Comparison and integration of MEG and fMRI

MEG directly measures the neuronal events and has greater temporal resolution than fMRI, which has limited temporal resolution mainly due to the larger timescale of the hemodynamic response. On the other hand fMRI has advantages in spatial resolution, while the localization results with MEG can be ambiguous due to the non-uniqueness of the electromagnetic inverse problem. Thus, these methods could provide complementary information and could be used to create both spatially and temporally accurate models of brain function. We investigated the degree of overlap, revealed by the two imaging methods, in areas involved in sensory or motor processing in healthy subjects and neurosurgical patients. Furthermore, we used the spatial information from fMRI to construct a spatiotemporal model of the MEG data in order to investigate the sensorimotor system and to create a spatiotemporal model of its function. We compared the localization results from the MEG and fMRI with invasive electrophysiological cortical mapping. We used a recently introduced method, contextual clustering, for hypothesis testing of fMRI data and assessed the the effect of neighbourhood information use on the reproducibility of fMRI results. Using MEG, we identified the ipsilateral primary sensorimotor cortex (SMI) as a novel source area contributing to the somatosensory evoked fields (SEF) to median nerve stimulation. Using combined MEG and fMRI measurements we found that two separate areas in the lateral fissure may be the generators for the SEF responses from the secondary somatosensory cortex region. The two imaging methods indicated activation in corresponding locations. By using complementary information from MEG and fMRI we established a spatiotemporal model of somatosensory cortical processing. This spatiotemporal model of cerebral activity was in good agreement with results from several studies using invasive electrophysiological measurements and with anatomical studies in monkey and man concerning the connections between somatosensory areas. In neurosurgical patients, the MEG dipole model turned out to be more reliable than fMRI in the identification of the central sulcus. This was due to prominent activation in non-primary areas in fMRI, which in some cases led to erroneous or ambiguous localization of the central sulcus.

Novel Mass Spectrometric Analysis Methods for Anabolic Androgenic Steroids in Sports Drug Testing

The feasibility of different modern analytical techniques for the mass spectrometric detection of anabolic androgenic steroids (AAS) in human urine was examined in order to enhance the prevalent analytics and to find reasonable strategies for effective sports drug testing. A comparative study of the sensitivity and specificity between gas chromatography (GC) combined with low (LRMS) and high resolution mass spectrometry (HRMS) in screening of AAS was carried out with four metabolites of methandienone. Measurements were done in selected ion monitoring mode with HRMS using a mass resolution of 5000. With HRMS the detection limits were considerably lower than with LRMS, enabling detection of steroids at low 0.2-0.5 ng/ml levels. However, also with HRMS, the biological background hampered the detection of some steroids. The applicability of liquid-phase microextraction (LPME) was studied with metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol. Factors affecting the extraction process were studied and a novel LPME method with in-fiber silylation was developed and validated for GC/MS analysis of the danazol metabolite. The method allowed precise, selective and sensitive analysis of the metabolite and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine without any other steps in sample preparation. Liquid chromatographic/tandem mass spectrometric (LC/MS/MS) methods utilizing electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were developed and applied for detection of oxandrolone and metabolites of stanozolol and 4-chlorodehydromethyltestosterone in urine. All methods exhibited high sensitivity and specificity. ESI showed, however, the best applicability, and a LC/ESI-MS/MS method for routine screening of nine 17-alkyl-substituted AAS was thus developed enabling fast and precise measurement of all analytes with detection limits below 2 ng/ml. The potential of chemometrics to resolve complex GC/MS data was demonstrated with samples prepared for AAS screening. Acquired full scan spectral data (m/z 40-700) were processed by the OSCAR algorithm (Optimization by Stepwise Constraints of Alternating Regression). The deconvolution process was able to dig out from a GC/MS run more than the double number of components as compared with the number of visible chromatographic peaks. Severely overlapping components, as well as components hidden in the chromatographic background could be isolated successfully. All studied techniques proved to be useful analytical tools to improve detection of AAS in urine. Superiority of different procedures is, however, compound-dependent and different techniques complement each other.

