Em pesquisa Genômica é de grande interesse o mapeamento de genes que controlam traços ou fenótipos quantitativos. Metodologias estatsticas para identicar genes que tenham efeitos sobre um unico traço são bem conhecidas na literatura e têm sido exaustivamente aplicadas no mapeamento genético de muitas doenças. Porem, na pratica, diferentes traços são correlacionados, como é o caso de hipertensão e obesidade, possivelmente, devido a aço de genes comuns envolvidos na sua regulação. Nestes casos, por meio de tecnicas estatísticas multivariadas, que exploram a estrutura de covariância entre os traços, é possvel identificar genes não detectados por analises univariadas, ganhar precisão nas estimativas dos efeitos e conhecer a posicão desses genes, alem de testar efeitos de pleiotropia (um mesmo gene controlando varios traços) e interacções gene-ambiente (os genes que controlam a pressão antes e depois de dieta com sal). Neste trabalho diferentes alternativas de analise estatstica são consideradas para explorar a informacão de vários tracos conjuntamente: modelo de regressão intervalar multivariado (Jiang & Zeng, 1995), mapeamento multivariado via a teoria espectral (Mangin et al.,1998), via medidas resumo relevantes (como a diferenca entre respostas antes e depois de uma exposição) e via ajustes por covariaveis. Também são introduzidas algumas abordagens graficas para o estudo do efeito de pleiotropia e interação geneambiente. As metodologias supracitadas são aplicadas a dados reais fornecidos pelo Laboratorio de Cardiologia e Genética Molecular do InCor/USP, que consideram várias medidas de pressão arterial em ratos provenientes de uma população F2. / In Genomic research, the mapping of genes which control quantitative traits has been of great interest. Statistical methods for detection of genes, in uencing a single trait, are well known in the literature and they have been exhaustive used in the genetic mapping of many diseases. However, in real situations, dierent kind of traits are correlated, such as hypertention and obesity, that would be due to the action of a set of commom genes involved in the regulation of these traits. In these cases, through of multivariate statistical techniques, which explore the covariance structure between the traits, it is possible to identify genes that are not detected by univariated analysis. In addition multivariate analysis are useful to obtain accurate estimates and to know the position of these genes, besides testing eects of pleiotropic (a gene controlling several traits) and geneenvironmental interations (genes that control the pressure before and after salt diet). In this work dierent alternatives from statistical analysis are considered to explore information of several traits jointly: Interval multivariate regression models (Jiang and Zeng, 1995); multivariate mapping through the espectral theory (Mangin et al. 1998), summary measures (for example, models formulated in terms of the dierence between two traits) and adjustments including covariates. Also, graphics procedures are introduced in order to study eects of pleiotropy and geneenvironmental interactions . The methodologies mentioned above are applied to real data set, supplied by the Cardiology and Molecular Genetic Laboratory of Heart institute (InCor-USP), that consider several measurements of blood pressure in rats that come from a F2 population.

Many organisms can acclimate to new environments through phenotypic plasticity, a complex trait that can be heritable, be subject to selection, and evolve. However, the rate and genetic basis of plasticity evolution remain largely unknown. Experimentally evolved populations of the nematode Caenorhabditis remanei were created by selecting for stress resistance under different environmental conditions. This resource was used to address key questions about how phenotypic plasticity evolves and what the genetic basis of plasticity is. Here, I highlight ways in which a fuller understanding of the environmental context influences our interpretation of the evolution of phenotypic plasticity. In a population selected to withstand heat stress, an apparent case of genetic assimilation did not show correlated changes in global gene regulation. However, further investigation revealed that the induced plasticity was not fixed across environments, but rather the threshold for the response was shifted over evolutionary time. Similarly, the past environment experienced by populations can play a role in directing the multivariate response to selection. Correlated responses to selection between traits and across environments were examined. The pattern of covariation in the evolutionary response among traits differed depending on the environment in which selection occurred, indicating that there exists variation in pleiotropy across the stress response network that is highly sensitive to the external environment. To understand how the patterns of pleiotropy are altered by environment and evolution, there is a pressing need to determine the structure of the molecular networks underlying plastic phenotypes. Using RNA-sequencing, the structure of the gene regulatory network is examined for a subset of evolved populations from one environment. Key modules within this network were identified that are strong candidates for the evolution of phenotypic plasticity in this system. Together, the data presented in this dissertation provide a comprehensive view of the myriad ways in which the environment shapes the genetic architecture of stress response phenotypes and directs the evolution of phenotypic plasticity. Additionally, the structure of transcriptional network provides valuable insight into the genetic basis of adaptation to environmental change and the evolution of phenotypic plasticity. This dissertation includes both previously published and co-authored material.

