Väitöskirjat

Recent Submissions

  • Keto, Antton (Helsingin yliopisto, 2017)
    Water-levels of lakes in Finland are artificially regulated for energy production, flood protection, navigation and recreational use. There is a need for changing water-level regulation practices, due to changing climate, increasing recreational use of lakes, and implementation of legally binding national targets for electrification of renewable sources. To obtain more knowledge-based assessments of new water management regulations, we need to develop water-level regulation assessment tools and to increase the sensitivity of ecological classification systems for hydromorphological pressures, as the European Union Water Framework Directive (WFD)requires. The main objectives of this study were to 1) develop criteria and threshold values for assessing the ecological status in regulated lakes, 2) identify both high-hydrological status and heavily modified lakes and 3) estimate the role of helophytes in the uppermost littoral zone. Impacts of water-level fluctuation on macrophytes, macrozoobenthos, and littoral fish fauna were clearly evident, and the threshold value between moderate and good ecological status was a 3.5 m winter water-level drawdown with the mean ecological quality ratio assessment method (1.8 m with the one-out-all-out principle). The vertical extension of Phragmites was most strongly associated with the water-level fluctuation of open water period (OWP), followed by Carex spp. and Equisetum. Overall, the RF models explained 4--41% of the variation observed in the helophytes zones. The models indicated that OWP fluctuation, slope, openness and cover of other macrophyte groups were key factors explaining the extent of the helophyte zones. The hydrological regime could be classified as having high hydrological status in 20% of the regulated lakes. Quite often, the ecological status was poorer, implying that high-hydrological status lakes often face other anthropogenic pressures, such as eutrophication that degrades high ecological status. Provisional designation with hydrological criteria seemed to work quite well, because 13 of the 15 lakes were estimated similarly with simple hydrological criteria, compared with the national HMWB designation only later produced by the environmental authorities.
  • Parkkonen, Olavi (Helsingin yliopisto, 2017)
    Takotsubo cardiomyopathy (TTC) is an acute cardiac condition resembling in symptoms acute coronary syndrome (ACS), but without obstructive coronary artery disease. TTC develops almost solely in post-menopausal women and usually after preceding stress. Of all patients with ACS symptoms, TTC incidence is 2%. Due to similar symptoms and findings, differential diagnosis requires coronary angiography (CAG). The pathophysiology of TTC is unknown. Even though the accumulated evidence suggests a causative role for a catecholamine surge, other theories exist. Aborted myocardial infarction (MI) produces similar electrocardiography (ECG) and biochemical findings as in TTC. In such cases, because of non-stenotic coronary artery plaques, a dissolved coronary thrombus might show no any signs in the CAG, which could lead to an assumption of non-atherothrombotic etiology for the heart attack. In ACS, altered levels of proteolytic enzyme called matrix metalloproteinase 8 (MMP-8), and its inhibitor, the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), associate to plaque rupture. Their direct comparison between ACS and TTC remains unknown. The purpose of this thesis is to test whether either of two non-invasive methods: the ECG (I) or MMP-8 and TIMP-1 (III), could differentiate TTC from ACS. We also set out to find whether, after all, what is responsible for the TTC is transient thrombosis in the coronary circulation (II). Both our prospective and retrospective collection of patients resulted in 92 TTC cases. The demographics of our material were similar to those of other TTC reports worldwide. In the ECG study (I), a review and comparison of 57 TTC and ACS acute ECGs resulted in criteria differentiating the two with 63% sensitivity and 93% specificity. In cases of suspected ST-elevation myocardial infarction, such accuracy is insufficient for a decision against coronary intervention. Blood samples from 45 TTC patients and matching numbers of ACS patients and controls were analyzed for coagulation markers. Despite the similar acute-phase reaction in TTC and ACS patients, the TTC d-dimer levels matched those of controls, and were lower and less frequently above reference level than with those in ACS. The blood samples were further analyzed for MMP-8 and TIMP-1 levels showing, on admission, better differentiation between TTC and ACS by TIMP-1 than by troponin T (TnT). In TTC, low ejection fraction (EF) correlated with low MMP-8/TIMP-1 ratio. In conclusion, ECG lacked the ability to differentiate TTC from ACS that would have allowed avoidance of invasive diagnostics. Secondly, the coagulation results supported catecholamine and argued against the thrombosis theory. Finally, TIMP-1 emerged as a potential future biomarker in differentiation between ACS and TTC. Furthermore, in some TTC patients MMP-8 and TIMP-1 levels may explain more severe left-ventricle (LV) impairment.
