'utilisation des gènes Rps. L'identification du gène d'avirulence reconnu par Rps8 permet également d'avoir une longueur d'avance dans la gestion de la maladie, en prévision de l'apparition inévitable de nouveaux pathotypes dans le futur. / The use of soybean lines with different Rps ("Resistant to Phytophthora sojae") genes remains the best control method to control Phytophthora root rot. The resistance conferred by Rps genes is based on the gene-for-gene concept where a relationship exists between a resistance gene (Rps gene) in the plant and a corresponding avirulence factor (Avr gene) in the pathogen. Seven Rps genes have been successfully introduced into commercial cultivars, namely Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps3a and Rps6. In the long term, the pathogen manages to by pass the resistance conferred by these different Rps genes by the genetic diversification of its avirulence genes, leading to the complexification of P. sojae pathotypes. To effectively exploit this type of resistance, it is therefore essential to be able to identify those pathotypes in order to know the interaction of the different strains of P. sojae with the Rps genes in use. This doctoral project aims to have a better understanding of the haplotypic diversity of P. sojae populations, concerning their interaction with the Rps genes of commercial interest, in order to allow producers to make an informed choice when the time comes to choose soybean lines resistant to the disease. In a first approach, our hypothesis stipulated that the analysis of the nucleotide and structural variations associated with the seven main avirulence genes of P. sojae would allow the identification of the spectrum of haplotypes associated with the phenotypes of the isolates, relative to their compatibility with the corresponding Rps resistance genes. The whole-genome-sequencing of 31 isolates representing the diversity of Canadian pathotypes made it possible to identify the different haplotypes of the seven main avirulence genes (1a, 1b, 1c, 1d, 1k, 3a and 6) and to associate a virulence profile for each corresponding Rps gene. The use of a hydroponic phenotyping method showed that the traditional hypocotyl inoculation test leads to a high proportion of false positives, which could have hampered the understanding of certain Rps-Avr interactions in the past. Our study found that the unique use of genomic signatures predicted 99.5% of the possible interactions and that these could therefore be used to accurately predict the virulence profile of P. sojae isolates. These results led to the second part of this doctoral project, which aimed to demonstrate that the discriminating markers identified in the first part of the study would make it possible to bypass the cumbersome phenotyping method by the development of a molecular tool capable of predicting the pathotype of P. sojae isolates. Specific primers for amplifying the alleles associated with avirulence towards each Rps gene were designed and multiplexed. The multiplex PCR developed demonstrated an efficiency of 97% when tested on a panel of isolates with an unknown virulence profile. The third objective of this project was to understand the interaction of P. sojae with the resistance gene Rps8. Our hypothesis suggested that the avirulence of P. sojae towards the Rps8 resistance gene product was determined by an RXLR effector encoded by a corresponding avirulence gene. Analysis of a segregating F2 progeny by using a genotyping-by-sequencing method coupled with the use of sequencing data from the 31 P. sojae isolates led to the identification of Avr3a as the best candidate gene. Using the CRISPR/Cas9-mediated genome editing method, we demonstrated that the complete knockout of the Avr3a gene in P. sojae induced a gain of virulence towards Rps8. We also demonstrated that a specific Avr3a allele is differentially recognized by Rps3a and Rps8, which results in the first clear distinction between those two resistance genes. This doctoral project provided new information about the complexity of Avr genes and demonstrated that their genomic signatures could be used to precisely predict the interaction of the pathogen with the various Rps genes in soybean. The development of a simple and precise molecular test offers to producers and breeders a concrete solution to effectively exploit the use of Rps genes. Identifying the avirulence gene recognized by Rps8 also provides a head start in disease management, in anticipation of the inevitable emergence of new pathotypes in the future.

No description available.