La levure S. cerevisiae est la seule espèce capable de terminer la fermentation alcoolique du jus de raisin qui est l’étape principale de la vinification. A cause de la forte variabilité technologique retrouvée chez cette espèce, des travaux de sélection sont réalisés dans le but d’utiliser des levains performants pour l’industrie. Ces souches montrent des différences importantes à la fois dans leurs cinétiques fermentaires et leur bilans en métabolites, ce qui impacte la qualité des vins. La réponse phénotypique des levures varie également de manière considérable et non homogène face aux variations environnementales. La compréhension des mécanismes génétiques expliquant cette réponse différenciée est une question scientifique non triviale. Elle revêt une importance particulière en oenologie, où les conditions de vinifications sont très changeantes (millésimes, cépages, terroirs, conduites de vinifications…). Afin de pouvoir proposer des levains garantissant le succès des fermentations dans un large éventail de conditions, nous proposons ici de mieux comprendre ces mécanismes d’interaction Gène x Environnement dans un contexte oenologique. L’identification de locus génétiques (Quantitative Trait Loci (QTL)) contrôlant des caractères quantitatifs est rendue possible par des approches de cartographie de QTLs. Celles-ci nécessitent l’étude d’une vaste descendance en ségrégation qui doit être caractérisée sur le plan génétique et phénotypique. L’établissement d’un lien statistique entre des marqueurs génétiques et un phénotype permet la localisation de QTLs influant les caractères étudiés. Au cours de cette thèse, une méthode de phénotypage pour suivre les fermentations de plusieurs centaines d’individus a été mise au point. Grâce à elle, les performances fermentaires de deux descendances génotypées par séquençage à haut débit ont été mesurées en faisant varier les conditions de fermentations. Cela a permis l’identification de nombreux QTLs et d’estimer leur impact sur la robustesse des souches. L’implication des allèles de trois gènes qui montrent une forte interaction avec l’environnement et qui possèdent des effets pléiotropiques liés au métabolisme du SO2 a été prouvée moléculairement. Les résultats obtenus font l’objet d’une discussion générale sur l’utilisation de QTLs pour la sélection de levures plus performantes. / The yeast S. cerevisiae is the only species able to complete the alcoholic fermentation of grape must which is the main step of the wine-making process. Because of the high technological variability found in this species, selection work is carried out with the aim of using efficient yeasts for the industry. These strains show important differences both in their fermentation kinetics and their metabolite yield which affects the wines quality. In addition to these important phenotypic variations, the phenotypic response of yeasts varies considerably and not homogeneously against environmental variations. Understanding the genetic mechanisms that explain these differentiated responses to environmental variations is a non-trivial scientific question. This is of particular importance in oenology, where the winemaking conditions are highly variable (vintages, grape variety, terroirs, oenological practices...). In order to obtain yeasts ensuring the success of the fermentations under a wide range of conditions, we propose here to better understand these mechanisms of interaction Gene x Environment in an oenological context. The identification of genetic locus (Quantitative Trait Loci (QTL)) controlling quantitative characters is made possible by QTL mapping approaches. These approaches require the study of a large progeny in segregation that must be characterized genetically and phenotypically. The establishment of a statistical link between genotype and phenotype allows the localization of QTLs that have an impact on phenotyped characters. During this thesis a phenotyping method that allows us to follow the fermentations of several hundred individuals has been developed. Thanks to it, two progenies genotyped by whole genome sequencing have been phenotyped in different environmental conditions. This lead to the identification of many QTLs and to the estimation of their impact in strain robustness. The implication of the alleles of 3 genes showing a strong interaction with environment and pleiotropic effects linked to the SO2 metabolism has been proved molecularly. The results achieved are discussed in the context of QTL exploitation for the selection of more efficient yeast.

Directional selection is prevalent in nature yet phenotypes tend to remain relatively constant, suggesting a limit to trait evolution. The genetic basis of evolutionary limits in unmanipulated populations, however, is generally not known. Given widespread pleiotropy, opposing selection on a focal trait may arise from the effects of the underlying alleles on other fitness components, generating net stabilizing selection on trait genetic variance and thus limiting evolution. Here, I look for the signature of stabilizing selection for a suite of cuticular hydrocarbons (CHCs) in Drosophila serrata. Despite strong directional sexual selection on CHCs, genetic variance differed between high and low fitness individuals and was greater among the low fitness males for seven of eight CHCs. Univariate tests of a difference in genetic variance were non-significant but have low power. My results implicate stabilizing selection, arising through pleiotropy, in generating a genetic limit to the evolution of CHCs in this species.

