O MHC é uma região genômica que contém genes de papel central na resposta imune adaptativa. Genes do MHC e, particularmente, genes HLA em humanos, estão envolvidos susceptibilidade e resistência a doenças infecciosas, na predisposição a doenças autoimunes e na rejeição de órgãos transplantados. Essas descobertas incentivaram uma série de estudos sobre padrões da variabilidade genética em genes HLA, que demonstraram possuir uma variação bastante distinta da expectativa neutra. Essa contundente evidência de seleção natural, ímpar no genoma humano, levanta uma série de perguntas a respeito das forças evolutivas específicas que agem nesses genes e as implicações genômicas para a evolução da região como um todo. O presente estudo investiga como a seleção natural afeta e é afetada pela diversidade de genes que estão ligados fisicamente a outros que constituem alvos de seleção. Nossa expectativa é que seleção balanceadora forte sobre genes HLA interfere na eficácia com que a seleção purificadora remove variantes deletérias em loci próximos. Especificamente, a partir da anotação funcional de variantes genéticas, testamos se grupos de genes ligados fisicamente aos genes HLA apresentam uma diversidade mais alta do que seria esperado na ausência de seleção balanceadora e de carona genética causada por ela, e se essa diversidade é enriquecida com variantes possivelmente deletérias. Por meio da análise de razão entre polimorfismos não-sinônimos e sinônimos (e diversas outras estatísticas relacionadas), fomos capazes de observar que loci próximos a genes HLA acumulam um excesso de variação não-sinônima (e portanto potencialmente deletéria). O grau de deleteriedade foi confirmado pelo emprego do software Polyphen 2, que utiliza como critério de classificação a conservação das sequências nucleotídicas e informação das estruturas protéicas, e pela análise de estatísticas como Pdel/Pn e Pdel/Ps. De acordo com testes de McDonald-Kreitman e o Índice de Neutralidade, entretanto, parte dessa variação deletéria se fixa em longo prazo, o que sugere que a seleção em genes HLA pode interferir tanto nos padrões de polimorfismos como de divergência / The MHC is a genomic region that contains genes with a central role in the adaptive immune response. Genes in the MHC region, in particular the HLA genes of humans, are involved in the differential susceptibility and resistance to infectious diseases, predisposition to autoimmune diseases and the rejection of transplanted organs. These findings have fueled a series of studies on patterns of genetic variation at HLA genes, which have conclusively demonstrated that their variation deviates from neutral expectations. Such strong evidence of natural selection, with few counterparts in the remainder of the human genome, raise a series of questions concerning the specific evolutionary forces acting on this region and their genomic implications for the evolution of the region as a whole. This work investigates how natural selection affects and is affected by the diversity of genes that are physically linked to those that are units of selection. Our expectation is that strong selection on HLA genes may interfere with the efficacy of selection in removing deleterious variants at closely linked loci. Specifically, by using functional annotations of genetic variants, we test whether sets of genes physically linked to the strongly selected HLA loci show a higher diversity than would be expected in the absence of balancing selection and genetic hitchhiking caused by it, and if this diversity is enriched for putatively deleterious variants. By analyzing the ratio of nonsynonymous to synonymous polymorphisms (and several related statistics) we were able to show that loci close to HLA genes are harboring an excess of nonsynonymous (and hence potentially deleterious) variation. The deleteriousness was confirmed by employing Polyphen 2 - a software that uses nucleotide sequence conservation and protein structure information to classify variants as deleterious or not - and computing statistics such as Pdel/Pn and Pdel/Ps. According to McDonald-Kreitman tests and the Neutrality Index, however, part of this putatively deleterious variation reaches fixation over long timespans, suggesting that selection at the HLA genes may be interfering with both the transient patterns of polymorphism and substitution processes

