Positive natural selection is recognized as the driving force underneath evolution. One of the surest signatures of recent positive selection is a local elevation of advantageous allele frequency and linkage disequilibrium (LD). This dissertation proposes a new test statistic to detect excess homozygosity based on a simple counting measure, which serves as a surrogate indicator of recent positive selection. Three tests are developed upon the new measure: (a) an extended genotype-based homozy- gosity test (EGHT), (b) a hidden Markov model test (HMMT), and (c) an extended haplotype-based homozygosity test (EHHT). The null hypotheses of all three tests assume random mating and Hardy-Weinberg equilibrium (HWE). They differ in how to treat LD under H0 . The EGHT assumes linkage equilibrium (LE) besides HWE while the EHHT allows arbitrary multi-locus LD. The HMMT stands between these two extremes and assumes pairwise but no higher-order disequilibrium interactions. We first conduct simulation study to compare the three score tests and verify that the EHHT is the most conservative one. We compare the performance of the EHHT with the prevailing detection methods and the EHHT has higher or similar power. We also evaluate the impact of simple demographic history on the EHHT and the simulation study suggests that the EHHT is resistant to the false-positive confounders resulting from simple demographic models. After extensive simulation studies, all three tests are then applied on HapMap Phase II data and we are able to replicate findings reported in the literature. We can also identify new candidate regions that may undergo recent selection through a set of filtering criteria including highest EHHT scores, high derived allele frequency and large population differentiation. Finally, we propose a cross-population comparison test statistic to detect chromosome regions in which there is no significant excess homozygosity in one population but homozygosity remains high in another population.

Repeated emergence of similar adaptations is often explained by parallel evolution of underlying genes. However, evidence of parallel evolution at amino acid level is limited. When the analyzed species are highly divergent, this can be due to epistatic interactions underlying the dynamic nature of the amino acid preferences: The same amino acid substitution may have different phenotypic effects on different genetic backgrounds. Distantly related species also often inhabit radically different environments, which makes the emergence of parallel adaptations less likely. Here, we hypothesize that parallel molecular adaptations are more prevalent between closely related species. We analyze the rate of parallel evolution in genome-size sets of orthologous genes in three groups of species with widely ranging levels of divergence: 46 species of the relatively recent lake Baikal amphipod radiation, a species flock of very closely related cichlids, and a set of significantly more divergent vertebrates. Strikingly, in genes of amphipods, the rate of parallel substitutions at nonsynonymous sites exceeded that at synonymous sites, suggesting rampant selection driving parallel adaptation. At sites of parallel substitutions, the intraspecies polymorphism is low, suggesting that parallelism has been driven by positive selection and is therefore adaptive. By contrast, in cichlids, the rate of nonsynonymous parallel evolution was similar to that at synonymous sites, whereas in vertebrates, this rate was lower than that at synonymous sites, indicating that in these groups of species, parallel substitutions are mainly fixed by drift.

Determining the genetic basis of reproductive isolation is a fundamental goal in evolutionary biology. Intrinsic reproductive isolation often arises due to epistasis between divergent interacting genes. The rapid evolution of hybrid male sterility is known to have several causes, including the exposure of recessive X-linked incompatibilities in males and the rapid evolution of male reproductive traits. Despite these insights, little is known about the genetics of reproductive isolation during the early stages of speciation. This deficiency inspired parallel studies on the molecular evolution of male reproduction in house mice and the genetic basis of hybrid male sterility between two mouse species, Mus domesticus and M. musculus. Evolutionary analysis of 946 genes showed that the intensity of positive selection varies across sperm development and acts primarily on phenotypes that develop late in spermatogenesis (Appendix A). Several reciprocal crosses between wild-derived strains of M. musculus and M. domesticus were used to examine F1 hybrid male sterility (Appendix B). These crosses revealed hybrid male sterility linked to the M. musculus X chromosome and a novel sterility polymorphism within M. musculus. A large introgression experiment was used to further dissect the genetic basis of X-linked incompatibilities between M. musculus and M. domesticus (Appendix C). Introgression of the M. musculus X chromosome into a M. domesticus genetic background produced male sterility and involved a minimum of four factors. No sterility factors were uncovered on the M. domesticus X chromosome. These data demonstrate the complex genetic basis of hybrid sterility in mice and provide numerous X-linked candidate sterility genes. The molecular evolution of five rapidly evolving candidate genes was examined using population and phylogenetic sampling in Mus (Appendix D). Four of these loci showed evidence of positive natural selection. One locus, 4933436I01Rik, showed divergent protein evolution between M. domesticus and M. musculus and was one of a handful of testis-expressed genes within a narrow interval involved in hybrid male sterility. In summary, these data demonstrate that hybrid male sterility has a complex genetic basis between two closely related species of house mice and provide a foundation for the identification of specific mutations that isolate these species.

