Made available in DSpace on 2014-08-20T13:32:58Z (GMT). No. of bitstreams: 1 dissertacao_carla_silveira.pdf: 1301704 bytes, checksum: e35505f057e1acb40d6e79b4da74308d (MD5) Previous issue date: 2011-05-26 / Oats (Avena sativa L.) are a winter cereal used for both humans and livestock, giving a great importance on the world stage. In the post-genomic era new sequenced genomes can be used to derive functions of organisms from the knowledge of these systems. In this work were apply this approach to the analysis of gene family of aquaporins in oats. The aquaporins are major intrinsic proteins (MIP) that have been characterized as facilitating the water flow. They are also known as water channels, glycerol facilitators and aquaglyceroporins, recent data suggest that they facilitate the movement of other molecular low weight metabolites. The presence of these genes is importance for the development and adaptation of plants, however, until now there are not studies reporting the identification of aquaporins in the oat genome. In this report we identified two sequences identified as potential aquaporins present in the genome of oat. Considering the number of aquaporin genes present in genome of rice, Arabidopsis, maize and wheat and the complexity of its polyploid genome, it is estimated that oats may have around 20 or more copies of these genes in each genome. Given the growing problems of flooding and drought, the need for cultivation in areas affected by salinity and the proven role of MIPs on plant response to stresses related to water availability, the investigations of these genes are essential in oats too. However a greater number of these genes can be identified in the future, this study was an important step in its characterization. / A aveia (Avena sativa L.) é um cereal de inverno utilizado tanto para a alimentação humana quanto animal, apresentando uma grande importância no cenário mundial. Na era pós-genômica novos genomas sequenciados podem ser utilizados para inferir funções de organismos a partir do conhecimento de outros sistemas. Neste trabalho foi aplicada essa abordagem à análise de genes da familia das aquaporinas em aveia. As aquaporinas são proteínas intrínsecas de membrana (MIP) que têm sido caracterizadas como facilitadoras do fluxo de água. São também conhecidas como canais de água, glicerol facilitadores e aquagliceroporinas. Dados recentes sugerem que elas facilitam a circulação de outros metabólitos de baixo peso molecular. A presença dos genes que codificam essas proteínas é de suma importância para o desenvolvimento e adaptação das plantas, contudo, até hoje não existiam estudos relatando a identificação de genes de aquaporinas no genoma da aveia. O sequenciamento e caracterização in silico das sequências obtidas neste trabalho propõem a presença de genes que codificam prováveis aquaporinas. Foram identificadas duas sequências como prováveis aquaporinas presentes no genoma da aveia. Considerando o número de genes de aquaporinas presentes no genoma do arroz, Arabidopsis, milho e trigo e a complexidade de seu genoma poliplóide, estimase que a aveia possua mais cópias desses genes. Tendo em vista os crescentes problemas de alagamento e/ou seca, a necessidade de cultivo em áreas afetadas pela salinidade, e o comprovado papel das MIPs na resposta das plantas a estresses relacionados com a disponibilidade de água, as investigações desses genes em aveia são essenciais. Contudo um número maior desses genes poderão ser identificados e caracterizados, esse estudo representa uma etapa importante para sua caracterização.

Duplications and deletions are a major source of genomic variation. Duplications, specifically, have a significant impact on gene genesis and dosage, and the malaria parasite P. falciparum has developed resistance to a growing number of anti-malarial drugs via gene duplication. It also contains highly duplicated families of antigenically variable allelic genes. While specific genes and families have been studied, a comprehensive analysis of duplications and deletions within the reference genome and population has not been performed. We analyzed the extent of segmental duplications (SD) in the reference genome for P. falciparum, primarily by a whole genome self alignment. We discovered that while 5% of the genome identified as SD, the distribution within the genome was partition clustered, with the vast majority localized to the subtelomeres. Within the SDs, we found an overrepresentation of genes encoding antigenically diverse proteins exposed to the extracellular membrane, specifically the var, rifin, and stevor gene families. To examine variation of duplications and deletions within the parasite populations, we designed a novel computational methodology to identify copy number variants (CNVs) from high throughput sequencing, using a read depth based approach refined with discordant read pairs. After validating the program against in vitro lab cultures, we analyzed isolates from Senegal for initial tests into clinical isolates. We then expanded our search to a global sample of 610 strains from Africa and South East Asia, identifying 68 CNV regions. Geographically, genic CNV were found on average in less than 10% of the population, indicating that CNV are rare. However, CNVs at high frequency were almost exclusively duplications associated with known drug resistant CNVs. We also identified the novel biallelic duplication of the crt gene – containing both the chloroquine resistant and sensitive allele. The synthesis of our SD and CNV analysis indicates a CNV conservative P. falciparum genome except where drug and human immune pressure select for gene duplication.