  • Lehtonen, Ilari (Helsingin yliopisto, 2017)
    The aim of this work was to study the climate change impact on two specific abiotic risks affecting forests in Finland: fires and heavy snow loads. Approximately 1000 forest fires occur annually in Finland, but thanks to effective fire suppression, the average size of fires is only about 0.5 ha. Occasionally, heavy snow loading causes forest damage, which reduces stand quality in boreal forests experiencing cold winters. In Finnish forests, snow damage occurs most commonly in the eastern and northern parts of the country. The basic tools used in this work to evaluate the climate change impact were climate models. In addition, observational weather data and fire statistics were used. In evaluating the forest fire risk, the Canadian Fire Weather Index (FWI) system was used. Snow load amounts were estimated mainly by applying a snow load model developed at the Finnish Meteorological Institute (FMI). The results indicate that forest fire risk will most likely increase in the future due to increasing temperature and enhanced evaporation. However, there is large uncertainty regarding the rate of change, which originates from the differences between climate model responses to the same radiative forcing. Moreover, an increase in forest fire risk will at the same time increase the risk of conflagrations. Crown snow loads were projected to become heavier in northern Finland and in the regions of Kainuu and North Karelia next to the Russian border. In southern and western Finland the risk of snow damage is expected to decrease. The largest decrease in the risk is projected to occur in coastal areas. In the areas expected to experience increased risk of snow damage, conditions favouring both heavy wet snow loading and rime accretion were predicted to become more common. The results of this work can be utilized when considering climatically-driven risks in forest management.
  • Hauptmann, Andreas (Helsingin yliopisto, 2017)
    Electrical impedance tomography (EIT) is a rather new approach to medical imaging that is motivated by using electricity to determine the inside of a body. The clear advantage lies in the usage of harmless electric currents, in contrast to the ionizing radiation of X-rays, whereas the mathematical problem is inherently more challenging. In EIT we seek to reconstruct an image of the inner organs by determining their conductivity, i.e. how well electricity is conducted. As a medical imaging modality it is most promising in pulmonary and cardiac imaging, due to considerably different conductivity values in the air filled lungs (low conductive) and the blood filled heart (high conductive). EIT is in principle capable of monitoring the respiratory process, detecting pathologies in the lungs, and monitoring the heart activity. The main focus of this work is on the partial-boundary problem in EIT, that means one has only access to a certain part of the boundary and data can only be collected there. In a hospital setting these situations can arise when monitoring a critical or unconscious patient and hence one can only access the front of the torso (ventral position). Furthermore, practical complications can arise due to faulty, dislocated, or dispatched electrodes and hence leading to incomplete data. The methods presented in this thesis are capable of dealing with such incomplete data. Following the tradition of mathematical research we are also interested in quantifying the error incomplete data introduces to the reconstruction. In a short summary, this thesis investigates how to improve EIT reconstructions from partial-boundary data by utilizing concepts from an ideal mathematical setting as well as how to apply these methods to real electrode models and measurement data.
  • Lorey, Martina (Helsingin yliopisto, 2017)
    The innate immune system is the first line of defence against microbial infections. Macrophages are the key cells of the innate immune system. Activated macrophages release inflammatory mediators through conventional and extracellular vesicle (EV)-mediated protein secretion. These protein secretion pathways are major components of intercellular communication in the immune system. Protein secretion is activated by inflammasomes in response to microbial components and endogenous danger signals. Inflammasomes are molecular platforms of the innate immune system that are critical to both local and systemic inflammation: they are involved in the pathogenesis of immune-mediated inflammatory diseases. Inflammasomes can be activated by both microbial stimuli as well as by endogenous danger signal molecules. The secretome of the cell is the pattern of all secreted molecules at a given time; it can provide important insights about the cell’s status as well as information about intercellular communication. The secretome can be studied by mass spectrometry (MS)-based proteomics; this technique provides a system-level overview of the current state of the cell. While traditional assays like ELISA or Luminex can quantify selected proteins in low pg/mL levels, they can only analyse a limited number of proteins. Proteomics offers the same sensitivity but supplemented with the capability of multiplex quantitative analysis of thousands of proteins in the same sample. The main goal of this work was to unravel the innate immune response of human macrophages activated by microbial stimuli with a focus on protein secretion using proteomics combined with bioinformatics and functional studies. The microbial stimuli used included Influenza A viruses (IAVs) and lipopolysaccharide (LPS), the cell wall components of Gram-negative bacteria. Both IAVs and Gram-negative bacteria are important human pathogens associated with aggressive infections. Influenza A virus and LPS are known to activate canonical NLRP3 inflammasome and non-canonical caspase-4/5 inflammasome, respectively. Inflammasome activation is followed by protein secretion which was studied in detail, with a special emphasis on unconventional protein secretion. In both studies, mediators of inflammation were released through EVs in response to influenza A virus infection and intracellular LPS stimulation. These included many danger signal proteins, cytokines and components of translational machinery. Our system-level characterization using modern mass spectrometry-based proteomics approach provides novel information how macrophages communicate to other cells through protein secretion. The results suggest that unconventional protein secretion is an essential part of the innate immune response to different activation stimuli.
