Bio- ja ympäristötieteellinen tiedekunta: Recent submissions

Now showing items 1-20 of 1057
  • Parri, Elina (Helsingin yliopisto, 2021)
    Dysregulated cellular signaling pathways provide cancer cells survival advantage and support their abnormal growth. In large granular lymphocytic leukemia and aggressive NK-cell malignancies, signal transducer and activator of transcription 3 (STAT3) is frequently mutated, resulting in a constitutively active protein, but do not alone explain the development of the disease. In addition, mutations in tumor suppressor p53 (TP53) worsen the prognosis of NK-cell malignancies. In cancers, activation of the STAT3 is associated with enhanced cellular transformation, increased metastasis and drug resistance. Direct therapeutic targeting of STAT3 has proven to be challenging and only a few direct STAT3 inhibitors have entered early phase clinical trials. So far, the most promising inhibitors of JAK/STAT-signaling are Janus-kinase (JAK) inhibitors, of which several are clinically approved. Using RNA-interference and drug screening applications, we identified new approaches for targeting hyperactive STAT3 and identified drug combinations for targeting JAK/STAT dependent malignant NK-cells. Furthermore, we have used biochemical screens for validating novel computationally predicted kinase-compound interactions. In study I, we identified novel kinases (CDC7, CSNK2, DDR2, CDK8, PI4KII, CSK) and a phosphatase (PTPRH) that regulate oncogenic STAT3 activity. By using inhibitors targeting identified kinases, we observed a reduction of STAT3 activity both at the transcriptional and phosphorylation level. In study II, using drug screens and extended drug perturbations, we found that malignant NK-cells did not develop resistance against drug combinations containing a JAK inhibitor ruxolitinib. Our data suggest that tipifarnib-ruxolitinib and idasanutlin-ruxolitinib combinations are effective depending on the STAT3 or TP53 mutational status of the malignant NK-cells. In contrast, dexamethasone-ruxolitinib was effective independently of the mutational status. Among these, the dexamethasone-ruxolitinib combination is under clinical evaluation for other hematological malignancies such as multiple myeloma. In studies III and IV, we validated computationally pre-screened kinase-compound interactions. Using a combined computational-experimental approach, we identified novel off-target effects for vascular endothelial growth factor receptor (VEGFR) inhibitor tivozanib and unveiled new lead-molecules targeting protein kinases. This thesis gives a new understanding on oncogenic STAT3 signaling, provides approaches for indirect STAT3 targeting and, based on in vitro data, suggests novel drug combinations for targeting NK-cell malignancies. The collaboration efforts of combining computational-experimental methods provide a better understanding of compound-kinase interactions and allow cost-effective in silico pre-screening tools for advancing identification of leads and off-target effects of kinase inhibitors.
  • Palojärvi, Ansa (Helsingin yliopisto, 2021)
    Agricultural productivity has improved significantly in recent decades. World food production has more than tripled through intensive crop production. In addition to improved food production and safety, environmental problems have also increased. However, due to global population growth, food production needs to be further intensified. This thesis is related to the sustainable intensification of crop production and the improvement of crop resilience. The study examined the effects of agricultural management practices on soil microbial communities and the ecosystem services they provide. In particular, the possibilities to improve the plant disease suppression of field microbiota and appearance of arbuscular mycorrhizal fungi (AMF) by tillage methods and crop diversity were in focus. In addition, the amount and location of different carbon fractions in the soil profile were investigated. The aim was also to identify useful indicators for beneficial soil microbial activity for soil quality monitoring. The studies utilized the field experiments and a cross-site comparison of several long-term tillage fields. The ability of soil microbiota to suppress fungi (fungistasis) and general disease suppression was often enhanced by the long-term reduced and no-tillage practices, compared with conventional mouldboard ploughing. The result could not be generalized to specific management but could be linked to the higher biomass of soil microbiota and fungi as well as the soil labile carbon content. Based on the results, the strengthening of soil disease suppression was reflected in lower prevalence of the test pathogen (F. culmorum) in the cereals, suggesting an impact on crop production. Reduced tillage was shown to alter the vertical distribution of carbon fractions and accumulate soil organic carbon (SOC), labile carbon, and microbial biomass in the topsoil layer. However, the SOC sequestration in the whole soil profile was not necessarily increased. Mycotrophy of host plants varied considerably between the special plants studied. Mycotrophy of the crop plant had a strong effect on the concentration of AMF in the rhizosphere and bulk soil. Field observations confirmed that the ecosystem services of microbiota could be enhanced by the choice of agricultural management although the effects of a specific management method may not be directly and generally related to the activity. The effects on the fungal community and crop performance should be considered in relation to the crop sequence used. In addition to this, potentially, single management practices have a combined effect on soil health. Soil microbiological ecosystem services need simple indicators to be used in developing and monitoring sustainable agricultural production. The intensity of disease suppression in the soil could be reliably assessed by a simple laboratory test. Soil labile carbon (POM-C, Cmic) is potentially a useful indicator for disease suppression. Cell membrane lipid assays (PLFA, NLFA) proved to be effective indicators for estimating AMF biomass in arable soil. Cultivation measures were shown to have a significant impact on the community structure and function of the field microbiota. In the light of these results, it is absolutely essential to take into account the functionality of the whole soil microbiome in the design of sustainable intensification of agricultural management practices.
  • Liu, Yixin (Helsingin yliopisto, 2021)
    The RET receptor tyrosine kinase is a versatile receptor which responds to multiple signalling cues. The signalling of RET plays a central role in cell proliferation, differentiation and maintenance. Its dysregulation contributes to various human diseases through gain- and loss-of-function, such as Hirschsprung’s disease and thyroid and lung cancers. Mutations at the cysteine-rich domain (CRD) of RET lead to constitutive activation through receptor dimerization via an intermolecular disulfide bond. However, due to the lack of functional and structural studies of RET and its ligands, the molecular mechanisms of RET activation under normal and pathological conditions is unclear. In this thesis project, a combination of biochemical and biophysical tools are used to characterize the activation of RET with an emphasize on three aspects: studying (1) its extracellular domain (ECD) complex formation with ephrin As (efn-As) and the growth and differentiation factor 15 (GDF15)/the glial cell-derived neurotrophic factor receptor a-like (GFRAL), (2) the oncogenic C634R mutation, which accounts for the majority of the multiple endocrine neoplasia type 2A (MEN2A) cases and (3) the solubilization and purification of full-length RET. To explore the binding between RET and efn-As, I first demonstrated that functional zebrafish RETECD (zRETECD) and efn-A5 (zefn-A5) can be expressed using insect cells. In contrast to previous cellular studies, I showed that zRETECD does not interact directly with zefn-As and postulated that another binding partner is required to mediate their interaction for the reverse signalling of efn-As. Unlike zRETECD, I found that human RETECD (hRETECD) expressed in insect cells is non-functional; therefore, I established a mammalian expression platform for functional expression of hRETECD, demonstrating its superiority to insect cell expression in which hRET is prone to misfold. Using recombinantly expressed proteins, I reconstituted the wild-type (WT) hRETECD/hGDF15/hGFRALECD complex and was able to reconstruct an 8-Å cryo-EM map of the complex, which revealed a “butterfly”-shaped tripartite complex. For the expression of the hRETECD(C634R) mutant, I used a C-terminal Fc tag to successfully drive the receptor dimer formation, as the mutant protein is otherwise monomeric. I studied the resulting apo-hRETECD(C634R) dimer and its complex with GDF15/GFRALECD using structural tools. Comparison of the cryo-EM structure of the hRETECD(WT)/hGDF15/hGFRALECD complex and the negative stain EM maps of the apo-hRETECD(C634R) dimer and the mutant hRETECD(C634R)/hGDF15/hGFRALECD complex revealed significant conformational changes that provide a structural model for the oncogenic activation of RET. Finally, I established a membrane protein production pipeline for full-length RET that will enable future structural studies in a more biologically native system.
