External signal-activated liposomal drug delivery systems

Modern drug discovery gives rise to a great number of potential new therapeutic agents, but in some cases the efficient treatment of patient may not be achieved because the delivery of active compounds to the target site is insufficient. Thus, drug delivery is one of the major challenges in current pharmaceutical research. Numerous nanoparticle-based drug carriers, e.g. liposomes, have been developed for enhanced drug delivery and targeting. Drug targeting may enhance the efficiency of the treatment and, importantly, reduce unwanted side effects by decreasing drug distribution to non-target tissues. Liposomes are biocompatible lipid-based carriers that have been studied for drug delivery during the last 40 years. They can be functionalized with targeting ligands and sensing materials for triggered activation. In this study, various external signal-assisted liposomal delivery systems were developed. Signals can be used to modulate drug permeation or release from the liposome formulation, and they provide accurate control of time, place and rate of activation. The study involved three types of signals that were used to trigger drug permeation and release: electricity, heat and light. Electrical stimulus was utilized to enhance the permeation of liposomal DNA across the skin. Liposome/DNA complex-mediated transfections were performed in tight rat epidermal cell model. Various transfection media and current intensities were tested, and transfection efficiency was evaluated non-invasively by monitoring the concentration of secreted reporter protein in cell culture medium. Liposome/DNA complexes produced gene expression, but electrical stimulus did not enhance the transfection efficiency significantly. Heat-sensitive liposomal drug delivery system was developed by coating liposomes with biodegradable and thermosensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate polymer. Temperature-triggered liposome aggregation and contents release from liposomes were evaluated. The cloud point temperature (CP) of the polymer was set to 42 °C. Polymer-coated liposome aggregation and contents release were observed above CP of the polymer, while non-coated liposomes remained intact. Polymer precipitates above its CP and interacts with liposomal bilayers. It is likely that this induces permeabilization of the liposomal membrane and contents release. Light-sensitivity was introduced to liposomes by incorporation of small (< 5 nm) gold nanoparticles. Hydrophobic and hydrophilic gold nanoparticles were embedded in thermosensitive liposomes, and contents release was investigated upon UV light exposure. UV light-induced lipid phase transitions were examined with small angle X-ray scattering, and light-triggered contents release was shown also in human retinal pigment epithelial cell line. Gold nanoparticles absorb light energy and transfer it into heat, which induces phase transitions in liposomes and triggers the contents release. In conclusion, external signal-activated liposomes offer an advanced platform for numerous applications in drug delivery, particularly in the localized drug delivery. Drug release may be localized to the target site with triggering stimulus that results in better therapeutic response and less adverse effects. Triggering signal and mechanism of activation can be selected according to a specific application.

The Mechanisms of Hedgehog Signal Transduction

Studies in both vertebrates and invertebrates have identified proteins of the Hedgehog (Hh) family of secreted signaling molecules as key organizers of tissue patterning. Initially discovered in Drosophila in 1992, Hh family members have been discovered in animals with body plans as diverse as those of mammals, insects and echinoderms. In humans three related Hh genes have been identified: Sonic, Indian and Desert hedgehog (Shh, Ihh and Dhh). Transduction of the Hh signal to the cytoplasm utilizes an unusual mechanism involving consecutive repressive interactions between Hh and its receptor components, Patched (Ptc) and Smoothened (Smo). Several cytoplasmic proteins involved in Hh signal transduction are known in Drosophila, but mammalian homologs are known only for the Cubitus interruptus (Ci) transcription factor (GLI(1-3)) and for the Ci/GLI-associated protein, Suppressor of Fused (Su(fu)). In this study I analyzed the mechanisms of how the Hh receptor Ptc regulates the signal transducer Smo, and how Smo relays the Shh signal from the cell surface to the cytoplasm ultimately leading to the activation of GLI transcription factors. In Drosophila, the kinesin-like protein Costal2 (Cos2) is required for suppression of Hh target gene expression in the absence of ligand, and loss of Cos2 causes embryonic lethality. Cos2 acts by bridging Smo to the Ci. Another protein, Su(Fu) exerts a weak suppressive influence on Ci activity and loss of Su(Fu) causes subtle changes in Drosophila wing pattern. This study revealed that domains in Smo that are critical for Cos2 binding in Drosophila are dispensable for mammalian Smo function. Furthermore, by analyzing the function of Su(Fu) and the closest mouse homologs of Cos2 by protein overexpression and RNA interference I found that inhibition of the Hh response pathway in the absence of ligand does not require Cos2 activity, but instead critically depends on the activity of Su(Fu). These results indicate that a major change in the mechanism of action of a conserved signaling pathway occurred during evolution, probably through phenotypic drift made possible by the existence in some species of two parallel pathways acting between the Hh receptor and the Ci/GLI transcription factors. In a second approach to unravel Hh signaling we cloned > 90% of all human full-length protein kinase cDNAs and constructed the corresponding kinase-activity deficient mutants. Using this kinome resource as a screening tool, two kinases, MAP3K10 and DYRK2 were found to regulate Shh signaling. DYRK2 directly phosphorylated and induced the proteasome dependent degradation of the key Hh-pathway regulated transcription factor, GLI2. MAP3K10, in turn, affected GLI2 indirectly by modulating the activity of DYRK2.

