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abstract: The complex life cycle and widespread range of infection of Plasmodium parasites, the causal agent of malaria in humans, makes them the perfect organism for the study of various evolutionary mechanisms. In particular, multigene families are considered one of the main sources for genome adaptability and innovation. Within Plasmodium, numerous species- and clade-specific multigene families have major functions in the development and maintenance of infection. Nonetheless, while the evolutionary mechanisms predominant on many species- and clade-specific multigene families have been previously studied, there are far less studies dedicated to analyzing genus common multigene families (GCMFs). I studied the patterns of natural selection and recombination in 90 GCMFs with diverse numbers of gene gain/loss events. I found that the majority of GCMFs are formed by duplications events that predate speciation of mammal Plasmodium species, with many paralogs being neutrally maintained thereafter. In general, multigene families involved in immune evasion and host cell invasion commonly showed signs of positive selection and species-specific gain/loss events; particularly, on Plasmodium species is the simian and rodent clades. A particular multigene family: the merozoite surface protein-7 (msp7) family, is found in all Plasmodium species and has functions related to the erythrocyte invasion. Within Plasmodium vivax, differences in the number of paralogs in this multigene family has been previously explained, at least in part, as potential adaptations to the human host. To investigate this I studied msp7 orthologs in closely related non-human primate parasites where homology was evident. I also estimated paralogs’ evolutionary history and genetic polymorphism. The emerging patterns where compared with those of Plasmodium falciparum. I found that the evolution of the msp7 multigene family is consistent with a Birth-and-Death model where duplications, pseudogenization and gene lost events are common. In order to study additional aspects in the evolution of Plasmodium, I evaluated the trends of long term and short term evolution and the putative effects of vertebrate- host’s immune pressure of gametocytes across various Plasmodium species. Gametocytes, represent the only sexual stage within the Plasmodium life cycle, and are also the transition stages from the vertebrate to the mosquito vector. I found that, while male and female gametocytes showed different levels of immunogenicity, signs of positive selection were not entirely related to the location and presence of immune epitope regions. Overall, these studies further highlight the complex evolutionary patterns observed in Plasmodium. / Dissertation/Thesis / Doctoral Dissertation Biology 2016

Retrotransposons comprise a large portion of mammalian genomes. They contribute to structural changes and more importantly to gene regulation. The expansion and diversification of gene families have been implicated as sources of evolutionary novelties. Given the roles retrotransposons play in genomes, their contribution to the evolution of gene families warrants further exploration. In this study, we found a significant association between two major retrotransposon classes, LINEs and LTRs, and lineage-specific gene family expansions in both the human and mouse genomes. The distribution and diversity differ between LINEs and LTRs, suggesting that each has a distinct involvement in gene family expansion. LTRs are associated with open chromatin sites surrounding the gene families, supporting their involvement in gene regulation, whereas LINEs may play a structural role promoting gene duplication. Our findings also suggest that gene family expansions, especially in the mouse genome, undergo two phases. The first phase is characterized by elevated deposition of LTRs and their utilization in reshaping gene regulatory networks. The second phase is characterized by rapid gene family expansion due to continuous accumulation of LINEs and it appears that, in some instances at least, this could become a runaway process. We provide an example in which this has happened and we present a simulation supporting the possibility of the runaway process. Altogether we provide evidence of the contribution of retrotransposons to the expansion and evolution of gene families. Our findings emphasize the putative importance of these elements in diversification and adaptation in the human and mouse lineages.

