Resumo: Comercialmente os híbridos de milho transgênico são hemizigotos para o evento que os define, ou seja, das linhagens parentais cruzadas, uma é transgênica e a outra convencional. No entanto, híbridos homozigotos também podem ser obtidos pelo do cruzamento de duas linhagens parentais transgênicas. Assim, uma vez que a influência de genes exógenos é pouco conhecida na morfologia, anatomia e fertilidade masculina de híbridos de milho, buscamos com esse trabalho verificar a influência do número de alelos transgênicos nestes caracteres. Foram utilizados cinco diferentes híbridos com o mesmo evento (TC1507xMON89034xNK603) e o isogênico convencional de um deles, além de um híbrido com três diferentes eventos (Bt11, MIR162 e a piramidação de ambos) e seu isogênico convencional. Totalizando 18 tratamentos, que foram conduzidos em casa de vegetação, elaborado em delineamento de blocos casualizados com duas repetições. Quando as plantas estavam em V3-V4 duas delas foram extraídas para avaliação dos caracteres anatômicos do colmo, as restantes foram conduzidas até V5-V6 e avaliados os caracteres anatômicos da folha e raiz, morfológicos da raiz, matéria fresca e matéria seca. Para a avaliação dos caracteres de fertilidade masculina foram instalados em campo o híbrido com os três diferentes eventos, com cinco repetições e utilizadas três plantas como parcela útil. No terceiro dia após a antese a viabilidade e a germinação desses grãos de pólen foram avaliados. Todos os híbridos foram difere... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Commercially the transgenic maize hybrids are hemizygous for the event that defines them, that is, for crossed parental lines, one is transgenic and the other conventional. However, homozygous hybrids can also be obtained by crossing two transgenic parent lines. Thus, since the influence of exogenous genes is not well known in the morphology, anatomy and male fertility of maize hybrids, we seek to evaluate the influence of the number of transgenic alleles in these traits. Five different hybrids were used with the same event (TC1507xMON89034xNK603) and the conventional isogenic of one of them, besides a hybrid with three different events (Bt11, MIR162 and pyramidation of both and its conventional isogenic. Totaling 18 treatments, they were conducted in greenhouse, elaborated in a randomized complete block design with two replicates. When the plants were in V3-V4, two of them were extracted to evaluate the anatomical traits of the stem, the remaining were conducted to V5-V6 then evaluated the anatomical traits of leaf and root and morphology of root, fresh matter and dry matter. For the evaluation of the male fertility traits, the hybrids with three events different, were installed in the field, with five replications and three plants were used as a useful plot. On the third day after the anthesis the viability and germination of these grains were evaluated. All hybrids were different in the anatomical traits, the morphological components and the viability of the pollen grain d... (Complete abstract click electronic access below) / Mestre

Submitted by Kian Eghrari Moraes null (kianem@gmail.com) on 2017-03-14T19:44:37Z No. of bitstreams: 1 Dissertação_Kian_Eghrari_Moraes.pdf: 1379647 bytes, checksum: 5a9daad8a0289711efcdab3175a3dd1e (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-21T19:54:29Z (GMT) No. of bitstreams: 1 moraes_ke_me_jabo.pdf: 1379647 bytes, checksum: 5a9daad8a0289711efcdab3175a3dd1e (MD5) / Made available in DSpace on 2017-03-21T19:54:29Z (GMT). No. of bitstreams: 1 moraes_ke_me_jabo.pdf: 1379647 bytes, checksum: 5a9daad8a0289711efcdab3175a3dd1e (MD5) Previous issue date: 2017-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os híbridos de milho transgênicos, em geral, apresentam o locus transgênico em hemizigose. O objetivo do trabalho foi avaliar o efeito do número de alelos transgênicos em híbridos de milho em relação à expressão de Cry1F nas folhas, ataque de Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) e a produtividade de grãos, utilizando cinco híbridos isogênicos nas versões transgênicas homozigota e hemizigota, além da versão convencional de um dos híbridos. O nível de expressão da proteína Cry1F (PRYF) foi quantificado pela técnica de ELISA. Nos experimentos de campo, conduzidos na primeira e segunda safras do ano agrícola 2015/2016, avaliou-se o ataque de S. frugiperda em campo (ALC), por infestação natural, e a produtividade de grãos (PG). Dois bioensaios foram realizados em laboratório para avaliar a sobrevivência