Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-04-08T14:22:20Z No. of bitstreams: 2 122Tese Roberta final Bib Central.pdf: 5487110 bytes, checksum: 1c6bb6e8403d263eac35107f5ca76519 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-08T14:22:20Z (GMT). No. of bitstreams: 2 122Tese Roberta final Bib Central.pdf: 5487110 bytes, checksum: 1c6bb6e8403d263eac35107f5ca76519 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012 / CAPES; FACEPE; PROPESQ-UFPE / A Degeneração Neuronal (DN) é um processo complexo e multifatorial, presente em várias desordens neuropsiquiátricas, como a Doença de Fahr (DF) e a Doença de Alzheimer (DA), entre outras. Este trabalho teve como objetivo, estudar fatores de risco genéticos, em dois modelos de doenças neurodegenerativas, através de sequencimentos e métodos de bioinformática. O screening de genes e variações candidatas foi realizado através de Reação em Cadeia da Polimerase (PCR), seguido de Sequenciamento Automático Direto do DNA. Para a DF, também conhecida como Calcificação Idiopática dos Gânglios da Base (IBGC) dois genes foram investigados, o gene MGEA6/CTAGE lócus IBGC1 e o gene SLC20A2 lócus IBGC3. As variações candidatas para DA foram selecionadas a partir de um pipeline de bioinformática, onde informações genéticas de diferentes bancos de dados foram integradas, sendo os principais genes estudados: COX-2, IL1a e CD83. Os resultados das genotipagens não identificaram, em ambos os grupos de controles e afetados, a variação Pro521Ala (rs36060072) / MEGEA6 lócus IBGC1. Essas genotipagens contribuíram para a diminuição da freqüência alélica mínima dessa variante, demonstrando o quanto ela é rara e ausente nessa amostra da população Brasileira. Para o segundo lócus analisado, IBGC3 gene SLC20A2, os achados tem mostrado novas variações em diferentes exons e estes estão associadas a mais de 40% das famílias com IBGC analisadas. No entanto, algumas famílias recrutadas foram excluídas para esses dois loci, sugerindo adicional heterogeneidade genética. Paralelamente, os resultados do ensaio experimental do screening das variações candidatas para DA, só foi permito em parte, pois apenas variações do tipo SNPs: “rs2745557”_COX2 (exon 1) e “rs17561”_IL1A (exon5), foram detectadas, sendo a grande maioria das variações preditas, INDELs (1 a 10pb), não encontradas. O desafio atual é aperfeiçoar os métodos de sequenciamentos, principalmente através do uso novas plataformas de sequenciamentos. Há uma grande expectativa de que um melhor entendimento, das bases biológicas destes dois distúrbios investigados, possa contribuir para a compreensão amplamente de outras doenças neuropsiquiátricas.

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Epigenetics are stable heritable traits that are not a result of the DNA sequence. Epigenetic modification of DNA cytosine plays a role in development and disease. The covalent bonding of a methyl group or a hydroxymethyl group to the 5-carbon of cytosine epigenetically modifies cytosine to 5-methylcytosine or 5-hydroxymethylcytosine. Upon PCR amplification, the bisulfite treatment of DNA converts unmethylated cytosine to thymine, while 5-methylcytosine, 5-hydroxymethylcytosine, and other bases remain unchanged. The resulting sequences can be mapped to a reference genome; however, this can be challenging due to sequencing technology complexity, low sequence complexity, and biases and errors introduced with bisulfite treatment. Once the short read is mapped, the identity of 5-methylcytosine or 5-hydroxymethylcytosine can be determined by comparing the mapped read to the aligned reference genome. Bisulfite DNA read mapping is characterized by mapping performance as low as 40%. This research improves bisulfite short read mapping quality. First, reads generated from the bisulfite hairpin PCR protocol are used to study mapping failure and solutions. A read may not map to the genome; it may map uniquely, or it may map to multiple locations. Sequence complexity correlates with these mapping categories. The hairpin protocol allows for a recovery, in some cases, of the original untreated read, and mapping this read with the regular read mapper Bowtie2 improved mapper performance by 10%. New bisulfite read mapping software called BisPin was created that calls BFAST (BLAT-like Fast Accurate Search Tool) for mapping. BisPin resolves ambiguously mapped reads with a rescoring strategy, which yields a statistically significant improvement. BFAST-Gap for Ion Torrent reads was developed, since Ion Torrent machines are less expensive than Illumina machines and since Ion Torrent reads are longer. There are few mappers for Ion Torrent data. BFAST-Gap uses homopolymer run length for contextual gap penalty functions, since homopolymer runs cause errors in Ion Torrent reads. In conjunction with BisPin, this software performed well on real and simulated bisulfite Ion Torrent data and Illumina data. InfoTrim, a read trimmer with an entropy term, was developed with competitive results. / Ph. D.