Over the last few decades, phylogenetics has emerged as a very promising field, facilitating a comparative framework to explain the genetic relationships among all the living organisms on earth. These genetic relationships are typically represented by a bifurcating phylogenetic tree — the tree of life. Reconstructing a phylogenetic tree is one of the central tasks in evolutionary biology. The different evolutionary processes, such as gene duplications, gene losses, speciation, and lateral gene transfer events, make the phylogeny reconstruction task more difficult. However, with the rapid developments in sequencing technologies and availability of genome-scale sequencing data, give us the opportunity to understand these evolutionary processes in a more informed manner, and ultimately, enable us to reconstruct genes and species phylogenies more accurately. This thesis is an attempt to provide computational methods for phylogenetic inference and give tools to conduct genome-scale comparative evolutionary studies, such as detecting homologous sequences and inferring gene families. In the first project, we present FastPhylo as a software package containing fast tools for reconstructing distance-based phylogenies. It implements the previously published efficient algorithms for estimating a distance matrix from the input sequences and reconstructing an un-rooted Neighbour Joining tree from a given distance matrix. Results on simulated datasets reveal that FastPhylo can handles hundred of thousands of sequences in a minimum time and memory efficient manner. The easy to use, well-defined interfaces, and the modular structure of FastPhylo allows it to be used in very large Bioinformatic pipelines. In the second project, we present a synteny-aware gene homology method, called GenFamClust (GFC) that uses gene content and gene order conservation to detect homology. Results on simulated and biological datasets suggest that local synteny information combined with the sequence similarity improves the detection of homologs. In the third project, we introduce a novel phylogeny-based clustering method, PhyloGenClust, which partitions a very large gene family into smaller subfamilies. ROC (receiver operating characteristics) analysis on synthetic datasets show that PhyloGenClust identify subfamilies more accurately. PhyloGenClust can be used as a middle tier clustering method between raw clustering methods, such as sequence similarity methods, and more sophisticated Bayesian-based phylogeny methods. Finally, we introduce a novel probabilistic Bayesian method based on the DLTRS model, to sample reconciliations of a gene tree inside a species tree. The method uses MCMC framework to integrate LGTs, gene duplications, gene losses and sequence evolution under a relaxed molecular clock for substitution rates. The proposed sampling method estimates the posterior distribution of gene trees and provides the temporal information of LGT events over the lineages of a species tree. Analysis on simulated datasets reveal that our method performs well in identifying the true temporal estimates of LGT events. We applied our method to the genome-wide gene families for mollicutes and cyanobacteria, which gave an interesting insight into the potential LGTs highways. / <p>QC 20161010</p>

The Lyme disease spirochete, B. burgdorferi is atypical in that a large portion of its genome is in the form of plasmids. Many of the plasmid-carried genes form extensive paralogous gene families and encode outer-surface proteins. In this report we have assessed the humoral immune response to proteins belonging to the paralogous protein family, family 163. We have cloned and expressed BBP39, BBO40, BBQ47 and BBN39 and used these recombinant proteins to monitor the temporal nature of the antibody response to these antigens during experimental infection of mice. The immunodominant regions of each protein have also been assessed through immunoblot analyses of a series of truncations of each protein. These analyses have led to the delineation of the targets of the antibody response during infection and of the specificity of the antibody response to family 163 proteins. In addition, we quantified the expression of each gene using real time RT-PCR.

Detailed gene family histories and reconciliations with species trees are a prerequisite for studying associations between genetic and phenotypic innovations. Even though the true evolutionary scenarios are usually unknown, they impose certain constraints on the mathematical structure of data obtained from simple yes/no questions in pairwise comparisons of gene sequences. Recent advances in this field have led to the development of methods for reconstructing (aspects of) the scenarios on the basis of such relation data, which can most naturally be represented by graphs on the set of considered genes. We provide here novel characterizations of best match graphs (BMGs) which capture the notion of (reciprocal) best hits based on sequence similarities. BMGs provide the basis for the detection of orthologous genes (genes that diverged after a speciation event). There are two main sources of error in pipelines for orthology inference based on BMGs. Firstly, measurement errors in the estimation of best matches from sequence similarity in general lead to violations of the characteristic properties of BMGs. The second issue concerns the reconstruction of the orthology relation from a BMG. We show how to correct estimated BMG to mathematically valid ones and how much information about orthologs is contained in BMGs. We then discuss implicit methods for horizontal gene transfer (HGT) inference that focus on pairs of genes that have diverged only after the divergence of the two species in which the genes reside. This situation defines the edge set of an undirected graph, the later-divergence-time (LDT) graph. We explore the mathematical structure of LDT graphs and show how much information about all HGT events is contained in such LDT graphs.

