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From Single Gene to Whole Genome Studies of Human Transcription RegulationRada-Iglesias, Alvaro January 2007 (has links)
<p>Transcriptional regulation largely determines which proteins and the protein levels that are found in a cell, and this is crucial in development, differentiation and responses to environmental stimuli. The major effectors of transcriptional regulation are a group of proteins known as transcription factors, which importance is supported by their frequent involvement in mendelian and complex diseases.</p><p>In paper I, we attempted to establish the importance of DNA sequence variation in transcriptional control, by analyzing the potential functionality of polymorphic short repetitive elements as cis-regulatory elements. However, the relevance of this study was constrained by the limited number of analyzed sequences and the <i>in vitro</i> nature of the experiments. To overcome these limitations, (paper II) we optimized an <i>in vivo</i> large-scale technology named ChIP-chip, which couples chromatin immunoprecipitation and microarray hybridization. We successfully identified the binding profiles of metabolic-disease associated transcription factors in 1% of the human genome, using a liver cellular model, and inferred the binding sites at base pair resolution.</p><p>Another important characteristic of transcriptional regulation is its plasticity, which allows adjusting the cellular transcriptome to cellular and environmental stimuli. In paper III, we investigated such plasticity by treating HepG2 cells with butyrate, a histone deacetylase inhibitor (HDACi) and interrogating the changes in histone H3 and H4 acetylation levels in 1% of the genome. Observation of frequent deacetylation around transcription start sites and hyperacetylation at the nuclear periphery challenges pre-assumed HDACi mechanisms of action.</p><p>Finally, in paper IV we extended the DNA binding profiles of the medically relevant transcription factors, USF1 and USF2, and H3 acetylation to the whole non-repetitive fraction of the human genome. Using motif finding tools and chromatin profiling, we uncovered the major determinants of USF-DNA interactions. Furthermore, USFs and H3ac were clearly localized around transcription start sites, frequently in the context of bidirectional promoters.</p>
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Structure-Function Studies of Bacteriophage P2 Integrase and Cox proteinEriksson, Jesper January 2005 (has links)
Probably no group of organisms has been as important as bacteriophages when it comes to the understanding of fundamental biological processes like transcriptional control, DNA replication, site-specific recombination, e.t.c. The work presented in this thesis is a contribution towards the complete understanding of these organisms. Two proteins, integrase, and Cox, which are important for the choice of the life mode of bacteriophage P2, are investigated. P2 is a temperate phage, i.e. it can either insert its DNA into the host chromosome (by site-specific recombination) and wait (lysogeny), or it can produce new progeny with the help of the host protein machinery and thereafter lyse the cell (lytic cycle). The integrase protein is necessary for the integration and excision of the phage genome. The Cox protein is involved as a directional factor in the site-specific recombination, where it stimulates excision and inhibits integration. It has been shown that the Cox protein also is important for the choice of the lytic cycle. The choice of life mode is regulated on a transcriptional level, where two mutually exclusive promoters direct whether the lytic cycle (Pe) or lysogeny (Pc) is chosen. The Cox pro-tein has been shown to repress the Pc promoter and thereby making tran-scription from the Pe promoter possible, leading to the lytic cycle. Further, the Cox protein can function as a transcriptional activator on the parasite phage, P4. P4 has gained the ability to adopt the P2 protein machinery to its own purposes. In this work the importance of the native size for biologically active integrase and Cox proteins has been determined. Further, structure-function analyses of the two proteins have been performed with focus on the protein-protein interfaces. In addition it is shown that P2 Cox and the P2 relative Wphi Cox changes the DNA topology upon specific binding. From the obtained results a mechanism for P2 Cox-DNA interaction is discussed. The results from this thesis can be used in the development of a gene delivery system based on the P2 site-specific recombination system.