The placement of genetic information within bacterial genomes is intentionally organized, creates predictable gradients of molecular properties along the origin-terminus of replication axis. Previous studies have reported that genes located near the origin of replication generally have a higher expression level, increased dosage, and are more conserved than genes located near the terminus of replication. Additionally, substitution rates usually increases with increasing distance from the origin of replication. However, the constant reorganization of genetic information is often overlooked when considering spatial molecular trends. Here, we explore the interplay of genomic reorganization along the origin and terminus of replication axis of gene expression and substitutions in Escherichia coli, Bacillus subtilis, Streptomyces, and Sinorhizobium meliloti. Using ancestral reconstruction to account for genome reorganization, we demonstrated that the correlation between the number of substitutions and distance from the origin of replication is significant but small and inconsistent in direction. In another study, we looked at the overall expression levels of all genes from the same bacteria, and confirmed that gene expression tends to decrease when moving away from the origin of replication. We looked specifically at how inversions - one type of genomic reorganization - impact gene expression between closely related strains of E. coli. Some inversions cause significant differences in gene expression compared to non-inverted regions, however, the variation in expression does not significantly differ between inverted and non-inverted regions. This change in gene expression may be due to the expression regulation properties of two nucleoid proteins, Histone-like Nucleoid-Structuring (H-NS) and Factor for inversion stimulation (Fis), who’s binding sites had a significant positive correlation with inverted regions. In conclusion, we highlight the impact that genomic rearrangements and location have on molecular trends in bacteria, illustrating the importance of considering spatial trends in molecular evolutionary analysis, and to ensure accurate generalization of previously determined trends. Assuming that molecular trends are exclusively in one direction can be problematic. / Dissertation / Doctor of Philosophy (PhD)

DNA methylation refers to an addition of a methyl group to the 5 position of the cytosine pyrimidine ring. As the best characterized epigenetic mark, DNA methylation plays an important role in a plethora of biological functions, including gene repression, genomic imprinting, silencing of retro-transposons and X chromosome inactivation. Genomic imprinting refers to the mono-allelic expression of certain genes according to their parent-of-origin. In mammals, the expression of imprinted genes is controlled by the cis-acting regulatory elements, termed imprinted control regions (ICRs). ICRs are marked by parent-of-origin-specific DNA methylation and loss of DNA methylation at ICRs also causes aberrant expression of imprinted genes. Therefore it is believed that the genomic imprinting is a DNA methylation-associated epigenetic phenomenon. As accurate expression of imprinted genes is essential for normal embryonic growth, energy homeostasis, development of the brain and behaviour and abnormal expression of imprinted genes leads to numerous clinical phenotype and human disorders, it is important to investigate how the imprinted DNA methylation is stably maintained in mammals. DNA methyltransferases (DNMTs) are the main enzymes that play a in the establishment and maintenance of imprinted DNA methylation. In primordial germ cells (PGCs), DNMT3A and DNMT3L are involved in the establishment of imprinted DNA methylation. Whereas once established, the imprinted DNA methylation is maintained by DNMT1, DNMT3A and DNMT3B, but mainly by DNMT1. In addition, some other enzymes and DNA binding proteins also play a role in this process. One of the best examples is ZFP57, which forms a complex with KAP1 and SETDB1. ZFP57 maintains imprinted DNA methylation by recognizing a methylated hexa-nucleotide and recruits DNMTs to the ICRs in mammalian embryonic stem (ES) cells. Interestingly, DNA methylation analysis combined with promoter microarrays carried out in our lab suggested that imprinted DNA methylation is absent from some of the maternal ICRs in ES cells genetically null for G9a, a histone H3 lysine 9 methylase. This indicates that G9a might also play a role in the maintenance of imprinted DNA methylation. In my work, I found that the repressive H3K9me2 and imprinted DNA methylation are absent from several analysed ICRs in embryonic stem (ES) cells genetically null for either G9a or its partner histone methyltransferase GLP. A knockdown of G9a in ES cells reproduced these observations suggesting that G9a/GLP complex is required for the maintenance of imprinted DNA methylation. I also found that neither wild type nor catalytically inactive G9a can restore the loss of imprinted DNA methylation in G9a-/- ES cells. Chromatin immunoprecipitation (ChIP) combined with bisulfite DNA sequencing showed that imprinted DNA methylation was present on the H3K9me2-marked allele indicating a direct role for G9a in maintenance of genomic imprinting. Using a pharmacological inhibitor of G9a and mutagenesis analyses, I found that G9a maintains the imprinted DNA methylation independently of its catalytic activity and recruits DNMTs to the ICRs via its ankyrin repeat domain. Dimerization of G9a with GLP is also essential for the maintenance of genomic imprinting in ES cells. In summary, in addition to establish H3K9me2, histone methyltransferases G9a and GLP also play an essential role in the maintenance of genomic methylation imprints in ES cells.

