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The hematopoietic transcription factor RUNX1 : a structural viewBäckström, Stefan January 2004 (has links)
The malfunction of the transcriptional regulator RUNX1 is the major cause of several variants of acute human leukemias and its normal function is to regulate the development of the blood system in concert with other transcriptional co-regulators. RUNX1 belongs to a conserved family of heterodimeric transcription factors that share a conserved DNA binding domain, the Runt domain (RD), named after the first member of this group – Runt - found in Drosophila melanogaster. The binding partner CBFβ serves as a regulator of RUNX by enhancing its DNA binding affinity through an allosteric mechanism. The main focus ofo my thesis work has been the crystallization and structural analysis of the RUNX1 RD and involved also more technical methodological aspects that can be applied to X-ray crystallography in general. The high resolution crystal structure of the free RD shows that this immunoglobulin-like molecule undergoes significant structural changes upon binding to both CBFβ and DNA. This involves a large flip of the L11 loop from a closed conformation in the free protein to an open conformation when CBFβ and/or DNA are bound. We refer to this transition as the “S-switch”. Smaller but significant conformational changes in other parts of the RD accompany the “S-switch”. We suggest that CBFβ triggers and stabilizes the “S-switch” which leads to the conversion of the RD into a conformation enhanced for DNA binding. During the structural analysis of the RD we identified two chloride ions that are coordinated by residues otherwise involved in DNA binding. In electrophoretic mobility-shift analyses (EMSA) we demonstrated a chloride ion concentration dependent stimulation of the DNA binding affinity of RUNX1. We further showed by NMR line width broadening experiments that the chloride binding occurred within the physiological range. A comparable DNA binding stimulation of RUNX1 was seen in the presence of negative amino acids. This suggests a regulation of the DNA binding activity of RUNX1 proteins through acidic amino acid residues possibly provided by activation domains of transcriptional co-regulators that interact with RUNX1. The use of the anomalous signal from halide ions has become a powerful technique for obtaining phase information. By replacing the sodium chloride with potassium bromide in the crystallisation conditions of the RD, we could demonstrate in a single wavelength anomalous diffraction (SAD) experiment that the anomalous signal from 2 bromide ions were sufficient to phase a 16 kDa protein. Due to lack of completeness in the low-resolution shells caused by overloaded intensities, density modification schemes failed and the resulting electron density maps were not interpretable. By combining the highresolution synchrotron data with low-resolution data from a native data set collected on a home X-ray source, the density modified bromide phases gave easily traceable maps.
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Genetic Analyses using Rolling Circle or PCR Amplified Padlock ProbesBanér, Johan January 2003 (has links)
Padlock probes are useful in a variety of genetic applications, some of which require that the probes are amplified in order to generate detectable signals. Two general padlock amplification methods, RCA and PCR, are discussed in this thesis. The isothermal rolling circle amplification (RCA) mechanism is described in detail as well as how a target strand affects primer extension. A mechanism to resolve the topological constraint imposed by the target strand, to which a padlock probe has been linked, is also discussed. We also present a more powerful amplification technique, termed serial circle amplification, which provides a highly precise tool for nucleic acid studies. Rolling circle products are digested to unit lengths, and each monomer converted to new circular oligonucleotides that can serve as templates in consecutive rounds of RCA. The final products are single-stranded DNA molecules, readily available for hybridization-based detection, for instance using molecular beacons or array hybridization. Padlock probes have the potential to be combined in large numbers for parallel gene analysis. A significant improvement of the level of multiplexed genotyping is presented using padlock probes and a molecular inversion strategy. Padlock probes containing common primer sequences along with locus-specific tag sequences were combined in multiplexed ligation reactions. After exonucleolytic selection for circular molecules, the probes were cleaved at uracil residues situated between the primer sequences, which facilitated release from the genomic DNA. A single PCR primer pair amplified all molecularly inverted probes, and the products were finally sorted on microarrays for simultaneous readout. Up to 1,500 genotypes could be detected in parallel, with sufficient signal strength for further scale-up. Finally, an application of the same parallel genotyping strategy is described where a set of padlock probes was used to study tumor induced immune responses. The distribution of TCR Vβ transcripts in tumor infiltrating T-cells and in normal control tissues were investigated in a microarray format.