From Polymorph Screening to Dissolution Testing - Solid Phase Analysis during Pharmaceutical Development and Manufacturing

Solid materials can exist in different physical structures without a change in chemical composition. This phenomenon, known as polymorphism, has several implications on pharmaceutical development and manufacturing. Various solid forms of a drug can possess different physical and chemical properties, which may affect processing characteristics and stability, as well as the performance of a drug in the human body. Therefore, knowledge and control of the solid forms is fundamental to maintain safety and high quality of pharmaceuticals. During manufacture, harsh conditions can give rise to unexpected solid phase transformations and therefore change the behavior of the drug. Traditionally, pharmaceutical production has relied on time-consuming off-line analysis of production batches and finished products. This has led to poor understanding of processes and drug products. Therefore, new powerful methods that enable real time monitoring of pharmaceuticals during manufacturing processes are greatly needed. The aim of this thesis was to apply spectroscopic techniques to solid phase analysis within different stages of drug development and manufacturing, and thus, provide a molecular level insight into the behavior of active pharmaceutical ingredients (APIs) during processing. Applications to polymorph screening and different unit operations were developed and studied. A new approach to dissolution testing, which involves simultaneous measurement of drug concentration in the dissolution medium and in-situ solid phase analysis of the dissolving sample, was introduced and studied. Solid phase analysis was successfully performed during different stages, enabling a molecular level insight into the occurring phenomena. Near-infrared (NIR) spectroscopy was utilized in screening of polymorphs and processing-induced transformations (PITs). Polymorph screening was also studied with NIR and Raman spectroscopy in tandem. Quantitative solid phase analysis during fluidized bed drying was performed with in-line NIR and Raman spectroscopy and partial least squares (PLS) regression, and different dehydration mechanisms were studied using in-situ spectroscopy and partial least squares discriminant analysis (PLS-DA). In-situ solid phase analysis with Raman spectroscopy during dissolution testing enabled analysis of dissolution as a whole, and provided a scientific explanation for changes in the dissolution rate. It was concluded that the methods applied and studied provide better process understanding and knowledge of the drug products, and therefore, a way to achieve better quality.

Molecular Genetics of Lactase Deficiencies

Congenital lactase deficiency (CLD) (MIM 223000) is a rare autosomal recessive gastrointestinal disorder characterized by watery diarrhea in infants fed with breast milk or other lactose-containing formulas. The CLD locus was previously assigned by linkage and linkage disequilibrium analyses on 2q21 in 19 Finnish families. In this study, the molecular background of this disorder is reported. The CLD locus was refined in 32 CLD patients in 24 families by using microsatellite and single nucleotide polymorphism (SNP) haplotypes. Mutation analyses were performed by direct sequencing. We identified 5 distinct mutations in the lactase (LCT) gene, encoding the enzyme that hydrolyzes lactose in the intestinal lumen. These findings facilitate genetic testing of CLD in clinical practice and enable genetic counseling. The present data also provide the basis for detailed characterization of the molecular pathogenesis of this disorder. Adult-type hypolactasia (MIM 223100) (lactase non-persistence, lactose intolerance) is an autosomal recessive gastrointestinal condition that is a result of a decline in the activity of lactase in the intestinal lumen after weaning. Adult-type hypolactasia is considered to be a normal phenomenon among mammals and symptoms are remarkably milder than experienced in CLD. Recently, a variant C/T-13910 was shown to associate with the adult-type hypolactasia trait, locating 13.9 kb upstream of the LCT gene. In this study, the functional significance of the C/T-13910 variant was determined by studying the LCT mRNA levels in intestinal biopsy samples in children and adults with different genotypes. RT-PCR followed by solid-phase minisequencing was applied to determine the relative expression levels of the LCT alleles using an informative SNP located in exon 1. In children, the C-13910 allele was observed to be downregulated after five years of age in parallel with lactase enzyme activity. The expression of the LCT mRNA in the intestinal mucosa in individuals with the T-13910 A-22018 alleles was 11.5 times higher than that found in individuals with the C-13910, G-22018 alleles. These findings suggest that the C/T-13910 associated with adult-type hypolactasia is associated with the transcriptional regulation of the LCT gene. The presence of the T-13910 A-22018 allele also showed significant elevation lactase activity. Galactose, the hydrolysing product of the milk sugar lactose, has been hypothesized to be poisonous to ovarian epithelial cells. Hence, consumption of dairy products and lactase persistence has been proposed to be a risk factor for ovarian carcinoma. To investigate whether lactase persistence is related to the risk of ovarian carcinoma the C/T-13910 genotype was determined in a cohort of 782 women with ovarian carcinoma 1331 individuals serving as controls. Lactase persistence did not associate significantly with the risk for ovarian carcinoma in the Finnish, in the Polish or in the Swedish populations. The findings do not support the hypothesis that lactase persistence increases the risk for ovarian carcinoma.