Understanding the genetic basis of adaptive evolution, and how natural selection has shaped patterns of polymorphism and divergence within and between species are enduring goals of evolutionary genetics. In this thesis, I used whole genome re-sequencing data to characterize the genomic signatures of natural selection along different evolutionary timescales in three Populus species: Populus tremula, P. tremuloides and P. trichocarpa. First, our study shows multiple lines of evidence suggesting that natural selection, due to both positive and purifying selection, has widely shaped patterns of nucleotide polymorphism at linked neutral sites in all three species. Differences in effective population sizes and rates of recombination largely explain the disparate magnitudes and signatures of linked selection that we observe among species. Second, we characterize the evolution of genomic divergence patterns between two recently diverged aspen species: P. tremula and P. tremuloides. Our findings indicate that the two species diverged ~2.2-3.1 million years ago, coinciding with the severing of the Bering land bridge and the onset of dramatic climatic oscillations during the Pleistocene. We further explore different mechanisms that may explain the heterogeneity of genomic divergence, and find that variation in linked selection and recombination likely plays a key role in generating the heterogeneous genomic landscape of differentiation between the two aspen species. Third, we link whole-genome polymorphic data with local environmental variables and phenotypic variation in an adaptive trait to investigate the genomic basis of local adaptation in P. tremula along a latitudinal gradient across Sweden. We find that a majority of single nucleotide polymorphisms (SNPs) (&gt;90%) identified as being involved in local adaptation are tightly clustered in a single genomic region on chromosome 10. The signatures of selection at this region are more consistent with soft rather than hard selective sweeps, where multiple adaptive haplotypes derived from standing genetic variation sweep through the populations simultaneously, and where different haplotypes rise to high frequency in different latitudinal regions. In summary, this thesis uses phylogenetic comparative approaches to elucidate how various evolutionary forces have shaped genome-wide patterns of sequence evolution in Populus. / <p>The research in this thesis was supported by the Swedish research council (to Pär K. Ingvarsson) and the JC Kempe Memorial Scholarship Foundation (to Jing Wang). The PhD study of Jing Wang in Sweden was funded by the State Scholarship from China Scholarship council.</p>

The stability of DNA is a critical factor for several diseases, the most prevalent of which is cancer. Several neurodegenerative and accelerated aging diseases are also characterized by genomic instability. The number and complexity of DNA repair pathways that human cells possess underscores the importance of genomic stability. These pathways ensure that damaged DNA is repaired and that a cells complement of DNA remains stable upon cell division. How one particular type of DNA alteration, a DNA loop, is processed in human cells was the focus of this study. We have employed an in vitro system to study defined DNA loop substrates by human nuclear extracts. The influence of either a 5 or 3 nick, the range of loop sizes processed, and the role of DNA mismatch repair, DNA nucleotide excision repair, and the Werner Syndrome helicase proteins were variables tested. The results indicate tha t DNA loops containing between 5 to 12 nucleotides are processed in a strand - specific manner when either a 5 or 3 nick is present , with repair being targeted solely to the nicked strand . This repair occurs by both mismatch repair dependent and independent pathways. The processing of DNA loops containing 30 nucleotides in length is directed either by a 5 nick, or by the loop itself, but not by a 3 nick. The nick independent pathway results solely in loop removal. The large loop pathway is independent of mismatch repair, nucleotide excision repair, and the WRN helicase/exonuclease protein. Both of the 5 nick directed pathways occur by excision that initiates at the pre- existing nick and proceeds towards the loop along the shortest path between the nick and loop. DNA resynthesis occurs using either DNA polymerase , , or and also initiates at the pre-existing 5 nick. The 3 nick directed intermediate loop repair pathway proceeds in a similar fashion, likely after a nick is made 5 to the loop region on the strand that contained the pre-existing nick. DNA synthesis inhibition has only a minor affect on the nick independent loop removal pathway as only a short tract of DNA surrounding the loop site is processed. In total, the results point to at least 3 novel pathways that process DNA loops that likely contribute to total genomic stability.

