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  • Fagerholm, Fabian (Helsingin yliopisto, 2007)
    Free and Open Source Software (FOSS) has gained increased interest in the computer software industry, but assessing its quality remains a challenge. FOSS development is frequently carried out by globally distributed development teams, and all stages of development are publicly visible. Several product and process-level quality factors can be measured using the public data. This thesis presents a theoretical background for software quality and metrics and their application in a FOSS environment. Information available from FOSS projects in three information spaces are presented, and a quality model suitable for use in a FOSS context is constructed. The model includes both process and product quality metrics, and takes into account the tools and working methods commonly used in FOSS projects. A subset of the constructed quality model is applied to three FOSS projects, highlighting both theoretical and practical concerns in implementing automatic metric collection and analysis. The experiment shows that useful quality information can be extracted from the vast amount of data available. In particular, projects vary in their growth rate, complexity, modularity and team structure.
  • Pryce, Peter (Helsingin yliopisto, 2006)
    Background Contemporary Finnish, spoken and written, reveals loanwords or foreignisms in the form of hybrids: a mixture of Finnish and foreign syllables (alumiinivalua). Sometimes loanwords are inserted into the Finnish sentence in their raw form just as they are found in the source language (pulp, after sales palvelu). Again, sometimes loanwords are calques, which appear Finnish but are spelled and pronounced in an altogether foreign manner (Protomanageri, Promenadi kampuksella). Research Questions What role does Finnish business translation play in the migration of foreignisms into Finnish if we consider translation "as a construct of solutions determined by the ideological constraints and conflicts characterizing the target culture" (Robyns 1992: 212)? What attitudes do the Finns display toward the presence of foreignisms in their language? What socio-economic or ideological conditions (Bassnett 1994: 321) are responsible for these attitudes? Are these conditions dynamic? What tools can be used to measure such attitudes? This dissertation set out to answer these and similar questions. Attitudes are imperialist (where otherness is both denied and transformed), defensive (where otherness is acknowledged, transformed, and vilified), transdiscursive (a neutral attitude to both otherness and transformation), or finally defective (where alien migration is acknowledged and "stimulated") (Robyns 1994: 60). Methodology The research method follows Rose's schema (1984: 8): (a) take an existing theory, (b) develop from it a proposition specific enough to be tested, (c) devise a scheme that tests this proposition, (d) carry through the scheme in practice, (e) draw up results and discuss conclusions in relation to the original theory. In other words, the method attempts an explanation of a Finnish social phenomenon based on systematic analyses of translated evidence (Lewins 1992: 4) whereby what really matters is the logical sequence that connects the empirical data to the initial research questions raised above and, ultimately to its conclusion (Yin 1984: 29). Results This research found that Finnish translators of the Nokia annual reports used a foreignism whenever possible such as komponentin instead of rakenneosa, or investoida instead of sijoittaa, and often without any apparent justification (Pryce 2003: 203-12) more than the translator's personal preference. In the old documents (minutes of meetings of the Board of Directors of Osakeyhtio H. Saastamoinen, Ltd. dated 5 July 1912-1917, a NOPSA booklet (1932), Enzo-Gutzeit-Tornator Oy document (1938), Imatra Steel Oy Annual Report 1964, and Nokia Oy Annual Report 1946), foreignisms under Haugen's (1950: 210-31) Classification #1 occurred an average of 0.6 times, while in the new documents (Nokia 1998 translated Annual Reports) they occurred an average of 6.5 times. That big difference, suggests transdiscursive and defective attitudes in Finnish society toward the other. In the 1850s, Finnish attitudes toward alien persons and cultures were hardened, intolerant and prohibitive because language politics were both nascent and emerging, and Finns adopted a defensive stance (Paloposki 2002: 102 ff) to protect their cultural and national treasures such as language and folklore. Innovation The innovation here is that no prior doctoral level research measured Finnish attitudes toward foreignisms using a business translation approach. This is the first time that Haugen's classification has been modified and applied in target language analysis. It is hoped that this method would be replicated in similar research in the future. Applications For practical applications, researchers with interest in languages, language development, language influences, language ideologies, and power structures that affect national language policies will find this thesis useful, especially the model for collecting, grouping, and analyzing foreignisms that has been demonstrated here. It is intended to document for posterity current attitudes of Finns toward the other as revealed in business translations from 1912-1964, and in 1998. This way, future language researchers would be able to explore a time-line of Finnish language development and attitudes toward the other. Communication firms may also find this research interesting. In future, could the model we adopted be used to analyze literary texts or religious texts for example? Future Trends Though business documents show transdiscursive attitudes, other segments of Finnish society may show defensive or imperialist attitudes. When the ideology of industrialization changes in the future, will Finnish attitudes toward the other change as well? Will it then be possible to use the same kind of analytical tools to measure Finnish attitudes? More broadly, will linguistic change continue in the same direction of transdiscursive attitudes, or will the change slow down or even reverse into xenophobic attitudes? Is this our model culture-specific or can it be used in the context of other cultures? Conclusion There is anger against foreignisms in Finland as newspaper publications and television broadcasts show, but research shows that a majority of Finns consider foreignisms and the languages from which they come as sources of enrichment for Finnish culture (Laitinen 2000, Eurobarometer series 41 of July 1994, 44 of Spring 1996, 50 of Autumn 1998). Ideologies of industrialization and globalization in Finland have facilitated transdiscursive tendencies. When Finland's political ideology was intolerant toward foreign influences in the 1850s because Finland was in the process of consolidating her nascent country and language, attitudes toward the importation of loanwords also became intolerant. Presently, when industrialization and globalization became the dominant ideologies, we see a shift in attitudes toward transdiscursive tendencies. Ideology is usually unseen and too often ignored by translation researchers. However, ideology reveals itself as the most powerful factor affecting language attitudes in a target culture. Key words Finnish, Business Translation, Ideology, Foreignisms, Imperialist Attitudes, Defensive Attitudes, Transdiscursive Attitudes, Defective Attitudes, the Other, Old Documents, New Documents.