Adult-type hypolactasia: Genotype-phenotype correlation

Adult-type hypolactasia (primary lactose malabsorption, lactase non-persistence) is the most common enzyme deficiency worldwide, and manifests with symptoms of lactose intolerance such as abdominal pain, gas formation and diarrhea. In humans with adult-type hypolactasia, lactase activity is high at birth, but declines during childhood to about one-tenth of the activity at birth. In 2002, a one base polymorphism C/T-13910, located 14 kilobases from the starting codon of the lactase-phlorizin hydrolase (LPH) gene was observed to be associated with the persistence of lactase activity. The T-13910 allele (C/T-13910 and T/T-13910 genotypes) associates with persistence of lactase activity throughout life, whereas the C/C-13910 genotype associates with adult-type hypolactasia. In this thesis work, the timing and mechanism of decline of lactase enzyme activity during development was studied using the C/T-13910 polymorphism as a molecular marker. We observed an excellent correlation between low lactase activity and the C/C-13910 genotype in all subjects > 12 years of age, irrespective their ethnicity. In children of African origin, the lactase activity declined somewhat earlier than among Finnish children. Furthermore, we observed an increasing imbalance in the relative lactase mRNA expression from the C-13910 and T-13910 alleles in Finnish children beginning from five years of age. The genetic test for adult-type hypolactasia showed a sensitivity of 93% and a specificity of 100% in the Finnish children and adolescents > 12 years of age. The relation of milk consumption and the milk-related abdominal complaints to the C/T-13910 genotypes associated with lactase persistence/non-persistence was studied by a questionnaire-based approach in > 2100 Finns. Both Finnish children and adults with the C/C-13910 genotype consumed significantly less dairy products compared to those with the C/T-13910 and T/T-13910 genotypes. Flatulence was the only of the abdominal symptoms of lactose intolerance that subjects with the C/C-13910 genotype reported significantly more often than those with the C/T-13910 and T/T-13910 genotypes. A minor proportion (<10%) of subjects with the C/C-13910 genotype, nevertheless, reported drinking milk without any symptoms afterwards. There was no association between cow's milk allergy starting as a newborn and adult-type hypolactasia. In an association study an increased risk of colorectal cancer was observed among those with molecular diagnosis of adult-type hypolactasia. It warrants further studies to clarify whether the increased risk observed in the Finnish population is associated with lactose or decreased intake of dairy products in these subjects.