Deciphering the genetic basis of phenotypic diversity is one of the central aims of biological research. Domestic animals provide a unique opportunity for making substantial progress towards this goal. Intense positive selection has lead to a rich reservoir of phenotypes and underlying genotypes that can be interrogated using genetic tools to gain insight into the genetic basis of phenotypic diversity. The dog is the most phenotypically diverse mammal. It was domesticated from the grey wolf 11-30,000 years ago. After domestication, a period of intense breeding has lead to the massive phenotypic diversity seen amongst dog breeds today. These two phases of strong positive selection at domestication and at breed creation are likely to have left their signature on the genome. In this thesis, we have analysed genome-wide patterns to detect genomic regions involved in selection in both of these phases. We used whole genome sequences from 60 dogs and 12 wolves, to detect dog domestication selective sweeps. We find evidence for genes involved in memory formation, neurotransmission and starch digestion. To decipher the genetic signals underlying breed diversity, we used genome-wide genotype data from >170,000 SNPs in 509 dogs from 46 different breeds. We find evidence for genes under selection in many breeds, and only a few breeds. In addition, we identify novel sweeps underlying morphology and behavior. Recombination can influence the configuration of alleles present on a haplotype, and can thus increase or decrease the efficiency of selection. The PRDM9 protein has been shown to be important for determining recombination hotspot locations in humans and other mammals, but of all the mammals studied so far the dog is the only one to have a non-functional PRDM9. We used the genome-wide genotype data described above to characterise the fine scale recombination map in dogs. We find that recombination hotspots exist in dogs despite the absence of PRDM9. Moreover, we show that these hotspots are enriched for GC rich peaks and that these peaks are getting stronger over time. Our results show that the absence of PRDM9 has lead to the stabilisation of the recombination landscape in dogs.

<p>The E-protein transcription factors E2A and HEB regulate various cell processes during the development of B and T lymphocytes, including cell differentiation, lineage commitment, recombination of immune receptor genes, proliferation, and survival. B cell development is dependent on E2A from the earliest stages whereas T cell development relies on the cooperative efforts of both E2A and HEB. Established work demonstrates that the timing and dosage of E-protein expression is critical for mediating these diverse functions. The goal of this dissertation is to develop and utilize new genetic tools to manipulate the timing and dosage of E2A and HEB expression in order to enhance our understanding of E-protein function. Here we develop two new mouse models to identify novel lineage and stage specific roles of E-proteins during B lineage commitment and thymocyte selection.</p><p>First, we have generated an E2A inducible mouse model to allow reversible regulation of E2A function and precise timing of induction at the protein level. This system was created by inserting a tamoxifen responsive region of the estrogen receptor ligand binding domain (ER) at the carboxyl end of the <italic>tcfe2a</italic> gene, encoding E2A, to generate E2AER fusion proteins. To our knowledge, the ER fusion system has not yet been tested from an endogenous locus in live animals. Using the E2AER system, we have demonstrated rapidly induced E2AER activity upon tamoxifen treatment that is capable of supporting B cell development in an <italic>ex vivo</italic> culture system. In addition to characterizing the kinetics and reversibility of this inducible system, we have utilized tamoxifen treatment of E2AER B cell progenitors to identify potential novel E2A target genes driving B lineage commitment.</p><p>Second, we have analyzed E-protein function during the double positive (DP) stage of alpha beta T cell development by using a Cre-loxp conditional deletion system. Here, E-protein dosage was manipulated by removal of both E2A and HEB, and the timing of deletion was controlled by using a CD4Cre transgene. During development, survival through the DP stage and initiation of differentiation to the subsequent single positive (SP) stage for generation mature alpha beta T cells is dependent on the production of a functional alpha beta T cell receptor (TCR). The mechanism that maintains cells at the DP stage prior to expression of a mature TCR remains unclear. In this study, we have shown that E2A and HEB together are required to maintain DP fate and regulate the transition to the SP stage. Loss of E2A and HEB in DP thymocytes was sufficient to trigger DP to SP differentiation, even in the absence of a TCR. Deletion of E2A and HEB allowed cells to bypass the requirement for a TCR-mediated positive selection signal. These findings identify E2A and HEB as key regulators enforcing thymocyte positive selection to ensure maturing T cells express a functional receptor.</p> / Dissertation