Phylogenetic inference relies heavily on statistical models that have been extended and refined over the past years into complex hierarchical models to capture the intricacies of evolutionary processes. The wealth of information in the form of fully sequenced genomes has led to the development of methods that are used to reconstruct the gene and species evolutionary histories in greater and more accurate detail. However, genes are composed of evolutionary conserved sequence segments called domains, and domains can also be affected by duplications, losses, and bifurcations implied by gene or species evolution. This thesis proposes an extension of evolutionary models, such as duplication-loss, rate, and substitution, that have previously been used to model gene evolution, to model the domain evolution. In this thesis, I am proposing DomainDLRS: a comprehensive, hierarchical Bayesian method, based on the DLRS model by Åkerborg et al., 2009, that models domain evolution as occurring inside the gene and species tree. The method incorporates a birth-death process to model the domain duplications and losses along with a domain sequence evolution model with a relaxed molecular clock assumption. The method employs a variant of Markov Chain Monte Carlo technique called, Grouped Independence Metropolis-Hastings for the estimation of posterior distribution over domain and gene trees. By using this method, we performed analyses of Zinc-Finger and PRDM9 gene families, which provides an interesting insight of domain evolution. Finally, a synteny-aware approach for gene homology inference, called GenFamClust, is proposed that uses similarity and gene neighbourhood conservation to improve the homology inference. We evaluated the accuracy of our method on synthetic and two biological datasets consisting of Eukaryotes and Fungal species. Our results show that the use of synteny with similarity is providing a significant improvement in homology inference. / <p>QC 20160904</p>

Made available in DSpace on 2014-08-20T13:32:58Z (GMT). No. of bitstreams: 1 tese_filipe_de_carvalho_victoria.pdf: 30767989 bytes, checksum: d4b2b8995ad70a6cbd4d6e6ff672fa95 (MD5) Previous issue date: 2011-05-27 / Iron is an essential element for plant development, involved in metabolic processes, such respiration and photosynthesis. However, data regarding the genotype by environment interaction are lacking. Comparative analysis with lower plant groups and crop plants can increase the understanding about these processes. The use of bryophytes as model plants rise as a promising strategy since they present simpler patterns of development. The present work aimed to identify the occurrence patterns of molecular markers in model plant species, as well as to infer about the phylogenetical relationships of gene families related with iron homoestasis in plants, allowing the development of tranfer strategies of genomic data across model and orphan species. Using bioinformatics tools, a survey analysis was performed to detect repetitive elements in EST banks of eleven plant species. To validate the SSR markers found, 100 primer pairs were developed on the microsatelite sequences obtained for Physcomitrella patens Brid. and tested against genomic DNA of Polytrichum juniperinum Hedw. Phylogenetic and divergence time analysis was performed for the gene families Iron Regulated Transporter (IRT), Ferric Redectase Oxidase (FRO), Nicotinamide synthase (NAS), Yellow Stripe-Like (YSL) and Natural Resistance-Associated Macrophage Protein (NRAMP), related to the iron homoestasis, with help of the Bayesian inference and using the rice, Arabidopsis and P. patens genes for the Blast search in distinct land plants species. Also, primers for transposable elements recognizably related to Ysl genes were developed and applied jointly with the SSR primers by the IRAP/REMAP technic searching to find microsatellite markers associated to copies of this gene family. A total of 13,133 SSR markers were discovered in non- redundant EST databases made for all eleven species chosen for this study. The dimer motifs are more frequent in lower plant species, such as green algae and mosses, and the trimer motifs are more frequent for the majority of higher plant groups, such as monocots and dicots. Thirty percent of EST-SSE were successfully transferred with a relative polimorphism information across Physcomitrella patens Brid. and P. juniperinum, being promising for mapping and comparative genome analyses in plants. A total of 243 iron uptake gene sequences for 30 plant species were found using rice and Arabidopsis thaliana (L.) Heynh. homologues as queries. The evolutionary fingerprinting analyses suggested a positive selective pressure on iron uptake genes for most of the plant