<p>Ultrastructural studies of airway epithelium in airway disease are important for diagnosis and understanding the underlying pathology which helps clinicians to improve the patients' treatment.</p><p>Airway biopsies from a 5-month old boy with respiratory problems and gastro-oesophageal reflux were studied by transmission electron microscopy (TEM). The tracheal columnar cells showed accumulation of lamellar bodies, indicative of lysosomal storage disease. The patient was diagnosed with Gaucher disease type 2.</p><p>Shedding of airway epithelial cells is commonly found in asthma. The attachment of these cells to the basal lamina was investigated by TEM of biopsies from patients with asthma and healthy controls. The contact area between columnar cells and basal lamina in asthmatics was significantly less than in controls. Attachment of columnar cells to the basal lamina occurs mainly indirectly, via desmosomal attachment to basal cells. </p><p>Primary ciliary dyskinesia (PCD) is a congenital disease. It is important to differentiate PCD from acquired (secondary) ciliary dyskinesia (SCD). The number of dynein arms determined by TEM was 1.5 and 1.4 for outer and inner dynein arms, respectively in PCD, versus 7.9 and 5.2 for controls and 8.1 and 5.9 in SCD. Compared to PCD patients, SCD patients have more structurally abnormal cilia. A significant difference was found in orientation of the central microtubule pair between PCD and SCD, but also overlap. </p><p>Leukotriene receptor antagonists are a new treatment for asthma. Both corticosteroids and montelukast caused apoptosis and necrosis of airway epithelial cells, and reduced the expression of intercellular adhesion molecule-1. Treatment of cells with tumor necrosis factor-α or interferon-γ reduced the fraction of the lateral cell membrane occupied by desmosomes and this effect was counteracted by corticosteroids. </p>

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.

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.

In this thesis, the shape of the distal humerus trochlea is analysed using landmark-based morphometrics and multivariate methods, with the aim of exploring locomotor evolution in carnivorans. Elbow joint morphology is used together with body size and craniodental morphology to characterize past and present carnivorans. Evolutionary implications are studied at the ordinal, familial, and species levels, testing specific hypotheses about scaling, morphological constraints, evolutionary trajectories, and potential for social pack-hunting behaviour. The circumference of the distal humerus trochlea is found to be highly correlated with body mass, and appears to scale similarly throughout the order Carnivora. A general predictive model for carnivoran bodymass is presented (a=0.601; b= 2.552; r2=0.952, SEE=0.136, p&lt;0001, n=92), which removes the need for the investigator to actively choose between the diverging estimates that different predictors and their equations often produce. At the elbow joint, manual manipulation and locomotion appear to be conflicting functions, thus suggesting mutually exclusive lifestyles involving either forelimb grappling or pursuit. At large body sizes, carnivorans are distributed over a strongly dichotomised pattern (grappling or locomotion), a pattern coinciding with the postulated threshold in predator-prey size ratio at 21.5-25 kg. This pattern is compared to that of two carnivoran faunas from the Tertiary. In the Oligocene (33.7-23.8 Myr BP), the overall pattern is remarkably similar to that observed for extant Carnivora. In the Miocene (23.8-11.2 Myr BP) carnivores show a similarly dichotomised pattern as the Oligocene and Recent, although the whole pattern is shifted towards larger body sizes. This difference is suggested to be a reflection of the extraordinary species richness of browsing ungulates in the early Miocene of North America. Such an increase in prey spectrum would create a unique situation, in which large carnivores need not commit to a cursorial habitus in order to fill their nutritional requirements. Finally, the elbow joints and craniodental morphology (14 measurements) of fossil canids were examined with the aim of assessing the potential for pack-hunting in fossil canids. It is clear that small and large members of the Recent Caninae share similar craniodental morphologies. However, this pattern is not present in Borophaginae and Hesperocyoninae. In the latter, large representatives are characterized by being short-faced, with reduced anterior premolars and enlarged posterior premolars, thus approaching a “pantherine-like” craniodental configuration. These traits are interpreted as an adaptation for killing prey with canine bites. It is similarly determined that, unlike recent Caninae, all analyzed species of borophagines and hesperocyonines have retained the ability to supinate their forearms. It is therefore likely that manual manipulation was part of their hunting behaviour, thus removing an essential part of the argument for social pack-hunting in these forms, as the benefits of such a strategy become less obvious.