Methods to improve gene signal: Application to cDNA microarrays

Microarrays are high throughput biological assays that allow the screening of thousands of genes for their expression. The main idea behind microarrays is to compute for each gene a unique signal that is directly proportional to the quantity of mRNA that was hybridized on the chip. A large number of steps and errors associated with each step make the generated expression signal noisy. As a result, microarray data need to be carefully pre-processed before their analysis can be assumed to lead to reliable and biologically relevant conclusions. This thesis focuses on developing methods for improving gene signal and further utilizing this improved signal for higher level analysis. To achieve this, first, approaches for designing microarray experiments using various optimality criteria, considering both biological and technical replicates, are described. A carefully designed experiment leads to signal with low noise, as the effect of unwanted variations is minimized and the precision of the estimates of the parameters of interest are maximized. Second, a system for improving the gene signal by using three scans at varying scanner sensitivities is developed. A novel Bayesian latent intensity model is then applied on these three sets of expression values, corresponding to the three scans, to estimate the suitably calibrated true signal of genes. Third, a novel image segmentation approach that segregates the fluorescent signal from the undesired noise is developed using an additional dye, SYBR green RNA II. This technique helped in identifying signal only with respect to the hybridized DNA, and signal corresponding to dust, scratch, spilling of dye, and other noises, are avoided. Fourth, an integrated statistical model is developed, where signal correction, systematic array effects, dye effects, and differential expression, are modelled jointly as opposed to a sequential application of several methods of analysis. The methods described in here have been tested only for cDNA microarrays, but can also, with some modifications, be applied to other high-throughput technologies. Keywords: High-throughput technology, microarray, cDNA, multiple scans, Bayesian hierarchical models, image analysis, experimental design, MCMC, WinBUGS.

Food intake, growth and social interactions of signal crayfish, Pacifastacus leniusculus (Dana)

The knowledge about the optimal rearing conditions, such as water temperature and quality, photoperiod and density, with the understanding of animal nutritional requirements forms the basis of economically stable aquaculture for freshwater crayfish. However, the shift from a natural environment to effective culture conditions induces several changes, not only at the population level, but also at the individual level. The social contacts between conspecifics increase with increasing animal density. The competition for limited resources (e.g. food, shelter, mates) is more severe with the presence of agonistic behaviour and may lead to unequal distribution of these. The objectives of this study were to: 1) study the distribution of a common food resource between communally reared signal crayfish (Pacifastacus leniusculus) and to assign potential feeding hierarchy on the basis of individual food intake measurements, 2) explore the possibilities of size distribution manipulations to affect population dynamics and food intake to improve growth and survival in culture and 3) study the effect of food ration and spatial distribution on food intake and to explore the effect of temperature and food ration on growth and body composition of freshwater crayfish. The feeding ranks between animals were assigned with a new method for individual food intake measurement of communally reared crayfish. This technique has a high feasibility and a great potential to be applied in crayfish aquaculture studies. In this study, signal crayfish showed high size-related variability in food consumption both among individuals within a group (inter-individual) and within individual day-to-day variation (intra-individual). Increased competition for food led to an unequal distribution of this resource and this may be a reason for large growth differences between animals. The consumption was significantly higher when reared individually in comparison with communal housing. These results suggest that communally housed crayfish form a feeding hierarchy and that the animal size is the major factor controlling the position in this hierarchy. The optimisation of the social environment ( social conditions ) was evaluated in this study as a new approach to crayfish aquaculture. The results showed that the absence of conspecifics (individual rearing vs. communal housing) affects growth rate, food intake and the proportion of injured animals, whereas size variation between animals influences the number and duration of agonistic encounters. In addition, animal size had a strong influence on the fighting success of signal crayfish reared in a social milieu with a wide size variation of conspecifics. Larger individuals initiated and won most of the competitions, which suggests size-based social hierarchy of P. leniusculus. This is further supported by the fact that the length and weight gain of smaller animals increased after size grading, maybe because of a better access to the food resource due to diminished social pressure. However, the high dominance index was not based on size under conditions of limited size variation, e.g. those characteristic of restocked natural populations and aquaculture, indicating the important role of behaviour on social hierarchy.