  • Lindén, Leena (Helsingin yliopisto, 2002)
  • Vainiola, Tarja (Helsingin yliopisto, 2014)
    Cost-utility analysis provides a means to determine the health benefit and economic burden of different health-care interventions. In cost-utility analyses, the benefit of care is measured in quality-adjusted life years (QALYs) gained. The calculation of QALYs requires knowledge of the change in health-related quality of life (HRQoL) and assumptions concerning when the benefit of care materialises and how long the benefit lasts. The gold standard for QALY calculations has not yet been defined and, as a consequence, the HRQoL instruments and calculation methods used vary from study to study. The aim of the current study was to clarify how much the differences in the components used for the calculation of QALYs are reflected in the end result, i.e., the number of QALYs gained in the critical care setting. The detailed aims were to study 1) the effect of the instrument used (the EQ-5D or the 15D) on the HRQoL score and the measured changes in it; 2) the effects of the baseline HRQoL and the assumptions concerning the progress of recovery on the number of QALYs; 3) how to estimate life expectancy in the critical care setting, and 4) which factors have an effect on the follow-up HRQoL. The results are based on two study populations. The first population comprises patients having been treated in an intensive care or high-dependency unit (n = 3600), and whose HRQoL was assessed using the EQ-5D and 15D HRQoL instruments 6 and 12 months after treatment. The second population consists of patients having underone treatment in a cardiac surgery intensive care unit (n = 980), and whose HRQoL was assessed using the 15D HRQoL instrument at baseline, when placed on a waiting list for surgery and 6 months after treatment. The results of the studies show that the HRQoL index score is dependent on the instrument used. The distribution of the patients HRQoL scores differed between instruments. The differences are explained, inter alia, by the ceiling effect of the EQ-5D i.e., for a significant proportion of the respondents, the instrument produced the best possible HRQoL score of 1 and by the negative scores of the EQ-5D i.e., for health states worse than death. The 15D produced higher mean HRQoL scores than the EQ-5D. The 15D was able to distinguish between a greater number of health states than the EQ-5D, thus showing a better discriminatory power. The choice of instrument was also reflected in the change observed in HRQoL. The two instruments classified patients according to the change in HRQoL (improved, remained stable, deteriorated) in a similar manner only in approximately half of the cases. The 15D was more sensitive to detecting a change than the EQ-5D. Consequently, both its discriminatory power and responsiveness to change were better than those for the EQ-5D. The assumptions concerning the progression of recovery and the baseline HRQoL score had an effect on the number of QALYs gained both within and between instruments and, consequently, on the cost per QALY ratio. The EQ-5D and the 15D performed differently under different calculation assumptions. The greatest difference in the number of QALYs gained was caused by the negative HRQoL scores observed with the EQ-5D enabling the accrual of more than 1 QALY per year. Patients having been treated in an intensive care unit showed long-lasting excess mortality and, as a consequence, a reduced life expectancy. By contrast, in cardiac surgery patients, the life expectancy was similar to or even better than that of the general population. In patient groups with excess mortality, neither the follow-up time nor the life expectancy of the general population can be regarded as optimal indicators for the duration of the benefit of care. In those patient groups, life expectancy should be extrapolated in relation to the observed excess mortality. In cardiac surgery patients, factors predicting mortality and morbidity are not able to accurately predict the follow-up HRQoL. Instead, patient experiences, such as restlessness and pain during intensive care, predicted poor post-treatment HRQoL. Given that these results are novel, future studies should be directed to patient experiences during treatment. They may be confounding factors in analyses concerning treatment effectiveness, and also diminish the effectiveness of treatment. QALY is not a universal measure, but is dependent on the HRQoL instrument used and on how the factors to be taken into account in the calculation of QALYs are chosen and defined. Furthermore, factors external to the interventions under evaluation, such as the patient s psychological experiences during treatment, may have an effect on the follow-up HRQoL. The ranking of different interventions in terms of their effectiveness calls for standardisation in the calculation of QALYs and more information on the effect of patient experiences during treatment on the follow-up HRQoL  
  • Keskitalo, Reijo (Helsingin yliopisto, 2009)
    The first quarter of the 20th century witnessed a rebirth of cosmology, study of our Universe, as a field of scientific research with testable theoretical predictions. The amount of available cosmological data grew slowly from a few galaxy redshift measurements, rotation curves and local light element abundances into the first detection of the cos- mic microwave background (CMB) in 1965. By the turn of the century the amount of data exploded incorporating fields of new, exciting cosmological observables such as lensing, Lyman alpha forests, type Ia supernovae, baryon acoustic oscillations and Sunyaev-Zeldovich regions to name a few. -- CMB, the ubiquitous afterglow of the Big Bang, carries with it a wealth of cosmological information. Unfortunately, that information, delicate intensity variations, turned out hard to extract from the overall temperature. Since the first detection, it took nearly 30 years before first evidence of fluctuations on the microwave background were presented. At present, high precision cosmology is solidly based on precise measurements of the CMB anisotropy making it possible to pinpoint cosmological parameters to one-in-a-hundred level precision. The progress has made it possible to build and test models of the Universe that differ in the way the cosmos evolved some fraction of the first second since the Big Bang. -- This thesis is concerned with the high precision CMB observations. It presents three selected topics along a CMB experiment analysis pipeline. Map-making and residual noise estimation are studied using an approach called destriping. The studied approximate methods are invaluable for the large datasets of any modern CMB experiment and will undoubtedly become even more so when the next generation of experiments reach the operational stage. -- We begin with a brief overview of cosmological observations and describe the general relativistic perturbation theory. Next we discuss the map-making problem of a CMB experiment and the characterization of residual noise present in the maps. In the end, the use of modern cosmological data is presented in the study of an extended cosmological model, the correlated isocurvature fluctuations. Current available data is shown to indicate that future experiments are certainly needed to provide more information on these extra degrees of freedom. Any solid evidence of the isocurvature modes would have a considerable impact due to their power in model selection.