  • Sun, Yan (Helsingin yliopisto, 2021)
    Soil pollution is one of the world’s biggest challenges. The most common soil pollutants include heavy metals and petroleum hydrocarbons. A great number of studies have developed strategies for pollutant remediation. For the removal of heavy metals including cadmium (Cd) in the soil, electrokinetic remediation has been rapidly developed in the recent decades as a both remediation and metal resource recovery method. However, field and full-scale applications of electrokinetic remediation in the removal of soil heavy metals are rare. As for organic pollutants including petroleum hydrocarbons, biostimulation with fertilization has been widely done and highly appreciated over physical and chemical methods ecologically and economically. Due to different soil physico-chemical properties and climates, fertilizers should be chosen carefully for the best outcome. Remediation strategies are often initially validated in the laboratory, which can still fail in the field tests or application usually due to soil heterogeneity that exists even within millimeters. A false conclusion may result from an improper experimental design in the laboratory, such as insufficient true replication (pseudoreplication) that fails to represent the sampling site, and composite sampling or randomized complete block design (RCBD) that only presents the average performance of the sampling site and can result in false positive or negative results for the component samples. This doctoral thesis presents three strategies in soil remediation, i.e. electrokinetic remediation of soil heavy metal Cd (paper I), biodegradation of soil gasoline aromatics stimulated by fertilization (paper II), and optimization of experimental design (paper III). Electrokinetic remediation of Cd was monitored for 14 days, in a pilot (4 m2) test and a full-scale (200 m2) application in paddy agricultural soil, at soil layers 0-10 cm, 10-20 cm, and 40-50 cm. A voltage of 20 V was applied at both scales. In the biostimulation study, three nitrogen fertilizers, i.e. inorganic NPK, urea, and methylene urea were compared in the biostimulation of gasoline aromatics removal in the soil collected from the high Arctic permafrost active layer and experimented in the laboratory at 10 °C for 28 days. Finally, the experimental design study that was conducted in the laboratory at 4 °C for 28 days illustrated how pseudoreplication and sole use of plot averages, would affect the conclusions drawn in biodegradation studies. In the experimental design study, three ecologically independent plots of soil were collected and processed independently in parallel. The soil was spiked with gasoline and divided for natural attenuation and biostimulation with methylene urea fertilization. Biodegradation results were analyzed individually within each plot of soil (pseudoreplication) as well as in a between-plot comparison by using the mean values of the three plots (plot averages). In 14 days, electrokinetic remediation showed a successful application at both scales, especially for the soil total Cd in the surface 0-10 cm layer, with a higher removal efficiency in the pilot test (87%) than the full-scale application (74%). The final concentration of soil total Cd was below the hazard threshold set for paddy agricultural soils in China. The higher removal efficiency in the pilot test can be due to the higher voltage gradient between the electrodes. The removal efficiency of the plant available Cd was lower than the soil total Cd, which can be due to the enhanced desorption of Cd cations through cation exchange and/or dissolution by lactic acid during electrokinetic remediation. In 28 days, biostimulation with urea fertilization managed to lower the concentration of the total gasoline aromatics below the initial level (by 47%). On day 7, the observed concentration of the total gasoline aromatics was higher than the initial level in the NPK and urea fertilized treatments, which indicated an enhanced desorption and extractability of soil gasoline aromatics. The enhanced desorption and thereafter bioaccessibility of gasoline aromatics in the NPK and urea fertilized treatments could be related to the enhanced biodegradation activity compared to natural attenuation and methylene urea fertilized treatment, mainly shown between day 7 and day 28. In contrast to the successful application of electrokinetic remediation and biostimulation with urea fertilization, the effect of methylene urea in biostimulation showed positive, negative, and negligible results. The random results of natural attenuation and biostimulation with methylene urea fertilization indicated the patchiness of biodegraders even within one plot of soil. Additionally, the results based on the between-plot comparison showed a dilution effect and could pose false positive and negative results for the component soil. Although electrokinetic remediation of heavy metal Cd and biodegradation of gasoline aromatics stimulated by urea fertilization showed success in the cases studied herein, the success of these remediation strategies cannot be generalized to other soil pollution cases due to soil heterogeneity at least, according to the third study. The results suggest that ecologically independent replication be adopted in the between-plot comparison and that parallel experiments and statistical analysis with within-plot replication for each replicate plot be performed to guide a more successful practical application of remediation strategies.
  • Leigh, Robert (Helsingin yliopisto, 2021)
    Disruption of chamber-specific gene regulatory networks underlies malformation of the heart and results in congenital heart disease. Re-activation of fetal gene regulatory networks in adults occurs during cardiac injury, and transcription factors composing these networks represent candidate therapeutic targets to impede heart failure progression. The identification of molecular markers and underlying regulators of cardiac chamber specification is thus an important step in understanding the aetiology of cardiovascular disease. Moreover, the examination of chemical modulation of molecular processes occurring during development allows for a more detailed understanding of the teratogenic effects of chemical compounds and could lead to the development of small molecule-based strategies for stimulating or impeding well-characterized developmental processes in the adult heart. To this end, this thesis is comprised of three studies related to the differentiation of atrial and ventricular cardiomyocytes and the selective modulation of this process. Study I led to the generation of an in vitro model of cardiomyocyte subtype specification of pluripotent stem cells for the use in studies II-III. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of native embryonic atrial and ventricular tissue confirmed the robust ventricular-specific expression of myosin light chain 2 (Myl2) and also indicated the lack of an endogenous atrial-specific marker during early embryogenesis. Genome editing was used to integrate a fluorescent reporter into the endogenous Myl2 locus to mark cells of the ventricular lineage, whereas atrial cells were traced by an atrial-specific transgene driven by the slow myosin heavy chain 3 (SMyHC3) promoter. Atrial and ventricular reporter expression were confirmed in vivo by laser-assisted morula injection of reporter mouse embryonic stem cells (mESCs) and microscopy of chimeric embryos. In addition to this in vivo validation, spontaneous differentiation of reporter mESCs was characterized by qRT-PCR, indicating dynamic expression of retinoic acid signalling components. Differentiation assays were developed based on chemical perturbation of undifferentiated progenitor cells and differentiated cardiomyocytes, respectively. Members of the retinoid family, known teratogens and modulators of anterior-posterior patterning, were tested for effects on the activation of atrial and ventricular reporter genes. Additionally, a directed-differentiation assay was developed based on highly pure multipotent progenitor cells and differentiation assessment in a 384-well format. In this assay, chemical inhibitors of Wnt and Transforming growth factor β (Tgfβ) pathways led to promotion of ventricular reporter expression when added at the multipotent progenitor stage, but not after the onset of spontaneous beating. Additionally, exogenous all-trans retinoic acid added to undifferentiated progenitors led to an inhibition of ventricular differentiation, whereas addition following the onset of spontaneous beating led to activation of the ventricular reporter gene. In addition to chemical probes described in study I, novel