Pathogen-Induced Defense Signaling and Signal Crosstalk in Arabidopsis

Erwinia carotovora subsp. carotovora is a bacterial phytopathogen that causes soft rot in various agronomically important crop plants. A genetically specified resistance to E. carotovora has not been defined, and plant resistance to this pathogen is established through nonspecific activation of basal defense responses. This, together with the broad host range, makes this pathogen a good model for studying the activation of plant defenses. Production and secretion of plant cell wall-degrading enzymes (PCWDE) are central to the virulence of E. carotovora. It also possesses the type III secretion system (TTSS) utilized by many Gram-negative bacteria to secrete virulence- promoting effector proteins to plant cells. This study elucidated the role of E. carotovora HrpN (HrpNEcc), an effector protein secreted through TTSS, and the contribution of this protein in the virulence of E. carotovora. Treatment of plants with HrpNEcc was demonstrated to induce a hypersensitive response (HR) as well as resistance to E. carotovora. Resistance induced by HrpNEcc required both salicylic acid (SA)- and jasmonate/ethylene (JA/ET)-dependent defense signaling in Arabidopsis. Simultaneous treatment of Arabidopsis with HrpNEcc and PCWDE polygalacturonase PehA elicited accelerated and enhanced induction of defense genes but also increased production of superoxide and lesion formation. This demonstrates mutual amplification of defense signaling by these two virulence factors of E. carotovora. Identification of genes that are rapidly induced in response to a pathogen can provide novel information about the early events occurring in the plant defense response. CHLOROPHYLLASE 1 (AtCLH1) and EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) are both rapidly triggered by E. carotovora in Arabidopsis. Characterization of AtCLH1 encoding chlorophyll-degrading enzyme chlorophyllase indicated that it might have a role in chlorophyll degradation during plant tissue damage. Silencing of this gene resulted in increased accumulation of reactive oxygen species (ROS) in response to pathogen infection in a light-dependent manner. This led to enhanced SA-dependent defenses and resistance to E. carotovora. Moreover, crosstalk between different defense signaling pathways was observed; JA-dependent defenses and resistance to fungal pathogen Alternaria brassicicola were impaired, indicating antagonism between SA- and JA-dependent signaling. Characterization of ERD15 suggested that it is a novel, negative regulator of abscisic acid (ABA) signaling in Arabidopsis. Overexpression of ERD15 resulted in insensitivity to ABA and reduced tolerance of the plants to dehydration stress. However, simultaneously, the resistance of the plants to E. carotovora was enhanced. Silencing of ERD15 improved freezing and drought tolerance of transgenic plants. This, together with the reducing effect of ABA on seed germination, indicated hypersensitivity to this phytohormone. ERD15 was hypothesized to act as a capacitor that controls the appropriate activation of ABA responses in Arabidopsis.

Spectral Tuning and Adaptation to Different Light Environments of Mysid Visual Pigments

In the present thesis, questions of spectral tuning, the relation of spectral and thermal properties of visual pigments, and evolutionary adaptation to different light environments were addressed using a group of small crustaceans of the genus Mysis as a model. The study was based on microspectrophotometric measurements of visual pigment absorbance spectra, electrophysiological measurements of spectral sensitivities of dark-adapted eyes, and sequencing of the opsin gene retrieved through PCR. The spectral properties were related to the spectral transmission of the respective light environments, as well as to the phylogentic histories of the species. The photoactivation energy (Ea) was estimated from temperature effects on spectral sensitivity in the long-wavelength range, and calculations were made for optimal quantum catch and optimal signal-to-noise ratio in the different light environments. The opsin amino acid sequences of spectrally characterized individuals were compared to find candidate residues for spectral tuning. The general purpose was to clarify to what extent and on what time scale adaptive evolution has driven the functional properties of (mysid) visual pigments towards optimal performance in different light environments. An ultimate goal was to find the molecular mechanisms underlying the spectral tuning and to understand the balance between evolutionary adaptation and molecular constraints. The totally consistent segregation of absorption maxima (λmax) into (shorter-wavelength) marine and (longer-wavelength) freshwater populations suggests that truly adaptive evolution is involved in tuning the visual pigment for optimal performance, driven by selection for high absolute visual sensitivity. On the other hand, the similarity in λmax and opsin sequence between several populations of freshwater M. relicta in spectrally different lakes highlights the limits to adaptation set by evolutionary history and time. A strong inverse correlation between Ea and λmax was found among all visual pigments studied in these respects, including those of M. relicta and 10 species of vertebrate pigments, and this was used to infer thermal noise. The conceptual signal-to-noise ratios thus calculated for pigments with different λmax in the Baltic Sea and Lake Pääjärvi light environments supported the notion that spectral adaptation works towards maximizing the signal-to-noise ratio rather than quantum catch as such. Judged by the shape of absorbance spectra, the visual pigments of all populations of M. relicta and M. salemaai used exclusively the A2 chromophore (3, 4-dehydroretinal). A comparison of amino acid substitutions between M. relicta and M. salemaai indicated that mysid shrimps have a small number of readily available tuning sites to shift between a shorter - and a longer -wavelength opsin. However, phylogenetic history seems to have prevented marine M. relicta from converting back to the (presumably) ancestral opsin form, and thus the more recent reinvention of marine spectral sensitivity has been accomplished by some other novel mechanism, yet to be found