  • Tammeorg, Priit (Helsingfors universitet, 2010)
    Meat and bone meal (MBM) is a valuable co-product of the rendering industry. Its feed and fertilizer uses were restricted in the EU in 2002 as a result of the occurrence of BSE crisis. Consequently, MBM was land filled, incinerated or used as an alternative fuel in cement production. The re-allowance of the fertilizer use of MBM in the EU since spring 2006 together with the rising prices of industrial nitrogen and phosphorus fertilizers have resulted in amplified research and use of MBM as a fertilizer. Additionally, growing consciousness of both finiteness of resources and deteriorating environmental effects of the artificial fertilizer use have contributed to growing popularity of nutrient recycling. The fertilizer effect and the nutrient uptake efficiencies of the Finnish MBM (N-P-K-Ca 8–6-0.5-15%) compared to mineral counterparts were tested for in present study. Additionally, the fertilizer effect of MBM in mixture with two potassium fertilizers was tested for. A greenhouse experiment with ryegrass (Lolium multiflorum) fertilized with five fertilizers (MBM, mineral fertilizers Y4PK and NK, two mixtures of MBM and potassium fertilizers) was conducted from 2007 to 2008 in Department of Agricultural Sciences greenhouses in University of Helsinki, Finland. Each of the fertilizers was applied on three nitrogen levels (80, 160 and 240 kg N ha-1). Four additional reference treatments (0 kg N ha-1 fertilizer, PK I, PK II and PK III) were included. Six cuts of ryegrass were cut and the yields were recorded. Post-harvest plant and soil analyses were conducted. MBM was shown to be a highly effective N and P fertilizer with fertilizer effect similar or even longer lasting than artificial Y4PK fertilizer. Additionally, unlike highest application levels of mineral fertilizer Y4PK and NK, meat and bone meal treatments did not lower the pH level of the soil. Therefore, it can be concluded that the relatively high Ca content of MBM is useful in preventing further costs for liming the soil. The nutrient uptake efficiencies of MBM were generally somewhat lower than for mineral counterparts. That could be partly attributable to possible immobilization of MBM-N to soil and partly to lower readily plant-available P content of MBM than mineral fertilizers. However, as significant amount of MBM-P is still available for following years, it is justified to use MBM only once in a crop rotation. That makes MBM an especially effective complementary N and P fertilizer for organic farming, where most of the nitrogen need in crop rotation is covered with legumes and manure. The fertilizing potential for mixture of MBM and K fertilizers was shown to be rather high. However, the lack of affordable potassium sources suitable for organic farming is a recognized challenge. Additional research is needed for convalescing MBM as NPK fertilizer.
  • Chen, Lin (Helsingfors universitet, 2008)
    Meat bone meal (MBM) contains considerable amount of nutrients (~8% N, ~5% P and ~10 Ca). So it can be a potential organic fertilizer for different crops. Traditionally, the production of mineral N and P fertilizers is unsustainable due to the reliance on fossil fuels in case on N, and limited mineral resource stocks in case of P. Using complementary fertilizers which originate from organic waste materials is gaining interest. On the other hand, organic farms are usually lacking nutrient sources if animals are not kept on farm at the same time. Whether MBM can achieve similar crop yield and quality as the mineral fertilizer was studied. To quantify and qualify MBM use as an N and P fertilizer, two field experiments were done on spring barley and oat in 2000 and 2001. MBM and two types fur animal manure based fertilizers (FAMB and FAMBCF) were compared to mineral fertilizer PellonY3 in three N levels: 60, 90 and 120 kg/ha. MBM and FAMBCF gave the same grain yield as PY3. There was no additional yield increase by increasing N rate from 90 to 120 kg/ha. Four aspects of grain quality, namely 1000grain weigh, test weigh, protein content and protein yield were tested. MBM, FAMBCF and PY3 tended to have similar effect while FAMB had lower effect. Since MBM has a low N/P ratio, when it is applied to meet crop's N demand, P will be at surplus. So using crop rotation and green manure for organic farm and only applying N fertilizer for conventional farm after using MBM is recommended. Adding K in MBM is also necessary when there is a lack of K in soil since MBM had only ~1% K.
  • Honkalammi, Johanna (Helsingin yliopisto, 2011)
    Drug-drug interactions may cause serious, even fatal clinical consequences. Therefore, it is important to examine the interaction potential of new chemical entities early in drug development. Mechanism-based inhibition is a pharmacokinetic interaction type, which causes irreversible loss of enzyme activity and can therefore lead to unusually profound and long-lasting consequences. The in vitro in vivo extrapolation (IVIVE) of drug-drug interactions caused by mechanism-based inhibition is challenging. Consequently, many of these interactions have remained unrecognised for many years. The concomitant use of the fibrate-class lipid-lowering agent gemfibrozil increases the concentrations of some drugs and their effects markedly. Even fatal cases of rhabdomyolysis occurred in patients administering gemfibrozil and cerivastatin concomitantly. One of the main mechanisms behind this effect is the mechanism-based inhibition of the cytochrome P450 (CYP) 2C8 enzyme by a glucuronide metabolite of gemfibrozil leading to increased cerivastatin concentrations. Although the clinical use of gemfibrozil has clearly decreased during recent years, gemfibrozil is still needed in some special cases. To enable safe use of gemfibrozil concomitantly with other drugs, information concerning the time and dose relationships of CYP2C8 inhibition by gemfibrozil should be known. This work was carried out as four in vivo clinical drug-drug interaction studies to examine the time and dose relationships of the mechanism-based inhibitory effect of gemfibrozil on CYP2C8. The oral antidiabetic drug repaglinide was used as a probe drug for measuring CYP2C8 activity in healthy volunteers. In this work, mechanism-based inhibition of the CYP2C8 enzyme by gemfibrozil was found to occur rapidly in humans. The inhibitory effect developed to its maximum already when repaglinide was given 1-3 h after gemfibrozil intake. In addition, the inhibition was shown to abate slowly. A full recovery of CYP2C8 activity, as measured by repaglinide metabolism, was achieved 96 h after cessation of gemfibrozil treatment. The dose-dependency of the mechanism-based inhibition of CYP2C8 by gemfibrozil was shown for the first time in this work. CYP2C8 activity was halved by a single 30 mg dose of gemfibrozil or by twice daily administration of less than 30 mg of gemfibrozil. Furthermore, CYP2C8 activity was decreased over 90% by a single dose of 900 mg gemfibrozil or twice daily dosing of approximately 100 mg gemfibrozil. In addition, with the application of physiological models to the data obtained in the dose-dependency studies, the major role of mechanism-based inhibition of CYP2C8 in the interaction between gemfibrozil and repaglinide was confirmed. The results of this work enhance the proper use of gemfibrozil and the safety of patients. The information related to time-dependency of CYP2C8 inhibition by gemfibrozil may also give new insights in order to improve the IVIVE of the drug-drug interactions of new chemical entities. The information obtained by this work may be utilised also in the design of clinical drug-drug interaction studies in the future.