The past two decades have produced a substantial amount of research about the teaching and learning of evolution; however, recent research often lacks a theoretical foundation. Application of a new theoretical framework could help fill the void and improve research about student concepts of evolution. This study seeks to show that a resource-based framework (Hammer et al., 2005) can improve research into student concepts of natural selection. Concepts of natural selection from urban community college students were assessed via qualitative (interviews, written open-response questions, and write/think aloud procedures) and quantitative methods (coded open response analysis, Concept Inventory for Natural Selection (CINS)(Anderson, Fisher, & Norman, 2002). Results showed that students demonstrate four important aspects of resource-based framework: the multi-faceted construction of concepts, context sensitivity/ concept flexibility, at-the-moment activation of resources, and perceptual frames. In open response assessment, evolutionary-gain responses produced significantly different responses than evolutionary-loss questions with: 1) significantly more correct answers for the gain than loss question (Wilcoxon signed rank test, z = -3.68, p=0.0002); 2) more Lamarckian responses to loss than the gain question (Fisher exact, p=0.0039); and significantly different distributions in expanded need vs basic need answers (Fishers exact, p = 0.02). Results from CINS scores showed significant differences in post activity scores between students that held different naive concepts associated with origin of variation, origin of species, differential reproduction, and limited survival suggesting that some naive ideas facilitate learning. Outcomes also suggest that an everyday or self-experience typological perceptual frame is an underlying source of many incorrect ideas about evolution. Interview and write/think aloud assessments propose four process resources applied by students as they explain evolutionary change: list what I know, why story, compare past to present, mapping self-experience. The study concludes that a resource-based framework is a valuable tool to advance the study student concepts of natural selection.

Directed evolution is a powerful tool that mimics the natural selection process to engineer biomolecules with improved and altered functionalities for a wide variety of applications. The advance of biological engineering based on directed evolution techniques depends upon selection assays that can practically search large, diverse libraries for the most improved variants. In Chapter 1, we begin by discussing the potential of growth selections to serve as accessible and robust assays for directed evolution. We then delve into the existing approaches to expand the scope of targets for growth selections beyond those that are intrinsically linked to growth and the strategies implemented to improve their throughput and sensitivity. The yeast three-hybrid (Y3H) assay is a versatile system that can expand the field of directed evolution if implemented as a growth selection for the search of large variant libraries. Although the Y3H assay has been successfully applied as a positive selection to evolve proteins with improved functions, its expansion into applications requiring a high-throughput, versatile selection against transcriptional activation has been hindered by its limited dynamic range as a counter selection. To address the limited dynamic range of the Y3H assay, we undertook a multi-pronged approach to reengineer our Y3H counter selection to have a high dynamic range. In Chapter 2, we discuss strategies to improve the dynamic range of the Y3H counter selection by maximizing the growth between cells with activated and basal reporter gene expression levels. Specifically, we elaborate on the development and characterization of two Y3H counter selections that were based on either reporter gene degradation or an alternative phototoxic reporter gene. In Chapter 3, we present our most successful strategy to improve the dynamic range of the Y3H counter selection that uses the dual tetracycline (Tet) system to increase transcriptional regulation of the reporter gene. We employed a guided strategy based on both rational design and library approaches to find the best Tet Y3H reporter gene construct with the highest dynamic range. We believe our method to engineer the best Tet Y3H reporter construct will be widely useful to synthetic biologists developing sophisticated in vivo assays that require fine-tuned reporter gene expression levels. Finally, in Chapter 4, we demonstrate the versatility of the Y3H system by developing a screen for the detection of natural product biosynthesis. This assay should have an impact for metabolic engineers that are employing directed evolution techniques to generate large metabolic pathway libraries for the overproduction of high-value small molecules in heterologous producer strains.

It is well known that nucleotide diversity varies across the genomes of eukaryotic species in ways consistent with the effects of natural selection. However, the contribution of selection on advantageous and deleterious mutations to the observed variation is not well understood. In this thesis, I aim to disentangle the contribution of background selection and selective sweeps to patterns of genetic diversity in the mouse genome, thus furthering our understanding of natural selection in mammals. In chapter 1, I introduce core concepts in evolutionary genetics and describe how recombination and selection interact to shape patterns of genetic diversity. I will then describe three projects in which I examine aspects of molecular evolution in house mice. In the first of these, I estimate the landscape of recombination rate variation in wild mice using population genomic data. In the second, I estimate the distribution of fitness effects for new mutations, based on the site frequency spectrum, then analyse population genomic simulations parametrized using my estimates. In the third, I use a model of selective sweeps to estimate and compare the strength of selection in protein-coding and regulatory regions of the mouse genome. This thesis demonstrates that selective sweeps are responsible for a large amount of the variation in genetic diversity across the mouse genome.