compounds targeting the protein-protein interaction of core cardiac transcription factors Gata transcription factor 4 (Gata4) and NK2 homeobox 5 (Nkx2-5) were examined in study II. Specifically, the effects of GATA-targeted compounds on the differentiation of atrial and ventricular cardiomyoyctes were explored. Lead compound 3i-1000 increased the proportion of atrial and ventricular reporter cells after 10-day treatment in the spontaneous differentiation assay. Further exploration of the effects of GATA4 targeted compounds revealed that a shorter treatment (2-day) of cells prior to the onset of spontaneous beating led to an upregulation of ventricular reporter genes in a directed differentiation assay. An acetyl-lysine like domain among active compounds, in addition to analysis of the GATA4 interactome by Bio-ID revealed the potential association of bromodomain-containing proteins with chamber-specific gene expression. This was further investigated by combinatorial treatment with the Bromo- and Extra- Terminal domain family (BET) bromodomain inhibitor JQ1 and GATA-targeted compounds in reporter gene assays. Finally, the effects of GATA compounds on cardiomyocyte maturation were explored by compound treatment and global run-on sequencing (GRO-seq) in primary cardiomyocytes, revealing the upregulation of several targets previously identified as regulators of cell fate determination and regeneration. Study III detailed the embryonic/cardiac expression of proCholecystokinin (proCCK), a classical gut and neuropeptide. Analysis of mRNA-seq data suggested that proCCK is a transcriptional target of the TBX5 transcription factor in mESC-derived cardiomyocytes. Native, endogenous mRNA levels were characterized in embryonic hearts by whole mount in situ hybridization and optical projection tomography, revealing that proCck mRNA is present prior to the linear heart tube stage and that it is upregulated in the ventricles compared to the atria in the newly formed embryonic heart. Interestingly, mRNA of proCck and its receptors Cckar/Cckbr is mostly restricted to the atrial chambers in neonatal stages, in line with its potential role in regulating cardiac rhythm. In silico analysis implicated both TBX5 and MEF2C as regulators of proCck transcription, and this regulation was confirmed by conducting in vitro reporter gene assays. Additionally, proCCK was induced by endothelin-1 (ET-1), another peptide associated with maladaptive remodelling during heart failure. Furthermore, proCck mRNA levels declined in the left ventricles of rats following myocardial infarction (MI). Finally, exogenous cholecystokinin octapeptide (CCK-8) exerted no effects on the differentiation process of pluripotent stem cells (PSCs) to the cardiomyocyte fate. Collectively, these studies led to the generation of new methodology for the study of chamber-specific cardiac gene regulatory networks. Additionally, they led to an improved understanding of the dynamics of chamber-specific marker localization and upstream transcription factors governing their expression, potentially important to biomarker development. Finally, these studies have indicated that specific chemical compounds are capable of influencing chamber-specific gene regulatory networks. This knowledge might be utilized to develop novel therapeutic strategies for the treatment of heart failure.
  • Översti, Sanni (Helsingin yliopisto, 2021)
    A mitochondrion is a cytoplasmic organelle responsible for the energy production of the eukaryotic cells. Mitochondria contain their own genome, mitochondrial DNA (mtDNA), which is a double-stranded circular molecule. Due to mitochondria’s essential role in metabolism, a cell can contain hundreds of thousands of copies of mitochondrial DNA, depending upon the cell’s energy requirements. In mammals, mtDNA is generally maternally inherited, meaning that it is transmitted from a mother to all of her descendants. Although mtDNA constitutes only a small fraction of the cell genome, it has several qualities which make it widely used in population genetic studies such as uniparental inheritance, and the fact that the mitochondrial genome does not recombine. Moreover, mtDNA has a mutation rate ten times higher than that of the nuclear genome and therefore allows us to trace back matrilineal lineages through generations and subsequently make inferences about maternal ancestors. The human mtDNA sequence consists of approximately 16,570 base pairs and also contains both a coding region and a non-coding control region, the latter constituting around 7% of the whole mtDNA genome. Since mtDNA does not undergo recombination, an individual’s mitochondrial haplotype can be determined simply by the direct sequencing of target amplicons. Haplotypes containing certain defining variants are considered to be descendants of a common ancestor and are classified into haplogroups. The geographical distribution of haplogroups among contemporary populations is well-known – for instance, the majority of Europeans exhibit mitochondrial lineages H, U, J, K, T and V. Ancient DNA research has uncovered that lineage U was already highly prevalent among the earliest hunter-gatherer settlers of Europe, whereas the gradual spread of agriculture from the Near East that started approximately 10,000 years ago brought along new people and hence also novel mitochondrial lineages (H, J, K and T). Previous studies have stated that compared with other European populations, contemporary Finns do not seem to be an exception in terms of mitochondrial genome pool. This is rather surprising, since other genetic markers have revealed that contemporary Finns are characterized by a strong Eastern genetic influence and are distinguishable from other Europeans. Moreover, the evident East-West distinction within Finland, apparent in Y-chromosomes and autosomes, has not been previously identified in the mtDNA. This thesis outlines the mitochondrial DNA variation among present-day Finns in a Bayesian framework. The aims were to evaluate if Finns display a homogeneous geographical distribution of haplogroups and if the mtDNA composition of Finns resembles that of other European populations, as previously suggested. While no spatial differences have previously been detected in the mitochondrial haplogroup frequencies within Finland, a clear geographical distinction arose when clustering haplogroups into ‘hunter-gatherer’ (U and V) and ‘farmer’ associated lineages (H, J, K and T). Whereas the farmer related haplogroups were notably more common in Southwestern Finland, the hunter-gatherer lineages had higher densities in the Northeastern parts compared to the Southwest. Furthermore, utilization of the complete mitochondrial genomes allowed for reassessing the Finnish mtDNA pool on a larger scale. One third of the subhaplogroups in Finland today were characteristic only of Finns, i.e. these lineages were virtually absent from other populations. When further partitioning the Finnish samples based on their inclusion in ‘local’ and ‘non-local’ lineages, two notably different demographic trajectories were obtained. The population history for Finn-characteristic lineages was more in accordance with what is known through other data types, such as Y-chromosomal and archaeological data. In general, the observed geographical within-country deviation in the Finnish mtDNA pool and the high proportion of Finn-characteristic lineages reflected the signals reported from other genetic markers. Alongside Finnish mtDNA, this thesis explores the molecular rate variation among the different subhaplogroups of lineage U. Unexpectedly, a noteworthy discrepancy emerged from the tip calibrated phylogenies: haplogroup U5b had a notably lower substitution rate when compared to U2, U4 and U5a. This lineage-specificity in the rates most likely arose, at least to some extent, from differences in past population dynamics. In particular, U4 and U5a have been associated with the rapid population expansion which occurred during the Bronze Age, whereas the frequency of U5b has remained rather stable. Subsequently, the observed rate of deviation influences the divergence estimates for subhaplogroups, suggesting that U5b emerged considerably earlier than U5a. Since molecular rates are fundamental to several population genetic analyses and the timing of divergence and demographic events relies heavily on the rate used, more attention should be paid to the interlineage molecular rate variation. The results of this thesis demonstrate not only the importance of using complete mtDNA genomes and the appropriate molecular rate, but also the relevance of approaching the data from new angles when assessing the demographic past of mitochondrial lineages.