This PhD seeks to explore the development of a bio-molecular (i.e., genomic) map as a sovereign resource in Mexico. The basic analytical thread of the dissertation is related to the circulation of genomic variability through the policy/legal and scientific social worlds that compose the Mexican medical-population genomics arena. It follows the construction of the Mexican Institute of Genomic Medicine (INMEGEN), the notion of genomic sovereignty, and the Mexican Genome Diversity Project (MGDP).The key argument for the construction of the INMEGEN relied in a nationalist policy framing, which considered the Mexican genome as a sovereign resource, coupling Mexican “uniqueness” to the very nature of genomic science. Nevertheless, the notion of genomic sovereignty was nothing similar to a paradigm, and was not based on shared visions of causality, since the very “nature” of the policy object —Mexican Genome— was, and still is, a disputed reality. It was through the rhetoric upon independence, emancipation and biopiracy: i.e. experiences of dispossession “in archaeology, botany or zoology” (IFS 2001: 25) that the novelty of population genomics became amenable to be understood as a sovereign matter. Therefore, the strategic reification of Mexicanhood fuelled the whole policy and the legal agenda of the INMEGEN as well, which permitted cooperation without consensus and opened the process of policy innovation. Conversely, scientists considered genomic sovereignty an unfounded exaggeration, but anyhow they cooperated and even created a new policy and scientific enterprise. Genomic sovereignty exemplifies the process of cooperation without consensus on its most extreme version .So, as the notion circulated and gradually became a law to protect Mexican genomic patrimony, the initial coalition of scientists, lawyers and policy makers disaggregated. Many of the original members of the coalition now think of genomic sovereignty as a strategy of the INMEGEN to monopolise genomic research in the country. This dissertation additionally explores the way in which the MGDP is constructed in mass media, in INMEGEN´s communication and in the laboratory practices. These different dimensions of the MGDP depict the difficulties that emerge between the probabilistic, relative and multiple constructions of population genomics and the rhetorical strategies to continually assert the existence of the unique “Mexican Genome”. I argue that the Mexican case study provides an entry point to what I and others (Benjamin 2009; Schwartz-Marin 2011) have identified as a postcolonial biopolitics in which the nation state is reasserted rather than diluted. However the relation between sovereignty, race and nation is not mediated by the biological purification of the nation (Agamben 1998; Foucault 2007), or the active participation of citizens looking to increase their vitality (Rose 2008, Rose & Rabinow 2006), but on an awareness of subalternity in the genomic arena and a collective desire to compete in the biomedical global economy.

Master of Science / Genetics Interdepartmental Program / Sanzhen Liu / The major component of complex genomes is repetitive elements, which remain recalcitrant to characterization. Using maize as a model system, we analyzed whole genome shotgun (WGS) sequences for the two maize inbred lines B73 and Mo17 using k-mer analysis to quantify the differences between the two genomes. Significant differences were identified in highly repetitive sequences, including centromere, 45S ribosomal DNA (rDNA), knob, and telomere repeats. Genotype specific 45S rDNA sequences were discovered. The B73 and Mo17 polymorphic k-mers were used to examine allele-specific expression of 45S rDNA in the hybrids. Although Mo17 contains higher copy number than B73, equivalent levels of overall 45S rDNA expression indicates that transcriptional or post-transcriptional regulation mechanisms operate for the 45S rDNA in the hybrids. Using WGS sequences of B73xMo17 doubled haploids, genomic locations showing differential repetitive contents were genetically mapped, revealing differences in organization of highly repetitive sequences between the two genomes. In an analysis of WGS sequences of HapMap2 lines, including maize wild progenitor, landraces, and improved lines, decreases and increases in abundance of additional sets of k-mers associated with centromere, 45S rDNA, knob, and retrotransposons were found among groups, revealing global evolutionary trends of genomic repeats during maize domestication and improvement.