Speciation – the process by which new species arise – is of fundamental importance in the biological sciences. The means by which new species arise, and the relationship among living species, has been a topic that has captivated both lay and scientific observers for centuries. In recent years, the study of speciation has enjoyed increased attention, resulting in significant advances in our understanding of how species form. Although our understanding of the processes that contribute to speciation has increased dramatically in recent years, our knowledge of how reproductive barriers accumulate as speciation proceeds is still limited. Thus, studies that evaluate trait divergence and its consequences at early verses late stages of divergence can provide valuable insight into the speciation process. Chapter 1 of my dissertation focuses on the role of animal personality in the speciation process. Animal personality – defined as consistent individual differences in behavioral tendencies – has been identified as a key player in several ecological and evolutionary processes, yet the role of personality in speciation remains unexplored. In this chapter I discuss the ways by which personality can contribute to a suite of reproductive barriers and drive the speciation process. Chapters 2 through 5 provide a case study evaluating how selection acts on traits at early and late stages of speciation, using the Neotropical Livebearing fish genus Brachyrhaphis as a model system. Brachyrhaphis is ideally suited for this research because several species pairs and population pairs within species occur in similarly divergent selective regimes. I first present results from a field demographic study that shows that the strength of divergent selection acting on life-history traits in populations from divergent predation environments diminishes as speciation proceeds. I then show that population pairs at different stages of divergence are evolving similar morphological patterns along parallel trajectories. At both early and late stages of divergence, populations from environments with dense predator populations have a body shape that appears to be optimized for burst-speed swimming, and important component of predator escape. In contrast, populations from environments lacking predators have a body shape optimized for endurance swimming ability, which is important in environments where competition for foods and mates is high. Next, I show that populations from divergent predation environments do indeed differ in their swimming abilities according to our predictions, reflecting a population level trade-off between burst and endurance swimming ability. Although population level trade-offs were strong, I found no evidence of within population level trade-offs, suggesting that populations have arrived at novel solutions to between population trade-offs that were not present within ancestral populations. Finally, I show that these specialized swimming modes are locally adaptive, and that divergent ecology selects against immigrants, effectively reducing gene flow between populations from divergent environments. Together, these studies provide a valuable glimpse into the repeatability and predictability of trait divergence at different stages of speciation.

Title from container. Includes bibliographies and indexes. Contributions to quantitative and population genetics -- The biochemical genetics of man -- The theory of plant breeding -- Natural selection and its constraints.

The expression level of a single gene can vary substantially within and between species, which might facilitate the emergence and fixation of novel expression patterns in the course of evolution. With rapidly accumulating data from genome-wide expression profiling, dense genotyping and individual genome re-sequencing, it is now possible to pinpoint the genetic loci that potentially give rise to gene expression variation. However, what remains elusive is how expression changes could be attributed to the differences in genetic elements, and our understanding of the phenotypic manifestation resulting from gene expression variation is far from comprehensive. In this thesis, I aim to answer these questions in budding yeast and in human. I first studied duplicated genes in budding yeast, which usually shared the identical expression patterns immediately upon duplication events. I searched for the cis-elements, whose divergence might explain the substantial expression variation between the extant paralogs, and established the role of nucleosome occupancy in driving expression differentiation between yeast duplicates. I next investigated the role of trans-factors in establishing species- or population-specific gene expression, and my study was specifically focused on primate microRNAs as a special class of regulators in trans. I first delineated the evolutionary trajectory of an X-linked primate microRNA cluster, and then proposed its function in regulating primate epididymal physiology. I extended this study to human by identifying several microRNAs with highly differentiated regulation among human populations, and such regulatory differentiation was driven by positive selection during recent human evolution. This study for the first time demonstrated high plasticity of the microRNA regulatory interactions in modulating expression variation of their target messengers. Beyond exploring the elements that control gene expression variation, I examined phenotypic manifestation of the observed expression variation in human populations, and my analysis revealed significant implication of expression variation towards differential disease susceptibility among individuals. Lastly, I examined gene expression variation at a micro scale among isogenic cell populations in budding yeast, which is termed “expression noise”. Though expression noise originates from stochasticity, my analysis demonstrated strong topological constraints on expression noise in yeast cellular networks, with which I was able to predict gene expression noise with high accuracy. These observations suggest that the seemingly stochastic gene expression may have been evolutionarily constrained. Taken together, my study presented in this thesis investigates the origin, consequence and evolutionary significance of gene expression variation in eukaryotes.