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Genetic Analyses of Bovid Remains and the Origin of Early European CattleAnderung, Cecilia January 2006 (has links)
The aurochs Bos primigenius, extinct since 1627, was the wild progenitor of cattle. It is believed that all European cattle originate from one domestication event in the Near East 10 000 years ago. However, it is evident from the archaeological record that the aurochs survived into historic time and spent many years existing alongside domestic cattle. Thus, a question posed is whether aurochsen were locally domesticated or incorporated into early domestic cattle stock. In this thesis, genetic techniques are applied to ancient and modern DNA from bovids in order to study questions relating to the origin of early European cattle. DNA from ancient specimens is fragmented and in greatly reduced quantity. Therefore mitochondrial DNA, present in many copies in the living cell, has long been dominating the ancient DNA research field. Analyses of ancient DNA presented in this work are based on both mitochondrial DNA and nuclear DNA, through the study of Single Nuclear Polymorohism (SNPs). A method for typing ancient SNPs was developed and applied to ancient cattle bones. Mitochondrial DNA of cattle is structured into five geographically distributed lineages, the dominant lineage in Europe is also found in the Near East where additional lineages are found. This pattern has been attributed to the proposed domestication event in the Near East from where cattle carrying the single lineage were brought to Europe. However, the results presented here show that cattle domestication was more complicated than previously suggested. SNP data from extant cattle and bones from cattle and aurochs point towards a hybridisation event. European cattle appear indeed to have been domesticated in the Near East and brought in to the European continent from there. However, once in Europe, hybridisation with local aurochsen took place. It appears therefore that today’s cattle descend both from both Anatolian and European aurochsen.
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Expression and function of Suppressor of zeste 12 in Drosophila melanogasterChen, Sa January 2009 (has links)
The development of animals and plants needs a higher order of regulation of gene expression to maintain proper cell state. The mechanisms that control what, when and where a gene should (or should not) be expressed are essential for correct organism development. The Polycomb group (PcG) is a family of genes responsible for maintaining gene silencing and Suppressor of zeste 12 (Su(z)12) is one of the core components in the PcG. The gene is highly conserved in organisms ranging from plants to humans, however, the specific function is not well known. The main tasks of this thesis was to investigate the function of Su(z)12 and its expression at different stages of Drosophila development. In polytene chromosomes of larval salivary glands, Su(z)12 binds to about 90 specific euchromatic sites. The binding along the chromosome arms is mostly in interbands, which are the most DNA de-condensed regions. The binding sites of Su(z)12 in polytene chromosomes correlate precisely with those of the Enhancer-of-zeste (E(z)) protein, indicating that Su(z)12 mainly exists within the Polycomb Repressive Complex 2 (PRC2). However, the binding pattern does not overlap well with Histone 3 lysine 27 tri-methylations (H3K27me3), the specific chromatin mark created by PRC2. The Su(z)12 binding to chromatin is dynamically regulated during mitotic and meiotic cell division. The two different Su(z)12 isoforms: Su(z)12-A and Su(z)12-B (resulting from alternative RNA splicing), have very different expression patterns during development. Functional analyses indicate that they also have different functions he Su(z)12-B form is the main mediator of silencing. Furthermore, a neuron specific localization pattern in larval brain and a giant larval phenotype in transgenic lines reveal a potential function of Su(z)12-A in neuron development. In some aspects the isoforms seem to be able to substitute for each other. The histone methyltransferase activity of PRC2 is due to the E(z) protein. However, Su(z)12 is also necessary for H3K27me3 methylation in vivo, and it is thus a core component of PRC2. Clonal over-expression of Su(z)12 in imaginal wing discs results in an increased H3K27me3 activity, indicating that Su(z)12 is a limiting factor for silencing. When PcG function is lost, target genes normally become de-repressed. The segment polarity gene engrailed, encoding a transcription factor, is a target for PRC2 silencing. However, we found that it was not activated when PRC2 function was deleted. We show that the Ultrabithorax protein, encoded by another PcG target gene, also acts as an inhibitor of engrailed and that de-regulation of this gene causes a continued repression of engrailed. The conclusion is that a gene can have several negative regulators working in parallel and that secondary effects have to be taken into consideration, when analyzing effects of mutants. PcG silencing affects very many cellular processes and a large quantity of knowledge is gathered on the overall mechanisms of PcG regulation. However, little is known about how individual genes are silenced and how cells “remember” their fate through cell generations.