Directional selection is prevalent in nature yet phenotypes tend to remain relatively constant, suggesting a limit to trait evolution. The genetic basis of evolutionary limits in unmanipulated populations, however, is generally not known. Given widespread pleiotropy, opposing selection on a focal trait may arise from the effects of the underlying alleles on other fitness components, generating net stabilizing selection on trait genetic variance and thus limiting evolution. Here, I look for the signature of stabilizing selection for a suite of cuticular hydrocarbons (CHCs) in Drosophila serrata. Despite strong directional sexual selection on CHCs, genetic variance differed between high and low fitness individuals and was greater among the low fitness males for seven of eight CHCs. Univariate tests of a difference in genetic variance were non-significant but have low power. My results implicate stabilizing selection, arising through pleiotropy, in generating a genetic limit to the evolution of CHCs in this species.

<p>Determining the genetic basis of adaptation has become a central focus of evolutionary biology, and the incorporation of increasingly sophisticated analytical tools from molecular biology has made identifying causal genes a practical reality. The work presented herein addresses the effects of pleiotropic constraint on evolutionary change at the level of individual genes and genetic networks. In the first chapter, I combine molecular phylogenetic analyses and direct assays of enzymatic function to determine the evolutionary processes following a gene duplication in the anthocyanin pathway. My results show that, prior to duplication, the DFR gene was constrained from functional improvement by its multiple enzymatic roles. Following duplication, this constraint was released and adaptive evolution proceeded along both paralog lineages. In the second chapter, I determine the molecular genetic basis of a flower color transition that is associated with change in pollinator attraction in morning glories. A regulatory change in a branching gene in the flavonoid biosynthetic pathway restricted flux down the cyanidin-producing branch, conferring nearly exclusive production of red pelargonidin pigment in flowers. I further demonstrate that this regulatory change was restricted to floral tissue, and that ancestral pathway flux predominates in vegetative tissues. I propose that deleterious pleiotropic effects prevented evolutionary change via enzymatic changes in the pathway due to the numerous essential products downstream of this branching point. Together, these two results show that evolutionary change may be constrained by the molecular genetic context in which prospective adaptive mutations occur.</p> / Dissertation

Humans and chimpanzees differ in a number of phenotypic traits chief among them being a larger sized human brain and an increased propensity for cancer in humans. Apoptosis or programmed cell death plays a role during brain development and disease progression to cancer. Results from my study, based on gene expression analysis, suggest that the apoptotic function may be generally reduced in humans relative to chimpanzees. In this thesis, I test the hypothesis that the apoptotic function is generally reduced in humans relative to chimpanzees by gene expression and experimental data. The experimental data are consistent with the hypothesis and also suggest that the apoptotic function may be reduced in humans relative to chimpanzees and macaques, suggesting that the reduced apoptotic function may be an evolutionary derived condition within the human lineage. I also evaluate the role of this reduced function in humans during brain development and disease progression to cancer. In addition, I also correlate Insertion/Deletion sequence variation between humans and chimpanzees with differences in gene expression between the two species.

Directional selection is prevalent in nature yet phenotypes tend to remain relatively constant, suggesting a limit to trait evolution. The genetic basis of evolutionary limits in unmanipulated populations, however, is generally not known. Given widespread pleiotropy, opposing selection on a focal trait may arise from the effects of the underlying alleles on other fitness components, generating net stabilizing selection on trait genetic variance and thus limiting evolution. Here, I look for the signature of stabilizing selection for a suite of cuticular hydrocarbons (CHCs) in Drosophila serrata. Despite strong directional sexual selection on CHCs, genetic variance differed between high and low fitness individuals and was greater among the low fitness males for seven of eight CHCs. Univariate tests of a difference in genetic variance were non-significant but have low power. My results implicate stabilizing selection, arising through pleiotropy, in generating a genetic limit to the evolution of CHCs in this species.