<p>Sex chromosomes are useful for the study of how factors such as mutation, selection, recombination and effective population size affect diversity and divergence.</p><p>A comparison of gametologous introns in seven different bird species revealed a complete lack of diversity on the female-specific W chromosome. In contrast, Z had at least one segregating site in all examined species. This can be explained by the lower mutation rate and lower effective population size of W but also suggests that selection affects diversity levels on the non-recombining W chromosome.</p><p>In a diverse set of chicken breeds, the Z chromosome showed reduced diversity compared to autosomes and significant heterogeneity in levels of variation. High variance in male reproductive success, leading to a reduced Z chromosome effective population size, can partly explain this observation. In addition, we suggest that selective sweeps frequently act on the Z chromosome and are responsible for a significant part of the observed Z reduction. </p><p>Differences in the mutation rate of Z and W chromosome sequences indicate that the time spent in male germ line is important for the mutation rate, but does not exclude a specifically reduced mutation rate on the Z chromosome. Estimates of mutation rate in autosomal, Z- and W-linked chicken and turkey sequences indicate a slight reduction in the rate on Z. However, due to rate heterogeneity among introns this reduction is not significant and we cannot exclude male biased mutation as the single cause of rate variation between the chromosomal classes.</p><p>Analysis of indel mutation rates in avian and mammalian gametologous introns show frequent occurrence of indels on both W and Y, excluding meiotic recombination as the only source of this type of mutation. The different indel rate patterns in birds (Z>W) and mammals (X=Y) suggest that indels are caused by both replication and recombination.</p>

Sex chromosomes are useful for the study of how factors such as mutation, selection, recombination and effective population size affect diversity and divergence. A comparison of gametologous introns in seven different bird species revealed a complete lack of diversity on the female-specific W chromosome. In contrast, Z had at least one segregating site in all examined species. This can be explained by the lower mutation rate and lower effective population size of W but also suggests that selection affects diversity levels on the non-recombining W chromosome. In a diverse set of chicken breeds, the Z chromosome showed reduced diversity compared to autosomes and significant heterogeneity in levels of variation. High variance in male reproductive success, leading to a reduced Z chromosome effective population size, can partly explain this observation. In addition, we suggest that selective sweeps frequently act on the Z chromosome and are responsible for a significant part of the observed Z reduction. Differences in the mutation rate of Z and W chromosome sequences indicate that the time spent in male germ line is important for the mutation rate, but does not exclude a specifically reduced mutation rate on the Z chromosome. Estimates of mutation rate in autosomal, Z- and W-linked chicken and turkey sequences indicate a slight reduction in the rate on Z. However, due to rate heterogeneity among introns this reduction is not significant and we cannot exclude male biased mutation as the single cause of rate variation between the chromosomal classes. Analysis of indel mutation rates in avian and mammalian gametologous introns show frequent occurrence of indels on both W and Y, excluding meiotic recombination as the only source of this type of mutation. The different indel rate patterns in birds (Z&gt;W) and mammals (X=Y) suggest that indels are caused by both replication and recombination.