larval de S. frugiperda de 1º instar (SL) alimentadas com as folhas dos híbridos. Os híbridos transgênicos, homozigotos e hemizigotos, não apresentaram silenciamento gênico. Os híbridos homozigotos apresentaram maior concentração de proteína Cry1F. Quando houve elevado ALC, na primeira safra, os híbridos transgênicos foram superiores à testemunha convencional na PG, entretanto, não houve diferença entre os híbridos homozigotos e hemizigotos. Os híbridos transgênicos também foram superiores à testemunha convencional nos bioensaios, sendo que os homozigotos apresentaram as menores médias de SL. A presença de um alelo transgênico a mais nos híbridos homozigotos propiciou comportamento genético aditivo para a expressão de Cry1F e controle de S. frugiperda, diminuindo ALC e SL, sem diminuir a capacidade produtiva das plantas. Diante do exposto, não há limitações para a utilização de híbridos de milho homozigotos para o transgene, pois apresentaram melhor controle de S. frugiperda em comparação com os híbridos hemizigotos. / Genetically modified (GM) maize hybrids, in general, possess the transgenic locus in a hemizygous state. The aim of the study was to assess the effect of the number of transgenic alleles in maize hybrids regarding the Cry1F leaf expression, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) attack and grain yield, through the evaluation of five isogenic hybrids in the homozygous and hemizygous transgenic versions and a non-GM hybrid. Cry1F protein expression levels (PRYF) were quantified by ELISA. In field experiments conducted during the summer and autumn seasons of 2015/2016, we assessed the leaf-feeding injury of S. frugiperda in the field (LFI) by natural infestation and grain yield (GY). Two bioassays were carried out in the laboratory to evaluate the survival of first-instar S. frugiperda larvae (SL) fed with the maize hybrids. Transgenic hybrids did not present gene silencing. Homozygous hybrids presented higher Cry1F expression levels than hemizygous hybrids. With high LFI during the summer season, transgenic hybrids were superior to the non-GM for GY, however, there was no difference between homozygous and hemizygous hybrids. The transgenic hybrids were superior to the non-GM hybrid in the bioassays, and the homozygotes caused the highest mortality of S. frugiperda. The addition of one transgenic allele in the homozygous hybrids provided an additive genetic effect, increasing PRYF and S. frugiperda control, whereas GY was not affected. In conclusion, there are no limitations to the use of transgenic homozygous maize hybrids, which presented better S. frugiperda control comparing to hemizygous hybrids.

Les cellules souches hématopoïétiques (CSH) assurent une production constante des cellules sanguines tout au long de la vie, mais sont vulnérables à l’acquisition de mutations pouvant mener à une transformation maligne. Les mutations qui confèrent un avantage de croissance entraîneront une prolifération clonale. L’étude de la clonalité est centrale à la compréhension de ces phénomènes. Historiquement, l’analyse de la clonalité a été possible grâce au principe de l’inactivation du chromosome X (ICX) chez les femmes qui entraîne la création de deux populations cellulaires, celle avec le X-paternel actif et celle avec le X-maternel actif. Une déviation (biais) de la proportion théorique de 1 :1 entre ces deux populations peut supposer une dominance clonale. Nous avons démontré un biais significatif de l’ICX chez les femmes avec l’âge. Ce phénomène peut être expliqué par plusieurs causes dont la sélection hémizygote (un des deux X possède des allèles plus forts que l’autre) et l’acquisition de mutations dans une CSH. Nous posons l’hypothèse que ces deux phénomènes coexistent et peuvent être distingués par une approche génomique. Nous avons recruté une cohorte de 2996 femmes canadiennes-françaises âgées entre 37 et 101 ans composée de 2172 individus issus de 321 familles et de 824 individus non apparentés. Deux tissus biologiques ont été recueillis : le sang périphérique (PMN, monocytes, lymphocytes T, lymphocytes B) et des cellules buccales. Le ratio de l’ICX a été déterminé par la méthode HUMARA, l’analyse de gènes associés à l’hématopoïèse clonale (19 gènes) a été faite par la méthode de séquençage NGS, et la cohorte a été génotypée à 700 625 loci polymorphiques de l’ADN (SNP). Des analyses bioinformatiques ont été - iv - appliquées pour étudier la contribution génétique au biais de l’ICX. Nous démontrons que : (i) le biais de l’ICX est plus prévalent dans les cellules sanguines