  • Weldatsadik, Rigbe Gebremichael (Helsingin yliopisto, 2021)
    Proteogenomics is an emerging field that combines genomic (transcriptomic) and proteomic data with the aim of improving gene models and identification of proteins. Technological advances in each domain increase the potential of the field in fostering further understanding of organisms. For instance, the current low cost and fast sequencing technologies have made it possible to sequence multiple representative samples of organisms thus improving the comprehensiveness of the organisms’ reference proteomes. At the same time, improvements in mass spectrometry techniques have led to an increase in the quality and quantity of proteomics data produced, which are utilized to update the annotation of coding sequences in genomes. Sequencing of pooled individual DNAs (Pool-seq) is one method for sequencing large numbers of samples cost effectively. It is a robust method that can accurately identify variations that exist between samples. Similar to other proteogenomics methods such as the sample specific databases derived from RNA-seq data, the variants from Pool-seq experiments can be utilized to create variant protein databases and improve the completeness of protein reference databases used in mass spectrometry (MS)-based proteomics analysis. In this thesis work, the efficiency of Pool-seq in identifying variants and estimating allele frequencies from strains of three β-hemolytic bacteria (GAS, GGS and GBS) is investigated. Moreover, in this work a novel Python package (‘PoolSeqProGen’) for creating variant protein databases from the Pool-seq experiments was developed. To our knowledge, this was the first work to use Pool-seq for sequencing large numbers of β-hemolytic bacteria and assess its efficiency on such genetically polymorphic bacteria. The ‘PoolSeqProGen’ tool is also the first and only tool available to create proteogenomic databases from Pool-seq data. For organisms such as the β-hemolytic bacteria GAS, GBS and GGS that have open pangenomes, the sequencing and annotation of multiple representative strains is paramount in advancing our understanding of these human pathogens and in developing mass spectrometry databases. Due to the increasing use of MS in diagnostics of infectious diseases, this in turn translates to better diagnosis and treatment of the diseases caused by the pathogens and alleviating their devastating burdens on the human population. In this thesis, it is demonstrated that Pool-seq can be used to cost effectively and accurately identify variations that exist among strains of these polymorphic bacteria. In addition, the utility of the tool developed to extend single genome based databases and thereby improve the completeness of the databases and peptide/protein identifications by using variants identified from Pool-seq experiments is illustrated.
  • Salo, Marja (Helsingin yliopisto, 2021)
    This dissertation studies how consumption data and applications such as carbon footprint calculators are used in steering household consumption (food, housing, travel, consumption of other goods and services). In addition to statistical data and analysis showing aggregated figures over populations, data and applications which monitor, estimate and provide feedback have been developed by various types of organisations. The tailored data on consumption are intended to inform and guide people on their carbon footprint and energy consumption. The study aims to address the research gap between the optimism that data-based applications can steer consumption and the critique presented of this view. To this end, the thesis examines the data-based applications and their use in the context of sustainable consumption policies. It draws on five articles that focus on household consumption patterns and applications which measure and steer consumption and related carbon footprints. The studies suggest that using the data as soft, information-based measures to persuade people to change their consumption patterns and doings provides novel opportunities for steering. At the same time, challenges such as the lack of long-term engagement with the applications, as shown in the studies, should also be taken seriously when considering the role of voluntary data-based measures in the sustainable consumption policy mix. The findings demonstrate that consumption and carbon footprint data have persuasive potential when they are used to support the activities and processes of committed actors. This is particularly the case when participants invest resources, time and effort in developing skills or adjusting the material environment to support more sustainable consumption and practices. Nevertheless, integrating the tools into the everyday lives and doings of ordinary people in order to steer them presents challenges. A novel contribution of this dissertation is to apply practice theory to unfolding these challenges. Practice thinking reveals how tension about, and resistance to, using footprint calculators and similar tools, then changing one’s doings according to the tailored advice, arises not only from the characteristics of the applications and interactions of people and applications: current taken-for-granted patterns of doing, perceptions of normal standards of comfort and convenience, the interlinked nature of everyday activities and competing priorities also hinder actions and ambition levels. Based on the findings, the dissertation provides recommendations for future practical initiatives and research on data-based applications to steer consumption from an environmental sustainability perspective. The results call for recognition of the prevalent forms of doings and circumstances instead of leaving them out of analysis of data-based applications and steering. The dissertation comprises critical reflection and discussion of the role and expected impact of the mechanisms of data-based steering and policies.
  • Rytteri, Susu (Helsingin yliopisto, 2021)
    Weather conditions are changing due to climate change. Average and extreme temperatures are warming and the variability of weather conditions is increasing. Warming winters shorten snow cover duration. Climate change together with other anthropogenic stressors, such as habitat loss and fragmentation, potentially have drastic ecological consequences. Insects as small, short-lived ectotherms are strongly affected by changing weather. Their complex life cycles make them particularly sensitive to the seasonal changes of weather. In this thesis, I utilised long-term data from two butterfly systems to study the ecological effects of changing weather and their implications for insect conservation. Firstly, metapopulation survey data from 1993-2019 on the Glanville fritillary (Melitaea cinxia) complemented with detailed field monitoring data and experimental data allowed the study on larval biology (chapters I, II). Secondly, mark-recapture data from 2000-2016 on a translocated population of the Clouded Apollo (Parnassius mnemosyne) enabled me to study the effects of weather on dispersal and colonisation in a novel landscape (chapters III, IV). Larval overwintering survival was enhanced by snow cover and late spring onset in an experiment (I). The detrimental effect of snowless overwintering conditions on individual level were not reflected by metapopulation growth in the wild. Instead, metapopulation growth was enhanced by increasing growing season precipitation, which largely determines the larval food availability. Microclimatic variation buffered populations against phenological asynchrony between post-diapause larvae and their host plants in a spring when larvae activated far prior to the onset of host plant growth (II). Larval growth was accelerated by warm microclimatic conditions, but survival was highest in cool microclimates. Flight season weather importantly altered annual dispersal rates of butterflies. Increasing solar radiation and proportion of exceptionally warm days increased emigration rates and shortened residence times in a natal patch (III). I simulated the spatial expansion of a translocated butterfly population in the novel release landscape based on prior estimates of habitat-specific dispersal rates in the source population. Dispersal rates were obtained from a model parameterised in a warm flight season, and consequently the simulated colonisation rates were overestimated in cold flight seasons. Warming flight season weather may enhance insect dispersal and colonisation on high-latitude and –altitude range margins with a potential to accelerate range expansions. On the other hand, increasing annual variation in weather may cause range retractions during years of unfavourable weather. Furthermore, increasingly variable weather makes insect conservation more difficult.
  • Eymann, Julia (Helsingin yliopisto, 2021)
    Vision is an important sensory modality for most vertebrates. The eye is a highly complex optical system at the center of which the neural retina acts as a sensory array. A stem cell population residing at the peripheral margin of the retina and Müller glia cells in the central retina mediate lifelong retinal growth and regeneration in fish and amphibians. The capacity for such retinal neurogenesis is reduced in birds and yet more limited in mammals. However, little is known about retinal development, postnatal growth and regeneration in squamate reptiles (lizards and snakes). Squamates occupy a central phylogenetic position among amniotes and display considerable morphological as well as functional diversity of the eye. This thesis explores various aspects of the squamate retina, particularly the neurogenic potential from embryonic development to postnatal growth and regeneration. First, the thesis elucidates the embryonic origins of the retinociliary junction (RCJ), a newly identified active stem/progenitor niche maintained in the postnatal retinal margin of squamates. The thesis then demonstrates a diverse spectrum of differing activity levels in the squamate RCJ. These variations in proliferative activity correlate with the diverse eye sizes, which is partly derived from differing ocular growth rates linked to RCJ activity. Next, although acute retinal damage does not stimulate a regenerative RCJ response, the thesis still highlights Müller glia cells as a potential source for regeneration in the squamate central retina. Finally, in the context of previously described vertebrate species, the thesis concludes that the squamate retina offers a new, more nuanced view on the field of vertebrate retinal growth and regeneration.