The expression level of a single gene can vary substantially within and between species, which might facilitate the emergence and fixation of novel expression patterns in the course of evolution. With rapidly accumulating data from genome-wide expression profiling, dense genotyping and individual genome re-sequencing, it is now possible to pinpoint the genetic loci that potentially give rise to gene expression variation. However, what remains elusive is how expression changes could be attributed to the differences in genetic elements, and our understanding of the phenotypic manifestation resulting from gene expression variation is far from comprehensive. In this thesis, I aim to answer these questions in budding yeast and in human. I first studied duplicated genes in budding yeast, which usually shared the identical expression patterns immediately upon duplication events. I searched for the cis-elements, whose divergence might explain the substantial expression variation between the extant paralogs, and established the role of nucleosome occupancy in driving expression differentiation between yeast duplicates. I next investigated the role of trans-factors in establishing species- or population-specific gene expression, and my study was specifically focused on primate microRNAs as a special class of regulators in trans. I first delineated the evolutionary trajectory of an X-linked primate microRNA cluster, and then proposed its function in regulating primate epididymal physiology. I extended this study to human by identifying several microRNAs with highly differentiated regulation among human populations, and such regulatory differentiation was driven by positive selection during recent human evolution. This study for the first time demonstrated high plasticity of the microRNA regulatory interactions in modulating expression variation of their target messengers. Beyond exploring the elements that control gene expression variation, I examined phenotypic manifestation of the observed expression variation in human populations, and my analysis revealed significant implication of expression variation towards differential disease susceptibility among individuals. Lastly, I examined gene expression variation at a micro scale among isogenic cell populations in budding yeast, which is termed “expression noise”. Though expression noise originates from stochasticity, my analysis demonstrated strong topological constraints on expression noise in yeast cellular networks, with which I was able to predict gene expression noise with high accuracy. These observations suggest that the seemingly stochastic gene expression may have been evolutionarily constrained. Taken together, my study presented in this thesis investigates the origin, consequence and evolutionary significance of gene expression variation in eukaryotes.

Human-induced eutrophication is indirectly affecting aquatic organisms by altering their environment. This brings on altered selective pressures and could thereby cause changes in the genetic composition of exposed populations. Since anthropogenic environmental changes are usually occurring at a much higher rate than naturally occurring changes, they force populations to adapt to the new conditions faster than normal. Here, I have studied populations of three-spined sticklebacks (Gasterosteus aculeatus) from four eutrophicated and four adjacent reference sites, along the coast of Finland, to investigate if this species has responded genetically to the human-induced eutrophication of the Baltic Sea. For this purpose I used amplified fragment length polymorphism (AFLP) and found distinctions in genetic composition between the two habitats, as well as similarities between populations from eutrophicated sites. This suggests a similar genetic response to eutrophicated conditions by stickleback populations from different geographical areas. Moreover I found a distinct geographic structure among three-spined sticklebacks in the Baltic Sea.

Human-induced eutrophication is indirectly affecting aquatic organisms by altering their environment. This brings on altered selective pressures and could thereby cause changes in the genetic composition of exposed populations. Since anthropogenic environmental changes are usually occurring at a much higher rate than naturally occurring changes, they force populations to adapt to the new conditions faster than normal. Here, I have studied populations of three-spined sticklebacks (Gasterosteus aculeatus) from four eutrophicated and four adjacent reference sites, along the coast of Finland, to investigate if this species has responded genetically to the human-induced eutrophication of the Baltic Sea. For this purpose I used amplified fragment length polymorphism (AFLP) and found distinctions in genetic composition between the two habitats, as well as similarities between populations from eutrophicated sites. This suggests a similar genetic response to eutrophicated conditions by stickleback populations from different geographical areas. Moreover I found a distinct geographic structure among three-spined sticklebacks in the Baltic Sea.