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Methods for Analysis of Disease Associated Genomic Sequence VariationLovmar, Lovisa January 2004 (has links)
In Molecular Medicine a wide range of methods are applied to analyze the genome to find genetic predictors of human disease. Apart from predisposing disease, genetic variations may also serve as genetic markers in the search for factors underlying complex diseases. Additionally, they provide a means to distinguish between species, analyze evolutionary relationships and subdivide species into strains. The development and improvement of laboratory techniques and computational methods was a spin-off effect of the Human Genome Project. The same techniques for analyzing genomic sequence variations may be used independent of organism or source of DNA or RNA. In this thesis, methods for high-throughput analysis of sequence variations were developed, evaluated and applied. The performance of several genotyping assays were investigated prior to genotyping 4000 samples in a co-operative genetic epidemiological study. Sequence variations in the estrogen receptor alpha gene were found to be associated with an increased risk of breast and endometrial cancer in Swedish women. Whole genome amplification (WGA) enables large scale genetic analysis of sparse amounts of biobanked DNA samples. The performance of two WGA methods was evaluated using four-color minisequencing on tag-arrays. Our in-house developed assay and “array of arrays” format allow up to 80 samples to be analyzed in parallel on a single microscope slide. Multiple displacement amplification by the Φ29 DNA polymerase gave essentially identical genotyping results as genomic DNA. To facilitate accurate method comparisons, a cluster quality assessment approach was established and applied to assess the performance of four commercially available DNA polymerases in the tag-array minisequencing assay. A microarray method for genotyping human group A rotavirus (HRV) was developed and applied to an epidemiological survey of infectious HRV strains in Nicaragua. The method combines specific capture of amplified viral sequences on microarrays with genotype-specific DNA-polymerase mediated extension of capture oligonucleotides with fluorescent dNTPs.
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Using Minisequencing Technology for Analysing Genetic Variation in DNA and RNAFredriksson, Mona January 2005 (has links)
In this thesis, the four-color fluorescence tag-microarray minisequencing system pioneered by our group was further developed and applied for analysing genetic variation in human DNA and RNA. A SNP marker panel representing different chromosomal regions was established and used for identification of informative SNP markers for monitoring chimerism after stem cell transplantation (SCT). The success of SCT was monitored by measuring the allelic ratios of informative SNPs in follow-up samples from nine patients with leukaemia. The results agreed with data obtained using microsatellite markers. Further the same SNP marker panel was used for evaluation of two whole genome amplification methods, primer extension preamplification (PEP) and multiple displacement amplification (MDA) in comparison with genomic DNA with respect to SNP genotyping success and accuracy in tag-array minisequencing. Identical results were obtained from MDA products and genomic DNA. The tag-microarray minisequencing system was also established for multiplexed quantification of imbalanced expression of SNP alleles. Two endothelial cell lines and a panel of ten coding SNPs in five genes were used as model system. Six heterozygous SNPs were genotyped in RNA (cDNA) from the cell lines. Comparison of the relative amounts of the SNPs alleles in cDNA to heterozygote SNPs in genomic DNA displayed four SNPs with significant imbalanced expression between the SNP alleles. Finally, the tag-array minisequencing system was modified for detection of splice variants in mRNA from five leukaemia cell lines. A panel of 20 cancer-related genes with 74 alternatively splice variants was screened. Over half of the splice variants were detected in the cell lines, and similar alternative splicing patterns were observed in each cell line. The results were verified by size analysis of the PCR product subjected to the minisequencing primer extension reaction. The data from both methods agreed well, evidencing for a high sensitivity of our system.
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Pharmacogenomics of Antihypertensive Treatment & Clinical Pharmacological Studies of Digoxin TreatmentHallberg, Pär January 2005 (has links)
In Part I we found that the CYP2C9 genotype appears to influence the diastolic blood pressure response to the angiotensin II-receptor antagonist irbesartan in patients with hypertension and left ventricular hypertrophy. Those with the *1/*2 genotype (slower metabolism) responded better than those with the *1/*1 genotype (normal metabolism), likely due to a slower elimination of the drug. We further found that a +9/-9 exon 1 polymorphism of the B2 bradykinin receptor gene – shown to affect mRNA expression - appears to influence the regression of left ventricular mass during therapy with irbesartan or the beta-blocker atenolol in the same patients. Subjects with the -9/-9 genotype (higher mRNA expression) had a greater regression than carriers of the +9 allele. In Part II we found that women on digoxin therapeutic drug monitoring have higher serum digoxin concentrations (SDCs) as compared to men (1.54±0.04 [nmol/L±SE] vs 1.20±0.05 [nmol/L±SE], p<0.001), which could be of importance since an SDC >1.4 nmol/L has been associated with increased mortality. We further found that coadministration of P-glycoprotein inhibitors with digoxin was common (47%) among the same patients, and that the SDC increased in a stepwise fashion with the number of P-glycoprotein inhibitors (20-60%). Lastly, we found that patients admitted to Swedish coronary care units with atrial fibrillation without heart failure and who had been given digoxin had a higher 1-year mortality than those not given digoxin (RR 1.44 [95% CI 1.29-1.60], adjustment made for potential confounders). In conclusion, Part I represents a further step in the pharmacogenomic prospect of tailoring antihypertensive therapy. Part II indicates that heightened attention to the digoxin-dose is warranted in women, that there is a need for awareness about P-glycoprotein interactions with digoxin, and that long-term therapy with digoxin is an independent risk factor for death among patients with atrial fibrillation without heart failure.