  • Lundberg, Piia (Helsingin yliopisto, 2021)
    My thesis combines conservation biology and conservation psychology to explore factors associated with environmental philanthropic behavior, and more specifically donating to conservation flagships aiming to improve their use in conservation marketing campaigns. Flagship species, usually charismatic and aesthetically appealing mammals and birds are used by environmental non-governmental organizations (ENGOs) in two ways: 1) when appealing to the potential donors to attract funds for conservation projects and 2) when raising conservation awareness of the public. In this thesis, I concentrate on flagship species’ usage from the conservation fundraising viewpoint. Because concentration on aesthetically pleasing species has also attracted criticism, there is a need to find additional ways to promote conservation projects. My aim is therefore to explore possibilities to use other donation targets in fundraising campaigns together with single species, as well as to find ways to help species and other targets, whose appearance is less attractive. This thesis builds upon a summary and four chapters. In Chapter I, I studied which kind of motivations and donor characteristics are associated with donating to ecosystems and species representing two taxonomic groups, mammals and birds. In the second chapter, I examined which flagship attributes are associated with the choices between flagships species and other flagship types including flagship fleets (a set of flagship species), ecosystems and biodiversity. The main aim of these two chapters was to study which kind of flagship types would be useful in fundraising campaigns, as well as to identify factors that make a conservation target appealing from the potential donors’ viewpoint. My goal was also to find out whether the potential donors can be divided into donor segments based on their preferences. Chapter III concentrates on real-life donation behavior. I measured both actual behavior by conducting a simple choice experiment and self-reported environmental philanthropic behavior by asking survey respondents questions about their past real-life donation behavior. The main aim was to study whether a variety of psychologic and sociodemographic variables are associated with donations of money and time, ENGO- membership and the amount donated. Chapter IV reviews literature on surrogacy analysis and willingness to pay studies of conservation flagships. The findings of my thesis emphasize the importance of segmenting environmental philanthropists based on their preferences. Common to all empirical studies in my thesis was that the potential donors favored both threatened targets as well as holistic flagship types that included biodiversity and ecosystems. According to these results, the range of donation targets in conservation fundraising campaigns could be wider than at present, although charismatic flagships also have their place as fundraising tools.
  • Herrero, Annika (Helsingin yliopisto, 2021)
    Large terrestrial carnivores are capable of long dispersal distances and thus have a potentially high rate of gene flow between populations. Even with such high mobility, discontinuous habitat and human-caused mortality may constrain dispersal and gene flow. Therefore, isolation of populations because of habitat fragmentation may cause genetic structuring in them because of genetic drift. In a continuous population, geographic barriers should not significantly affect dispersal and gene flow, so the effects of social, ecological and evolutionary forces are easier to detect. In large carnivores, males generally disperse more often and earlier than females and their dispersal distances are longer than those of females. The direction of sex-bias in dispersal is commonly explained by inbreeding avoidance, polygynous mating system and male-male competition. Remaining in, or near, the natal home range is explained by kin selection and inclusive fitness. Molecular evidence reveals the spatial genetic structure and clustering of relatives and family lines that may underlie these traits. We studied the spatial genetic relatedness, family structure, movement patterns and sex- bias of dispersal in the Eurasian lynx (Lynx lynx) in an unfragmented population in southern Finland. We used GIS and resource selection functions to analyse telemetry data of dispersing lynx and genetic methods to analyse data obtained from hunted lynx. Dispersal onset age, duration, distance, route or route linearity did not differ statistically between males and females that dispersed. However, the small number of females and the high variation in all dispersal parameters likely affected the outcome of analysis. Linear distance between the start and the end comprised only 20 % of the total dispersal route. Lynx selected their habitat non-randomly. During daylight hours lynx were more discerning in their habitat selection, while most of the traveling took place at night, reflecting the crepuscular and nocturnal activity of the lynx. According to the results of genetic analyses, the majority of females stayed close to their natal home range after reaching independence. Males dispersed and settled randomly in space. This led to genetic differentiation and spatial clustering of related females but not of males. Females form the backbone of the local populations, and genetic evidence is in line with the idea that females facilitate the settling of related females. In contrast to females, for males, relatedness is inversely important to avoid inbreeding. Hunting of adult lynx may disturb the forming of matrilineages and decrease genetic variation. Hunting should aim at mimicking a natural mortality pattern, which means hunting mostly young lynx, as the natural adult survival in Eurasian lynx is high.
  • Yan, Yan (Helsingin yliopisto, 2021)
    Blue and UV radiation are environmental cues or sources of information that can shape the morphology and development of plants. It was hypothesized that: H1) long-term treatments of solar blue (400–500 nm), long-UV (350–400 nm) and short-UV (290–350 nm) radiation (starting before seedling emergence) are perceived as different and can trigger distinct morphological, physiological and molecular responses; H2) parental long-term exposure to short-UV radiation before flowering affects response patterns to blue and UV radiation in the offspring; H3) long-term exposure to solar blue, long-UV and/or short-UV radiation enhances drought tolerance; H4) the responses in H1, H2 and H3 are accession-dependent and related to the environments where the accessions originate. To test these hypotheses, three experiments assessed morphological, physiological and molecular responses of accessions of two legume species, faba bean (Vicia faba L.) (I, II) and barrel medic (Medicago truncatula Gaertn.) (III). To impose radiation treatments by attenuating different wavebands of sunlight, four types of plastic filters were used in experiments I and III outdoors. Through pairwise filter comparisons, three different solar wavebands were assessed: blue, long-UV and short-UV radiation. In experiment I, two accessions of V. faba (Aurora; ILB938) originating from contrasting UV environments (southern Sweden; Andean region of Colombia and Ecuador) were grown under the four filters in sunlight. To study the transgenerational effect of solar short-UV radiation, experiment II was established using seeds produced by plants from experiment I and a factorial experiment design combining the two V. faba accessions, two parental UV treatments (full sunlight and exclusion of short-UV radiation) and four offspring light treatments, from the factorial combination of UVB and blue radiation manipulations in a controlled environment. In experiment III, the effect of long-term exposure to solar blue, long-UV and short-UV radiation during growth on the tolerance of subsequent progressive drought was studied in three M. truncatula accessions using the same filter treatments as in experiment I combined with progressive drought treatments imposed by withholding watering for 2 and 7 days to half the plants starting 40 days after sowing. The three M. truncatula accessions, Jemalong A17, HM006 and HM020, originate from Australia, France and Tunisia, respectively. After long-term natural light treatments (I, III), blue light but not long-UV or short-UV radiation, significantly regulated plant morphology and transcript abundance. In contrast, both solar blue and short-UV radiation, but not long-UV radiation, induced the accumulation of total flavonoids in leaves of V. faba (I) and M. truncatula (III). Moreover, simultaneous exposure to blue and UVB radiation had a synergetic effect on the induction of flavonoid accumulation (II). In V. faba, the variations of flavonoid composition and gene expression between the two accessions were consistent throughout the two successive generations (I, II). In V. faba, the transgenerational effect of short-UV radiation altered the morphological responses of the progenies to blue light, and it also affected flavonoid accumulation of the offspring in response to UVB radiation. Moreover, the transgenerational effects differed in the two accessions (II): in Aurora, the parental exposure to solar short-UV radiation led to a near-doubling of total quercetin concentration in response to UVB radiation in the progeny, while this was not observed in ILB938. The difference of responses to blue and UV radiation in these two accessions are consistent with adaptation to contrasting UV environments. In M. truncatula, long-term exposure to both solar blue and UV radiation pre-acclimated plants to subsequent slowly imposed drought, as observed in the transcriptomic result in accession Jemalong A17 that drought (2 and 7 days without watering) did not regulate differentially expressed genes (DEGs) under the filter transmitting blue and UV radiation. In contrast, drought increased transcript abundance of several previously described stress-inducible genes under all other filters. In the light of transcriptomic and flavonoid responses to filter and drought treatments, two processes potentially contribute to light-driven acclimation to drought: 1) increased flavonoid accumulation under blue and short-UV radiation could enhance the capability to scavenge drought-induced reactive oxygen species (ROS); 2) down-regulation of genes involved in light reactions of photosynthesis by blue light could reduce the generation of ROS when stomata close. In conclusion, under long-term sunlight treatment, blue light modified plants’ morphology and transcript change while both blue and short-UV radiation induced the accumulation of flavonoids; a transgenerational effect of short-UV radiation influenced offspring responses to blue and UVB radiation differently in the two accessions; both blue and UV radiation contributed to pre-acclimation toward subsequent drought by functioning as environmental cues rather than stressors even if the specific responses differed among accessions. Thus, the results support the four hypotheses.