Molecular Tuning of Rhodopsins for High Visual Sensitivity in Different Light Environments : Variation in Absorbance Spectrum and Opsin Sequence within and between Species

Visual pigments of different animal species must have evolved at some stage to match the prevailing light environments, since all visual functions depend on their ability to absorb available photons and transduce the event into a reliable neural signal. There is a large literature on correlation between the light environment and spectral sensitivity between different fish species. However, little work has been done on evolutionary adaptation between separated populations within species. More generally, little is known about the rate of evolutionary adaptation to changing spectral environments. The objective of this thesis is to illuminate the constraints under which the evolutionary tuning of visual pigments works as evident in: scope, tempo, available molecular routes, and signal/noise trade-offs. Aquatic environments offer Nature s own laboratories for research on visual pigment properties, as naturally occurring light environments offer an enormous range of variation in both spectral composition and intensity. The present thesis focuses on the visual pigments that serve dim-light vision in two groups of model species, teleost fishes and mysid crustaceans. The geographical emphasis is in the brackish Baltic Sea area with its well-known postglacial isolation history and its aquatic fauna of both marine and fresh-water origin. The absorbance spectrum of the (single) dim-light visual pigment were recorded by microspectrophotometry (MSP) in single rods of 26 fish species and single rhabdoms of 8 opossum shrimp populations of the genus Mysis inhabiting marine, brackish or freshwater environments. Additionally, spectral sensitivity was determined from six Mysis populations by electroretinogram (ERG) recording. The rod opsin gene was sequenced in individuals of four allopatric populations of the sand goby (Pomatoschistus minutus). Rod opsins of two other goby species were investigated as outgroups for comparison. Rod absorbance spectra of the Baltic subspecies or populations of the primarily marine species herring (Clupea harengus membras), sand goby (P. minutus), and flounder (Platichthys flesus) were long-wavelength-shifted compared to their marine populations. The spectral shifts are consistent with adaptation for improved quantum catch (QC) as well as improved signal-to-noise ratio (SNR) of vision in the Baltic light environment. Since the chromophore of the pigment was pure A1 in all cases, this has apparently been achieved by evolutionary tuning of the opsin visual pigment. By contrast, no opsin-based differences were evident between lake and sea populations of species of fresh-water origin, which can tune their pigment by varying chromophore ratios. A more detailed analysis of differences in absorbance spectra and opsin sequence between and within populations was conducted using the sand goby as model species. Four allopatric populations from the Baltic Sea (B), Swedish west coast (S), English Channel (E), and Adriatic Sea (A) were examined. Rod absorbance spectra, characterized by the wavelength of maximum absorbance (λmax), differed between populations and correlated with differences in the spectral light transmission of the respective water bodies. The greatest λmax shift as well as the greatest opsin sequence difference was between the Baltic and the Adriatic populations. The significant within-population variation of the Baltic λmax values (506-511 nm) was analyzed on the level of individuals and was shown to correlate well with opsin sequence substitutions. The sequences of individuals with λmax at shorter wavelengths were identical to that of the Swedish population, whereas those with λmax at longer wavelengths additionally had substitution F261F/Y in the sixth transmembrane helix of the protein. This substitution (Y261) was also present in the Baltic common gobies and is known to redshift spectra. The tuning mechanism of the long-wavelength type Baltic sand gobies is assumed to be the co-expression of F261 and Y261 in all rods to produce ≈ 5 nm redshift. The polymorphism of the Baltic sand goby population possibly indicates ambiguous selection pressures in the Baltic Sea. The visual pigments of all lake populations of the opossum shrimp (Mysis relicta) were red-shifted by 25 nm compared with all Baltic Sea populations. This is calculated to confer a significant advantage in both QC and SNR in many humus-rich lakes with reddish water. Since only A2 chromophore was present, the differences obviously reflect evolutionary tuning of the visual protein, the opsin. The changes have occurred within the ca. 9000 years that the lakes have been isolated from the Sea after the most recent glaciation. At present, it seems that the mechanism explaining the spectral differences between lake and sea populations is not an amino acid substitution at any other conventional tuning site, but the mechanism is yet to be found.