par rapport aux cellules épithéliales et maximal dans les cellules myéloïdes; (ii) le biais augmente avec l’âge seulement dans les cellules sanguines et que cette influence est plus marquée pour les neutrophiles; (iii) la concordance du biais est très importante pour les différents types cellulaires sanguins, suggérant un mécanisme opérant au niveau de la CSH ; (iv) il y a une composante héréditaire liée au biais de l’ICX; (v) la présence de mutations acquises (TET2, DNMT3A, etc.) explique seulement une partie du biais ; (vi) à l’aide d’analyses par liaison génétique la présence d’une région sur le chromosome X à Xq21 (LOD score 4.9) qui est associée au biais des lymphocytes T et une autre sur le chromosome 1 à 1q21 (LOD score 6) qui est associée au biais des neutrophiles. Nous avons départagé la contribution liée à l’acquisition de mutations somatiques et identifié pour la première fois des régions liées à une prédisposition génétique. Nos travaux se poursuivront d’une part par l’analyse de gènes candidats dans les régions identifiées, et d’autre part nous tenterons d’identifier les cibles génétiques qui confèrent un potentiel de transformation maligne en utilisant une approche basée sur l’analyse du méthylome, de l’hydroxyméthylome et du transcriptome que nous venons de valider. Notre étude démontre la complexité de l’adaptation de l’hématopoïèse au vieillissement et ouvre des portes sur l’identification de facteurs prédisposant aux cancers hématologiques. / Hematopoietic stem cells (HSC) ensure a constant lifelong production of blood cells, but are vulnerable to acquisition of mutations, which may lead to malignant transformation. Mutations that confer a growth advantage will lead to clonal derivation of cells. The study of clonality is central to the understanding of hematopoiesis adaptation to aging. Historically, the first clonality assays were based on the principle of X-chromosome inactivation (XCI) in women. Women are mosaics with half the cells with the paternal X active and the other half with the maternal one. A skewing from the theoretical 1:1 ratio between these two populations of cells could infer clonal derivation of cells. More than 20 years ago, our team demonstrated, through analysis of (XCI) in women, that skewing increases with age. This intriguing phenomenon can be explained by several etiology including hemizygous selection (one of the 2 Xs has stronger alleles) or the acquisition of mutations giving a growth advantage. The first etiology is genetically predetermined and the second, acquired in somatic cells of bone marrow. We hypothesize that these two phenomena coexist and can be distinguished with a genomic approach. To test our hypothesis, we investigated skewing in a cohort of 2996 French-Canadian women aged 37 to 101 comprised of 2172 related individuals from 321 families and 824 unrelated individuals. We analyzed XCI ratios at the HUMARA locus in epithelial cells, neutrophils, T-cells, monocytes, B-lymphocytes. We genotyped the cohort for clonal hematopoiesis and looked for germline heritable components by genome wide association studies and linkage analyses. We document that skewing was more prevalent in blood cells than in epithelial cells, and maximal in myeloid cells. Skewing increases with age only in blood cells. Intra- vi - individual correlation of skewing blood cell types was strongly correlated, suggesting selection influences operating at the HSC. Sibship analyses demonstrated heritability which was strongest when parental origin of skewing was taken into account. Clonal hematopoiesis accounted only for a small proportion of the skewing trait but its importance increased in the very old. Linkage analysis identified a region at Xq21 for skewing occurring in T-cells (LOD score 4.9) suggesting a hemizygous cell selection influence. We also identified a region at 1q21 for skewing in neutrophils (LOD score 6) suggesting a gene-gene interaction with Xlinked genes. We have demonstrated that age-associated skewing is a complex trait caused in part by acquired mutations and genetic predisposition variants. We will pursue our investigation using a candidate gene approach in the two identified regions and will try to identify genetic targets of oncogenic potential by a method based on analysis of the methylome, hydroxymethylome and transcriptome that was have validated in this cohort. This thesis demonstrates the complexity of the adaptation mechanisms of hematopoiesis to aging and set the stage to identification of factors predisposing to hematological cancers.