  • Pussila, Marjaana (Helsingin yliopisto, 2017)
    Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in the Western world and interactions between genetic and environmental factors, including diet, are suggested to play a critical role in its etiology. Yet, the mechanisms by which diet impacts colorectal tumorigenesis remain largely unknown. Colorectal cancer evolves as a multistep process, which requires a series of genetic and epigenetic alterations in growth regulatory genes. The process is accelerated in individuals with inherited cancer predisposition such as Lynch syndrome (LS) which is one of the most common inherited cancer susceptibility syndromes and caused by inherited mutation in one of the DNA mismatch repair (MMR) genes. CRC is thought to develop via the so called adenoma-carcinoma sequence. However, the early events that occur in colon mucosa prior to polyp formation remain unknown. The research presented here investigates the gene expression changes arising in histologically normal colonic mucosa as putative cancer-preceding events available for early detection. This was achieved by pursuing a long-term feeding experiment in the mouse model for human Lynch syndrome (Mlh1+/-), and the wild type (Mlh1+/+) littermates, fed with either Western-style (WD) diet or healthy AIN-93G control diet. Carcinomas developed mainly in WD fed mice. WD also accelerated the progression of carcinogenesis. In the first study, the expression of 94 growth-regulatory genes previously linked to human CRC was studied for 5 weeks and 12 months old mice. Promoter CpG island methylation status was also studied for the genes which showed reduced expression. In mice fed for 12 months with WD, proximal colon mucosa, the predominant site of cancer formation in LS, exhibited a significant expression decrease in several tumor suppressor genes. Furthermore, a reduced mRNA expression was accompanied by an increased CpG dinucleotide promoter methylation of the respective genes suggesting a cause for the mRNA down regulation. The strongest expression decrease together with a significant increase in its promoter methylation was seen in Dkk1, an antagonist of the canonical Wnt signaling pathway. Furthermore, the inactivation of Dkk1 seemed to predispose to neoplasias in the proximal colon. The findings suggest that the inactivation of Dkk1 is a prominent early marker for colon oncogenesis. In study 2 the aim was to comprehensively clarify the role of Mlh1 expression during colon tumorigenesis, which is usually associated with Lynch syndrome and MSI. Here, the same mouse model and diets were used to study cancer-preceding expression changes in the colon mucosa of 12 and 18-month-old mice. The Mlh1 protein expression and MSI status were studied in the colon carcinomas, and the effect of inherited predisposition (Mlh1+/) and Western-style diet on those. CRC development always includes a lack of genomic integrity and the different types of genomic instabilities, such as chromosomal instability and MSI are thought to reflect distinct cancer initiating mechanisms. In the present study neither wildtype Mlh1+/+ nor heterozygote Mlh1+/- mice lacked the Mlh1 protein or showed MSI in their CRCs, while Mlh1 RNA expression was already significantly decreased in their normal mucosa. Instead, CRC mice showed a distinct expression profile with shortage of Mlh1 and several other chromosomal segregation gene-specific transcripts in mucosa and aberrant mitosis in tumors. The genome wide expression profiling experiment demonstrated that cancer-preceding changes are already seen in histologically normal colon mucosa and a that decreased expression of Mlh1 together with other chromosomal segregation genes may form a field-defect in mucosa and trigger MMR-proficient, chromosomally unstable CRC.
  • Tirkkonen, Birger Taneli (Helsingin yliopisto, 2017)
    More than 150 species of mycobacteria are described, most being opportunistic pathogens and all representing a risk for human and animal health. Human infections derived from environmental mycobacteria are increasing in both industrialized and developing countries. The most susceptible groups are children, the elderly and those, including animals, with immunocompressive conditions. Drug therapy for mycobacteriosis is difficult and not always successful. Infections caused by drug-resistant mycobacteria can be life threatening also for healthy adults and thus represent a real risk for humans. Environmental mycobacterial infections of pigs are usually without clinical signs and the lesions are mainly detected at slaughter. Mycobacterium-infected pork can pass for human consumption due to the poor sensitivity of visual meat control at slaughterhouses, and mycobacteria in pigs also cause economic losses due to condemnation of carcasses. The main challenge is represented by evaluation of the hygiene risk associated with using mycobacteria-contaminated pork. Most environmental mycobacteria species have been isolated from sources such as water, swimming pools, soil, plants and bedding material. In our study mycobacterial growth in piggeries was identified in all bedding materials, sawdust, straw, peat and wood chips in most cases, and water and food samples in many cases, and only occasionally in dust and on wall surfaces. The maximum number of mycobacteria was almost 1 billion (109) per gram of bedding, which is close to the maximum concentration in any growth media. Mycobacteria can multiply in piggeries and contaminate feed and water. Isolation of mycobacteria from pig faeces can be considered an indicator for risk of human infection. Environmental mycobacteriosis in humans and pigs is mainly caused by M. avium subsp. hominissuis. There is little evidence of direct transmission from animals to humans, but particular strains can be recovered from both humans and pigs. In our studies, identical mycobacteria RFLP and MIRU-VNTR fingerprints of porcine and human origins were evident. Interspecies clusters were more common than intraspecies clusters using both methods. Therefore, we concluded that pigs act as a reservoir for virulent M. avium strains and the vector for transmission of infections in humans to pigs, and vice versa, may have an identical source of infection. Culturing mycobacteria is the gold standard for diagnosis, but detection of environmental mycobacteria based on cultivation and biochemical methods can take several weeks. Culture-independent, rapid and accurate techniques for detecting mycobacteria in food and feed chains are urgently needed. In this work we developed a rapid and accurate real-time quantitative PCR for detecting environmental mycobacteria in bedding materials and pig organs. Conclusion: Mycobacteria can multiply in bedding materials and the consequent heavy contamination can cause simultaneous infections in pigs. Mycobacterial DNA was found in pig organ samples, including those without lesions, and similar strains were found from humans and pig organ samples, which suggests that mycobacteria can be transmitted between humans and pigs.