Design and testing of stand-specific bucking instructions for use on modern cut-to-length harvesters

This study addresses three important issues in tree bucking optimization in the context of cut-to-length harvesting. (1) Would the fit between the log demand and log output distributions be better if the price and/or demand matrices controlling the bucking decisions on modern cut-to-length harvesters were adjusted to the unique conditions of each individual stand? (2) In what ways can we generate stand and product specific price and demand matrices? (3) What alternatives do we have to measure the fit between the log demand and log output distributions, and what would be an ideal goodness-of-fit measure? Three iterative search systems were developed for seeking stand-specific price and demand matrix sets: (1) A fuzzy logic control system for calibrating the price matrix of one log product for one stand at a time (the stand-level one-product approach); (2) a genetic algorithm system for adjusting the price matrices of one log product in parallel for several stands (the forest-level one-product approach); and (3) a genetic algorithm system for dividing the overall demand matrix of each of the several log products into stand-specific sub-demands simultaneously for several stands and products (the forest-level multi-product approach). The stem material used for testing the performance of the stand-specific price and demand matrices against that of the reference matrices was comprised of 9 155 Norway spruce (Picea abies (L.) Karst.) sawlog stems gathered by harvesters from 15 mature spruce-dominated stands in southern Finland. The reference price and demand matrices were either direct copies or slightly modified versions of those used by two Finnish sawmilling companies. Two types of stand-specific bucking matrices were compiled for each log product. One was from the harvester-collected stem profiles and the other was from the pre-harvest inventory data. Four goodness-of-fit measures were analyzed for their appropriateness in determining the similarity between the log demand and log output distributions: (1) the apportionment degree (index), (2) the chi-square statistic, (3) Laspeyres quantity index, and (4) the price-weighted apportionment degree. The study confirmed that any improvement in the fit between the log demand and log output distributions can only be realized at the expense of log volumes produced. Stand-level pre-control of price matrices was found to be advantageous, provided the control is done with perfect stem data. Forest-level pre-control of price matrices resulted in no improvement in the cumulative apportionment degree. Cutting stands under the control of stand-specific demand matrices yielded a better total fit between the demand and output matrices at the forest level than was obtained by cutting each stand with non-stand-specific reference matrices. The theoretical and experimental analyses suggest that none of the three alternative goodness-of-fit measures clearly outperforms the traditional apportionment degree measure. Keywords: harvesting, tree bucking optimization, simulation, fuzzy control, genetic algorithms, goodness-of-fit

UV-induced NOy emissions in gas-exchange chambers enclosing Scots pine shoots: an analysis on their origin and significance

It is essential to have a thorough understanding of the sources and sinks of oxidized nitrogen (NOy) in the atmosphere, since it has a strong influence on the tropospheric chemistry and the eutrophication of ecosystems. One unknown component in the balance of gaseous oxidized nitrogen is vegetation. Plants absorb nitrogenous species from the air via the stomata, but it is not clear whether plants can also emit them at low ambient concentrations. The possible emissions are small and difficult to measure. The aim of this thesis was to analyse an observation made in southern Finland at the SMEAR II station: solar ultraviolet radiation (UV) induced NOy emissions in chambers measuring the gas exchange of Scots pine (Pinus sylvestris L.) shoots. Both measuring and modelling approaches were used in the study. The measurements were performed under noncontrolled field conditions at low ambient NOy concentrations. The chamber blank i.e. artefact NOy emissions from the chamber walls, was dependent on the UV irradiance and increased with time after renewing the Teflon film on chamber surfaces. The contribution of each pine shoot to the total NOy emissions in the chambers was determined by testing whether the emissions decrease when the shoots are removed from their chambers. Emissions did decrease, but only when the chamber interior was exposed to UV radiation. It was concluded that also the pine shoots emit NOy. The possible effects of transpiration on the chamber blank are discussed in the summary part of the thesis, based on previously unpublished data. The possible processes underlying the UV-induced NOy emissions were reviewed. Surface reactions were more likely than metabolic processes. Photolysis of nitrate deposited on the needles may have generated the NOy emissions; the measurements supported this hypothesis. In that case, the emissions apparently would consist mainly of nitrogen dioxide (NO2), nitric oxide (NO) and nitrous acid (HONO). Within studies on NOy exchange of plants, the gases most frequently studied are NO2 and NO (=NOx). In the present work, the implications of the emissions for the NOx exchange of pine were analysed with a model including both NOy emissions and NOy absorption. The model suggested that if the emissions exist, pines can act as an NOx source rather than a sink, even under relatively high ambient concentrations.