  • Laine, Mikaela (Helsingin yliopisto, 2021)
    Psychiatric disorders are very common, with anxiety disorders being the most prevalent (16 % lifetime prevalence). While moderately heritable, their incidence is also strongly influenced by environmental risk factors, chiefly psychosocial stress. However, our knowledge of how, in neurobiological terms, these disorders arise is lacking, slowing down the development of efficient treatments. The aim of this thesis was firstly to identify which brain regions are recruited by chronic psychosocial stress, by using mice as model organisms. To model psychosocial stress we used chronic social defeat stress (CSDS), which involves short confrontations between intruder and resident-aggressor male mice, repeated daily for 10 consecutive days. A week after stress exposure C57BL/6NCrl (B6) mice had a higher number of cells expressing ΔFOSB, a marker of repeated neural activation, in several stress-related brain regions. These included the bed nucleus of the stria terminalis (BNST) and ventral hippocampus (vHPC). We also found significant correlations in the numbers of ΔFOSB+ cells between medial prefrontal cortex (mPFC) subregions (infralimbic and prelimbic cortices) and the vHPC of stress-exposed but not non-stressed control mice. Our second aim was to discover, by using unbiased RNA-sequencing (RNA-seq) of stress-related brain regions (the mPFC and vHPC), biological pathways perturbed by CSDS. Additionally, we used mice from two inbred strains, representing different levels of baseline anxiety-like behaviour: B6 (low-anxiety) and DBA/2NCrl (D2, high-anxiety). This enabled us to explore how genetic background modulates the response to stress. While some mice exposed to CSDS show social avoidance (called stress-susceptibility), others are behaviourally similar to non-stressed control mice. This phenomenon, known as resilience, is also observed in humans. We found that B6 and D2 mice showed vastly different behavioural responses to CSDS. B6 mice displayed a predominantly resilient phenotype (66.1 % of CSDS-exposed mice across several cohorts), while the majority (85.0 %) of D2 mice were susceptible. Pathway analysis of RNA-seq data suggested that differentially expressed genes (DEGs) were enriched in genes related to oligodendrocytes (OLGs), the myelin-producing cells of the CNS. For example, genes encoding myelin components were downregulated in the mPFC and vHPC of B6 susceptible mice compared to controls. Myelin is a lipid-rich ensheathment around axons, and it enables both fast nerve conduction and adjustment thereof via myelin plasticity. We followed up the gene expression findings with a structural analysis of myelinated axons using transmission electron microscopy (TEM). We found that in the vHPC, B6 susceptible mice had thinner myelin sheaths than controls. In the mPFC, B6 resilient mice had thicker myelin than controls, restricted to axons with small diameters. By contrast, D2 resilient mice had thinner myelin in this region than susceptible mice, indicating potential bidirectional dynamics of myelin plasticity in resilience. Lastly, we performed RNA-seq of enriched OLGs and myelin from the mPFC. The aim of this experiment was to identify pathways affected by CSDS specifically in these cells and in myelin. Additionally, we used this dataset to identify genes (24.8 % of all genes and 18.8 % of expressed micro-RNAs [miRNAs]) which were enriched in the myelin fraction compared to OLGs in nonstressed control mice. This suggests selective transport of certain mRNAs and microRNAs into the myelin sheath, potentially for local regulation. When comparing CSDS-exposed mice and controls, we found lower expression of myelin-related genes in B6 susceptible, D2 susceptible and D2 resilient mice compared to same-strain controls. In the B6 strain this was found in the myelin fraction, while in the D2 strain these genes were differentially expressed only in OLGs. Ingenuity Pathway Analysis (IPA) predicted TCF7L2, a transcription factor, to be an upstream regulator of these DEGs. In summary, we showed involvement of a broad but selective network of brain regions in CSDS. Genetic background had a large influence on the response to stress, highlighting a source of individual variation with implications for understanding human stress-resilience and -susceptibility. This moderation by genetic background was also seen at the level of gene expression in the brain. Finally, our findings suggest that myelin plasticity is an important part of the chronic response to stress. Future work will identify by which mechanisms stress influences myelin, and how this could be best harnessed for improving therapeutic strategies.