  • Grönthal, Thomas (Helsingin yliopisto, 2017)
    Staphylococcus pseudintermedius (SP) is part of the normal microbiota of dogs and cats. Since the mid-1980s, an ever-increasing number of methicillin-resistant SP (MRSP) isolates have been reported. In the mid-2000s, two predominant MRSP clones, ST71 (sequence type 71) and ST68, spread through Europe and North America, respectively. MRSP isolates are commonly multidrug resistant (MDR), and are thus capable of causing infections that do not respond to routinely used antimicrobials. MRSP appeared in the small animal population of Finland in the late 2000s, also causing numerous infections at the Veterinary Teaching Hospital (VTH) of the University of Helsinki. No data on the epidemiology of MRSP in Finland have been published. This thesis study aimed to explore the epidemiology of MRSP in the Finnish small animal population. This was done by investigating and describing the MRSP outbreak at the VTH, and investigating risk factors for patients being colonized or infected by MRSP in the hospital during the outbreak. The prevalence of MRSP and the risk factors for MRSP carriage were investigated in a canine subpopulation at the Guide Dog School for the Visually Impaired. The susceptibility of SP isolates to antimicrobials in 2011–2015 was also investigated using data from the Clinical Microbiology Laboratory (CML) of the Faculty of Veterinary Medicine, University of Helsinki. Risk factors for an SP isolate being MRSP, as well as for a screening specimen revealing MRPS, were also investigated. Furthermore, the molecular epidemiology of all MRSP isolates stored in 2010–2014 was investigated using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing. Antimicrobial therapy, whether previous or ongoing during sampling, was a risk factor for MRSP in all studies. Furthermore, a prolonged hospital stay and veterinary visits were risk factors among guide dogs. An SP isolate originating from a private clinic (versus the VTH) was a significant risk factor for MRSP among clinical specimens. The same could be seen among screening specimens from patients with risk factors for MRSP. In addition, it was noted that a sizeable proportion (~20–60%) of animals in the studies had been or were being treated with antimicrobials. During the outbreak at the VTH, rigorous hygiene and barrier measures were necessary to achieve control. ST71, the MRSP clone that caused the outbreak, was the predominant clone in 2010–2011, accounting for over 50% of MRSP isolates, even among non-outbreak-related isolates. By 2014 the situation had changed, as ST71 represented only ~10% of MRSP isolates. MRSP clones belonging to CC45 (clonal complex 45) and CC258, and other unrelated STs, dominated the MRSP population by that time. SCCmec type IV was detected in a majority of different STs, indicating the horizontal spread of resistance genes. The prevalence of MRSP was only 3% among guide dogs. The proportion of clinical specimens from small animals that revealed MRSP was similar, 2.5%. However, 9% of screening specimens from high-risk patients revealed MRSP. Overall, 14% of SP isolates were MRSP. Roughly 30–40% of isolates were not susceptible to alternative antimicrobials, such as lincosamides, macrolides, or tetracyclines. MRSP in feline specimens was rare (<1% in all specimens after 2011). Our results give further credence to the hypothesis that antimicrobial therapy and contact with the veterinary environment are risk factors for MRSP in small animals. However, cats do not appear to be a significant source of MRSP. Our data suggest that the epidemiology of MRSP has changed from a predominantly clonal spread to a mix of clonal spread and the spread of genetic elements. The resistance rates among SP are at an alarming level. Decisive action, including the use of non-antimicrobial treatments whenever feasible and more prudent use of antimicrobials, is required to improve the situation.