Breath Testing by Fourier Transform Infrared Spectroscopy for Solvent Intoxication Diagnostics

Technical or contaminated ethanol products are sometimes ingested either accidentally or on purpose. Typical misused products are black-market liquor and automotive products, e.g., windshield washer fluids. In addition to less toxic solvents, these liquids may contain the deadly methanol. Symptoms of even lethal solvent poisoning are often non-specific at the early stage. The present series of studies was carried out to develop a method for solvent intoxication breath diagnostics to speed up the diagnosis procedure conventionally based on blood tests. Especially in the case of methanol ingestion, the analysis method should be sufficiently sensitive and accurate to determine the presence of even small amounts of methanol from the mixture of ethanol and other less-toxic components. In addition to the studies on the FT-IR method, the Dräger 7110 evidential breath analyzer was examined to determine its ability to reveal a coexisting toxic solvent. An industrial Fourier transform infrared analyzer was modified for breath testing. The sample cell fittings were widened and the cell size reduced in order to get an alveolar sample directly from a single exhalation. The performance and the feasibility of the Gasmet FT-IR analyzer were tested in clinical settings and in the laboratory. Actual human breath screening studies were carried out with healthy volunteers, inebriated homeless men, emergency room patients and methanol-intoxicated patients. A number of the breath analysis results were compared to blood test results in order to approximate the blood-breath relationship. In the laboratory experiments, the analytical performance of the Gasmet FT-IR analyzer and Dräger 7110 evidential breath analyzer was evaluated by means of artificial samples resembling exhaled breath. The investigations demonstrated that a successful breath ethanol analysis by Dräger 7110 evidential breath analyzer could exclude any significant methanol intoxication. In contrast, the device did not detect very high levels of acetone, 1-propanol and 2-propanol in simulated breath. The Dräger 7110 evidential breath ethanol analyzer was not equipped to recognize the interfering component. According to the studies the Gasmet FT-IR analyzer was adequately sensitive, selective and accurate for solvent intoxication diagnostics. In addition to diagnostics, the fast breath solvent analysis proved feasible for controlling the ethanol and methanol concentration during haemodialysis treatment. Because of the simplicity of the sampling and analysis procedure, non-laboratory personnel, such as police officers or social workers, could also operate the analyzer for screening purposes.

Ventricular repolarization during cardiovascular autonomic function testing

The autonomic nervous system is an important modulator of ventricular repolarization and arrhythmia vulnerability. This study explored the effects of cardiovascular autonomic function tests on repolarization and its heterogeneity, with a special reference to congenital arrhythmogenic disorders typically associated with stress-induced fatal ventricular arrhythmias. The first part explored the effects of standardized autonomic tests on QT intervals in a 12-lead electrocardiogram and in multichannel magnetocardiography in 10 healthy adults. The second part studied the effects of deep breathing, Valsalva manouvre, mental stress, sustained handgrip and mild exercise on QT intervals in asymptomatic patients with LQT1 subtype of the hereditary long QT syndrome (n=9) and in patients with arrhythmogenic right ventricular dysplasia (ARVD, n=9). Even strong sympathetic activation had no effects on spatial QT interval dispersion in healthy subjects, but deep respiratory efforts and Valsalva influenced it in ways that were opposite in electrocardiographic and magnetocardiographic recordings. LQT1 patients showed blunted QT interval and sinus nodal responses to sympathetic challenge, as well as an exaggerated QT prolongation during the recovery phases. LQT1 patients showed a QT interval recovery overshoot in 2.4 ± 1.7 tests compared with 0.8 ± 0.7 in healthy controls (P = 0.02). Valsalva strain prolonged the T wave peak to T wave end interval only in the LQT1 patients, considered to reflect the arrhythmogenic substrate in this syndrome. ARVD patients showed signs of abnormal repolarization in the right ventricle, modulated by abrupt sympathetic activation. An electrocardiographic marker reflecting interventricular dispersion of repolarization was introduced. It showed that LQT1 patients exhibit a repolarization gradient from the left ventricle towards the right ventricle, significantly larger than in controls. In contrast, ARVD patients showed a repolarization gradient from the right ventricle towards the left. Valsalva strain amplified the repolarization gradient in LQT1 patients whereas it transiently reversed it in patients with ARVD. In conclusion, intrathoracic volume and pressure changes influence regional electrocardiographic and magnetocardiographic QT interval measurements differently. Especially recovery phases of standard cardiovascular autonomic functions tests and Valsalva manoeuvre reveal the abnormal repolarization in asymptomatic LQT1 patients. Both LQT1 and ARVD patients have abnormal interventricular repolarization gradients, modulated by abrupt sympathetic activation. Autonomic testing and in particular the Valsalva manoeuvre are potentially useful in unmasking abnormal repolarization in these syndromes.