  • Ismail, Shamel (Helsingin yliopisto, 2020)
    Endoscopic retrograde cholangiopancreatography (ERCP) is a minimally invasive procedure for biliary and pancreatic disorders. It has evolved to predominantly therapeutic procedure with the diagnosis of primary sclerosing cholangitis (PSC) being one of the main exceptions. ERCP has the high potential for complications, like post ERCP pancreatitis (PEP), bleeding, cholangitis and perforation. Therefore, the data concerning the risk factors of ERCP is essential. The roles of preoperative laboratory testing and the new criteria of difficult biliary cannulation are poorly studied. ERCP plays also the major role in the endoscopic removal of papillary tumours (European Society of Gastrointestinal Endoscopy, European Association for the Study of the Liver, 2017). The main purpose of this study is to assess the safety of ERCP for different groups of patients, and the adverse effects that can follow. Additionally, the study examines the effect of using different tools during the procedure on the complication rate and the means to minimize the complication rates in the future. The main complications after ERCP are post ERCP pancreatitis (PEP), bleeding, cholangitis and perforation. The risk factors of ERCP complications in 441 consecutive PCS patients were retrospectively analysed. The outcome of 61 endoscopic papillectomies was retrospectively studied in study II. In the prospective study (III) of 821 consecutive patients with native papilla were evaluated the complication rates in difficult cannulation cases. To evaluate the relationship between ERCP complications and the need for routine preoperative laboratory tests (RPLT) before the ERCP procedure, we conducted prospective study (IV) with 1196 patients. In PSC patients (study I) the PEP and cholangitis rates were 7 and 1.4 %, respectively. PEP predictive factors are female sex (Odds ratio 2.6, p=0.015), and a guide wire slipping in the pancreatic duct (PD) (Odds ratio 8.2, p<0.01). The risk of PEP is directly proportional to the number of times the guide wire slips into the pancreatic duct. A previous endoscopic biliary sphincterotomy (EST) was found to be a protective factor from PEP (Odds ratio 0.28, p= 0.02). In benign tumours of the papilla (study II), the recurrence rate after resection was 25.5% (study II). Altogether 5 (9.5%) patients underwent a pancreaticoduodenectomy, and 46 out of 51 (90.5%) were treated endoscopically. Obstruction of the bile duct and jaundice as a manifestation were risk factors for malignancy (p<0.001). PEP occurred in 6 cases (9.8%). After a benign tumour resection and placing a stent, the PEP rate decreased (p=0.045). The bleeding rate was 18%. In study (III), when the primary cannulation method used to cannulate the bile duct succeeded, the PEP rate was 2.3%, but when the surgeon used advanced methods to achieve the cannulation, the PEP rate increased up to 13.5%. The primary cannulation success rate was 79% in the absence of difficult cannulation criteria compared to 20.4% when at least one criteria was present (p<0.001). Broad RPLT could not predict the adverse effects of ERCP in study (IV). In conclusion, the new criteria of difficult biliary cannulation are important because the complication risk increases with the complexity of cannulation. Endoscopic papillectomy for benign tumours is feasible and relatively safe. The role of RPLT in safety of ERCP is minimal.
  • Backer Johnsen, Hermanni (Helsingin yliopisto, 2020)
    This thesis studies the ecosystem approach (EA) which is a concept used in environmental science, policy, and law. It is widely referred to in protection of the world’s seas and oceans, including regional cooperation such as the work of The Baltic Marine Environment Protection Commission (HELCOM). In general, the ecosystem approach reflects the idea that management should be more integrated across different human activities but also that it should be based on, usually scientific, knowledge of ecosystems. More specifically, however, its substance remains a subject of debate. This thesis aims at contributing to the understanding of the ecosystem approach by studying and outlining a HELCOM interpretation as emerging from the implementation process 2003-2018. The thesis is based on the findings of five articles, four of which provide case studies on different aspects of EA implementation at HELCOM based on meeting records, literature research and first-hand knowledge. The fifth article is a study of HELCOM work 2003-2018 based on attendance and topics of 724 international meetings organised during the implementation period, types of organizational output and the outcomes of Ministerial Meetings. The summary derives a conceptual framework for EA with three elements, to report and discuss the findings of the five Articles. The thesis concludes that, as implemented within HELCOM, EA can be characterised by an element of quantification consisting of definitions and regulatory use of scientific targets of ecosystem quality, with roots in EU regulatory approaches and a different focus compared to the EA as defined within the Convention on Biological Diversity (CBD). A second element of integration is manifested in a significant expansion of new HELCOM activities in the fields of MSP, Fisheries, Agriculture and regional linkages to EU and global policies. However, regarding the third element, EA resulting in concrete management measures, has been a central component of implementation activities, but the main result seems to be in providing a more elaborate ways for proposing, justifying, specifying, and reporting the achievement of management action decided in other processes than HELCOM. The results of the fifth article indicate that the annual HELCOM work has doubled in volume during 2003-2018, measured as hours spent in meetings. This increase can be attributed to a general dilation, across all activities, as well as the new fields of work. Comparing the types of documents adopted by HELCOM in the beginning and end of the period 2003-2018 there is some evidence that a shift has taken place from technical specifications and concrete emission standards to more assessment products & indicators. Based on the data collected it is not possible to conclude whether these developments are a result of EA implementation as such, or primarily due to other factors such as the parallel, and closely intertwined, implementation of the EU Marine Strategy Framework Directive (MSFD). Due to its scientific nature the EA element of quantification is more an evolution of the traditional HELCOM monitoring and assessment work, however the integration element involves expansion of cooperation to new substance areas, professional groups, and worldviews. This work requires diplomatic effort and innovation for joint solutions to the environmental problems of the Baltic Sea. More focus on relevant human activities, drivers of change and monitoring progress in the implementation on key management measures affecting the state of the marine environment, but also scenarios and foresight could provide new avenues for the protection of the Baltic Sea. Keywords: sea, marine, ecosystem management, regional seas cooperation, Helsinki Convention, Baltic Sea, EU, Marine Strategy Framework Directive, marine spatial planning, MSP, international organization, meeting
  • Karki, Sudeep (Helsingin yliopisto, 2020)
    Synaptic adhesion molecules play a key role in the regulation of synapse development and maintenance. Several families of leucine-rich repeat (LRR) domain containing synaptic adhesion molecules have been characterized, including synaptic adhesion-like molecules (SALMs), and the LRR transmembrane (LRRTM) proteins. These proteins localize mostly in postsynaptic neurons in excitatory synapses and interact with presynaptic adhesion protein families; neurexin (NRXN) and leukocyte common antigen-related protein tyrosine phosphatases (LAR-RPTPs). Dysfunction of the synaptic adhesion molecules is linked to cognitive disorders, such as autism spectrum disorders and schizophrenia. This thesis work comprises the structural and functional study of SALM3 and SALM5 proteins from the SALM family, and their interaction with PTPσ from the LAR-RPTPs family. In addition, this thesis includes the work on the development of an inhibitory screening assay for synaptic adhesion molecules interactions, here targeting the LRRTM2-NRXN interaction. The SALM family proteins include five members, SALM1-5. We have solved the crystal structures of the mouse SALM5 LRR-Ig, and SALM3 LRR constructs at 3.1 Å and 2.8 Å resolution, respectively. Both the structures show the LRR domain mediating the dimerization, also verified by biophysical studies. We determined the binding affinity of SALM3 and SALM5 as 2-23 µM towards PTPσ, and solved the solution structure of the SALM3-PTPσ complex using small-angle X-ray scattering (SAXS), revealing a 2:2 complex formation similar to that observed for SALM5 and PTP. Based on our structure-function studies, SALM3 dimerization is vital for the SALM3-PTPσ complex to exert synaptogenic activity. We also developed an inhibitor screening method for the adhesion proteins interactions, focusing on the LRRTM2-NRXN interaction. We utilized the AlphaScreen technology to identify inhibitors with moderate IC50-values and established an orthogonal in-cell western blot assay to verify the obtained hits. This paves the way for the future development of high affinity compounds by further high throughput screening of larger compound libraries. The studies conducted in this thesis and the results have contributed to a better understanding of the role of SALM proteins in synapse formation, and possibly these structure-based studies will help to understand their implications in disease. The inhibitor screening assay developed can be adapted towards other synaptic adhesion interactions, and the inhibitors obtained could be used for functional studies, or towards drug discovery.