  • Davidsson, Pär (Helsingin yliopisto, 2017)
    Necrotrophic phytopathogens, such as soft rot bacteria, cause large losses in agriculture. Unlike biotrophic pathogens, these typically rely on toxins and plant cell wall-degrading enzymes (PCWDEs) to kill and degrade the host tissue. As such, the methods utilised by the plants to defend themselves against biotrophs, such as the hypersensitivity response (HR), could instead be beneficial for necrotrophic pathogens. One key component in plant defence against necrotrophic pathogens is the recognition of oligogalacturonides (OGs), a breakdown product of the pectin in the plant cell wall, formed by the action of PCWDEs. Similar to direct recognition of the pathogen itself, recognition of OGs trigger a wide array of defence responses, resulting in improved protection against pathogens. Long OGs with a degree of polymerisation (DP) between 10 and 20 have been well studied. In this study, we explored the role of the relatively less understood short OGs (DP < 9). We utilised trimeric OGs to study the changes induced by short OGs on the transcriptome of Arabidopsis thaliana. We established that, similarly to long OGs, short OGs up-regulate genes related to defence and down-regulate genes related to plant growth and development. Phenotypic assays confirmed that pre-treatment with short OGs could improve resistance in A. thaliana against the soft rot bacteria Pectobacterium carotovorum, to the same degree as long OGs. Furthermore, we showed that treatment with both types of OGs results in seedling growth retardation. As part of investigating the signalling triggered by short OGs, we confirmed that trimeric OGs do not trigger the characteristic initial ROS (reactive oxygen species) burst, but do trigger expression of a large set of peroxidases. Similar to long OGs, part of the signalling in response to short OGs goes via phosphorylation of Mitogen-activated protein kinases (MAPKs). Our results show that short OGs are indeed biologically active elicitors of plant defence, with a signalling pathway that appears to be in part distinct from long OG signalling. We used the established trade-off between plant defence and plant growth and development to develop screens for mutants with altered OG sensitivity. One mutant line exhibiting hypersensitivity to OGs, resistance to the necrotrophic pathogens Botrytis cinerea and P. carotovorum, as well as sensitivity to the hemibiotrophic pathogen Pseudomonas syringae, was chosen for further studies. We established that the observed phenotypes were due to overexpression a cell wall-localised apoplastic peroxidase (class III peroxidase, CIII Prx) – PEROXIDASE 57 (PER57). We detected increased levels of ROS and increased cuticle permeability, associated with downregulation of genes involved in cutin formation and biosynthesis. We also observed a priming of OG related response genes. The phenotypes could be recaptured by overexpression of several CIII Prxs, indicating a general phenomenon. ABA treatment of these lines restored the phenotypes to wild-type. This appears to be mediated via removal of ROS. Noticeably, the peroxidase activity remained high in the peroxidase overexpression lines, indicating that while exogenous application of ABA was able to remove the ROS produced by the peroxidases it only had a minor direct effect on the activity of the peroxidases. Our results, combined with previous research on cuticular and ABA mutants, led us to propose that cuticle integrity is influenced by a positive feed-back loop. A disturbed cuticle leads to elevated ROS levels via increased peroxidase activity, which in turn impairs cuticle formation and biosynthesis. Under normal circumstances this loop is regulated by ABA. In the situation where a necrotrophic pathogen is invading the plant recognition of cell-wall derived DAMPs, such as OGs, it leads to activation of peroxidases that further promote resistance signalling via the creation of ROS.
  • Jiang, Ping (Helsingin yliopisto, 2017)
    The high-level cognitive abilities such as attention and working memory develop throughout childhood and adolescence. Neuroimaging studies have shown that several brain regions including areas in the prefrontal (PFC) and parietal cortices play an important role in cognitive control. The prolonged maturation of the PFC and related networks may underlie the immature cognitive control abilities in children. In this thesis, functional magnetic resonance imaging and 1-back WM tasks were used to investigate 1) top-down modulation of brain activity in cortical areas related to visual information processing and 2) functional connectivity of resting state and task-related brain networks in healthy 7-11-year-old children and young adults. The tasks required the subjects to attend to either face or scene stimuli and to ignore distracting scene or face images, respectively. Studies I and II of this thesis found weaker or otherwise immature top-down modulation of the face processing-related visual association cortices that could partially be explained by the observed weaker functional connectivity between the PFC and the visual association cortex in the typically developing 7–11-year-old children compared to the young adults. Moreover, there were age-dependent differences in the recruitment of the PFC during visual working memory tasks. These age-dependent differences between the two groups are in line with the observed differences in the performance of the working memory tasks that was poorer in children than young adults. Study III showed that the 7-11-year-old children have already established an adult-like pattern of resting state networks, but especially during task performance, the functional connectivity within and between these networks differed from that in young adults. The group differences observed in the brain activation and functional connectivity are likely partly related to the morphological developmental state of the grey- and white matter in the 7–11-year-old children (i.e., the ongoing synaptic pruning and myelination of axons that continue up to young adulthood). The findings of this thesis conform to the suggestion that during development, the function of brain regions, especially the PFC, and the functional connectivity of brain networks, undergo dynamic changes, and that the same cognitive function may rely on different brain networks at different ages.