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.

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.

Ultrastructural studies of airway epithelium in airway disease are important for diagnosis and understanding the underlying pathology which helps clinicians to improve the patients' treatment. Airway biopsies from a 5-month old boy with respiratory problems and gastro-oesophageal reflux were studied by transmission electron microscopy (TEM). The tracheal columnar cells showed accumulation of lamellar bodies, indicative of lysosomal storage disease. The patient was diagnosed with Gaucher disease type 2. Shedding of airway epithelial cells is commonly found in asthma. The attachment of these cells to the basal lamina was investigated by TEM of biopsies from patients with asthma and healthy controls. The contact area between columnar cells and basal lamina in asthmatics was significantly less than in controls. Attachment of columnar cells to the basal lamina occurs mainly indirectly, via desmosomal attachment to basal cells. Primary ciliary dyskinesia (PCD) is a congenital disease. It is important to differentiate PCD from acquired (secondary) ciliary dyskinesia (SCD). The number of dynein arms determined by TEM was 1.5 and 1.4 for outer and inner dynein arms, respectively in PCD, versus 7.9 and 5.2 for controls and 8.1 and 5.9 in SCD. Compared to PCD patients, SCD patients have more structurally abnormal cilia. A significant difference was found in orientation of the central microtubule pair between PCD and SCD, but also overlap. Leukotriene receptor antagonists are a new treatment for asthma. Both corticosteroids and montelukast caused apoptosis and necrosis of airway epithelial cells, and reduced the expression of intercellular adhesion molecule-1. Treatment of cells with tumor necrosis factor-α or interferon-γ reduced the fraction of the lateral cell membrane occupied by desmosomes and this effect was counteracted by corticosteroids.

Traditionally, response to treatment in hematological malignancies is evaluated by light microscopy of bone marrow (BM) smears, but due to more effective therapies more sensitive methods are needed. Today, detection of minimal residual disease (MRD) using immunological and molecular techniques can be 100 times more sensitive than morphology. The main aim of this thesis was to compare and evaluate three currently available MRD methods in childhood acute lymphoblastic leukemia (ALL): (i) real-time quantitative PCR (RQ-PCR) of rearranged antigen receptor genes, (ii) multicolor flow cytometry (FCM) of leukemia-associated immunophenotypes and (iii) real-time quantitative PCR of fusion gene transcripts (RT-PCR). In paper I, we assessed the applicability of RQ-PCR in a population-based cohort of childhood ALL diagnosed in Sweden between 2002-2006. Clonal IG/TCR rearrangements were identified in the 96% of the 279 ALL cases. Using RQ-PCR, the quantitative range of 10-3 was reached in 93% of B-cell precursor (BCP) ALL and 86% of T-cell ALL (T-ALL) by at least one target gene. In paper II, we compared MRD detection using both RQ-PCR and FCM in the context of NOPHO ALL-2000 protocol. By applying the stratification threshold of ≥0.1% MRD late during induction therapy (day 29), we could demonstrate that both methods can predict the risk of BM relapse but not extramedullary relapse. However, the threshold of ≥0.2% MRD appears to be more optimal using RQ-PCR in BCP ALL, whilst in T-ALL, the results indicate that RQ-PCR is preferable for MRD assessment. The stability of RNA in vitro is a critical factor when using sensitive molecular techniques such as MRD detection. In paper III, we evaluated the influence on MRD detection when blood is collected in tubes with RNA stabilization reagents (PAX gene Vacutatiner®) compared to collection in EDTA-tubes (non-stabilized). We analyzed 68 matched samples from chronic myeloid leukemia patients and the results indicated that non-stabilized blood processed within 30 hours is preferable for MRD detection. In paper IV, follow-up samples from eight children with Philadelphia positive (Ph+) ALL were evaluated with the three available MRD methods. MRD measured by the fusion gene transcripts (BCR-ABL1) appeared to be the most sensitive method, however, precise quantification can be difficult and the other methods are thus complementary. In conclusion, all three applied MRD methods are useful and correlate to each other, although not necessary exchangeable in individual patients. We also conclude that MRD assessment by RQ-PCR, based on rearranged IG/TCR genes and multicolor FCM are predictive for identification of high risk childhood ALL patients.

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&lt;0.001), which could be of importance since an SDC &gt;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.

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.