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Readout Strategies for Biomolecular AnalysesGöransson, Jenny January 2008 (has links)
This thesis describes three readout formats for molecular analyses. A common feature in all works is probing techniques that upon specific target recognition ideally results in equimolar amounts of DNA circles. These are then specifically amplified and detected using any of the techniques presented herein. The first paper presents a method that enables homogeneous digital detection and enumeration of biomolecules, represented as fluorescence-labelled DNA macromolecules. This method offers precise measurements to be performed with a wide linear dynamic range. As an application, two closely related bacterial species were selectively detected. The second paper further investigates and optimizes the properties of the technique presented in paper one. The third paper demonstrates a platform that enables simultaneous quantitative analysis of large numbers of biomolecules. The array format and decoding scheme together propose a digital strategy for decoding of biomolecules. The array and the decoding procedure were characterized and evaluated for gene copy-number measurements. The fourth paper examines a new strategy for non-optical measurements of biomolecules. Characteristics of this technique are investigated, and compared to its optical equivalent, fluorescence polarization.
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Genome and Transcriptome Comparisons between Human and ChimpanzeeWetterbom, Anna January 2010 (has links)
The chimpanzee is humankind’s closest living relative and the two species diverged ~6 million years ago. Comparative studies of the human and chimpanzee genomes and transcriptomes are of great interest to understand the molecular mechanisms of speciation and the development of species-specific traits. The aim of this thesis is to characterize differences between the two species with regard to their genome sequences and the resulting transcript profiles. The first two papers focus on indel divergence and in particular, indels causing premature termination codons (PTCs) in 8% of the chimpanzee genes. The density of PTC genes is correlated with both the distance to the telomere and the indel divergence. Many PTC genes have several associated transcripts and since not all are affected by the PTC we propose that PTCs may affect the pattern of expressed isoforms. In the third paper, we investigate the transcriptome divergence in cerebellum, heart and liver, using high-density exon arrays. The results show that gene expression differs more between tissues than between species. Approximately 15% of the genes are differentially expressed between species, and half of the genes show different splicing patterns. We identify 28 cassette exons which are only included in one of the species, often in a tissue-specific manner. In the fourth paper, we use massive parallel sequencing to study the chimpanzee transcriptome in frontal cortex and liver. We estimate gene expression and search for novel transcribed regions (TRs). The majority of TRs are located close to genes and possibly extend the annotations. A subset of TRs are not found in the human genome. The brain transcriptome differs substantially from that of the liver and we identify a subset of genes enriched with TRs in frontal cortex. In conclusion, this thesis provides evidence of extensive genomic and transcriptomic variability between human and chimpanzee. The findings provide a basis for further studies of the underlying differences affecting phenotypic divergence between human and chimpanzee.
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High Content Analysis of Proteins and Protein Interactions by Proximity LigationLeuchowius, Karl-Johan January 2010 (has links)
Fundamental to all biological processes is the interplay between biomolecules such as proteins and nucleic acids. Studies of interactions should therefore be more informative than mere detection of expressed proteins. Preferably, such studies should be performed in material that is as biologically and clinically relevant as possible, i.e. in primary cells and tissues. In addition, to be able to take into account the heterogeneity of such samples, the analyses should be performed in situ to retain information on the sub-cellular localization where the interactions occur, enabling determination of the activity status of individual cells and allowing discrimination between e.g. tumor cells and surrounding stroma. This requires assays with an utmost level of sensitivity and selectivity. Taking these issues into consideration, the in situ proximity-ligation assay (in situ PLA) was developed, providing localized detection of proteins, protein-protein interactions and post-translational modifications in fixed cells and tissues. The high sensitivity and selectivity afforded by the assay's requirement for dual target recognition in combination with powerful signal amplification enables visualization of single protein molecules in intact single cells and tissue sections. To further increase the usefulness and application of in situ PLA, the assay was adapted to high content analysis techniques such as flow cytometry and high content screening. The use of in situ PLA in flow cytometry offers the possibility for high-throughput analysis of cells in solution with the unique characteristics offered by the assay. For high content screening, it was demonstrated that in situ PLA can enable cell-based drug screening of compounds affecting post-translational modifications and protein-protein interactions in primary cells, offering superior abilities over current assays. The methods presented in this thesis provide powerful new tools to study proteins in genetically unmodified cells and tissues, and should offer exciting new possibilities for molecular biology, diagnostics and drug discovery.