  • Deptula, Paulina (Helsingin yliopisto, 2017)
    Vitamin B12 is the most complex vitamin in existence and one of the most complex non-polymeric molecules occurring in nature. It is predominantly present in animal-derived products, which places vegetarians and people with limited access to animal-derived foods at risk for developing vitamin B12 deficiency. With the current trend of limiting the consumption of foods of animal origin, the deficiency may also affect other populations. In situ fortification of foods through microbial fermentation with food-grade bacteria is a viable method for the introduction of vitamin B12 into foods, if the microorganism is capable of synthesising the active vitamin form. Here, the capability of Propionibacterium freudenreichii to produce active vitamin B12 was explored with the use of a combination of microbiological and molecular approaches. First, the activity of the heterogolously expressed and purified enzyme BluB/CobT2 was investigated. The results showed that the novel fusion enzyme was responsible for biosynthesis of 5,6-dimethylbenzimidazole (DMBI) base and its activation for attachment as the lower ligand of vitamin B12. The enzyme’s inability to activate adenine, the lower ligand of pseudovitamin B12, revealed a mechanism favouring production of active vitamin B12 in P. freudenreichii. The in vivo study showed that formation of DMBI is oxygen dependent as no vitamin B12 was produced under strictly anaerobic atmosphere. Exogenous DMBI was incorporated into the vitamin molecule under both microaerobic and anaerobic conditions, with a clear preference over incorporation of adenine. In the following study, the capability of 27 P. freudenreichii and 3 Acidipropionibacterium acidipropionici strains to produce active vitamin B12 was examined by UHPLC. The yields obtained from growth in whey-based medium enriched in cobalt and supplemented with either DMBI, with the precursors of DMBI- riboflavin and nicotinamide, or without supplementation. A. acidipropionici strains required supplementation of DMBI to produce small amounts of active vitamin B12 (<0.2 µg/mL), while all of the P. freudenreichii strains were able to produce active vitamin B12 in all conditions tested. The yields of active vitamin B12 produced by P. freudenreichii and responses to supplementation were strain dependent and ranged from 0.2 to 5.3 µg/mL. Subsequently, the active vitamin B12 production by the strain P. freudenreichii 2067 without addition of cobalt or DMBI was tested. The experiments were performed in a medium mimicking cheese environment as well as in the whey-based medium. The production of other key metabolites was examined by HPLC, while the global protein production was compared by gel-based proteomics. The results showed that regardless of different effects of the media on the metabolic state of the cells, which was reflected by distinct metabolite and protein production patterns, P. freudenreichii produced nutritionally relevant levels of active vitamin B12. Finally, whole genome sequencing was employed to better characterise the species through a comparative genomics study. The use of PacBio sequencing platform, a PCR-free method producing long reads, resulted in discovery of additional circular elements: two novel, putative conjugative plasmids and three active, lysogenic bacteriophages. The long reads also permitted characterisation and classification of two distinct types of CRISPR-Cas systems. In addition, the use of PacBio sequencing platform allowed for identification of DNA modifications, which led to characterisation of Restriction-Modification systems together with their recognition motifs, many of which were reported for the first time. Genome mining suggested surface piliation in the strain P. freudenreichii JS18, which was confirmed by transmission electron microscopy and assessment of specific mucus binding.
  • Tarkiainen, Katriina (Helsingin yliopisto, 2017)
    Carboxylesterases (CESs) catalyze the hydrolysis of a variety of ester- and amide containing compounds to their respective free acids. The main CES isozymes involved in drug metabolism are carboxylesterases 1 and 2 (CES1 and CES2). CES1 contributes to an estimated 80 to 95% of the total hydrolytic activity in the human liver. A CES1 c.428G>A (p.G143E, rs71647871) single nucleotide variation (SNV) markedly decreases the catalytic efficiency of CES1 in vitro. Possible effects of this variant on drug pharmacokinetics in vivo in humans, however, have not been systematically investigated. Furthermore, only a few studies have investigated the effects of CES1 variants on its expression and/or activity. Therefore, this thesis aimed to investigate genetic variability in the CES1 gene in the Finnish population and their effects on drug pharmacokinetics and pharmacodynamics in humans. The frequency distribution of the CES1 c.428G>A SNV was investigated in 860 healthy Finnish volunteers. The effects of the CES1 c.428G>A SNV on the pharmacokinetics of oseltamivir, clopidogrel, quinapril, and enalapril were investigated in 40 healthy volunteers. The CES1 gene and its flanking regions were sequenced in 192 healthy volunteers to identify previously unknown variants affecting CES1 whole blood expression. The findings were replicated in another set of 88 healthy volunteers. Furthermore, the effects of the detected variants on CES1 liver expression were investigated in 177 liver samples and on clopidogrel pharmacokinetics in 106 healthy volunteers from previous pharmacokinetic studies on clopidogrel. The CES1 c.428G>A variant allele was found with a minor allele frequency of 2.2%. The c.428G>A SNV reduced the hydrolysis of oseltamivir to the active oseltamivir carboxylate. The oseltamivir carboxylate to oseltamivir area under the plasma concentration-time curve (AUC) ratio was 23% smaller in heterozygous carriers than noncarriers. The c.428G>A SNV reduced the hydrolysis of clopidogrel to the inactive carboxylic acid metabolite. Consequently, the AUC of the parent clopidogrel was about 120% higher and that of the active metabolite about 70% higher in carriers than in noncarriers. Consistently, the c.428G>A SNV markedly enhanced the platelet inhibitory effect of clopidogrel. The average percentage inhibition of platelet aggregation at 0-12 hours was 19% higher in carriers than in noncarriers. The c.428G>A SNV significantly reduced the hydrolysis of enalapril to active enalaprilat. The AUC of enalaprilat was 20% lower in carriers than in noncarriers. The c.428G>A SNV had no observable effect on the pharmacokinetics of quinapril. Two intronic CES1 rs12443580 and rs8192935 SNVs were discovered to have a major effect on CES1 expression in whole blood, but not the liver. Moreover, these two SNVs had no effect on clopidogrel pharmacokinetics. In conclusion, the CES1 c.428G>A SNV reduces the bioactivation of oseltamivir, markedly increases the clopidogrel active metabolite plasma concentrations and antiplatelet effects, and reduces the bioactivation of enalapril in vivo in humans. The two intronic CES1 rs12443580 and rs8192935 SNVs have tissue-specific effects on CES1 expression. This could lead to substrate-dependent effects of these SNVs on drug biotransformation.