Molecular genetics of RECQL4 syndromes

RAPADILINO syndrome is an autosomally resessively inherited condition that belongs to a group of rare syndromes more common in Finland than in other parts of the world. RAPADILINO is characterized by pre- and postnatal growth retardation, radial ray defects, diarrhoea of unknown aetiology during chilhood, a facial resemblance with other patients and normal intelligence. In Finland, 15 patients with this condition have been found which compares with only five patients in other parts of the world. We found RECQL4 gene mutations in RAPADILINO patients and proved this syndrome to be allelic with a subgroup of Rothmund-Thomson syndrome (RTS). Later we found RECQL4 mutations in patients with Baller-Gerold syndrome (BGS). These three syndromes share clinical findings and differential diagnostics rely on poikiloderma and craniosynostosis not seen in RAPADILINO syndrome. We found five different mutations in the Finnish RAPADILINO patients. The g.2545delT mutation is the founder mutation in the Finnish population as all the patients are either homozygotes or compound heterozygotes for it. This mutation leads to the inframe skipping of exon seven from mRNA. The protein encoded by this mutant mRNA lacks the nuclear retention signal and thus leads to the mislocalization of the mutant protein. The genotype-phenotype correlation is not straightforward but it seems that RAPADILINO could be due to alteration in protein function and truncating mutations in both alleles are more common among RTS patients. RTS patients with RECQL4 mutations have an elevated risk for osteosarcoma, but their risk to develop other types of malignancies is not increased.Two Finnish RAPADILINO patients have been diagnosed with osteosarcoma, but in addition to this we have found an excess of lymphoma cases among the Finnish RAPADILINO patients. This difference between cancer types could be due to different mutations found in these syndromes. The mutation screening of the patients will help to differentiate patients who have RECQL4 mutations and thus the elevated cancer risk. Patients will benefit from the follow up since early detection of malignancies is important for the treatment.

Atrial Electric Signal During Sinus Rhythm in Lone Paroxysmal Atrial Fibrillation