  • Lisal, Jiri (Helsingin yliopisto, 2006)
    Molecular motors are proteins that convert chemical energy into mechanical work. The viral packaging ATPase P4 is a hexameric molecular motor that translocates RNA into preformed viral capsids. P4 belongs to the ubiquitous class of hexameric helicases. Although its structure is known, the mechanism of RNA translocation remains elusive. Here we present a detailed kinetic study of nucleotide binding, hydrolysis, and product release by P4. We propose a stochastic-sequential cooperative model to describe the coordination of ATP hydrolysis within the hexamer. In this model the apparent cooperativity is a result of hydrolysis stimulation by ATP and RNA binding to neighboring subunits rather than cooperative nucleotide binding. Simultaneous interaction of neighboring subunits with RNA makes the otherwise random hydrolysis sequential and processive. Further, we use hydrogen/deuterium exchange detected by high resolution mass spectrometry to visualize P4 conformational dynamics during the catalytic cycle. Concerted changes of exchange kinetics reveal a cooperative unit that dynamically links ATP binding sites and the central RNA binding channel. The cooperative unit is compatible with the structure-based model in which translocation is effected by conformational changes of a limited protein region. Deuterium labeling also discloses the transition state associated with RNA loading which proceeds via opening of the hexameric ring. Hydrogen/deuterium exchange is further used to delineate the interactions of the P4 hexamer with the viral procapsid. P4 associates with the procapsid via its C-terminal face. The interactions stabilize subunit interfaces within the hexamer. The conformation of the virus-bound hexamer is more stable than the hexamer in solution, which is prone to spontaneous ring openings. We propose that the stabilization within the viral capsid increases the packaging processivity and confers selectivity during RNA loading. Finally, we use single molecule techniques to characterize P4 translocation along RNA. While the P4 hexamer encloses RNA topologically within the central channel, it diffuses randomly along the RNA. In the presence of ATP, unidirectional net movement is discernible in addition to the stochastic motion. The diffusion is hindered by activation energy barriers that depend on the nucleotide binding state. The results suggest that P4 employs an electrostatic clutch instead of cycling through stable, discrete, RNA binding states during translocation. Conformational changes coupled to ATP hydrolysis modify the electrostatic potential inside the central channel, which in turn biases RNA motion in one direction. Implications of the P4 model for other hexameric molecular motors are discussed.
  • Hannula, Minna (Helsingin yliopisto, 2010)
    In industrialized countries, campylobacteriosis is the most common cause of human gastroenteric infection. It is mainly caused by Campylobacter jejuni and Campylobacter coli, but the pathogenic role of other species, such as Campylobacter hyointestinalis, remains unclear. The predominant antimicrobial agents for treating Campylobacter infections are erythromycin and ciprofloxacin. However, during the past few decades resistant C. jejuni and C. coli strains have rapidly emerged. The aim of this thesis was to study the various resistance mechanisms of C. jejuni, C. coli and C. hyointestinalis towards antimicrobial agents. When susceptibility to 12 different antimicrobials was tested, it was concluded that Finnish C. hyointestinalis strains remain susceptible to most antimicrobials of veterinary importance. All the 24 reindeer strains included in the study proved susceptible, but resistance to streptomycin and sulphonamides was observed in 32% and 24% of the 25 bovine strains, respectively. The difference in the susceptibility profile of the bovine and reindeer strains most probably reflects the veterinary use of these substances in bovine husbandry, but not in reindeer. Unlike C. jejuni and C. coli, C. hyointestinalis is inherently resistant to nalidixic acid, but the mutations in the quinolone resistance-determining region (QRDR) of gyrA involved in the acquired quinolone resistance of C. jejuni were not present in C. hyointestinalis. An efflux pump system, CmeABC, has been established to play an important role in the resistance of C. jejuni and C. coli to various antimicrobial agents. The effect of putative efflux pump inducers and inhibitors on the minimum inhibitory concentration (MIC) values of C. jejuni, C. coli and C. hyointestinalis was evaluated. It was concluded that the most potent was an efflux pump inhibitor, PAβN, which decreased inherent resistance of C. hyointestinalis to nalidixic acid 2- to 8-fold, and of C. jejuni and C. coli to rifampicin 8- to 64-fold. It also increased inherent susceptibility of C. jejuni and C. coli to erythromycin 8- to 32-fold. Another inhibitor, NMP, had similar but smaller effect on erythromycin, rifampicin and tetracycline MIC values of C. jejuni and C. coli. The two tested putative efflux pump inducers, sodium salicylate and bile salts, did not have any major effect on the MIC values of the studied antimicrobials. Since target mutation is an important resistance mechanism in C. jejuni and C. coli, the mutation frequencies of C. jejuni and C. coli strains were determined. The obtained values varied from hypomutable to strongly hypermutable. The relatively large proportion (25%) of hypermutable strains may facilitate the adaptation of C. jejuni and C. coli to selective environments. When originally ciprofloxacin-susceptible C. jejuni strains were subjected to low-level (0.125 or 1 mg/l) ciprofloxacin, the MIC values for these strains increased up to 32 mg/l. The high MIC values persisted even when the strains were repeatedly subcultured in the absence of ciprofloxacin. Subsequent sequencing of the QRDR revealed multiple peaks at nucleotide positions 256 and 267, but some variants had ciprofloxacin MIC levels of up to 16 mg/l, even though no peaks corresponding to mutated nucleotides in the QRDR were observed; this suggests the presence of a QRDR-independent resistance mechanism in these strains.