Hematopoietic stem cell transplantation (HSCT) is used to treat patient with hematological diseases such as leukemia and genetic conditions such as sickle cell anemia. After HSCT the patients are supervised for signs of relapse of disease or rejection of transplanted cells. This is done by using chimerism analysis. At the department of clinical genetics at Akademiska sjukhuset fragment analysis of short tandem repeats is used for chimerism analysis, which is to be replaced by a Next generation sequencing (NGS) based method called Devyser chimerism, which includes an IVDR labelled kit. The aim of this project was to validate the new method for chimerism analysis. DNA samples from twelve HSCT patients and their donors were analyzed with Devyser chimerism and the results were compared to the results from the current method. The sensitivity of the new method was tested by analysis of artificial chimerism samples from blood donors. The results from the comparison showed a good correlation between methods (R2 = 0,9864) and the sensitivity of the method was confirmed to be 0,1% mixed chimerism. There was some difficulty in identifying enough informative markers for re-transplanted patients two had separate donors. This is a known problem for chimerism analysis in general and not a specific problem to the new method and will not be a hindrance for the implementation of Devyser chimerism at the clinical laboratory.

A fundamental problem in biology is the reconstruction of the relatedness of all (extant) species. Traditionally, systematists employ visually recognizable characters of organisms for classification and evolutionary analysis. Recent developments in molecular and computational biology, however, lead to a whole different perspective on how to address the problem of inferring relatedness. The discovery of molecules, carrying genetic information, and the comparison of their primary structure has, in a rather short period of time, revolutionized our understanding of the phylogenetic relationship of many organisms. These novel approaches, however, turned out to bear similar problems as previous techniques. Moreover, they created new ones. Hence, taxonomists came to realize that even with this new type of data not all problematic relationships could be unambiguously resolved. The search for complementary approaches has led to the utilization of rare genomic changes and other characters which are largely independent from the primary structure of the underlying sequence(s). These “higher order” characters are thought to be evolutionary conserved in certain lineages and largely unaffected by primary sequence data-based problems, allowing for a better resolution of the Tree of Life. The central aim of this thesis is the utilization of molecular characters of higher order in connection with their consistent and comparable extraction from a given data set. Two novel methods are presented that allow such an inference. This is complemented with the search for and analysis of known and novel molecular characteristics to study the relationships among Metazoa, both intra- as well as interspecific. The first method tackles a common problem in phylogenetic analyses: the inference of reliable data set. As part of this thesis a pipeline was created for the automated annotation of metazoan mitochondrial genomes. Data thus obtained constitutes a reliable and standardized starting point for all downstream analyses, e.g. genome rearrangement studies. The second method utilizes a subclass of gaps, namely those which define an approximate split of a given data set. The definition and inference of such split-inducing indels (splids) is based on two basic principles. First, indels at the same position, i.e. sharing the same end points in two sequences, are likely homologous. Second, independent single-residue insertions and deletions tend to occur more frequently than multi-residue indels. It is shown that trees based on splids recover most of the undisputed monophyletic groups while influence of the underlying alignment algorithm is relatively small. Mitochondrial markers are a valuable tool for the understanding of small and large scale population structure. The non-coding control region of mitochondrial DNA (mtDNA) often contains a higher amount of variability compared to genes encoding proteins and non-coding RNAs. A case study on a small scale population structure investigates the control region of the European Fire-bellied Toad in order to find highly variable parts which are of potential importance to develop informative genetic markers. A particular focus is placed on the investigation of the evolutionary dynamics of the repetitive region at an inter- and intraspecific level. This includes understanding mechanisms underlying its evolution, i.e. by exploring the impact of secondary structure on slipped strand mispairing during mtDNA replication. The 7SK RNA is a key player in the regulation of polymerase II (Pol-II) transcription, interacting with at least three known proteins: It mediates the inhibition of the Positive Transcription Elongation Factor b (P-TEFb) by the HEXIM1/2 proteins, thereby repressing transcript elongation by Pol-II. A highly specific interaction with LARP7 (La-Related Protein 7), on the other hand, regulates its stability. 7SK RNA is capped at its 5’ end by a highly specific methyltransferase MePCE (Methylphosphate Capping Enzyme). Employing sequence and structure similarity it is shown that the 7SK RNA as well as its protein binding partners have a much earlier evolutionary origin than previously expected. Furthermore, this study presents a good illustration of the pitfalls of using markers of higher order for phylogenetic inference.