Atrial fibrillation (AF) is the most common tachyarrhythmia and is associated with substantial morbidity, increased mortality and cost. The treatment modalities of AF have increased, but results are still far from optimal. More individualized therapy may be beneficial. Aiming for this calls improved diagnostics. Aim of this study was to find non-invasive parameters obtained during sinus rhythm reflecting electrophysiological patterns related to propensity to AF and particularly to AF occurring without any associated heart disease, lone AF. Overall 240 subjects were enrolled, 136 patients with paroxysmal lone AF and 104 controls (mean age 45 years, 75% males). Signal measurements were performed by non-invasive magnetocardiography (MCG) and by invasive electroanatomic mapping (EAM). High-pass filtering techniques and a new method based on a surface gradient technique were adapted to analyze atrial MCG signal. The EAM was used to elucidate atrial activation in patients and as a reference for MCG. The results showed that MCG mapping is an accurate method to detect atrial electrophysiologic properties. In lone paroxysmal AF, duration of the atrial depolarization complex was marginally prolonged. The difference was more obvious in women and was also related to interatrial conduction patterns. In the focal type of AF (75%), the root mean square (RMS) amplitudes of the atrial signal were normal, but in AF without demonstrable triggers the late atrial RMS amplitudes were reduced. In addition, the atrial characteristics tended to remain similar even when examined several years after the first AF episodes. The intra-atrial recordings confirmed the occurrence of three distinct sites of electrical connection from right to left atrium (LA): the Bachmann bundle (BB), the margin of the fossa ovalis (FO), and the coronary sinus ostial area (CS). The propagation of atrial signal could also be evaluated non-invasively. Three MCG atrial wave types were identified, each of which represented a distinct interatrial activation pattern. In conclusion, in paroxysmal lone AF, active focal triggers are common, atrial depolarization is slightly prolonged, but with a normal amplitude, and the arrhythmia does not necessarily lead to electrical or mechanical dysfunction of the atria. In women the prolongation of atrial depolarization is more obvious. This may be related to gender differences in presentation of AF. A significant minority of patients with lone AF lack frequent focal triggers, and in them, the late atrial signal amplitude is reduced, possibly signifying a wider degenerative process in the LA. In lone AF, natural impulse propagation to LA during sinus rhythm goes through one or more of the principal pathways described. The BB is the most common route, but in one-third, the earliest LA activation occurs outside the BB. Susceptibility to paroxysmal lone AF is associated with propagation of the atrial signal via the margin of the FO or via multiple pathways. When conduction occurs via the BB, it is related with prolonged atrial activation. Thus, altered and alternative conduction pathways may contribute to pathogenesis of lone AF. There is growing evidence of variability in genesis of AF also within lone paroxysmal AF. Present study suggests that this variation may be reflected in cardiac signal pattern. Recognizing the distinct signal profiles may assist in understanding the pathogenesis of AF and identifying subgroups for patient-tailored therapy.

Exploiting ground-based measurements of the Global Positioning System for numerical weather prediction

Data assimilation provides an initial atmospheric state, called the analysis, for Numerical Weather Prediction (NWP). This analysis consists of pressure, temperature, wind, and humidity on a three-dimensional NWP model grid. Data assimilation blends meteorological observations with the NWP model in a statistically optimal way. The objective of this thesis is to describe methodological development carried out in order to allow data assimilation of ground-based measurements of the Global Positioning System (GPS) into the High Resolution Limited Area Model (HIRLAM) NWP system. Geodetic processing produces observations of tropospheric delay. These observations can be processed either for vertical columns at each GPS receiver station, or for the individual propagation paths of the microwave signals. These alternative processing methods result in Zenith Total Delay (ZTD) and Slant Delay (SD) observations, respectively. ZTD and SD observations are of use in the analysis of atmospheric humidity. A method is introduced for estimation of the horizontal error covariance of ZTD observations. The method makes use of observation minus model background (OmB) sequences of ZTD and conventional observations. It is demonstrated that the ZTD observation error covariance is relatively large in station separations shorter than 200 km, but non-zero covariances also appear at considerably larger station separations. The relatively low density of radiosonde observing stations limits the ability of the proposed estimation method to resolve the shortest length-scales of error covariance. SD observations are shown to contain a statistically significant signal on the asymmetry of the atmospheric humidity field. However, the asymmetric component of SD is found to be nearly always smaller than the standard deviation of the SD observation error. SD observation modelling is described in detail, and other issues relating to SD data assimilation are also discussed. These include the determination of error statistics, the tuning of observation quality control and allowing the taking into account of local observation error correlation. The experiments made show that the data assimilation system is able to retrieve the asymmetric information content of hypothetical SD observations at a single receiver station. Moreover, the impact of real SD observations on humidity analysis is comparable to that of other observing systems.