  • Sinha, Vishal (Helsingin yliopisto, 2020)
    The genetic aetiology of schizophrenia has been extensively studied in a nationwide collection of Finnish families since the late 1980s. Linkage and association studies of families ascertained for schizophrenia in Finland (SCZ) have continually identified the 1q42 region, and the Disrupted in Schizophrenia 1 (DISC1) gene in particular, as an important locus. Within the same SCZ cohort, an association at the genes for DISC1 interacting partners, the DISC1 network, including NDE1, PDE4D, PDE4B, and NDEL1, has also been observed, however, any true causal variants are yet to be identified. Thus, our research aims to identify variants at the DISC1 network of 27 genes, including DISC1, that can help us understand the origins of major mental illnesses, principally SCZ in Finland. We have used a three-stage sequencing and genotyping strategy to identify, validate, and replicate variants of interest. These were later studied in other cohorts ascertained for major mental illness, and in phenotypes with alternative traits. In total, 19,844 variants were identified through sequencing, and 93 variants were selected for genotyping in the larger SCZ cohort (n=1122) based on the association evidence and potential functional consequences. Variants associated in this larger cohort were again genotyped the rest of the SCZ cohort (n=1696), and in other major mental illness cohorts (n=2733). Through this work, we report a functional variant in the regulatory region of PDE4D associated with schizophrenia in a replicable manner, particularly in the psychotic and cognitive domains, with this variant significantly altering the amount PDE4D is expressed in the brain. We have further identified another sex-dependent functional variant in NDE1 gene associated within the females in the schizophrenia cohort, and is an eQTL for the NDE1 gene according to the GTEx database. Furthermore, a significant number of the gene expression changes are also significantly altered between the sexes, shedding light on to sex differences in psychiatric disorders and the sex-dependent association for NDE1. The analysis of DISC1 is currently ongoing, however, our initial findings have identified an exonic variant in DISC1 associating to both schizophrenia and anxiety disorders.
  • Virolainen-Arne, Eija (Helsingin yliopisto, 2020)
    Flooding stress and concomitant anoxia stress of plants has been in the focus of abiotic stress research for several decades. Despite that, there are several poorly studied areas in the field of flooding stress research such as responses of the anoxia-tolerant wild plant species and the function of plant mitochondria under oxygen deprivation stress. Contents and redox states of the small molecular antioxidants, that is ascorbate, glutathione and tocopherols, were determined in roots or rhizomes of four anoxia-tolerant and -intolerant plant species under anoxia and reoxygenation. Results of the study demonstrate that no correlation could be detected between the anoxia-tolerances of individual plant species and in the contents of given antioxidant induced by anoxia-reoxygenation stress but some correlation was observed between the redox states of glutathione pool and anoxia tolerances of the iris species of the study. Permeability transition was induced under high calcium ion concentration in wheat root mitochondria. Properties of the permeability transition in wheat mitochondria were characterized and results of the study demonstrated several similarities with the properties of the mammalian permeability transition. However, several features of the permeability transition observed in wheat root mitochondria diverge from the features found in plant mitochondria of different species or origin of tissue. Anaerobic ATP use of mitochondria isolated from roots of anoxia-sensitive wheat and anoxia-tolerant yellow flag iris was determined. Results of the study demonstrate that wheat mitochondria show high ATP hydrolyzing activity while mitochondria of yellow flag iris hydrolyze ATP molecules at a low rate under anoxia. Results of the study correlate well with the anoxia tolerances of the species referring to divergent survival strategies of the species under oxygen deprivation. While mitochondria of yellow flag iris, a species tolerating long-term anoxia, consume ATP at a sparing rate thus avoiding depletion of the ATP pool under anoxia, wheat mitochondria consume rapidly ATP molecules leading to abrupt ATP depletion and cell death. Investigating the mechanisms underlying plant anoxia-tolerance would benefit breeding of cultivated plants in order to have more flooding tolerant cultivars in future. Several aspects of plant anoxia-tolerance are still unknown and should be investigated such as properties and function of mitochondria in anoxia-tolerant wild plant species and function of the plant F1Fo-ATP synthase under anoxia.
  • Elovaara, Samu (Helsingin yliopisto, 2020)
    Cell lysis, as a consequence of adverse conditions, has been recognized as an important loss process among phytoplankton, in addition to the well-known loss processes of grazing and sinking. Cell lysis has been connected to increased release of carbon fixed by phytoplankton as dissolved organic carbon (DOC), the primary carbon source for pelagic heterotrophic bacteria. This has the potential to enhance pelagic remineralization at the cost of reduced sedimentation of organic carbon. Cell lysis may, therefore, have global consequences as the ratio of pelagic remineralization to sedimentation widely determines whether oceans function as a source or a sink of atmospheric carbon. However, the subject has been studied predominantly in oceans and oligotrophic marine regions. The Baltic Sea is different from these environments and the causes and consequences of phytoplankton cell lysis may, therefore, be expected to differ. The studies included in this thesis are the first attempt to study phytoplankton cell lysis and its effect on carbon cycling in the Baltic Sea. The focus of the thesis is mainly on elucidating the abiotic and biological controls of cell lysis and its relationship with pelagic DOC concentration. These were studied on a spatial scale during a spring bloom on an area covering the Gulf of Finland, the Åland Sea and the Baltic Proper, and on a temporal scale during a two-year long monitoring campaign in an estuary in the northern Gulf of Finland. In both studies the proportion of cells undergoing lysis was measured using a membrane impermeable nucleic acid stain to indicate cells with compromised membrane integrity. The spatial monitoring study revealed considerable variation in the proportion of cells undergoing lysis with generally higher proportion of dying cells in deep water (1-10 m: average 84%, range 67-91%; 30 m: average 77%, range 62-90%; 60 m: average 71%, range 58-86%) and among nanophytoplankton (surface water average: 64%), as compared to smaller eukaryotic picophytoplankton (surface water average: 88%) and picocyanobacteria (surface water average: 82%). No clear correlations between cell lysis and nutrient concentrations were found, although there was a weak correlation between the proportion of intact eukaryotic picophytoplankton and phosphate concentration (R2 = 0.13, p = 0.029). No connection between cell lysis and DOC concentration was found. Also during the temporal monitoring campaign variation of cells undergoing lysis was high (surface water average: 62%, range 18-97%). Again, no correlation between nutrient concentrations and cell lysis was found, although this time there was a weak negative relationship between the proportion of cells undergoing lysis and DOC concentration (R2 = 0.15, p = 0.0185). In both studies some indication was found that phytoplankton lysis is less prevalent in conditions where interspecific phytoplankton competition is low. Details of the flow of carbon from phytoplankton to pelagic heterotrophic bacteria was studied experimentally using two phytoplankton species (a dinoflagellate Apocalathium malmogiense and a cryptophyte Rhodomonas marina). Contrasting species specific differences were found in their ability to transfer carbon from the inorganic pool via DOC to bacterial biomass and in the composition of the emerging bacterial community. The smaller R. marina released more bioavailable DOC and attracted a bacterial community mainly consisting of copiotrophs (bacteria thriving when DOC is abundant and highly bioavailable), which likely directs more carbon towards microbial loop. The DOC released by the larger A. malmogiense was less bioavailable. If these results can be generalized to other taxa of similar size, the fast consumption of DOC released by R. marina may partially explain why no relationship between the lysis of small phytoplankton and DOC concentration was found. The overarching conclusion from the two field studies is that the environmental conditions, such as nutrient limitation, that have been shown to promote cell lysis in oligotrophic marine regions are not the main determinants of cell lysis in the Baltic Sea. Also, the high ambient DOC concentration and terrestrial runoff in the Baltic Sea seem to mask the effect of cell lysis on DOC concentration. The group and species specific differences in both cell lysis and carbon cycling indicate that investigating cell lysis on lower taxonomic levels will help to connect cell lysis to carbon cycling.