  • Virtaniemi, Matti-Pekka (Helsingin yliopisto, 2017)
    The purpose of this study is to investigate the dimensions of the existential process manifested in the narratives of ALS patients. ALS is a progressive, neurodegenerative disease to which there is no curative treatment and that leads to death typically within a few years due to respiratory failure. The existential process is both psychological and spiritual. The research material of this study was collected by interviewing six ALS patients, three times each, within one year. This narrative study seeks to find answers to the following questions: 1.) What kinds of existential processes do ALS patients undergo during their illness? 2.) How does one create meaning of life and find meaning in life when ALS threatens one’s existence? 3.) How does religious spirituality contribute to an ALS patient’s perceived meaningfulness of life? A combination of three narrative analysis methods allowed the study to understand the existential process of an ALS patient. The thematic analysis revealed the fundamental issues in life, or existential dimensions. Existential dimensions were further divided into two groups: first, the ultimate concerns in life and second, meaning in life and meaninglessness of life. The first group involved anxiety of fate, the choice (of the permanent ventilator) concerning existence, loss of autonomy, responsibility and existential guilt, functional loss and shame for disability, agony of failing respiration, menacing future and death. The things that the interviewees considered most important in their lives are in the present study called sources of meaning in life: close relationships, work, helping others, nature, life in itself, personal growth, hope, and connection to the transcendent. The experience of the meaningfulness of life correlates with the amount of the sources of meaning in life. Six life narratives were construed into four types of illness narrative by help of the holistic form analysis. Each type of illness narrative demonstrates a different existential process. The mini stories of the interviewees as well as their structural and meaning analysis revealed the changes in the existential dimensions manifesting in the existential process. Mini stories also provided insight into how religious spirituality changes and how it contributes to one’s perceived meaningfulness of life. The effect was positive in three of the four narrative types. The threefold narrative analysis provides an integrated view of the existential process of an ALS patient. The existential process consists of two separate but nested spiritual processes: one involves processing the ultimate concerns in life, while the other involves processing meaning in life and meaninglessness of life. The ultimate concerns may transform, but they will remain present until the end of life. This study found that addressing these ultimate concerns does not prevent one from perceiving life as meaningful and even strengthening this view. One may need either to reconstruct the sources of meaning in life, or to find new ones or create new meanings. Thus the study sheds light on the puzzling finding: quality of life in ALS is maintained while physical function declines.