<p>Currently centered on molecular data, bacterial and archaeal relationships are often based on their relative branching in 16S rRNA based phylogenetic trees. The availability of numerous bacterial genome sequences over the past two decades has provided new information for insights previously inaccessible to the field of taxonomy. Through utilization of comparative genomics, numerous molecular markers in the form of insertions and deletions within conserved regions of proteins, also known as Conserved Signature Indels or CSIs, have been discovered for various prokaryotic taxa. Using these techniques, we have analyzed relationships among the bacterial phyla of Thermotogae and Synergistetes and the conglomeration of bacterial organisms known as the PVC super-phylum. Through identification of large numbers of CSIs we have described the phyla Thermotogae and Synergistetes, and their sub-groups, in molecular terms for the first time. The identified molecular markers support a reconstruction of the current taxonomic divisions of these phyla. Similarly, previously only observed to group in phylogenetic trees, we have identified molecular markers for the PVC clade of bacterial phyla which are indicative of their shared ancestry. Further, in response to recent suggestions of extensive lateral gene transfer masking evolutionary relationships, an argument in favour of Darwinian mode of evolution for prokaryotic organisms is made using the identified molecular markers identified here along with markers previously identified in similar studies. Due to their taxonomic specificity, the markers that we have discovered provide useful tools for biochemical tests aiming for an understanding of the unique characteristics of the bacterial groups to which they are specific.</p> / Master of Science (MSc)

In many organisms, the most fundamental event during embryogenesis is differentiating between germline cells and specialized somatic cells. In C. elegans, PIE-1 functions to protect the germline from somatic differentiation and appears to do so by blocking transcription and by preventing chromatin remodeling in the germline during early embryogenesis. Yet the molecular mechanisms by which PIE-1 specifies germline remain poorly understood. Our work shows that SUMOylation facilitates PIE-1-dependent germline maintenance and specification. In vivo SUMO purification in various CRISPR strains revealed that PIE-1 is SUMOylated at lysine 68 in the germline and that this SUMOylation is essential for forming NuRD complex and preserving HDA-1 activity. Moreover, HDA-1 SUMOylation is dependent on PIE-1 and enhanced by PIE-1 SUMOylation, which is required for protecting germline integrity. Our results suggest the importance of SUMOylation in the germline maintenance and exemplify simultaneous SUMOylation of proteins in the same functional pathway.

Microsatellites are repetitive DNA characterized by tandem repeats of short motifs (2 – 5 bp). High mutation rates make them ideal for population level studies. Microsatellite allele genesis is generally attributed to strand slippage, and it is assumed that alleles are caused only by changes in repeat number. Most analyses are limited to alleles (electromorphs) scored by mobility only, and models of evolution rarely account for homoplasy in allele length. Additionally, insertion/deletion events (indels) in the flanking region or interruptions in the repeat can obfuscate the accuracy of genotyping. Many investigators use microsatellites, designed for a focal species, to screen for genetic variation in non-focal species. Comparative studies have shown different mutation rates of microsatellites in different species, and even individuals. Recent studies have used reciprocal comparisons to assess the level of polymorphism of microsatellites between pairs of taxa. In this study, I investigated the evolution of microsatellites within a phylogenetic context, using comparisons within the rodent family Bathyergidae. Bathyergidae represents a monophyletic group endemic to sub-Saharan Africa and relationships are well supported by morphological and molecular data. Using mitochondrial and nuclear DNA, a robust phylogeny was generated for the Bathyergidae. From my results, I proposed the new genus, Coetomys. I designed species-specific genotyping and microsatellite flanking sequence (MFS) primers for each genus. Sequencing of the MFS provided direct evidence of the evolutionary dynamics of the repeat motifs and their flanking sequence, including rampant electromorphic homoplasy, null alleles, and indels. This adds to the growing body of evidence regarding problems with genotype scores from fragment analysis. A number of the loci isolated were linked with repetitive elements (LTRs and SINEs), characterized as robust phylogenetic characters. Results suggest that cryptic variation in microsatellite loci are not trivial and should be assessed in all studies. The phylogenetic utility of the nucleotide variation of the MFS was compared to the well-resolved relationships of this family based on the 12S/TTR phylogeny. Variation observed in MFS generated robust phylogenies, congruent with results from 12S/TTR. Finally, a number of the indels within the MFS provided a suite of suitable phylogenetic characters.