homologues analyzed, enabling an optimization and maintenance of gene function. The divergence time analysis indicates IRT as the most ancient gene family and FRO as the most recent. NRAMP and YSL genes appear as a close branch in the evolution of iron uptake gene families. No recent duplication in grasses were found based in the bayesian inference, and paralogue copies were only observed for dicot species. The Nramp cis-acting homology search indicated an ancestral duplication hypothesis for this gene family in grasses. Using IRAP/REMAP techniques, it was observed that YSL homologues in Physcomitrella are surrounded by copia-like retrotransposons as occurs in the maize ZmYSL1 copy. Also Polytrchum juniperinum Hedw. in vitro cultures were estabilished using spores as explants. Protonemal and gametophyte development were obtained using a growth regulator free culture medium. / O ferro é um elemento essencial para o crescimento e desenvolvimento das plantas, envolvido em processos metabólicos essenciais, como fotossíntese e respiração. Porém, são poucos os dados relacionando a interação entre diferentes genótipos e ambientes. Análises comparativas entre plantas inferiores e plantas cultivadas podem possibilitar o melhor entendimento destes processos. O uso de briófitas como modelo para estudos de processos biológicos em plantas surge como uma estratégia promissora devido ao padrão relativamente simples de desenvolvimento destas plantas. O presente trabalho objetivou identificar padrões de ocorrência de marcadores moleculares em plantas modelo, bem como inferir acerca da filogenia das famílias gênicas envolvidas na homoestase do ferro em plantas, possibilitando a criação de estratégias de transferência de informação genômica entre espécies modelo e espécies órfãs. Utilizando ferramentas de bioinformática foram realizadas análises exploratórias para detectar as ocorrências de elementos repetitivos em bancos de ESTs de onze espécies de plantas. Para a validação destes marcadores moleculares foram desenvolvidos 100 conjuntos de iniciadores a partir das sequências contendo microssatélites obtidas para Physcomitrella patens Brid. e testadas contra o DNA genômico de Polytrichum juniperinum Hedw. Foram realizadas análises filogenéticas e de divergência das famílias gênicas Iron Regulated Transporter (IRT), Ferric Redectase Oxidase (FRO), Nicotinamide synthase (NAS), Yellow Stripe-Like (YSL) e Natural Resistance-Associated Macrophage Protein (NRAMP), envolvidas na homoestase de ferro por meio de inferência bayesiana, utilizando genes de arroz, Arabidopsis e Physcomitrella patens Brid. na busca de homólogos em diferentes espécies de plantas terrestres, com o auxílio da ferramenta Blast (NCBI). Também foram desenvolvidos iniciadores para elementos transponíveis reconhecidamente associados a genes Ysl de milho e utilizados conjuntamente com os iniciadores EST-SSR por meio da técnica IRAP/REMAP buscando encontrar marcadores microssatélites associados a cópias desta familia gênica. Como resultados foram identificados 13.133 marcadores microssatélites em bancos de dados não redundantes de regiões expressas (EST) de onze espécies de plantas. Os motivos dinucleotídeos foram mais frequentes em espécies basais, enquanto os motivos trinucleotídeos foram mais frequentes em espécies derivadas. Em 30% dos conjuntos de iniciadores EST SSR testados contra o DNA de P. juniperinum, foi obtido bandas polimórficas promissoras para estudos de mapeamento comparativo e de diversidade genética. Foram encontrados 243 homólogos de genes relacionados as famílias gênicas envolvidas com a homoestase de ferro em trinta espécies de plantas. A análise de fingerprinting realizada sugere que a maioria destes genes estão submetidos a seleção positiva, indicando acúmulo de mutações adaptativas, essencial para a manutenção e otimização da resposta gênica. A análise de tempo de divergência indica que os genes IRT são mais basais e os genes FRO os mais recentes entre as familias gênicas estudadas. As famílias NRAMP e YSL são evolutivamente próximas. A análise bayesiana das sequências e de regiões promotoras dos genes NRAMP não indica duplicações recentes em gramíneas, sendo as duplicações provenientes de divergência ancestral a origem do grupo. Parálogos foram identificados somente em dicotiledôneas. Por meio da transferência de marcadores IRAP/REMAP é observado que genes YSL de P. patens estão cercados por retroelementos do tipo cópia, a exemplo do que ocorre com o gene ZmYSL1 em milho. Também foi estabelecido o cultivo, em condições axênicas, de Polytrichum juniperinum Hedw. utilizando esporos como explantes, onde foi observado que protonemas são obtidos utilizando meio de cultura livre de fitorreguladores, regenerando gametófitos em cultivo in vitro.