  • Myllykylä, Emmi (VTT, 2017)
    Currently, the preferred option for the long-term disposal of spent nuclear fuel (SNF) and potentially for future thorium-based fuels in Finland and Sweden is disposal in a geological repository. In deep bedrock, the release of Th, U and other radionuclides through man-made barriers and the geo- and biosphere will be controlled by the dissolution of the fuel by groundwater. Thorium dioxide is isostructural to uranium dioxide, sharing the same fluorite structure (space group Fm3m) and making it a useful analogue material for nuclear fuel, which mainly consists of UO2 (>95%). This thesis aimed to investigate the dissolution of ThO2, which was synthesised to approximate as closely as possible the microstructure of UO2 in a nuclear fuel matrix. The investigation consists of dissolution studies conducted using pellets, fragments (2 to 4 mm) and particles (80 to 160 µm) of 232ThO2. The evolution of dissolving surfaces and grain boundaries were examined by combining different microscope imaging techniques (scanning electron microscopy (SEM), atomic force microscopy (AFM), SEM with electron backscattering diffraction detector (SEM-EBSD) and profilometer imaging). Part of the dissolution experiments were conducted in the presence of a 229Th tracer to gain additional data on the dissolution and precipitation by following the change in isotopic ratio 229Th/232Th. Furthermore, the pellets from these experiments were measured with direct alpha spectrometry to estimate the contents and thickness of the 229Th-rich layer formed on the pellet surface. The results of all dissolution studies showed a relatively fast release of thorium during the early stage of the experiment followed by a slow decrease in the thorium concentration and suppression of the dissolution rate as the experiments continued over 100 days. Microscopic studies revealed that the grain boundaries play a significant role in the initial release of thorium. It was also observed that the different surfaces of thorium dioxide particles behave differently either dissolving, precipitating or showing an almost inert nature, most likely due to the different surface energies of the heterogeneous material. High-resolution (sector field) inductively coupled plasma mass spectrometry (SF/HR-ICP-MS) was used for the thorium isotope analyses of leached solutions. The developed method was powerful for analysing thorium isotopes. In addition, alpha spectrometry was used for thorium analysis for comparative purposes. The alpha spectrometry yielded a lower detection limit for 229Th and a higher detection limit for 232Th than SF-ICP-MS, which was 1 x 10-12 mol for both isotopes. Thus, these methods provided comparable results for the analysed 229Th concentration. However, the chemical separation needed before alpha spectrometry is very time consuming compared to the sample preparation necessary for HR-ICP-MS. When combined with simulations, direct alpha analysis confirmed that during leaching a new layer, with a maximum thickness of 0.1 µm, formed on the surface of ThO2 pellets. Alpha spectrometry also provided interesting insight into the dissolution and co-precipitation behaviour of 229Th and 232Th decay series daughter nuclides. The surface layer contained not only 229Th and its daughters, but also an elevated concentration of daughters from the 232Th decay series, indicating that they were first released from the bulk during the leaching experiment, subsequently co-precipitating or adsorbing onto the surface of the pellet.
  • Hotta, Jaakko (Helsingin yliopisto, 2017)
    Complex regional pain syndrome (CRPS) causes disabling and severe limb pain that is difficult to treat. The pain typically increases during motor actions, but is present also at rest. The pathophysiology of CRPS is incompletely understood. Some of the symptoms suggest involvement of the central nervous system, and accordingly, patients have been shown to display alterations in, for instance, the primary sensorimotor cortex (SM1) and indications of neuroinflammation. More thorough pathophysiological knowledge of CRPS would help to develop better treatments and diagnostics. My thesis includes four studies exploring the cerebral basis of CRPS. All patients participating in these studies suffered from unilateral upper-limb CRPS. Studies I and II explored the complex couplings between pain, motor actions and visual information in CRPS. In healthy persons, observation and imagery of motor actions are known to elicit brain activation in similar brain areas as does action execution. CRPS patients experience pain while they execute or imagine actions and at the same time their brain activity is abnormal. However, these phenomena have not previously been studied for action observation in CRPS. In Study I, ratings of subjective experiences (e.g. pain) during observation of others’ actions were compared between 19 CRPS patients and 19 healthy control subjects. We found that CRPS patients experienced observation of motor actions as unpleasant and painful, and they overestimated the force applied in the observed actions. In Study II, the brain responses to action observation, measured with functional magnetic resonance imaging (fMRI), were compared between 13 CRPS patients and 13 healthy control subjects using multivariate pattern analysis (MVPA). During painful action-observation, the patients displayed abnormal activity in, for example, the representation area of the painful limb in the SM1 as well as in other brain areas important for pain experience and motor actions. With MVPA, these abnormal activations reliably discriminated CRPS patients from healthy subjects, indicating potential of fMRI responses as biomarkers for CRPS. Together Studies I and II build the grounds for future studies on using action observation as an add-on treatment for CRPS. Study III elucidated alterations of brain functioning during spontaneous pain in CRPS. Comparison of resting-state fMRI between 12 CRPS patients and 17 healthy control subjects showed disrupted functional connectivity of the SM1 in CRPS, especially for the representation areas of the painful and contralateral non-painful limb. These results suggest that pain alters SM1 functions even during rest, disrupting normal brain processes that could e.g. support motor learning. In Study IV, volumes of brain structures, measured with magnetic resonance imaging, were compared between 12 CRPS patients, 8 non-CRPS pain patients and 12 healthy control subjects. In the CRPS patients, the right-ventricle choroid plexus was about one-fifth larger than in healthy subjects and about one-eight larger than in non-CRPS patients. Choroid plexus is important in e.g. neuroinflammation and this finding may open new lines in research for the pathogenesis and treatment of CRPS. In conclusion, these studies revealed novel brain-derived symptoms and signs of CRPS. Brain changes involved especially areas that have already previously been studied in CRPS (e.g. the representation areas of the painful and contralateral non-painful limb in the SM1) but also other areas, such as choroid plexus. The findings further corroborate central nervous system involvement in CRPS.