  • Ylikallio, Emil (Helsingin yliopisto, 2011)
    Defects in mitochondrial DNA (mtDNA) maintenance cause a range of human diseases, including autosomal dominant progressive external ophthalmoplegia (adPEO). This study aimed to clarify the molecular background of adPEO. We discovered that deoxynucleoside triphosphate (dNTP) metabolism plays a crucial in mtDNA maintenance and were thus prompted to search for therapeutic strategies based on the modulation of cellular dNTP pools or mtDNA copy number. Human mtDNA is a 16.6 kb circular molecule present in hundreds to thousands of copies per cell. mtDNA is compacted into nucleoprotein clusters called nucleoids. mtDNA maintenance diseases result from defects in nuclear encoded proteins that maintain the mtDNA. These syndromes typically afflict highly differentiated, post-mitotic tissues such as muscle and nerve, but virtually any organ can be affected. adPEO is a disease where mtDNA molecules with large-scale deletions accumulate in patients tissues, particularly in skeletal muscle. Mutations in five nuclear genes, encoding the proteins ANT1, Twinkle, POLG, POLG2 and OPA1, have previously been shown to cause adPEO. Here, we studied a large North American pedigree with adPEO, and identified a novel heterozygous mutation in the gene RRM2B, which encodes the p53R2 subunit of the enzyme ribonucleotide reductase (RNR). RNR is the rate-limiting enzyme in dNTP biosynthesis, and is required both for nuclear and mitochondrial DNA replication. The mutation results in the expression of a truncated form of p53R2, which is likely to compete with the wild-type allele. A change in enzyme function leads to defective mtDNA replication due to altered dNTP pools. Therefore, RRM2B is a novel adPEO disease gene. The importance of adequate dNTP pools and RNR function for mtDNA maintenance has been established in many organisms. In yeast, induction of RNR has previously been shown to increase mtDNA copy number, and to rescue the phenotype caused by mutations in the yeast mtDNA polymerase. To further study the role of RNR in mammalian mtDNA maintenance, we used mice that broadly overexpress the RNR subunits Rrm1, Rrm2 or p53R2. Active RNR is a heterotetramer consisting of two large subunits (Rrm1) and two small subunits (either Rrm2 or p53R2). We also created bitransgenic mice that overexpress Rrm1 together with either Rrm2 or p53R2. In contrast to the previous findings in yeast, bitransgenic RNR overexpression led to mtDNA depletion in mouse skeletal muscle, without mtDNA deletions or point mutations. The mtDNA depletion was associated with imbalanced dNTP pools. Furthermore, the mRNA expression levels of Rrm1 and p53R2 were found to correlate with mtDNA copy number in two independent mouse models, suggesting nuclear-mitochondrial cross talk with regard to mtDNA copy number. We conclude that tight regulation of RNR is needed to prevent harmful alterations in the dNTP pool balance, which can lead to disordered mtDNA maintenance. Increasing the copy number of wild-type mtDNA has been suggested as a strategy for treating PEO and other mitochondrial diseases. Only two proteins are known to cause a robust increase in mtDNA copy number when overexpressed in mice; the mitochondrial transcription factor A (TFAM), and the mitochondrial replicative helicase Twinkle. We studied the mechanisms by which Twinkle and TFAM elevate mtDNA levels, and showed that Twinkle specifically implements mtDNA synthesis. Furthermore, both Twinkle and TFAM were found to increase mtDNA content per nucleoid. Increased mtDNA content in mouse tissues correlated with an age-related accumulation of mtDNA deletions, depletion of mitochondrial transcripts, and progressive respiratory dysfunction. Simultaneous overexpression of Twinkle and TFAM led to a further increase in the mtDNA content of nucleoids, and aggravated the respiratory deficiency. These results suggested that high mtDNA levels have detrimental long-term effects in mice. These data have to be considered when developing and evaluating treatment strategies for elevating mtDNA copy number.
  • Järvinen, Elina (Helsingin yliopisto, 2008)
    In most non-mammalian vertebrates, such as fish and reptiles, teeth are replaced continuously. However, tooth replacement in most mammals, including human, takes place only once and further renewal is apparently inhibited. It is not known how tooth replacement is genetically regulated, and little is known on the physiological mechanism and evolutionary reduction of tooth replacement in mammals. In this study I have attempted to address these questions. In a rare human condition cleidocranial dysplasia, caused by a mutation in a Runt domain transcription factor Runx2, tooth replacement is continued. Runx2 mutant mice were used to investigate the molecular mechanisms of Runx2 function. Microarray analysis from dissected embryonic day 14 Runx2 mutant and wild type dental mesenchymes revealed many downstream targets of Runx2, which were validated using in situ hybridization and tissue culture methods. Wnt signaling inhibitor Dkk1 was identified as a candidate target, and in tissue culture conditions it was shown that Dkk1 is induced by FGF4 and this induction is Runx2 dependent. These experiments demonstrated a connection between Runx2, FGF and Wnt signaling in tooth development and possibly also in tooth replacement. The role of Wnt signaling in tooth replacement was further investigated by using a transgenic mouse model where Wnt signaling mediator β-catenin is continuously stabilized in dental epithelium. This stabilization led to activated Wnt signaling and to the formation of multiple enamel knots. In vitro and transplantation experiments were performed to examine the process of extra tooth formation. We showed that new teeth were continuously generated and that new teeth form from pre-existing teeth. A morphodynamic activator-inhibitor model was used to simulate enamel knot formation. By increasing the intrinsic production rate of the activator (β-catenin), the multiple enamel knot phenotype was reproduced by computer simulations. It was thus concluded that β-catenin acts as an upstream activator of enamel knots, closely linking Wnt signaling to the regulation of tooth renewal. As mice do not normally replace teeth, we used other model animals to investigate the physiological and genetic mechanisms of tooth replacement. Sorex araneus, the common shrew was earlier reported to have non-functional tooth replacement in all antemolar tooth positions. We showed by histological and gene expression studies that there is tooth replacement only in one position, the premolar 4 and that the deciduous tooth is diminished in size and disappears during embryogenesis without becoming functional. The growth rates of deciduous and permanent premolar 4 were measured and it was shown by competence inference that the early initiation of the replacement tooth in relation to the developmental stage of the deciduous tooth led to the inhibition of deciduous tooth morphogenesis. It was concluded that the evolutionary loss of deciduous teeth may involve the early activation of replacement teeth, which in turn suppress their predecessors. Mustela putorius furo, the ferret, has a dentition that resembles that of the human as ferrets have teeth that belong to all four tooth families, and all the antemolar teeth are replaced once. To investigate the replacement mechanism, histological serial sections from different embryonic stages were analyzed. It was noticed that tooth replacement is a process which involves the growth and detachment of the dental lamina from the lingual cervical loop of the deciduous tooth. Detachment of the deciduous tooth leads to a free successional dental lamina, which grows deeper into the mesenchyme, and later buds the replacement tooth. A careful 3D analysis of serial histological sections was performed and it was shown that replacement teeth are initiated from the successional dental lamina and not from the epithelium of the deciduous tooth. The molecular regulation of tooth replacement was studied and it was shown by examination of expression patterns of candidate regulatory genes that BMP/Wnt inhibitor Sostdc1 was strongly expressed in the buccal aspect of the dental lamina, and in the intersection between the detaching deciduous tooth and the successional dental lamina, suggesting a role for Sostdc1 in the process of detachment. Shh was expressed in the enamel knot and in the inner enamel epithelium in both generations of teeth supporting the view that the morphogenesis of both generations of teeth is regulated by similar mechanisms. In summary, histological and molecular studies on different model animals and transgenic mouse models were used to investigate tooth replacement. This thesis work has significantly contributed to the knowledge on the physiological mechanisms and molecular regulation of tooth replacement and its evolutionary suppression in mammals.