Quantifying Time Awake Posturographically

This thesis focuses on the issue of testing sleepiness quantitatively. The issue is relevant to policymakers concerned with traffic- and occupational safety; such testing provides a tool for safety legislation and -surveillance. The findings of this thesis provide guidelines for a posturographic sleepiness tester. Sleepiness ensuing from staying awake merely 17 h impairs our performance as much as the legally proscribed blood alcohol concentration 0.5 does. Hence, sleepiness is a major risk factor in transportation and occupational accidents. The lack of convenient, commercial sleepiness tests precludes testing impending sleepiness levels contrary to simply breath testing for alcohol intoxication. Posturography is a potential sleepiness test, since clinical diurnal balance testing suggests the hypothesis that time awake could be posturographically estimable. Relying on this hypothesis this thesis examines posturographic sleepiness testing for instrumentation purposes. Empirical results from 63 subjects for whom we tested balance with a force platform during wakefulness for maximum 36 h show that sustained wakefulness impairs balance. The results show that time awake is posturographically estimable with 88% accuracy and 97% precision which validates our hypothesis. Results also show that balance scores tested at 13:30 hours serve as a threshold to detect excessive sleepiness. Analytical results show that the test length has a marked effect on estimation accuracy: 18 s tests suffice to identify sleepiness related balance changes, but trades off some of the accuracy achieved with 30 s tests. The procedure to estimate time awake relies on equating the subject s test score to a reference table (comprising balance scores tested during sustained wakefulness, regressed against time awake). Empirical results showed that sustained wakefulness explains 60% of the diurnal balance variations, whereas the time of day explains 40% of the balance variations. The latter fact implies that time awake estimations also must rely on knowing the local times of both test and reference scores.

Irradiation of silicon particle detectors with MeV-protons

Silicon particle detectors are used in several applications and will clearly require better hardness against particle radiation in the future large scale experiments than can be provided today. To achieve this goal, more irradiation studies with defect generating bombarding particles are needed. Protons can be considered as important bombarding species, although neutrons and electrons are perhaps the most widely used particles in such irradiation studies. Protons provide unique possibilities, as their defect production rates are clearly higher than those of neutrons and electrons, and, their damage creation in silicon is most similar to the that of pions. This thesis explores the development and testing of an irradiation facility that provides the cooling of the detector and on-line electrical characterisation, such as current-voltage (IV) and capacitance-voltage (CV) measurements. This irradiation facility, which employs a 5-MV tandem accelerator, appears to function well, but some disadvantageous limitations are related to MeV-proton irradiation of silicon particle detectors. Typically, detectors are in non-operational mode during irradiation (i.e., without the applied bias voltage). However, in real experiments the detectors are biased; the ionising proton generates electron-hole pairs, and a rise in rate of proton flux may cause the detector to breakdown. This limits the proton flux for the irradiation of biased detectors. In this work, it is shown that, if detectors are irradiated and kept operational, the electric field decreases the introduction rate of negative space-charges and current-related damage. The effects of various particles with different energies are scaled to each others by the non-ionising energy loss (NIEL) hypothesis. The type of defects induced by irradiation depends on the energy used, and this thesis also discusses the minimum proton energy required at which the NIEL-scaling is valid.