Submitted by YURI TANI UTSUNOMIYA null (yuri.tani@yahoo.com.br) on 2018-01-08T12:45:05Z No. of bitstreams: 1 UTSUNOMIYA.pdf: 11784469 bytes, checksum: 9fc7d258b6e5e683b47b78ad25853acc (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-01-09T09:34:15Z (GMT) No. of bitstreams: 1 utsunomiya_yt_dr_jabo.pdf: 11784469 bytes, checksum: 9fc7d258b6e5e683b47b78ad25853acc (MD5) / Made available in DSpace on 2018-01-09T09:34:15Z (GMT). No. of bitstreams: 1 utsunomiya_yt_dr_jabo.pdf: 11784469 bytes, checksum: 9fc7d258b6e5e683b47b78ad25853acc (MD5) Previous issue date: 2017-12-06 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O gene 1 do adenoma pleomórfico (PLAG1) apresenta evidência de seleção positiva recente e associação com tamanho corporal e fertilidade em um grande número de raças bovinas ao redor do mundo. Tendo em vista sua recentemente descoberta função como fator de transcrição para o gene do fator de crescimento semelhante à insulina 2 (IGF2), o PLAG1 possui papel emergente como um dos principais reguladores do crescimento e da reprodução em bovinos. Apesar de sua importância, a variante de sequência de DNA responsável pelos efeitos pleiotrópicos atribuídos ao PLAG1 em bovinos permanece desconhecida. Também não está claro se a mesma mutação explica as associações fenótipo-genótipo encontradas em diferentes populações bovinas. Além disso, ainda é incerto onde e quando ocorreu a pressão de seleção responsável pelo aumento da frequência da mutação do PLAG1. No presente trabalho, reportamos o desenvolvimento de um pacote para o software estatístico R, o qual é direcionado à análise de haplótipos como preditores para variantes genéticas não observadas. Através da aplicação desta ferramenta a dados genômicos de bovinos oriundos de diversas regiões do mundo, encontramos evidência indicando que um único alelo derivado do PLAG1 aumentou em frequência rapidamente em bovinos Bos taurus do noroeste europeu entre os séculos XVI e XVIII. Este período é reconhecido como a última onda de aumento de estatura em bovinos por meio de registros arqueológicos. Os dados também sugerem que o alelo foi introgredido em B. taurus não europeu e raças Bos indicus entre os séculos XIX e XX, adquirindo uma distribuição quase global no último século. Análises de DNA antigo revelaram que esta mutação segrega em gado do noroeste europeu há pelo menos 1.000 anos. Em conjunto, estes resultados implicam um papel central da mutação do PLAG1 em recentes mudanças de tamanho corporal em bovinos. / The pleomorphic adenoma gene 1 (PLAG1) presents both evidence of recent positive selection and association with body size and fertility in a wide range of worldwide cattle breeds. Considering its recently uncovered function as a transcription factor for the insulin-like growth factor 2 gene (IGF2), PLAG1 is emerging as a major regulator of bovine growth and reproduction. In spite of its importance, the causal DNA sequence variant underlying the pleiotropic effects of PLAG1 in cattle remains unknown. It is also unclear whether the same mutation accounts for the phenotype-genotype associations detected across different cattle populations. Furthermore, when and where the selective pressure responsible for increasing the frequency of the PLAG1 mutation occurred is still uncertain. Here, we report the development of a package for the R statistical software to analyze haplotypes as surrogates for unobserved genetic variants. By applying this tool to genomic data of worldwide cattle breeds, we found evidence that a single bovine PLAG1 derived allele increased rapidly in frequency in Northwestern European Bos taurus populations between the 16th and 18th centuries. This period is recognized as the last wave of increase in bovine stature from archaeological data. The data also suggested that the allele was introgressed into non-European B. taurus and Bos indicus breeds towards the 19th and 20th centuries, achieving an almost global distribution in the last century. Ancient DNA analyses further revealed that this mutation has been segregating in Northwestern European cattle for at least 1,000 years. Altogether, these results implicate a major role of the PLAG1 mutation in recent changes in body size in cattle. / 2014/01095-8 / 2016/07531-0

abstract: The pattern and strength of genetic covariation is shaped by selection so that it is strong among functionally related characters and weak among functionally unrelated characters. Genetic covariation is expressed as phenotypic covariation within species and acts as a constraint on evolution by limiting the ability of linked characters to evolve independently of one another. Such linked characters are "constrained" and are expected to express covariation both within and among species. In this study, the pattern and magnitude of covariation among aspects of dental size and shape are investigated in anthropoid primates. Pleiotropy has been hypothesized to play a significant role in derivation of derived hominin morphologies. This study tests a series of hypotheses; including 1) that negative within- and among-species covariation exists between the anterior (incisors and canines) and postcanine teeth, 2) that covariation is strong and positive between the canines and incisors, 3) that there is a dimorphic pattern of within-species covariation and coevolution for characters of the canine honing complex, 4) that patterns of covariation are stable among anthropoids, and 5) that genetic constraints have been a strong bias on the diversification of anthropoid dental morphology. The study finds that patterns of variance-covariance are conserved among species. Despite these shared patterns of variance-covariance, dental diversification has frequently occurred along dimensions not aligned with the vector of genetic constraint. As regards the canine honing complex, there is no evidence for a difference in the pleiotropic organization or the coevolution of characters of the complex in males and females, which undermines arguments that the complex is selectively important only in males. Finally, there is no evidence for strong or negative pleiotropy between any dental characters, which falsifies hypotheses that predict such relationships between incisors and postcanine teeth or between the canines and the postcanine teeth. / Dissertation/Thesis / Ph.D. Anthropology 2011