  • Golubtsov, Andrey (Edita, 2008)
    Replication and transcription of the RNA genome of alphaviruses relies on a set of virus-encoded nonstructural proteins. They are synthesized as a long polyprotein precursor, P1234, which is cleaved at three processing sites to yield nonstructural proteins nsP1, nsP2, nsP3 and nsP4. All the four proteins function as constitutive components of the membrane-associated viral replicase. Proteolytic processing of P1234 polyprotein is precisely orchestrated and coordinates the replicase assembly and maturation. The specificity of the replicase is also controlled by proteolytic cleavages. The early replicase is composed of P123 polyprotein intermediate and nsP4. It copies the positive sense RNA genome to complementary minus-strand. Production of new plus-strands requires complete processing of the replicase. The papain-like protease residing in nsP2 is responsible for all three cleavages in P1234. This study addressed the mechanisms of proteolytic processing of the replicase polyprotein in two alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) representing different branches of the genus. The survey highlighted the functional relation of the alphavirus nsP2 protease to the papain-like enzymes. A new structural motif the Cys-His catalytic dyad accompanied with an aromatic residue following the catalytic His was described for nsP2 and a subset of other thiol proteases. Such an architecture of the catalytic center was named the glycine specificity motif since it was implicated in recognition of a specific Gly residue in the substrate. In particular, the presence of the motif in nsP2 makes the appearance of this amino acid at the second position upstream of the scissile bond a necessary condition for the cleavage. On top of that, there were four distinct mechanisms identified, which provide affinity for the protease and specifically direct the enzyme to different sites in the P1234 polyprotein. Three factors RNA, the central domain of nsP3 and the N-terminus of nsP2 were demonstrated to be external modulators of the nsP2 protease. Here I suggest that the basal nsP2 protease specificity is inherited from the ancestral papain-like enzyme and employs the recognition of the upstream amino acid signature in the immediate vicinity of the scissile bond. This mechanism is responsible for the efficient processing of the SFV nsP3/nsP4 junction. I propose that the same mechanism is involved in the cleavage of the nsP1/nsP2 junction of both viruses as well. However, in this case it rather serves to position the substrate, whereas the efficiency of the processing is ensured by the capability of nsP2 to cut its own N-terminus in cis. Both types of cleavages are demonstrated here to be inhibited by RNA, which is interpreted as impairing the basal papain-like recognition of the substrate. In contrast, processing of the SIN nsP3/nsP4 junction was found to be activated by RNA and additionally potentiated by the presence of the central region of nsP3 in the protease. The processing of the nsP2/nsP3 junction in both viruses occurred via another mechanism, requiring the exactly processed N-terminus of nsP2 in the protease and insensitive to RNA addition. Therefore, the three processing events in the replicase polyprotein maturation are performed via three distinct mechanisms in each of two studied alphaviruses. Distinct sets of conditions required for each cleavage ensure sequential maturation of P1234 polyprotein: nsP4 is released first, then the nsP1/nsP2 site is cut in cis, and liberation of the nsP2 N-terminus activates the cleavage of the nsP2/nsP3 junction at last. The first processing event occurs differently in SFV and SIN, whereas the subsequent cleavages are found to be similar in the two viruses and therefore, their mechanisms are suggested to be conserved in the genus. The RNA modulation of the alphavirus nonstructural protease activity, discovered here, implies bidirectional functional interplay between the alphavirus RNA metabolism and protease regulation. The nsP2 protease emerges as a signal transmitting moiety, which senses the replication stage and responds with proteolytic cleavages. A detailed hypothetical model of the alphavirus replicase core was inferred from the data obtained in the study. Similar principles in replicase organization and protease functioning are expected to be employed by other RNA viruses.