Twenty-five different iso-chromosomal III lines of Drosophila melanogaster, with known haplotypes, were mixed and selected on malaoxon over twelve generations in order to find the importance of variants already present in the Drosophila genome relative to new mutations in the evolution of insecticide resistance. Measurements of mixed function oxidase and acetylcholinesterase activities, as well as insecticide resistance, were made over the twelve generations of selection and on the the iso-chromasomal III lines extracted after the selection was completed. These measurements were compared to those made of the twenty-five lines before selection. This comparison indicated that 52A, one of the origional twenty-five lines, may have been selected during this experiment. Comparisons of a possible cytochrome P-450 produced by a previously selected line of Drosophila called D23, and the DNA which is probably responsible for the production of this P-450, with the microsomal proteins and total genomic DNA of the selected lines were made using the techniques of Western and Southern blotting. The results of these procedures suggested that the mechanism of resistance used by the selected lines was not the same mechanism of resistance used by the D23 line. / Thesis / Master of Science (MSc)

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Orientador: Celso Luís Marino / Banca: Fábio Tebaldi Silveira Nogueira / Banca: Mario Luiz Teixeira de Moraes / Resumo: Florestas de eucalipto no Brasil estão entre os ecossistemas mais produtivos do mundo. A madeira do eucalipto é principalmente utilizada pelas empresas de papel e celulose. Contudo, fatores abióticos e bióticos afetam a expansão dessas florestas. A disponibilidade de água é uma das maiores limitações para o desenvolvimento das espécies, afetando a produção de biomassa. O hormônio ácido abscísico (ABA) desencadeia respostas de resistência a estresses abióticos, em particular a seca. O objetivo deste trabalho foi identificar Single Nucleotide Polymorphisms (SNPs) no gene NCED (nove-cis-epoxicarotenóide dioxigenase), da rota biossintética do ácido abscísico (ABA), em Eucalyptus. Através da utilização da sequencia de aminoácido da enzima de Arabidopsis (AtNCED3) e do banco de dados de Expressed Sequence Tags (ESTs) de Eucalyptus foi possível o desenvolvimento de três oligonucleotídeos, os quais amplificaram a região desejada do gene. Então, primers específicos foram desenhados e os produtos de amplificação de diferentes indivíduos submetidos ao sequenciamento direto. O alinhamento e as análises das sequencias revelaram a ocorrência de sete SNPs no gene NCED, em uma região de 1230 pares de bases. A razão transição/transversão foi 1.33. Após a predição da proteína, no site Softberry e Expasy, foram verificados cinco SNPs presentes na região codificadora, os quais geraram mutações sinônimas. Através do software DnaSP, sete haplótipos foram encontrados na amostra de 65 indivíduos gerando 15 genótipos, distribuídos entre as espécies E. grandis, E. urophylla e no híbrido Urograndis. Para os sete sítios polimórficos foram desenhados conjuntos de primers específicos que permitirão a genotipagem em larga escala para estudos de genética de população e em programas de melhoramento assistido / Abstract: Eucalyptus plantations in Brazil are among the most productive ecosystems in the world. The eucalyptus wood has its principal use in the paper and cellulose industry. However, abiotic and biotic factors affect the expansion of forest plantation. Water availability is the major limitation to development of species affecting biomass production. The plant hormone abscisic acid (ABA) triggers resistance responses to abiotic stresses, in particular to drought. The objective of this study was to identify Single Nucleotide Polymorphism (SNPs) in nine-cis-epoxycarotenoid dioxygenase (NCED), enzyme of ABA biosynthetic pathway, in Eucalyptus. Through the utilization of amino acid sequence of this enzyme of Arabidopsis (AtNCED3) and using availability Expressed Sequence Tags (ESTs) database of Eucalyptus, as possible the development of three PCR primers that amplified the desirable regions of the gene. Thus, specifics primers were designed and amplification products of different individuals submitted to direct sequencing. The alignment and analysis of sequences revealed the occurence of seven SNPs in NCED gene, in a region of 1230 base pairs. The rate of transition/transversion was 1.33. After the prediction of protein, by the sites Softberry and Expasy, it was verified five SNPs, in coding region, that generated synonymous substitutions. Using the DnaSP program, seven haplotypes were found in a sample of 65 individuals, consisting of the species E. grandis, E. urophylla and the hybrid Urograndis. For these seven polymorphic sites were designed SNPs markers sets that will allow large-scale genotyping for population genetic studies and assisted breeding programs / Mestre