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PCR-RFLP analys av mt-DNA hos Öring (Salmo trutta) i Gävleborgs län.Björkbom, Tommy January 2010 (has links)
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Investigation of phylogenetic relationships using microRNA sequences and secondary structuresDnyansagar, Rohit January 2010 (has links)
MicroRNAs are important biomolecules for regulating biological processes. Moreover, the secondary structure of microRNA is important for its activity and has been used previously as a mean for finding unknown microRNAs. A phylogenetic study of the microRNA secondary structure reveals more information than its primary sequence, because the primary sequence can undergo mutations that give rise to different phylogenetic relationships, whereas the secondary structure is more robust against mutations and therefore sometimes more informative. Here we constructed a phylogenetic tree entirely based on microRNA secondary structures using tools PHYLIP (Felsenstein, 1995) and RNAforester (Matthias Höchsmann, 2003, Hochsmann et al., 2004), and compared the overall topology and clusters with the phylogenetic tree constructed using microRNA sequence. The purpose behind this comparison was to investigate the sequence and structure similarity in phylogenetic context and also to investigate if functionally similar microRNA genes are closer in their structure-derived phylogenetic tree. Our phylogenetic comparison shows that the sequence similarity has hardly any effect on the structure similarity in the phylogenetic tree. MicroRNAs that have similar function are closer in the phylogenetic tree based on secondary structure than its respective sequence phylogeny. Hence, this approach can be very useful in predicting the functions of the new microRNAs whose function is yet to be known, since the function of the miRNAs heavily relies on its secondary structure.
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Ancient DNA as a Means to Investigate the European NeolithicMalmström, Helena January 2007 (has links)
The transition from a hunter-gatherer lifestyle to a farming lifestyle, i.e. the Neolithisation, is arguably the most important event in human prehistory. While the geography and dating of the Neolithisation is well known, the process is still under debate, especially if it occurred through diffusion of ideas or with migrating farmers. The process accelerated when alternative use of domesticated animals increased. Especially the use of dairy products, and the consumption of unprocessed milk, appears to be of importance. As milk consumption (lactose digestion) is dependent upon genetic components, it is debated whether the genetic disposition allowed for dairy production to evolve, or if the usages of dairy products added selection pressure that eventually lead to present day allele frequencies. Molecular genetics have the potential to solve this and similar questions, but only if the contamination problem, where authentic DNA can be distinguished from modern contaminating DNA, can be resolved. Here I investigate the nature and extent of contamination with modern human DNA in museum specimens and explore several approaches to minimise this contamination and to authenticate DNA results from ancient humans. I use real-time quantification, pyrosequencing and FLX-generated clonal sequencing assays to generate data on ancient humans and ancient dogs. I further use the techniques to study the development of lactase persistence and the nature of animal domestication. The results presented show that sample-based contamination is extensive, but can be minimised if treated with bleach. I retrieved authentic HVSI sequences from 30 Neolithic hunter-gatherers and farmers from Sweden, of which eighteen also yielded nuclear data indicating that the farmers had a higher frequency of the allele linked to lactase persistence compared to the hunter-gatherers. I conclude that genetic data from ancient humans as well as from ancient animals can be retrieved and used, but only under high stringency.
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Molecular Screening for Target Discovery in CancerFryknäs, Mårten January 2006 (has links)
Cancer is one of the major causes of death in the western world. Resistance to anti-cancer drugs and diagnostic difficulties are major obstacles to successful treatment. This thesis describes studies based on microarray expression analysis and high-throughput compound screening for identification of resistance mechanisms, drug targets and diagnostic markers. In paper I-IV, we applied global expression analysis and measurements of drug response in a human tumor cell line panel to identify drug targets and resistance mechanisms. In paper I, we identified gene transcript levels that correlate with drug resistance and sensitivity. Both well known and new potential markers and mechanisms were identified. In paper II, we showed that STAT1 activity is associated with cross-resistance to both doxorubicin and radiation in vitro and that fludarabine can counteract STAT1 activity and reduce resistance. In Paper III-IV, cell lines were exposed to a compound library consisting of more than thousand different substances in a high-throughput screening effort. These studies revealed that cell line models of squamous cell carcinoma (Paper III) and drug resistant myeloma (Paper IV) are sensitive to phosphodiesterase inhibitors and glucocorticoids respectively. The target molecules for these drugs were over-expressed at the mRNA level and constitute likely explanations for the observed drug potency. In paper V, we identified mRNA markers for the distinction between two types of thyroid tumors, thyroid follicular adenomas and thyroid follicular carcinomas, by means of microarray expression analysis. Our results indicated that distinction between the two tumor types is possible with a small number of markers.