  • Syväranta, Suvi (Helsingin yliopisto, 2013)
    Aortic valve stenosis (AVS) is the most common valvular disease in Western countries. Pharmacological prevention of AVS having proved unsuccessful, its current treatment is still valve replacement. The etiology of AVS is multifactorial, both genetic and external risk factors predisposing to the active pathological process eventually leading to clinically manifest stenosis. Histological features of the disease resemble those of atherosclerosis, including the accumulation and modification of lipoproteins, inflammation, extracellular matrix remodeling, and calcification. Furthermore, valvular interstitial cells undergo phenotypic differentiation into actively proliferating myofibroblasts, which contribute to the local inflammatory response as well as extracellular matrix remodeling in stenotic aortic valves. Blood vessels also grow into the normally avascular valve leaflets already in the early stages of the disease. This thesis aimed at elucidating the mechanisms behind the pathological neovascularization of the stenotic aortic valves. Furthermore, we characterized valvular lymphangiogenesis and investigated potential factors contributing to the balance between valvular angiogenesis and lymphangiogenesis, focusing on the role of valvular myofibroblasts and mast cells. For this purpose, we studied a total of 117 stenotic valves obtained at valve replacement surgery, and 49 control valves obtained at cardiac transplantations, at valve replacement surgery due to aortic valve regurgitation, or from deceased organ donors whose hearts were unsuitable for use as grafts. The valve leaflets were either used freshly for myofibroblast cell culture or frozen for e.g. PCR and immunohistochemical analyses. First, we assessed the adverse extracellular matrix remodeling of stenotic aortic valves, a process necessary for angiogenic sprouting to occur. We found that the mRNA expression levels of cathepsins S, K, and V, and their inhibitor cystatin C were higher in stenotic aortic valves than in control valves. Furthermore, the total activity of such cathepsins was increased in AVS. In immunohistochemical stainings, the expressions of cathepsin S, cathepsin V, and cystatin C localized to valvular macrophages, chondroblast-like cells, and endothelial cells lining both the valvular surface and the neovessels in the stenotic valves. Next, we characterized the neovessels and lymphatic vessels in stenotic aortic valves and control valves using immunohistochemistry. We found that in addition to immature microvessels, the stenotic aortic valves contained organized arterioles, indicating an advanced stage of angiogenesis. Lymphatic vessels correlated with valvular blood vessels, but were present in much fewer numbers. Valvular mast cells resided close to neovessels and secreted the angiogenic Vascular endothelial growth factor A (VEGF-A). Furthermore, we showed that the lymphangiogenic growth factors VEGF-C and VEGF-D are locally produced in the aortic valves, and that the receptors for all these VEGFs, VEGFR-2 and VEGFR-3, are upregulated in AVS. We also identified several factors that induce VEGF-A secretion in cultured valvular myofibroblasts. These include mast cell-derived components, the inflammatory cytokine TNF-α, hypoxia, and cigarette smoke. Myofibroblasts were also able to promote VEGF-A secretion by cultured human mast cells, suggesting potential angiogenic interplay between these two valvular cell types. Interestingly, mast cell-derived proteases also efficiently degraded the lymphangiogenic growth factor VEGF-C. Thus, by secreting VEGF-A, by urging myofibroblasts to produce VEGF-A, and by releasing VEGF-C-degrading proteases, mast cells may strongly influence the observed imbalance between valvular blood vessels and lymphatic vessels. Finally, we investigated the potential effects of oxidized low-density lipoprotein (oxLDL) on valvular angiogenesis. We found that oxLDL induces the expression of several inflammatory cytokines in cultured myofibroblasts. Moreover, we identified oxLDL-binding scavenger receptors to be locally expressed in the aortic valves. The mRNA expression levels of scavenger receptor class A type 1 (SR-A1) and Lectin-like oxidized LDL receptor-1 (LOX-1) were increased in AVS, whereas CD36 was downregulated in stenotic valves. Furthermore, the expression of LOX-1 in cultured valvular myofibroblasts increased in response to mast cell-derived components and TNF-α. The observed changes in valvular scavenger receptor expression particularly favor inflammation and angiogenesis. In conclusion, several angiogenic factors were found to be activated in stenotic aortic valves. Furthermore, valvular mast cells and myofibroblasts were identified as potential players promoting valvular angiogenesis and contributing to the pathological imbalance between valvular angiogenesis and lymphangiogenesis. This imbalance, in turn, could facilitate the harmful infiltration of inflammatory cells and lipoproteins into the stenotic aortic valves and ultimately contribute to the progression of the disease.
  • Siikala, Emilia (Helsingin yliopisto, 2011)
    Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS1) is an autoimmune disease caused by a loss-of function mutation in the autoregulator gene (AIRE). Patients with APECED suffer from chronic mucocutaneous candidosis (CMC) of the oral cavity and oesophagus often since early childhood. The patients are mainly colonized with Candida albicans and decades of exposure to antifungal agents have lead to the development of clinical and microbiological resistance in the treatment of CMC in the APECED patient population in Finland. A high incidence of oral squamous cell carcinoma is associated with oral CMC lesions in the APECED patients over the age of 25. The overall aim of this study was firstly, to investigate the effect of long-term azole exposure on the metabolism of oral C. albicans isolates from APECED patients with CMC and secondly, to analyse the specific molecular mechanisms that are responsible for these changes. The aim of the first study was to examine C. albicans strains from APECED patients and the level of cross-resistance to miconazole, the recommended topical compound for the treatment of oral candidosis. A total of 16% of the strains had decreased susceptibility to miconazole and all of these isolates had decreased susceptibility to fluconazole. Miconazole MICs also correlated with MICs to voriconazole and posaconazole. A significant positive correlation between the years of miconazole exposure and the MICs to azole antifungal agents was also found. These included azoles the patients had not been exposed to. The aim of our second study was to determine if the APECED patients are continuously colonized with the same C. albicans strains despite extensive antifungal treatment and to gain a deeper insight into the genetic changes leading to azole resistance. The strains were typed using MLST and our results confirmed that all patients were persistently colonized with the same or a genetically related strain despite antifungal treatment between isolations. No epidemic strains were found. mRNA expression was analysed by Northern blotting, protein level by western blotting, and TAC1 and ERG11 genes were sequenced. The main molecular mechanisms resulting in azole resistance were gain-of-function mutations in TAC1 leading to over expression of CDR1 and CDR2, genes linked to azole resistance. Several strains had also developed point mutations in ERG11, another gene linked to azole resistance. In the third study we used gas chromatography to test whether the level of carcinogenic acetaldehyde produced by C. albicans strains isolated from APECED patients were different from the levels produced by strains isolated from healthy controls and oral carcinoma patients. Acetaldehyde is a carcinogenic product of alcohol fermentation and metabolism in microbes associated with cancers of the upper digestive tract. In yeast, acetaldehyde is a by-product of the pyruvate bypass that converts pyruvate into acetyl-CoA during fermentation. Our results showed that strains isolated from APECED patients produced mutagenic levels of acetaldehyde in the presence of glucose (100mM, 18g/l) and the levels produced were significantly higher than those from strains isolated from controls and oral carcinoma patients. All strains in the study, however, were found to produce mutagenic levels of acetaldehyde in the presence of ethanol (11mM). The glucose and ethanol levels used in this study are equivalent to those found in food and beverages and our results highlight the role of dietary sugars and ethanol on carcinogenesis. The aims of our fourth study were to research the effect of growth conditions in the levels of acetaldehyde produced by C. albicans and to gain deeper insight into the role of different genes in the pyruvate-bypass in the production of high acetaldehyde levels. Acetaldehyde production in the presence of glucose increased by 17-fold under moderately hypoxic conditions compared to the levels produced under normoxic conditions. Under moderately hypoxic conditions acetaldehyde levels did not correlate with the expression of ADH1 and ADH2, genes catalyzing the oxidation of ethanol to acetaldehyde, or PDC11, the gene catalyzing the oxidation of pyruvate to acetaldehyde but correlated with the expression of down-stream genes ALD6 and ACS1. Our results highlight a problem where indiscriminate use of azoles may influence azole susceptibility and lead to the development of cross-resistance. Despite clinically successful treatment leading to relief of symptoms, colonization by C. albicans strains is persistent within APECED patients. Microevolution and point mutations that occur in strains may lead to the development of azole-resistant isolates and metabolic changes leading to increased production of carcinogenic acetaldehyde.