<p>Deciphering the molecular mechanisms driving the phenotypic differences between humans and primates remains a daunting challenge. Mutations found in protein coding DNA alone has not been able to explain these phenotypic differences. The hypothesis that mutations in non-coding regulatory DNA are responsible for altered gene expression leading to these phenotypic changes has now been widely supported by differential gene expression experiments. Yet, comprehensive identification of all regulatory DNA elements across different species has not been performed. To identify the genetic source of regulatory change, genome-wide DNaseI hypersensitivity assays, marking all types of active gene regulatory element sites, were performed in human, chimpanzee, macaque, orangutan, and mouse. Many DNaseI hypersensitive (DHS) sites were conserved among all 5 species, but we also identified hundreds of novel human- and chimpanzee-specific DHS gains and losses that showed signatures of positive selection. Species-specific DHS gains were enriched in distal non-coding regions, associated with active histone modifications, and positively correlated with increased expression - indicating that these are likely to be functioning as enhancers. Comparison to mouse DHS data indicate that human or chimpanzee DHS gains are likely to have been a result of single events that occurred primarily on the human- or chimpanzee-specific branch, respectively. In contrast, DHS losses are associated with events that occurred on multiple branches. At least one mechanism contributing to DHS gains and losses are species-specific variants that lead to sequence changes at transcription factor binding motifs, affecting the binding of TFs such as AP1. These variants were functionally verified by DNase footprinting and ChIP-qPCR analyses.</p> / Dissertation

The ability to link a particular phenotype to its causative genotype is one of the most challenging objectives for biological research. Although the genetic code provides an explicit formula for determining the sequence of amino acid phenotypes produced by a given nucleotide sequence, identifying specific residues that are functionally important remains problematic. Many computational approaches have been developed that use patterns observed in DNA sequences to identify these critical sites. However, very few research studies have used empirical data to test whether these approaches are truly able to identify sites of interest.In most empirical studies, the actual protein function and selective pressures are unknown; thus it is difficult to assess whether computational approaches are correctly identifying critical sites. Here I present two studies that utilize well-characterized empirical systems to evaluate and compare the performance of several computational approaches. In both cases, the proteins under study have specific amino acid substitutions that are confirmed to alter protein function and expected to be constrained by natural selection. In chapter 2, I examine functional variants in angiopoietin-like protein 4 (ANGPTL4), a protein involved in regulating plasma triglyceride levels; loss-of-function variants in this gene are believed to decrease the risk of cardiovascular disease. I apply several computational approaches to identify functional variants, including phylogenetic approaches for detecting positive selection. In chapter 3, I investigate the emergence of drug-resistance in HIV-1 during the course of antiretroviral drug therapy. I compare the performance of eight selection detection methods in identifying drug-resistant mutations in 109 intrapatient datasets with HIV-1 sequences isolated at multiple timepoints throughout drug treatment.It is critical that we develop methods to detect positively selected sites. The ability to detect these sites in silico, without the need for expensive and time consuming assays, would be invaluable to researchers in evolutionary biology, human genetics, and medicine. Through the research presented in this thesis, I hope to provide insight into the strengths and weaknesses of current approaches, thereby facilitating future research towards the development and improvement of evolutionary models.