L'empreinte génomique est un mécanisme épigénétique qui conduit à l'expression d'un seul des deux allèles parentaux pour une centaine de gènes autosomaux chez les mammifères. La majorité des gènes soumis à l'empreinte est regroupée en clusters et tous ces gènes sont sous le contrôle de séquences discrètes appelées ICR (Imprinting Control Region). Les ICRs sont marquées épigénétiquement par une méthylation d'ADN et des modifications des histones alléliques. La méthylation d'ADN au niveau de ces ICRs est un facteur clé de l'empreinte et va être établie dans les lignées germinales suivant le sexe de l'embryon. Après fécondation, le nouvel embryon portera les empreintes paternelles et maternelles, ces empreintes devront alors être maintenues pendant tout le développement et interprétés dans le but de conduire à l'expression allélique des gènes soumis à l'empreinte. Cependant, la méthylation d'ADN ne peut expliquer à elle seule tous les aspects de l'empreinte génomique. Ainsi, d'autres marques épigénétiques doivent agir dans le maintien et la lecture de ces empreintes. Nous avons mis en évidence dans un premier temps que le contrôle de l'expression allélique dans le cerveau de Grb10 repose sur la résolution d'un domaine bivalent allélique spécifiquement dans le cerveau. Ces résultats mettent en avant pour la première fois un domaine bivalent dans le contrôle de l'expression des gènes soumis à l'empreinte et propose un nouveau mécanisme dans l'expression tissu spécifique de ces gènes. D'autre part, bien que des études en cellules ES aient démontré un rôle de G9a dans le maintien des empreintes au cours du développement embryonnaire, nos données suggèrent que G9a ne serait pas essentielle a ce maintien dans un contexte in vivo. / Genomic imprinting is a developmental mechanism which leads to parent-of-origin-specific expression for about one hundred genes in mammals. Most of imprinted genes are clustered and all are under control of sequence of few kilobases called Imprinting Control Region or ICR. ICRs are epigenetically marked by allelic DNA methylation and histone modifications. DNA methylation on ICRs is a key factor which is established in germ cells according to the sex of the embryo. After fecundation, the new embryo will harbored both paternal and maternal imprints which have to be maintained during the development and read to lead to allelic expression of imprinted genes. However, allelic DNA methylation alone cannot explain every aspect of genomic imprinting. Thus, there should be other epigenetic marks which act in the maintaining and reading of the imprints.Our data first indicate that bivalent chromatin, in combination with neuronal factors, controls the paternal expression of Grb10 in brain, the bivalent domain being resolved upon neural commitment, during the developmental window in which paternal expression is activated. This finding highlights a novel mechanism to control tissue-specific imprinting. On an other hand, although previous studies in ES cells show a role for G9a in the maintaining of imprints during embryonic development, our data suggest that G9a would not be essential in an in vivo model.

Doctor of Philosophy / Department of Agronomy / Guihua Bai / Allan K. Fritz / Wheat pre-harvest sprouting (PHS), germination of physiologically matured grains in a wheat spike before harvesting, can cause significant reduction in grain yield and end-use quality. Many quantitative trait loci (QTL) for PHS resistance have been reported in different sources. To determine the genetic architecture of PHS resistance and its relationship with grain color (GC) in US hard winter wheat, a genome-wide association study (GWAS) on both PHS resistance and GC was conducted using in a panel of 185 U.S. elite breeding lines and cultivars and 90K wheat SNP arrrays. PHS resistance was assessed by evaluating sprouting rates in wheat spikes harvested from both greenhouse and field experiments. Thirteen QTLs for PHS resistance were identified on 11 chromosomes in at least two experiments, and the effects of these QTLs varied among different environments. The common QTLs for PHS resistance and GC were identified on the long arms of the chromosome 3A and 3D, indicating pleiotropic effect of the two QTLs. Significant QTLs were also detected on chromosome arms 3AS and 4AL, which were not related to GC, suggesting that it is possible to improve PHS resistance in white wheat. To identify markers closely linked to the 4AL QTL, genotyping-by-sequencing (GBS) technology was used to analyze a population of recombinant inbred lines (RILs) developed from a cross between two parents, “Tutoumai A” and “Siyang 936”, contrasting in 4AL QTL. Several closely linked GBS SNP markers to the 4AL QTL were identified and some of them were coverted to KASP for marker-assisted breeding. To investigate effects of the two non-GC related QTLs on 3AS and 4AL, both QTLs were transferered from “Tutoumai A” and “AUS1408” into a susceptible US hard winter wheat breeding line, NW97S186, through marker-assisted backcrossing using the gene marker TaPHS1 for 3AS QTL and a tightly linked KASP marker we developed for 4AL QTL. The 3AS QTL (TaPHS1) significantly interacted with environments and genetic backgrounds, whereas 4AL QTL (TaMKK3-A) interacted with environments only. The two QTLs showed additive effects on PHS resistance, indicating pyramiding these two QTLs can increase PHS resistance. To improve breeding selection efficiency, genomic prediction using genome-wide markers and marker-based prediction (MBP) using selected trait-linked markers were conducted in the association panel. Among the four genomic prediction methods evaluated, the ridge regression best linear unbiased prediction (rrBLUP) provides the best prediction among the tested methods (rrBLUP, BayesB, BayesC and BayesC0). However, MBP using 11 significant SNPs identified in the association study provides a better prediction than genomic prediction. Therefore, for traits that are controlled by a few major QTLs, MBP may be more effective than genomic selection.