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Genetic Analyses of Multiple Sclerosis and Systemic Lupus Erythematosus : From Single Markers to Genome-Wide DataSandling, Johanna K January 2010 (has links)
In autoimmune diseases an individual’s immune system becomes targeted at the body’s own healthy cells. The aim of this thesis was to identify genetic risk factors for the two autoimmune diseases multiple sclerosis (MS) and systemic lupus erythematosus (SLE). In Study I, we found that genetic variation in the interferon regulatory factor 5 gene (IRF5), previously shown to be associated with SLE, rheumatoid arthritis and inflammatory bowel diseases, was associated also with MS. An insertion/deletion polymorphism in the first intron of IRF5 is as a good functional candidate for this association. IRF5, together with the signal transducer and activator of transcription 4 gene (STAT4), are the most important genetic risk factors for SLE, outside the HLA region. In Study II we showed using a family-based study design that genetic variation in STAT4 is associated with SLE also in the Finnish population. In Study III, we investigated a STAT4 risk allele for SLE for its association with cardiovascular disease in SLE patients. The risk allele of STAT4 proved to be strongly associated with ischemic cerebrovascular disease and anti-phospholipid antibodies in SLE patients. A possible mechanism for this association is that the risk allele leads to increased production of pro-thrombotic anti-phospholipid antibodies, which in turn increases the risk for stroke. Both IRF5 and STAT4 are involved in signalling of the type I interferon system. In Study IV, we investigated 78 additional genes in this system for their association with SLE in a Swedish cohort. The most promising results were followed up in additional patients and controls from Sweden and the US. Two novel SLE genes were identified. In Study V a large follow-up of a genome-wide association study was performed. Five new SLE loci were identified: TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10. A number of genes previously shown to be associated with other autoimmune diseases were also tested for association with SLE. This analysis identified the type I interferon system gene IFIH1 as a novel SLE risk locus. These studies confirms the central role of the type I interferon system in SLE and further suggests common genetic risk factors in autoimmunity.
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Application of Padlock Probe Based Nucleic Acid Analysis In SituHenriksson, Sara January 2010 (has links)
The great variation displayed by nucleic acid molecules in human cells, and the continuous discovery of their impact on life, consequently require continuous refinements of molecular analysis techniques. Padlock probes and rolling circle amplification offer single nucleotide discrimination in situ, a high signal-to-noise ratio and localized detection within cells and tissues. In this thesis, in situ detection of nucleic acids with padlock probes and rolling circle amplification was applied for detection of DNA in the single cell gel electrophoresis assay to detect nuclear and mitochondrial DNA. This assay is used to measure DNA damage and repair. The behaviour of mitochondrial DNA in the single cell gel electrophoresis assay has earlier been controversial, but it was shown herein that mitochondrial DNA diffuses away early in the assay. In contrast, Alu repeats remain associated with the nuclear matrix throughout the procedure. A new twelve gel approach was also developed with increased throughput of the single cell gel electrophoresis assay. DNA repair of three genes OGG1, XPD and HPRT and of Alu repeats after H2O2 induced damage was further monitored. All three genes and Alu repeats were repaired faster than total DNA. Finally, padlock probes and rolling circle amplification were applied for detection of the single stranded RNA virus Crimean Congo hemorrhagic fever virus. The virus was detected by first reverse transcribing RNA into cDNA.. The virus RNA together with its complementary RNA and the nucleocapsid protein were detected in cultured cells. The work presented here enables studies of gene specific damage and repair as well as viral infections in situ. Detection by ligation offers high specificity and makes it possible to discriminate even between closely related molecules. Therefore, these techniques will be useful for a wide range of applications within research and diagnostics.
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