No description available.

As taxas de resposta viral ao tratamento com Interferon- associado com Ribavirina ainda não estão bem definidas. Muitos estudos associam vários fatores virais e do hospedeiro, tais como a idade, sexo, peso, etnia, nível de enzimas hepáticas, estágio de fibrose, genótipo do HCV com os níveis de RNA viral. Outros estudos associam as células CD8+ específicas para epítopos do HCV com o controle da viremia. O objetivo desse trabalho foi verificar se há aumento na pressão seletiva contra o vírus nos pacientes em tratamento com Interferon- associado à Ribavirina respondedores em comparação com os pacientes não respondedores e associar esse aumento com a ação antiviral do tratamento e/ou com o aumento da resposta imune devido à ação imunomoduladora do Interferon-, utilizando-se modelos de máxima verossimilhança, de cálculo da razão dN/dS e realizando o mapeamento dos epítopos das proteínas NS5A. Nossos resultados demonstram que usando modelos par a par não foi detectado evidência de seleção positiva, entretanto utilizando modelos de máxima verossimilhança nós observamos evidência no grupo que não responde a terapia. O mapeamento dos epítopos revelou que o epítopo VLSDFKTWL estava associado com a resposta efetiva do tratamento com suporte estatístico. Estes resultados indicam que a resposta imunológica aumenta durante o período de tratamento, auxiliando na eliminação do vírus. Além das análises, foi desenvolvida uma ferramenta de mapeamento automático dos epítopos do HCV e análise de seleção natural aplicando modelos de máxima verossimilhança / The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Several studies suggest that viral load is closely associated to viral and host factors, such age, sex, body weight, transaminase levels and HCV genotype. Moreover, an strong CD8+ T-cell immunity in acute resolving hepatitis C is matched by strong and sustained CD4+ T-cell proliferation to multiple recombinant structural and non-structural viral proteins is responsible for the control of viraemia in HCV infection. Here, we investigate the differences in CD8 epitopes frequencies from Los Alamos database between groups of patients that showed distinct response to pegylated alpha interpheron with ribavirin therapy, and test for evidence of natural selection on virus in treatment failure group, using five maximum likelihood evolutionary models. Our results indicated no evidence of positive selection by using pairwise models, however we identify evidence of positive selection in non responder group by applying maximum likelihood models. The epitope mapping showed that the epitope VLSDFKTWL was associated with efective therapy outcome, with statistical support. Those results suggest that the immune response increase during the treatment period, allowing the virus clearance. We also developed a tool for automatic mapping of epitopes of HCV that tests for evidence of natural selection by applying maximum likelihood models

<p>The mutability of transcription factors (<strong>TF</strong>) is thought to be of high importance for the evolutionary change of living organisms. Transcription factors, coactivators, coregulators, kinases, chromatin remodelers conditional factors and other proteins together govern the timing and level of gene expression. About 10% of the genes in the human genome are predicted to be TFs and mutational changes in these genes or in the target regulatory sequences they bind will potentially give rise to evolutionary advantages or malfunctions for the organism. Recent research has suggested that the parts of the transcription factors that are not structurally defined in solution, so called intrinsically disordered regions (IDRs), have a higher potential for evolutionary diversification than more structurally rigid regions. This suggests that these domains that earlier have been considered mostly unimportant may have an important potential for evolutionary diversification. This project aimed to further evaluate evidence supporting the hypothesis that variable-structured domains in transcription factors are of significant importance for functional diversification. This was be done by comparing the rate of synonymous and non-synonymous genetic variation in the coding regions of 12 selected TFs within a highly conserved clade of European wine yeasts and by comparing this variation to divergent phenotypic patterns within the strains. The frequency of non-synonymous mutations was much greater than for synonymous mutations indicating an important role of positive selection acting on these TFs during diversification of the different strains. No significant connections were discovered between the distribution of DNA variation and phenotypic patterns.</p>