  • Helmy, Mohamed (Helsingin yliopisto, 2014)
    Birth asphyxia is a major cause of infant and childhood death, disability and neurodevelopmental delay worldwide. During birth, impairment of respiration is reflected in elevated levels of CO2 and diminished levels of O2 in the neonate. The fundamental presentation and diagnostic criterion of birth asphyxia is severe acidosis, most commonly measured in umbilical blood. Resuscitation is associated with normalization of blood pH values and arterial blood gases. Within hours of a moderate or severe asphyxic insult during birth, severe seizures are triggered. In the present study, asphyxic conditions during birth are modeled as an induced hypoxia and hypercapnia in postnatal day 6 rat pups. Respiratory conditions are altered so that pups breathe 20 % CO2 with either 9 % O2 or 4 % O2 (N2 balanced) for 60 or 45 minutes, respectively. Brain extracellular and intraneuronal pH became rapidly acidotic during asphyxic conditions. After experimental asphyxia, immediate restoration of normoxia and normocapnia was associated with a large seizure burden. Seizures in the postasphyxia period were tightly correlated with a recovery and alkaline overshoot in brain pH. Enhanced acid extrusion from the brain was attributed to increased Na/H exchange across the blood-brain barrier. Pharmacologic blockade of Na/H exchange in the blood-brain barrier with amiloride or its analog abolished brain alkalosis and seizures. These findings suggest that a brain-confined alkalosis is generated by Na/H exchangers in the blood-brain barrier when normocapnic conditions are immediately restored after experimental birth asphyxia. A putative therapeutic strategy was tested, where the CO2 level of inhaled air in the postasphyxic period was reduced in steps, so that normocapnic conditions are gradually restored. This graded restoration of normocapnia was achieved by exposing the pup to 10 % CO2 in air for 30 minutes, followed by 5 % CO2 in air for a further 30 minutes, and finally with room air. A dramatic attenuation of brain alkalosis and seizures was induced by graded restoration of normocapnia. Immediate restoration of normocapnia after asphyxia was associated with adverse outcome in juvenile and adult rats, manifest as compromised sensorimotor coordination, altered emotional reactivity to acute stress, diminished inhibition of fear-motivated behavior, impaired memory and learning, abnormal social interaction, and increased seizure susceptibility. Graded restoration of normocapnia after asphyxia was associated with significant and favorable improvement of outcome, such that behavioral deficits were rescued, and seizure threshold was not significantly different from control animals. The findings of the this study suggest a central role for Na/H exchange in the blood-brain barrier in mediating the postasphyxia brain alkalosis as measured in the present study as well as in human babies. Importantly, the findings also suggest a putative therapeutic strategy in which recovery from acidosis during neonatal resuscitation is controlled through a graded restoration of normocapnia.
  • Ludwig, Anastasia (Helsingin yliopisto, 2008)
    Gamma-aminobutyric acid (GABA) acting through ionotropic GABAA receptors plays a crucial role in the activity of the central nervous system (CNS). It triggers Ca2+ rise providing trophic support in developing neurons and conducts fast inhibitory function in mature neuronal networks. There is a developmental change in the GABAA reversal potential towards more negative levels during the first two postnatal weeks in rodent hippocampus. This change provides the basis for mature GABAergic activity and is attributable to the developmental expression of the neuron-specific potassium chloride cotransporter 2 (KCC2). In this work we have studied the mechanisms responsible for the control of KCC2 developmental expression. As a model system we used hippocampal dissociated cultures plated from embryonic day (E) 17 mice embryos before the onset of KCC2 expression. We showed that KCC2 was significantly up-regulated during the first two weeks of culture development. Interestingly, the level of KCC2 upregulation was not altered by chronic pharmacological blockage of action potentials as well as GABAergic and glutamatergic synaptic transmission. By in silico analysis of the proximal KCC2 promoter region we identified 10 candidate transcription factor binding sites that are highly conserved in mammalian KCC2 genes. One of these transcription factors, namely early growth response factor 4 (Egr4), had similar developmental profile as KCC2 and considerably increased the activity of mouse KCC2 gene in neuronal cells. Next we investigated the involvement of neurotrophic factors in regulation of Egr4 and KCC2 expression. We found that in immature hippocampal cultures Egr4 and KCC2 levels were strongly up-regulated by brain derived neurotrophic factor (BDNF)and neurturin. The effect of neurotrophic factors was dependent on the activation of a mitogen activated protein kinase (MAPK) signal transduction pathway. Intact Egr4-binding site in proximal KCC2 promoter was required for BDNF-induced KCC2 transcription. In vitro data were confirmed by several in vivo experiments where we detected an upregulation of KCC2 protein levels after intrahippocampal administration of BDNF or neurturin. Importantly, a MAPK-dependent rise in Egr4 and KCC2 expression levels was also observed after a period of kainic acid-induced seizure activity in neonatal rats suggesting that neuronal activity might be involved in Egr4-mediated regulation of KCC2 expression. Finally we demonstrated that the mammalian KCC2 gene (alias Slc12a5) generated two neuron-specific isoforms by using alternative promoters and first exons. A novel isoform of KCC2, termed KCC2a, differed from the previously known KCC2b isoform by 40 unique N-terminal amino acid residues. KCC2a expression was restricted to CNS,remained relatively constant during postnatal development, and contributed 20 50% of total KCC2 mRNA expression in the neonatal mouse brainstem and spinal cord. In summary, our data provide insight into the complex regulation of KCC2 expression during early postnatal development. Although basal KCC2 expression seems to be intrinsically regulated, it can be further augmented by neurotrophic factors or by enhanced activity triggering MAPK phosphorylation and Egr4 induction. Additional KCC2a isoform, regulated by another promoter, provides basal KCC2 level in neonatal brainstem and spinal cord required for survival of KCC2b knockout mice.