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Genome Variation in Human Populations : Exploring the Effects of Demographic History and the Potential for Mapping of Complex TraitsJohansson, Åsa January 2006 (has links)
A major challenge in human genetics is to understand the genetic variation underlying common diseases. In this thesis, I focus on forces creating differences between individuals and genomic regions, methods for characterizing genomic variation, and the association between genomic and phenotypic variation. Genetic markers are widely used to locate genes associated with different phenotypes. In my first paper, I describe novel algorithms for automatic genotype determination of microsatellite markers, a procedure which is currently both time-consuming and error prone. The co-segregation of genetic markers in a population leads to non-random association of alleles at different loci - linkage disequilibrium (LD). LD varies throughout the genome and differs between populations due to factors such as their demographic history. In my second paper, I discuss the increased power, for mapping of human traits, that results from studying a population with appreciable levels of LD such as is found in the Swedish Sami population. Lately, large-scale analyses of single nucleotide polymorphisms (SNPs) have become available and efforts have been made to identify a set of SNPs, which captures most of the genome variation in a population (tagSNPs). In my third paper, I describe the limitations of this approach when applied to data from an independent population sample of randomly ascertained SNPs. The transferability of tagSNPs between populations is poor, presumably due to variation in allele frequencies and the bias towards common SNPs used in most studies. The level of genomic variation is influenced by population structure, recombination and mutation rate, as well as natural selection. During the exodus from Africa, humans have adapted to new environmental conditions. In my fourth paper, I describe a new method for identifying genomic regions carrying signatures of recent positive selection and apply this to an available dataset of millions of SNPs.
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From Single Gene to Whole Genome Studies of Human Transcription RegulationRada-Iglesias, Alvaro January 2007 (has links)
Transcriptional regulation largely determines which proteins and the protein levels that are found in a cell, and this is crucial in development, differentiation and responses to environmental stimuli. The major effectors of transcriptional regulation are a group of proteins known as transcription factors, which importance is supported by their frequent involvement in mendelian and complex diseases. In paper I, we attempted to establish the importance of DNA sequence variation in transcriptional control, by analyzing the potential functionality of polymorphic short repetitive elements as cis-regulatory elements. However, the relevance of this study was constrained by the limited number of analyzed sequences and the in vitro nature of the experiments. To overcome these limitations, (paper II) we optimized an in vivo large-scale technology named ChIP-chip, which couples chromatin immunoprecipitation and microarray hybridization. We successfully identified the binding profiles of metabolic-disease associated transcription factors in 1% of the human genome, using a liver cellular model, and inferred the binding sites at base pair resolution. Another important characteristic of transcriptional regulation is its plasticity, which allows adjusting the cellular transcriptome to cellular and environmental stimuli. In paper III, we investigated such plasticity by treating HepG2 cells with butyrate, a histone deacetylase inhibitor (HDACi) and interrogating the changes in histone H3 and H4 acetylation levels in 1% of the genome. Observation of frequent deacetylation around transcription start sites and hyperacetylation at the nuclear periphery challenges pre-assumed HDACi mechanisms of action. Finally, in paper IV we extended the DNA binding profiles of the medically relevant transcription factors, USF1 and USF2, and H3 acetylation to the whole non-repetitive fraction of the human genome. Using motif finding tools and chromatin profiling, we uncovered the major determinants of USF-DNA interactions. Furthermore, USFs and H3ac were clearly localized around transcription start sites, frequently in the context of bidirectional promoters.
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